Combining Optical Coherence Tomography with Fluorescence Molecular Imaging: Towards Simultaneous Morphology and Molecular Imaging

PubMed Central

Yuan, Shuai; Roney, Celeste A.; Wierwille, Jerry; Chen, Chao-Wei; Xu, Biying; Jiang, James; Ma, Hongzhou; Cable, Alex; Summers, Ronald M.; Chen, Yu

2010-01-01

Optical coherence tomography (OCT) provides high-resolution, cross-sectional imaging of tissue microstructure in situ and in real-time, while fluorescence molecular imaging (FMI) enables the visualization of basic molecular processes. There are great interests in combining these two modalities so that the tissue's structural and molecular information can be obtained simultaneously. This could greatly benefit biomedical applications such as detecting early diseases and monitoring therapeutic interventions. In this research, an optical system that combines OCT and FMI was developed. The system demonstrated that it could co-register en face OCT and FMI images with a 2.4 × 2.4 mm field of view. The transverse resolutions of OCT and FMI of the system are both ~10 μm. Capillary tubes filled with fluorescent dye Cy 5.5 in different concentrations under a scattering medium are used as the phantom. En face OCT images of the phantoms were obtained and successfully co-registered with FMI images that were acquired simultaneously. A linear relationship between FMI intensity and dye concentration was observed. The relationship between FMI intensity and target fluorescence tube depth measured by OCT images was also observed and compared with theoretical modeling. This relationship could help in correcting reconstructed dye concentration. Imaging of colon polyps of APCmin mouse model is presented as an example of biological applications of this co-registered OCT/FMI system. PMID:20009192

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilson, Matthew D., E-mail: Matt.Wilson@stfc.ac.uk; Seller, Paul; Veale, Matthew C.

A novel, “single-shot” fluorescence imaging technique has been demonstrated on the B16 beamline at the Diamond Light Source synchrotron using the HEXITEC energy dispersive imaging detector. A custom made furnace with 200µm thick metal alloy samples was positioned in a white X-ray beam with a hole made in the furnace walls to allow the transmitted beam to be imaged with a conventional X-ray imaging camera consisting of a 500 µm thick single crystal LYSO scintillator, mirror and lens coupled to an AVT Manta G125B CCD sensor. The samples were positioned 45° to the incident beam to enable simultaneous transmission andmore » fluorescence imaging. The HEXITEC detector was positioned at 90° to the sample with a 50 µm pinhole 13 cm from the sample and the detector positioned 2.3m from pinhole. The geometric magnification provided a field of view of 1.1×1.1mm{sup 2} with one of the 80×80 pixels imaging an area equivalent to 13µm{sup 2}. Al-Cu alloys doped with Zr, Ag and Mo were imaged in transmission and fluorescence mode. The fluorescence images showed that the dopant metals could be simultaneously imaged with sufficient counts on all 80x80 pixels within 60 s, with the X-ray flux limiting the fluorescence imaging rate. This technique demonstrated that it is possible to simultaneously image and identify multiple elements on a spatial resolution scale ~10µm or higher without the time consuming need to scan monochromatic energies or raster scan a focused beam of X-rays. Moving to high flux beamlines and using an array of detectors could improve the imaging speed of the technique with element specific imaging estimated to be on a 1 s timescale.« less

Fluorescence Behavioral Imaging (FBI) Tracks Identity in Heterogeneous Groups of Drosophila

PubMed Central

Ramdya, Pavan; Schaffter, Thomas; Floreano, Dario; Benton, Richard

2012-01-01

Distinguishing subpopulations in group behavioral experiments can reveal the impact of differences in genetic, pharmacological and life-histories on social interactions and decision-making. Here we describe Fluorescence Behavioral Imaging (FBI), a toolkit that uses transgenic fluorescence to discriminate subpopulations, imaging hardware that simultaneously records behavior and fluorescence expression, and open-source software for automated, high-accuracy determination of genetic identity. Using FBI, we measure courtship partner choice in genetically mixed groups of Drosophila. PMID:23144871

Fluorescence behavioral imaging (FBI) tracks identity in heterogeneous groups of Drosophila.

PubMed

Ramdya, Pavan; Schaffter, Thomas; Floreano, Dario; Benton, Richard

2012-01-01

Distinguishing subpopulations in group behavioral experiments can reveal the impact of differences in genetic, pharmacological and life-histories on social interactions and decision-making. Here we describe Fluorescence Behavioral Imaging (FBI), a toolkit that uses transgenic fluorescence to discriminate subpopulations, imaging hardware that simultaneously records behavior and fluorescence expression, and open-source software for automated, high-accuracy determination of genetic identity. Using FBI, we measure courtship partner choice in genetically mixed groups of Drosophila.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shcheslavskiy, V. I.; Institute of Biomedical Technologies, Nizhny Novgorod State Medical Academy, Minin and Pozharsky Square, 10/1, Nizhny Novgorod 603005; Neubauer, A.

We present a lifetime imaging technique that simultaneously records the fluorescence and phosphorescence lifetime images in confocal laser scanning systems. It is based on modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multidimensional time-correlated single photon counting board. We demonstrate our technique on the recording of the fluorescence/phosphorescence lifetime images of human embryonic kidney cells at different environmental conditions.

Fluorescence imaging in the upper gastrointestinal tract for the detection of dysplasic changes

NASA Astrophysics Data System (ADS)

Sukowski, Uwe; Ebert, Bernd; Ortner, Marianne; Mueller, Karsten; Voderholzer, W.; Weber-Eibel, J.; Dietel, M.; Lochs, Herbert; Rinneberg, Herbert H.

2001-10-01

During endoscopy of the esophagus fluorescence images were recorded at a delay of 20 ns after pulsed laser excitation simultaneously with conventional reflected white light images. To label malignant cells (dysplasia, tumor) 5-aminolaevulinic acid was applied prior to fluorescence guided bi-opsy. In this way pre-malignant and malignant lesions were detected not seen previously during routine endoscopy.

In vivo tomographic imaging of lung colonization of tumour in mouse with simultaneous fluorescence and X-ray CT.

PubMed

Zhang, Bin; Gao, Fuping; Wang, Mengjiao; Cao, Xu; Liu, Fei; Wang, Xin; Luo, Jianwen; Wang, Guangzhi; Bai, Jing

2014-01-01

Non-invasive in vivo imaging of diffuse and wide-spread colonization within the lungs, rather than distinct solid primary tumors, is still a challenging work. In this work, a lung colonization mouse model bearing A549 human lung tumor was simultaneously scanned by a dual-modality fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) system in vivo. A two steps method which incorporates CT structural information into the FMT reconstruction procedure is employed to provide concurrent anatomical and functional information. By using the target-specific fluorescence agent, the fluorescence tomographic results show elevated fluorescence intensity deep within the lungs which is colonized with diffuse and wide-spread tumors. The results were confirmed with ex vivo fluorescence reflectance imaging and histological examination of the lung tissues. With FMT reconstruction combined with the CT information, the dual-modality FMT/micro-CT system is expected to offer sensitive and noninvasive imaging of diffuse tumor colonization within the lungs in vivo. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous fluorescence and quantitative phase microscopy with single-pixel detectors

NASA Astrophysics Data System (ADS)

Liu, Yang; Suo, Jinli; Zhang, Yuanlong; Dai, Qionghai

2018-02-01

Multimodal microscopy offers high flexibilities for biomedical observation and diagnosis. Conventional multimodal approaches either use multiple cameras or a single camera spatially multiplexing different modes. The former needs expertise demanding alignment and the latter suffers from limited spatial resolution. Here, we report an alignment-free full-resolution simultaneous fluorescence and quantitative phase imaging approach using single-pixel detectors. By combining reference-free interferometry with single-pixel detection, we encode the phase and fluorescence of the sample in two detection arms at the same time. Then we employ structured illumination and the correlated measurements between the sample and the illuminations for reconstruction. The recovered fluorescence and phase images are inherently aligned thanks to single-pixel detection. To validate the proposed method, we built a proof-of-concept setup for first imaging the phase of etched glass with the depth of a few hundred nanometers and then imaging the fluorescence and phase of the quantum dot drop. This method holds great potential for multispectral fluorescence microscopy with additional single-pixel detectors or a spectrometer. Besides, this cost-efficient multimodal system might find broad applications in biomedical science and neuroscience.

Simultaneous dual-color fluorescence microscope: a characterization study.

PubMed

Li, Zheng; Chen, Xiaodong; Ren, Liqiang; Song, Jie; Li, Yuhua; Zheng, Bin; Liu, Hong

2013-01-01

High spatial resolution and geometric accuracy is crucial for chromosomal analysis of clinical cytogenetic applications. High resolution and rapid simultaneous acquisition of multiple fluorescent wavelengths can be achieved by utilizing concurrent imaging with multiple detectors. However, such class of microscopic systems functions differently from traditional fluorescence microscopes. To develop a practical characterization framework to assess and optimize the performance of a high resolution and dual-color fluorescence microscope designed for clinical chromosomal analysis. A dual-band microscopic imaging system utilizes a dichroic mirror, two sets of specially selected optical filters, and two detectors to simultaneously acquire two fluorescent wavelengths. The system's geometric distortion, linearity, the modulation transfer function, and the dual detectors' alignment were characterized. Experiment results show that the geometric distortion at lens periphery is less than 1%. Both fluorescent channels show linear signal responses, but there exists discrepancy between the two due to the detectors' non-uniform response ratio to different wavelengths. In terms of the spatial resolution, the two contrast transfer function curves trend agreeably with the spatial frequency. The alignment measurement allows quantitatively assessing the cameras' alignment. A result image of adjusted alignment is demonstrated to show the reduced discrepancy by using the alignment measurement method. In this paper, we present a system characterization study and its methods for a specially designed imaging system for clinical cytogenetic applications. The presented characterization methods are not only unique to this dual-color imaging system but also applicable to evaluation and optimization of other similar multi-color microscopic image systems for improving their clinical utilities for future cytogenetic applications.

Dynamic Measurement of Tumor Vascular Permeability and Perfusion using a Hybrid System for Simultaneous Magnetic Resonance and Fluorescence Imaging.

PubMed

Ren, Wuwei; Elmer, Andreas; Buehlmann, David; Augath, Mark-Aurel; Vats, Divya; Ripoll, Jorge; Rudin, Markus

2016-04-01

Assessing tumor vascular features including permeability and perfusion is essential for diagnostic and therapeutic purposes. The aim of this study was to compare fluorescence and magnetic resonance imaging (MRI)-based vascular readouts in subcutaneously implanted tumors in mice by simultaneous dynamic measurement of tracer uptake using a hybrid fluorescence molecular tomography (FMT)/MRI system. Vascular permeability was measured using a mixture of extravascular imaging agents, GdDOTA and the dye Cy5.5, and perfusion using a mixture of intravascular agents, Endorem and a fluorescent probe (Angiosense). Dynamic fluorescence reflectance imaging (dFRI) was integrated into the hybrid system for high temporal resolution. Excellent correspondence between uptake curves of Cy5.5/GdDOTA and Endorem/Angiosense has been found with correlation coefficients R > 0.98. The two modalities revealed good agreement regarding permeability coefficients and centers-of-gravity of the imaging agent distribution. The FMT/dFRI protocol presented is able to accurately map physiological processes and poses an attractive alternative to MRI for characterizing tumor neoangiogenesis.

Detecting fluorescence hot-spots using mosaic maps generated from multimodal endoscope imaging

NASA Astrophysics Data System (ADS)

Yang, Chenying; Soper, Timothy D.; Seibel, Eric J.

2013-03-01

Fluorescence labeled biomarkers can be detected during endoscopy to guide early cancer biopsies, such as high-grade dysplasia in Barrett's Esophagus. To enhance intraoperative visualization of the fluorescence hot-spots, a mosaicking technique was developed to create full anatomical maps of the lower esophagus and associated fluorescent hot-spots. The resultant mosaic map contains overlaid reflectance and fluorescence images. It can be used to assist biopsy and document findings. The mosaicking algorithm uses reflectance images to calculate image registration between successive frames, and apply this registration to simultaneously acquired fluorescence images. During this mosaicking process, the fluorescence signal is enhanced through multi-frame averaging. Preliminary results showed that the technique promises to enhance the detectability of the hot-spots due to enhanced fluorescence signal.

Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection

PubMed Central

Bates, Mark; Dempsey, Graham T; Chen, Kok Hao; Zhuang, Xiaowei

2012-01-01

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution. PMID:22213647

Single-Shot Optical Sectioning Using Two-Color Probes in HiLo Fluorescence Microscopy

PubMed Central

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-01-01

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. PMID:21641327

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, E.S.; Taylor, J.A.

1996-03-12

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis. 14 figs.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, E.S.; Taylor, J.A.

1994-06-28

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis. 14 figures.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, Edward S.; Taylor, John A.

1996-03-12

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, Edward S.; Taylor, John A.

1994-06-28

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis.

Intraoperative Fluorescence Cerebral Angiography by Laser Surgical Microscopy: Comparison With Xenon Microscopy and Simultaneous Observation of Cerebral Blood Flow and Surrounding Structures.

PubMed

Ito, Yuhei; Suzuki, Kyouichi; Ichikawa, Tsuyoshi; Watanabe, Yoichi; Sato, Taku; Sakuma, Jun; Saito, Kiyoshi

2018-06-12

Laser surgical microscopes should enable uniform illumination of the operative field, and require less luminous energy compared with existing xenon surgical microscopes. To examine the utility of laser illumination in fluorescence cerebral angiography. Fluorescein sodium (fluorescein) was used as a fluorescent dye. We first compared the clarity of cerebral blood flow images collected by fluorescence angiography between the laser illumination and xenon illumination methods. We then assessed use of the laser illuminator for simultaneous observation of blood flow and surrounding structures during fluorescence angiography. Furthermore, the study was designed to evaluate usefulness of the thus determined excitation light in clinical cases. Fluorescence angiography using blue light laser for excitation provided higher clarity and contrast blood flow images compared with using blue light generated from a xenon lamp. Further, illumination with excitation light consisting of a combination of 3 types of laser (higher level of blue light, no green light, and lower level of red light) enabled both blood flow and surrounding structures to be observed through the microscope directly by the surgeon. Laser-illuminated fluorescence angiography provides high clarity and contrast images of cerebral blood flow. Further, a laser providing strong blue light and weak red light for excitation light enables simultaneous visual observation of fluorescent blood flow and surrounding structures by the surgeon using a surgical microscope. Overall, these data suggest that laser surgical microscopes are useful for both ordinary operative manipulations and fluorescence angiography.

Multispectral laser-induced fluorescence imaging system for large biological samples

NASA Astrophysics Data System (ADS)

Kim, Moon S.; Lefcourt, Alan M.; Chen, Yud-Ren

2003-07-01

A laser-induced fluorescence imaging system developed to capture multispectral fluorescence emission images simultaneously from a relatively large target object is described. With an expanded, 355-nm Nd:YAG laser as the excitation source, the system captures fluorescence emission images in the blue, green, red, and far-red regions of the spectrum centered at 450, 550, 678, and 730 nm, respectively, from a 30-cm-diameter target area in ambient light. Images of apples and of pork meat artificially contaminated with diluted animal feces have demonstrated the versatility of fluorescence imaging techniques for potential applications in food safety inspection. Regions of contamination, including sites that were not readily visible to the human eye, could easily be identified from the images.

Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue

PubMed Central

Scolaro, Loretta; Lorenser, Dirk; Madore, Wendy-Julie; Kirk, Rodney W.; Kramer, Anne S.; Yeoh, George C.; Godbout, Nicolas; Sampson, David D.; Boudoux, Caroline; McLaughlin, Robert A.

2015-01-01

Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue. PMID:26137379

Co-registered photoacoustic and fluorescent imaging of a switchable nanoprobe based on J-aggregates of indocyanine green

NASA Astrophysics Data System (ADS)

Dumani, Diego S.; Brecht, Hans-Peter; Ivanov, Vassili; Deschner, Ryan; Harris, Justin T.; Homan, Kimberly A.; Cook, Jason R.; Emelianov, Stanislav Y.; Ermilov, Sergey A.

2018-02-01

We introduce a preclinical imaging platform - a 3D photoacoustic/fluorescence tomography (PAFT) instrument augmented with an environmentally responsive dual-contrast biocompatible nanoprobe. The PAFT instrument was designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct co-registration of the two imaging modalities. The nanoprobe was based on liposomes loaded with J-aggregates of indocyanine green (PAtrace). Once PAtrace interacts with the environment, a transition from J-aggregate to monomeric ICG is induced. The subsequent recovery of monomeric ICG is characterized by dramatic changes in the optical absorption spectrum and reinstated fluorescence. In the activated state, PAtrace can be simultaneously detected by both imaging modes of the PAFT instrument using 780 nm excitation and fluorescence detection at 810 nm. The fluorescence imaging component is used to boost detection sensitivity by providing lowresolution map of activated nanoprobes, which are then more precisely mapped in 3D by the photoacoustic imaging component. Activated vs non-activated particles can be distinguished based on their different optical absorption peaks, removing the requirements for complex image registration between reference and detection scans. Preliminary phantom and in vivo animal imaging results showed successful activation and visualization of PAtrace with high sensitivity and resolution. The proposed PAFT-PAtrace imaging platform could be used in various functional and molecular imaging applications including multi-point in vivo assessment of early metastasis.

Hyperspectral imaging to monitor simultaneously multiple protein subtypes and live track their spatial dynamics: a new platform to screen drugs for CNS diseases

NASA Astrophysics Data System (ADS)

Labrecque, S.; Sylvestre, J.-P.; Marcet, S.; Mangiarini, F.; Verhaegen, M.; De Koninck, P.; Blais-Ouellette, S.

2015-03-01

In the past decade, the efficacy of existing therapies and the discovery of innovative treatments for Central Nervous System (CNS) diseases have been limited by the lack of appropriate methods to investigate complex molecular processes at the synaptic level. In order to better understand the fundamental mechanisms that regulate diseases of the CNS, a fast fluorescence hyperspectral imaging platform was designed to track simultaneously various neurotransmitter receptors trafficking in and out of synapses. With this hyperspectral imaging platform, it was possible to image simultaneously five different synaptic proteins, including subtypes of glutamate receptors (mGluR, NMDAR, AMPAR), postsynaptic density proteins, and signaling proteins. This new imaging platform allows fast simultaneous acquisitions of at least five fluorescent markers in living neurons with a high spatial resolution. This technique provides an effective method to observe several synaptic proteins at the same time, thus study how drugs for CNS impact the spatial dynamics of these proteins.

Fluorescence optical imaging in anticancer drug delivery.

PubMed

Etrych, Tomáš; Lucas, Henrike; Janoušková, Olga; Chytil, Petr; Mueller, Thomas; Mäder, Karsten

2016-03-28

In the past several decades, nanosized drug delivery systems with various targeting functions and controlled drug release capabilities inside targeted tissues or cells have been intensively studied. Understanding their pharmacokinetic properties is crucial for the successful transition of this research into clinical practice. Among others, fluorescence imaging has become one of the most commonly used imaging tools in pre-clinical research. The development of increasing numbers of suitable fluorescent dyes excitable in the visible to near-infrared wavelengths of the spectrum has significantly expanded the applicability of fluorescence imaging. This paper focuses on the potential applications and limitations of non-invasive imaging techniques in the field of drug delivery, especially in anticancer therapy. Fluorescent imaging at both the cellular and systemic levels is discussed in detail. Additionally, we explore the possibility for simultaneous treatment and imaging using theranostics and combinations of different imaging techniques, e.g., fluorescence imaging with computed tomography. Copyright © 2016 Elsevier B.V. All rights reserved.

A Dual-Modality System for Both Multi-Color Ultrasound-Switchable Fluorescence and Ultrasound Imaging

PubMed Central

Kandukuri, Jayanth; Yu, Shuai; Cheng, Bingbing; Bandi, Venugopal; D’Souza, Francis; Nguyen, Kytai T.; Hong, Yi; Yuan, Baohong

2017-01-01

Simultaneous imaging of multiple targets (SIMT) in opaque biological tissues is an important goal for molecular imaging in the future. Multi-color fluorescence imaging in deep tissues is a promising technology to reach this goal. In this work, we developed a dual-modality imaging system by combining our recently developed ultrasound-switchable fluorescence (USF) imaging technology with the conventional ultrasound (US) B-mode imaging. This dual-modality system can simultaneously image tissue acoustic structure information and multi-color fluorophores in centimeter-deep tissue with comparable spatial resolutions. To conduct USF imaging on the same plane (i.e., x-z plane) as US imaging, we adopted two 90°-crossed ultrasound transducers with an overlapped focal region, while the US transducer (the third one) was positioned at the center of these two USF transducers. Thus, the axial resolution of USF is close to the lateral resolution, which allows a point-by-point USF scanning on the same plane as the US imaging. Both multi-color USF and ultrasound imaging of a tissue phantom were demonstrated. PMID:28165390

Simultaneous imaging of fuel vapor mass fraction and gas-phase temperature inside gasoline sprays using two-line excitation tracer planar laser-induced fluorescence.

PubMed

Zigan, Lars; Trost, Johannes; Leipertz, Alfred

2016-02-20

This paper reports for the first time, to the best of our knowledge, on the simultaneous imaging of the gas-phase temperature and fuel vapor mass fraction distribution in a direct-injection spark-ignition (DISI) spray under engine-relevant conditions using tracer planar laser-induced fluorescence (TPLIF). For measurements in the spray, the fluorescence tracer 3-pentanone is added to the nonfluorescent surrogate fuel iso-octane, which is excited quasi-simultaneously by two different excimer lasers for two-line excitation LIF. The gas-phase temperature of the mixture of fuel vapor and surrounding gas and the fuel vapor mass fraction can be calculated from the two LIF signals. The measurements are conducted in a high-temperature, high-pressure injection chamber. The fluorescence calibration of the tracer was executed in a flow cell and extended significantly compared to the existing database. A detailed error analysis for both calibration and measurement is provided. Simultaneous single-shot gas-phase temperature and fuel vapor mass fraction fields are processed for the assessment of cyclic spray fluctuations.

A pH-sensitive red fluorescent protein compatible with hydrophobic resin embedding

NASA Astrophysics Data System (ADS)

Guo, Wenyan; Gang, Yadong; Liu, Xiuli; Zhou, Hongfu; Zeng, Shaoqun

2017-02-01

pH sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EYFP or EGFP improved from GFP in jellyfish are good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is of urgent need. Here a pH sensitive red fluorescent protein, pHuji, is selected and verified to be compatible with hydrophobic resin embedding and thus may be promising for dual-colour chemical reactivation imaging in conjunction with EGFP or EYFP.

In Vivo Deep Tissue Fluorescence and Magnetic Imaging Employing Hybrid Nanostructures.

PubMed

Ortgies, Dirk H; de la Cueva, Leonor; Del Rosal, Blanca; Sanz-Rodríguez, Francisco; Fernández, Nuria; Iglesias-de la Cruz, M Carmen; Salas, Gorka; Cabrera, David; Teran, Francisco J; Jaque, Daniel; Martín Rodríguez, Emma

2016-01-20

Breakthroughs in nanotechnology have made it possible to integrate different nanoparticles in one single hybrid nanostructure (HNS), constituting multifunctional nanosized sensors, carriers, and probes with great potential in the life sciences. In addition, such nanostructures could also offer therapeutic capabilities to achieve a wider variety of multifunctionalities. In this work, the encapsulation of both magnetic and infrared emitting nanoparticles into a polymeric matrix leads to a magnetic-fluorescent HNS with multimodal magnetic-fluorescent imaging abilities. The magnetic-fluorescent HNS are capable of simultaneous magnetic resonance imaging and deep tissue infrared fluorescence imaging, overcoming the tissue penetration limits of classical visible-light based optical imaging as reported here in living mice. Additionally, their applicability for magnetic heating in potential hyperthermia treatments is assessed.

Simultaneous optical and electrical recording of a single ion-channel.

PubMed

Ide, Toru; Takeuchi, Yuko; Aoki, Takaaki; Yanagida, Toshio

2002-10-01

In recent years, the single-molecule imaging technique has proven to be a valuable tool in solving many basic problems in biophysics. The technique used to measure single-molecule functions was initially developed to study electrophysiological properties of channel proteins. However, the technology to visualize single channels at work has not received as much attention. In this study, we have for the first time, simultaneously measured the optical and electrical properties of single-channel proteins. The large conductance calcium-activated potassium channel (BK-channel) labeled with fluorescent dye molecules was incorporated into a planar bilayer membrane and the fluorescent image captured with a total internal reflection fluorescence microscope simultaneously with single-channel current recording. This innovative technology will greatly advance the study of channel proteins as well as signal transduction processes that involve ion permeation processes.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

NASA Astrophysics Data System (ADS)

Duman, M.; Pfleger, M.; Zhu, R.; Rankl, C.; Chtcheglova, L. A.; Neundlinger, I.; Bozna, B. L.; Mayer, B.; Salio, M.; Shepherd, D.; Polzella, P.; Moertelmaier, M.; Kada, G.; Ebner, A.; Dieudonne, M.; Schütz, G. J.; Cerundolo, V.; Kienberger, F.; Hinterdorfer, P.

2010-03-01

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ~ 25 to ~ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

PubMed

Duman, M; Pfleger, M; Zhu, R; Rankl, C; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Mayer, B; Salio, M; Shepherd, D; Polzella, P; Moertelmaier, M; Kada, G; Ebner, A; Dieudonne, M; Schütz, G J; Cerundolo, V; Kienberger, F; Hinterdorfer, P

2010-03-19

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

An endoscopic fluorescence imaging system for simultaneous visual examination and photodetection of cancers

NASA Astrophysics Data System (ADS)

Wagnières, Georges A.; Studzinski, André P.; van den Bergh, Hubert E.

1997-01-01

We describe the design and performance tested during six years of clinical trials of a fluorescence endoscope for the detection and delineation of cancers in several hollow organs. The apparatus is based on the imaging of the laser-induced fluorescence that differs between a tumor and its surrounding normal tissue. The tests are carried out in the upper aerodigestive tract, the tracheobronchial tree, the esophagus, and the colon. In the three former cases an exogenous dye is used (Photofrin II), whereas in the latter case fluorescein molecules conjugated with monoclonal antibodies directed against carcinoembryonic antigen are injected. The decrease of native tissue autofluorescence observed in early cancers is also used for detecting lesions in the tracheobronchial tree. The fluorescence contrast between the tumor and surrounding normal tissue is enhanced by real time image processing. This is done by simultaneously recording the fluorescence image in two spectral domains, after which these two images are digitized and manipulated with a mathematical operator (look-up table) at video frequency. Moreover, the device that is described below allows for an immediate observation of the endoscopic area under white light illumination during fluorescence detection in order to localize the origin of the "positive" fluorescence signals. Typical results obtained in the tracheobronchial tree and in the colon are presented and the sources of false positives and false negatives are evaluated in terms of the fluorescent dye, tissue optical properties, and illumination optics.

Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer

PubMed Central

Elliott, Amicia D.; Gao, Liang; Ustione, Alessandro; Bedard, Noah; Kester, Robert; Piston, David W.; Tkaczyk, Tomasz S.

2012-01-01

Summary The development of multi-colored fluorescent proteins, nanocrystals and organic fluorophores, along with the resulting engineered biosensors, has revolutionized the study of protein localization and dynamics in living cells. Hyperspectral imaging has proven to be a useful approach for such studies, but this technique is often limited by low signal and insufficient temporal resolution. Here, we present an implementation of a snapshot hyperspectral imaging device, the image mapping spectrometer (IMS), which acquires full spectral information simultaneously from each pixel in the field without scanning. The IMS is capable of real-time signal capture from multiple fluorophores with high collection efficiency (∼65%) and image acquisition rate (up to 7.2 fps). To demonstrate the capabilities of the IMS in cellular applications, we have combined fluorescent protein (FP)-FRET and [Ca2+]i biosensors to measure simultaneously intracellular cAMP and [Ca2+]i signaling in pancreatic β-cells. Additionally, we have compared quantitatively the IMS detection efficiency with a laser-scanning confocal microscope. PMID:22854044

A portable near-infrared fluorescence image overlay device for surgical navigation (Conference Presentation)

NASA Astrophysics Data System (ADS)

McWade, Melanie A.

2016-03-01

A rise in the use of near-infrared (NIR) fluorescent dyes or intrinsic fluorescent markers for surgical guidance and tissue diagnosis has triggered the development of NIR fluorescence imaging systems. Because NIR wavelengths are invisible to the naked eye, instrumentation must allow surgeons to visualize areas of high fluorescence. Current NIR fluorescence imaging systems have limited ease-of-use because they display fluorescent information on remote display monitors that require surgeons to divert attention away from the patient to identify the location of tissue fluorescence. Furthermore, some systems lack simultaneous visible light imaging which provides valuable spatial context to fluorescence images. We have developed a novel, portable NIR fluorescence imaging approach for intraoperative surgical guidance that provides information for surgical navigation within the clinician's line of sight. The system utilizes a NIR CMOS detector to collect excited NIR fluorescence from the surgical field. Tissues with NIR fluorescence are overlaid with visible light to provide information on tissue margins directly on the surgical field. In vitro studies have shown this versatile imaging system can be applied to applications with both extrinsic NIR contrast agents such as indocyanine green and weaker sources of biological fluorescence such as parathyroid gland tissue. This non-invasive, portable NIR fluorescence imaging system overlays an image directly on tissue, potentially allowing surgical decisions to be made quicker and with greater ease-of-use than current NIR fluorescence imaging systems.

Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells

NASA Astrophysics Data System (ADS)

Li, Ke; Zhang, Zhi-Ping; Luo, Ming; Yu, Xiang; Han, Yu; Wei, Hong-Ping; Cui, Zong-Qiang; Zhang, Xian-En

2011-12-01

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging αvβ3 integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging αvβ3 integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging. Electronic supplementary information (ESI) available. See DOI: 10.1039/c1nr11132a

Fluorescent probes in biology and medicine: measurement of intracellular pH values in individual cells

NASA Astrophysics Data System (ADS)

Slavik, Jan; Cimprich, Petr; Gregor, Martin; Smetana, Karel, Jr.

1997-12-01

The application possibilities of fluorescent probes have increased dramatically in the last few years. The main areas are as follows (Slavik, 1994, 1996, 1998). Intracellular ionic cell composition: There are selective ion-sensitive dyes for H+, Ca2+, Mg2+, K+, Na+, Fe3+, Cl-, Zn2+, Cd2+, Hg2+, Pb2+, Ba2+, La3+. Membrane potential: Using the so-called slow (Nernstian dyes) or electrochromic dyes one can assess the value of the transmembrane potential. Membrane fluidity: Fluorescent probes inform about the freedom of rotational and translational movement of membrane proteins and lipids. Selective labeling: Almost any object of interest inside the cell or on its surface can be selectively fluorescently labeled. There are dyes specific for DNA, RNA, oligonucleotides (FISH), Golgi, endoplasmic reticulum, mitochondria, vacuoles, cytoskeleton, etc. Using fluorescent dyes specific receptors may be localized, their conformational changes followed and the polarity of corresponding binding sites accessed. The endocytic pathway may be followed, enzymes and their local enzymatic activity localized. For really selective labeling fluorescent labeled antibodies exist. Imaging: One of the main advantages of fluorescence imaging is its versatility. It allow choice among ratio imaging in excitation, ratio imaging in emission and lifetime imaging. These approaches can be applied to both the classical wide-field fluorescence microscopy and to the laser confocal fluorescence microscopy, one day possibly to the scanning near field optical microscopy. Simultaneous application of several fluorescent dyes: The technical progress in both excitation sources and in detectors allows to extend the excitation deeper in the blue and ultraviolet side and the detection further in the NIR and IR. Consequently, up to 6 peaks in excitation and up to 6 peaks in emission can be followed without any substantial difficulties. Application of dyes such with longer fluorescence lifetimes such as rare earth dyes gives chance for the separated detection of another six peak pairs. The literature data on simultaneous applications of several fluorescent dyes are rare, usually it is only pH and calcium, pH and membrane potential or pH and cytoskeleton changes that are mentioned. Nevertheless, I am sure that in the near future it will be quite common to employ several fluorescent dyes simultaneously. So, in a few years, you may expect to be comfortably seated in an armchair in front of the monitor screen, sip your coffee and follow simultaneously several physiological parameters trying to find out new relations among them. In this respect the potential of fluorescent probes is unsurpassed if you just recall only the discovery of calcium waves and calcium spikes during the past years.

Single-shot optical sectioning using two-color probes in HiLo fluorescence microscopy.

PubMed

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-06-08

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Differential high-speed digital micromirror device based fluorescence speckle confocal microscopy.

PubMed

Jiang, Shihong; Walker, John

2010-01-20

We report a differential fluorescence speckle confocal microscope that acquires an image in a fraction of a second by exploiting the very high frame rate of modern digital micromirror devices (DMDs). The DMD projects a sequence of predefined binary speckle patterns to the sample and modulates the intensity of the returning fluorescent light simultaneously. The fluorescent light reflecting from the DMD's "on" and "off" pixels is modulated by correlated speckle and anticorrelated speckle, respectively, to form two images on two CCD cameras in parallel. The sum of the two images recovers a widefield image, but their difference gives a near-confocal image in real time. Experimental results for both low and high numerical apertures are shown.

Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

NASA Astrophysics Data System (ADS)

Jahn, Karolina; Buschmann, Volker; Hille, Carsten

2015-09-01

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

PubMed

Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

2017-05-01

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

Near-field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores.

PubMed

Mahieu-Williame, L; Falgayrettes, P; Nativel, L; Gall-Borrut, P; Costa, L; Salehzada, T; Bisbal, C

2010-04-01

We have coupled a spectrophotometer with a scanning near-field optical microscope to obtain, with a single scan, simultaneously scanning near-field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.

Indocyanine green fluorescence imaging in the surgical management of liver cancers: current facts and future implications.

PubMed

Lim, C; Vibert, E; Azoulay, D; Salloum, C; Ishizawa, T; Yoshioka, R; Mise, Y; Sakamoto, Y; Aoki, T; Sugawara, Y; Hasegawa, K; Kokudo, N

2014-04-01

Imaging detection of liver cancers and identification of the bile ducts during surgery, based on the fluorescence properties of indocyanine green, has recently been developed in liver surgery. The principle of this imaging technique relies on the intravenous administration of indocyanine green before surgery and the illumination of the surface of the liver by an infrared camera that simultaneously induces and collects the fluorescence. Detection by fluorescence is based on the contrast between the (fluorescent) tumoral or peri-tumoral tissues and the healthy (non-fluorescent) liver. Results suggest that indocyanine green fluorescence imaging is capable of identification of new liver cancers and enables the characterization of known hepatic lesions in real time during liver resection. The purpose of this paper is to present the fundamental principles of fluorescence imaging detection, to describe successively the practical and technical aspects of its use and the appearance of hepatic lesions in fluorescence, and to expose the diagnostic and therapeutic perspectives of this innovative imaging technique in liver surgery. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

PubMed Central

Moffitt, Jeffrey R.; Zhuang, Xiaowei

2016-01-01

Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

Medical imaging systems

DOEpatents

Frangioni, John V [Wayland, MA

2012-07-24

A medical imaging system provides simultaneous rendering of visible light and fluorescent images. The system may employ dyes in a small-molecule form that remains in a subject's blood stream for several minutes, allowing real-time imaging of the subject's circulatory system superimposed upon a conventional, visible light image of the subject. The system may also employ dyes or other fluorescent substances associated with antibodies, antibody fragments, or ligands that accumulate within a region of diagnostic significance. In one embodiment, the system provides an excitation light source to excite the fluorescent substance and a visible light source for general illumination within the same optical guide that is used to capture images. In another embodiment, the system is configured for use in open surgical procedures by providing an operating area that is closed to ambient light. More broadly, the systems described herein may be used in imaging applications where a visible light image may be usefully supplemented by an image formed from fluorescent emissions from a fluorescent substance that marks areas of functional interest.

Integrated ultrasonic particle positioning and low excitation light fluorescence imaging

NASA Astrophysics Data System (ADS)

Bernassau, A. L.; Al-Rawhani, M.; Beeley, J.; Cumming, D. R. S.

2013-12-01

A compact hybrid system has been developed to position and detect fluorescent micro-particles by combining a Single Photon Avalanche Diode (SPAD) imager with an acoustic manipulator. The detector comprises a SPAD array, light-emitting diode (LED), lenses, and optical filters. The acoustic device is formed of multiple transducers surrounding an octagonal cavity. By stimulating pairs of transducers simultaneously, an acoustic landscape is created causing fluorescent micro-particles to agglomerate into lines. The fluorescent pattern is excited by a low power LED and detected by the SPAD imager. Our technique combines particle manipulation and visualization in a compact, low power, portable setup.

Simultaneous mapping of pan and sentinel lymph nodes for real-time image-guided surgery.

PubMed

Ashitate, Yoshitomo; Hyun, Hoon; Kim, Soon Hee; Lee, Jeong Heon; Henary, Maged; Frangioni, John V; Choi, Hak Soo

2014-01-01

The resection of regional lymph nodes in the basin of a primary tumor is of paramount importance in surgical oncology. Although sentinel lymph node mapping is now the standard of care in breast cancer and melanoma, over 20% of patients require a completion lymphadenectomy. Yet, there is currently no technology available that can image all lymph nodes in the body in real time, or assess both the sentinel node and all nodes simultaneously. In this study, we report an optical fluorescence technology that is capable of simultaneous mapping of pan lymph nodes (PLNs) and sentinel lymph nodes (SLNs) in the same subject. We developed near-infrared fluorophores, which have fluorescence emission maxima either at 700 nm or at 800 nm. One was injected intravenously for identification of all regional lymph nodes in a basin, and the other was injected locally for identification of the SLN. Using the dual-channel FLARE intraoperative imaging system, we could identify and resect all PLNs and SLNs simultaneously. The technology we describe enables simultaneous, real-time visualization of both PLNs and SLNs in the same subject.

Innovative molecular-based fluorescent nanoparticles for multicolor single particle tracking in cells

NASA Astrophysics Data System (ADS)

Daniel, Jonathan; Godin, Antoine G.; Palayret, Matthieu; Lounis, Brahim; Cognet, Laurent; Blanchard-Desce, Mireille

2016-03-01

Based on an original molecular-based design, we present bright and photostable fluorescent organic nanoparticles (FONs) showing excellent colloidal stability in various aqueous environments. Complementary near-infrared emitting and green emitting FONs were prepared using a simple, fast and robust protocol. Both types of FONs could be simultaneously imaged at the single-particle level in solution as well as in biological environments using a monochromatic excitation and a dual-color fluorescence microscope. No evidence of acute cytotoxicity was found upon incubation of live cells with mixed solutions of FONs, and both types of nanoparticles were found internalized in the cells where their motion could be simultaneously tracked at video-rate up to minutes. These fluorescent organic nanoparticles open a novel non-toxic alternative to existing nanoparticles for imaging biological structures, compatible with live-cell experiments and specially fitted for multicolor single particle tracking.

Enhancing early bladder cancer detection with fluorescence-guided endoscopic optical coherence tomography

NASA Astrophysics Data System (ADS)

Pan, Y. T.; Xie, T. Q.; Du, C. W.; Bastacky, S.; Meyers, S.; Zeidel, M. L.

2003-12-01

We report an experimental study of the possibility of enhancing early bladder cancer diagnosis with fluorescence-image-guided endoscopic optical coherence tomography (OCT). After the intravesical instillation of a 10% solution of 5-aminolevulinic acid, simultaneous fluorescence imaging (excitation of 380-420 nm, emission of 620-700 nm) and OCT are performed on rat bladders to identify the photochemical and morphological changes associated with uroepithelial tumorigenesis. The preliminary results of our ex vivo study reveal that both fluorescence and OCT can identify early uroepithelial cancers, and OCT can detect precancerous lesions (e.g., hyperplasia) that fluorescence may miss. This suggests that a cystoscope combining 5-aminolevulinic acid fluorescence and OCT imaging has the potential to enhance the efficiency and sensitivity of early bladder cancer diagnosis.

Optical imaging-guided cancer therapy with fluorescent nanoparticles

PubMed Central

Jiang, Shan; Gnanasammandhan, Muthu Kumara; Zhang, Yong

2010-01-01

The diagnosis and treatment of cancer have been greatly improved with the recent developments in nanotechnology. One of the promising nanoscale tools for cancer diagnosis is fluorescent nanoparticles (NPs), such as organic dye-doped NPs, quantum dots and upconversion NPs that enable highly sensitive optical imaging of cancer at cellular and animal level. Furthermore, the emerging development of novel multi-functional NPs, which can be conjugated with several functional molecules simultaneously including targeting moieties, therapeutic agents and imaging probes, provides new potentials for clinical therapies and diagnostics and undoubtedly will play a critical role in cancer therapy. In this article, we review the types and characteristics of fluorescent NPs, in vitro and in vivo imaging of cancer using fluorescent NPs and multi-functional NPs for imaging-guided cancer therapy. PMID:19759055

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

NASA Astrophysics Data System (ADS)

Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

2014-09-01

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

A 3D imaging system integrating photoacoustic and fluorescence orthogonal projections for anatomical, functional and molecular assessment of rodent models

NASA Astrophysics Data System (ADS)

Brecht, Hans P.; Ivanov, Vassili; Dumani, Diego S.; Emelianov, Stanislav Y.; Anastasio, Mark A.; Ermilov, Sergey A.

2018-03-01

We have developed a preclinical 3D imaging instrument integrating photoacoustic tomography and fluorescence (PAFT) addressing known deficiencies in sensitivity and spatial resolution of the individual imaging components. PAFT is designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct registration of the two imaging modalities. Orthogonal photoacoustic projections are utilized to reconstruct large (21 cm3 ) volumes showing vascularized anatomical structures and regions of induced optical contrast with spatial resolution exceeding 100 µm. The major advantage of orthogonal fluorescence projections is significant reduction of background noise associated with transmitted or backscattered photons. The fluorescence imaging component of PAFT is used to boost detection sensitivity by providing low-resolution spatial constraint for the fluorescent biomarkers. PAFT performance characteristics were assessed by imaging optical and fluorescent contrast agents in tissue mimicking phantoms and in vivo. The proposed PAFT technology will enable functional and molecular volumetric imaging using fluorescent biomarkers, nanoparticles, and other photosensitive constructs mapped with high fidelity over robust anatomical structures, such as skin, central and peripheral vasculature, and internal organs.

pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

PubMed Central

Shen, Yi; Rosendale, Morgane

2014-01-01

Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultaneous two-color imaging of clathrin-mediated internalization of both the transferrin receptor and the β2 adrenergic receptor. These experiments reveal that the two receptors are differentially sorted at the time of endocytic vesicle formation. PMID:25385186

Handheld Fluorescence Microscopy based Flow Analyzer.

PubMed

Saxena, Manish; Jayakumar, Nitin; Gorthi, Sai Siva

2016-03-01

Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

Parallel, confocal, and complete spectrum imager for fluorescent detection of high-density microarray

NASA Astrophysics Data System (ADS)

Bogdanov, Valery L.; Boyce-Jacino, Michael

1999-05-01

Confined arrays of biochemical probes deposited on a solid support surface (analytical microarray or 'chip') provide an opportunity to analysis multiple reactions simultaneously. Microarrays are increasingly used in genetics, medicine and environment scanning as research and analytical instruments. A power of microarray technology comes from its parallelism which grows with array miniaturization, minimization of reagent volume per reaction site and reaction multiplexing. An optical detector of microarray signals should combine high sensitivity, spatial and spectral resolution. Additionally, low-cost and a high processing rate are needed to transfer microarray technology into biomedical practice. We designed an imager that provides confocal and complete spectrum detection of entire fluorescently-labeled microarray in parallel. Imager uses microlens array, non-slit spectral decomposer, and high- sensitive detector (cooled CCD). Two imaging channels provide a simultaneous detection of localization, integrated and spectral intensities for each reaction site in microarray. A dimensional matching between microarray and imager's optics eliminates all in moving parts in instrumentation, enabling highly informative, fast and low-cost microarray detection. We report theory of confocal hyperspectral imaging with microlenses array and experimental data for implementation of developed imager to detect fluorescently labeled microarray with a density approximately 103 sites per cm2.

In Vivo Dual Fluorescence Imaging to Detect Joint Destruction.

PubMed

Cho, Hongsik; Bhatti, Fazal-Ur-Rehman; Lee, Sangmin; Brand, David D; Yi, Ae-Kyung; Hasty, Karen A

2016-10-01

Diagnosis of cartilage damage in early stages of arthritis is vital to impede the progression of disease. In this regard, considerable progress has been made in near-infrared fluorescence (NIRF) optical imaging technique. Arthritis can develop due to various mechanisms but one of the main contributors is the production of matrix metalloproteinases (MMPs), enzymes that can degrade components of the extracellular matrix. Especially, MMP-1 and MMP-13 have main roles in rheumatoid arthritis and osteoarthritis because they enhance collagen degradation in the process of arthritis. We present here a novel NIRF imaging strategy that can be used to determine the activity of MMPs and cartilage damage simultaneously by detection of exposed type II collagen in cartilage tissue. In this study, retro-orbital injection of mixed fluorescent dyes, MMPSense 750 FAST (MMP750) dye and Alexa Fluor 680 conjugated monoclonal mouse antibody immune-reactive to type II collagen, was administered in the arthritic mice. Both dyes were detected with different intensity according to degree of joint destruction in the animal. Thus, our dual fluorescence imaging method can be used to detect cartilage damage as well as MMP activity simultaneously in early stage arthritis. © 2016 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Spectroscopic imaging using acousto-optic tunable filters

NASA Astrophysics Data System (ADS)

Bouhifd, Mounir; Whelan, Maurice

2007-07-01

We report on novel hyper-spectral imaging filter-modules based on acousto-optic tuneable filters (AOTF). The AOTF functions as a full-field tuneable bandpass filter which offers fast continuous or random access tuning with high filtering efficiency. Due to the diffractive nature of the device, the unfiltered zero-order and the filtered first-order images are geometrically separated. The modules developed exploit this feature to simultaneously route both the transmitted white-light image and the filtered fluorescence image to two separate cameras. Incorporation of prisms in the optical paths and careful design of the relay optics in the filter module have overcome a number of aberrations inherent to imaging through AOTFs, leading to excellent spatial resolution. A number of practical uses of this technique, both for in vivo auto-fluorescence endoscopy and in vitro fluorescence microscopy were demonstrated. We describe the operational principle and design of recently improved prototype instruments for fluorescence-based diagnostics and demonstrate their performance by presenting challenging hyper-spectral fluorescence imaging applications.

Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography

NASA Astrophysics Data System (ADS)

Patalay, Rakesh; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Breunig, Hans Georg; König, Karsten; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Christopher

2011-07-01

Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.

Development of a Time Domain Fluorimeter for Fluorescent Lifetime Multiplexing Analysis

PubMed Central

Weissleder, Ralph; Mahmood, Umar

2009-01-01

We show that a portable, inexpensive USB-powered time domain fluorimeter (TDF) and analysis scheme were developed for use in evaluating a new class of fluorescent lifetime multiplexed dyes. Fluorescent proteins, organic dyes, and quantum dots allow the labeling of more and more individual features within biological systems, but the wide absorption and emission spectra of these fluorophores limit the number of distinct processes which may be simultaneously imaged using spectral separation alone. By additionally separating reporters in a second dimension, fluorescent lifetime multiplexing provides a means to multiply the number of available imaging channels. PMID:19830273

Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy

NASA Astrophysics Data System (ADS)

Descloux, A.; Grußmayer, K. S.; Bostan, E.; Lukes, T.; Bouwens, A.; Sharipov, A.; Geissbuehler, S.; Mahul-Mellier, A.-L.; Lashuel, H. A.; Leutenegger, M.; Lasser, T.

2018-03-01

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.

Imaging of molecular hydrogen and oxygen by single and two-photon fluorescence using laser and flashlamp sources

NASA Technical Reports Server (NTRS)

Diskin, Glenn S.; Lempert, Walter R.; Miles, Richard B.; Kumar, Vinod; Glesk, Ivan

1991-01-01

Two flow visualization techniques, i.e., simultaneous two-dimensional fluorescence imaging of H2 and O2 in a diffusion flame, and quasi-linear fluorescence imaging of O2, are presented. The first uses an injection-locked argon-fluoride excimer laser and a partial overlap of a two-photon ground state absorption in H2 with a single photon absorption from a vibrational level in O2. The second uses a simple, high-intensity ultraviolet flashlamp which provides a flux of photons in the 180-195 nm range, sufficient to produce a quasi-one-dimensional fluorescence image of hot/room temperature oxygen. Both techniques do not require that a seed material be introduced into the flow, they can image major flow constituents, and provide an instantaneous snapshot of the flow.

Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells.

PubMed

Li, Ke; Zhang, Zhi-Ping; Luo, Ming; Yu, Xiang; Han, Yu; Wei, Hong-Ping; Cui, Zong-Qiang; Zhang, Xian-En

2012-01-07

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging α(v)β(3) integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.

Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins

NASA Astrophysics Data System (ADS)

Krumholz, Arie; Shcherbakova, Daria M.; Xia, Jun; Wang, Lihong V.; Verkhusha, Vladislav V.

2014-02-01

Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo.

Folic acid-targeted magnetic Tb-doped CeF3 fluorescent nanoparticles as bimodal probes for cellular fluorescence and magnetic resonance imaging.

PubMed

Ma, Zhi-Ya; Liu, Yu-Ping; Bai, Ling-Yu; An, Jie; Zhang, Lin; Xuan, Yang; Zhang, Xiao-Shuai; Zhao, Yuan-Di

2015-10-07

Magnetic fluorescent nanoparticles (NPs) have great potential applications for diagnostics, imaging and therapy. We developed a facile polyol method to synthesize multifunctional Fe3O4@CeF3:Tb@CeF3 NPs with small size (<20 nm), high water solubility and good biocompatibility. The NPs were modified by ligand exchange reactions with citric acid (CA) to obtain carboxyl-functionalized NPs (Fe3O4@CeF3:Tb@CeF3-COOH). Folic acid (FA) as an affinity ligand was then covalently conjugated onto NPs to yield Fe3O4@CeF3:Tb@CeF3-FA NPs. They were then applied as multimodal imaging agents for simultaneous in vitro targeted fluorescence imaging and magnetic resonance imaging (MRI) of HeLa cells with overexpressed folate receptors (FR). The results indicated that these NPs had strong luminescence and enhanced T2-weighted MR contrast and would be promising candidates as multimodal probes for both fluorescence and MRI imaging.

Simultaneous Denoising, Deconvolution, and Demixing of Calcium Imaging Data

PubMed Central

Pnevmatikakis, Eftychios A.; Soudry, Daniel; Gao, Yuanjun; Machado, Timothy A.; Merel, Josh; Pfau, David; Reardon, Thomas; Mu, Yu; Lacefield, Clay; Yang, Weijian; Ahrens, Misha; Bruno, Randy; Jessell, Thomas M.; Peterka, Darcy S.; Yuste, Rafael; Paninski, Liam

2016-01-01

SUMMARY We present a modular approach for analyzing calcium imaging recordings of large neuronal ensembles. Our goal is to simultaneously identify the locations of the neurons, demix spatially overlapping components, and denoise and deconvolve the spiking activity from the slow dynamics of the calcium indicator. Our approach relies on a constrained nonnegative matrix factorization that expresses the spatiotemporal fluorescence activity as the product of a spatial matrix that encodes the spatial footprint of each neuron in the optical field and a temporal matrix that characterizes the calcium concentration of each neuron over time. This framework is combined with a novel constrained deconvolution approach that extracts estimates of neural activity from fluorescence traces, to create a spatiotemporal processing algorithm that requires minimal parameter tuning. We demonstrate the general applicability of our method by applying it to in vitro and in vivo multineuronal imaging data, whole-brain light-sheet imaging data, and dendritic imaging data. PMID:26774160

A multi-signal fluorescent probe for simultaneously distinguishing and sequentially sensing cysteine/homocysteine, glutathione, and hydrogen sulfide in living cells† †Electronic supplementary information (ESI) available: Experimental details for chemical synthesis of all compounds, chemical structure characterization, supplementary spectra of probe, and fluorescence imaging methods and data. See DOI: 10.1039/c7sc00423k Click here for additional data file.

PubMed Central

He, Longwei; Yang, Xueling; Xu, Kaixin; Kong, Xiuqi

2017-01-01

Biothiols, which have a close network of generation and metabolic pathways among them, are essential reactive sulfur species (RSS) in the cells and play vital roles in human physiology. However, biothiols possess highly similar chemical structures and properties, resulting in it being an enormous challenge to simultaneously discriminate them from each other. Herein, we develop a unique fluorescent probe (HMN) for not only simultaneously distinguishing Cys/Hcy, GSH, and H2S from each other, but also sequentially sensing Cys/Hcy/GSH and H2S using a multi-channel fluorescence mode for the first time. When responding to the respective biothiols, the robust probe exhibits multiple sets of fluorescence signals at three distinct emission bands (blue-green-red). The new probe can also sense H2S at different concentration levels with changes of fluorescence at the blue and red emission bands. In addition, the novel probe HMN is able to discriminate and sequentially sense biothiols in biological environments via three-color fluorescence imaging. We expect that the development of the robust probe HMN will provide a powerful strategy to design fluorescent probes for the discrimination and sequential detection of biothiols, and offer a promising tool for exploring the interrelated roles of biothiols in various physiological and pathological conditions. PMID:28989659

Multimodal optoacoustic and multiphoton fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sela, Gali; Razansky, Daniel; Shoham, Shy

2013-03-01

Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The multimodal system may provide complementary structural and functional information to the fluorescently labeled tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits, pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR spectrum.

Combined SRCT & FXCT - The next steps

NASA Astrophysics Data System (ADS)

Hall, C.; Acres, R. G.; Winnett, A.; Wang, F.

2016-03-01

One of the goals in developing synchrotron radiation x-ray computed tomography (SRCT) for biomedical specimens, is allowing particular tissues and cell types to be marked in the images. This is equivalent to the staining in histology, which enables researchers to visualise and measure tissue structure and biochemical processes within the specimen. Some progress in this direction for SRCT is being made, using a variety of contrast agents that alter the natural x-ray attenuation of the marked tissue [1]. However there are limits to the usefulness of these attenuation altering techniques. Often high concentrations of potentially disruptive chemicals are required with reduced compatibility for in-vivo studies. Another image highlighting technique which might prove more sensitive is x-ray fluorescence imaging. In this case usually endogenous elemental markers are visualised. We would like to develop a lower resolution, but wider field of view means of three-dimensional (3-D) fluorescence imaging compatible with SRCT. We have previously proposed a technique in which x-ray fluorescence CT (FXCT) and SRCT data can be collected simultaneously [2]. This work resulted in proof of concept modelling, and a simple experiment test system. We show data here which demonstrate a two-dimensional (2-D) reconstruction of an iodine fluorescence map from a phantom. Measurements were performed with a fixed beam modulating mask using the Imaging and Medical beam line (IMBL) at the Australian Synchrotron. Fluorescence data was obtained during a CT scan using a single point detector, while transmission data was simultaneously collected using an area detector. A maximum likelihood expectation maximisation (MLEM) iterative algorithm was used to reconstruct the fluorescence map. We report on technique development and now believe compressive sensing (CS) imaging techniques suit SRCT and may overcome the issues encountered so far in combining SRCT and FXCT.

Multispectral scanning laser ophthalmoscopy combined with optical coherence tomography for simultaneous in vivo mouse retinal imaging

NASA Astrophysics Data System (ADS)

Zhang, Pengfei; Zam, Azhar; Jian, Yifan; Wang, Xinlei; Burns, Marie E.; Sarunic, Marinko V.; Pugh, Edward N.; Zawadzki, Robert J.

2015-03-01

A compact, non-invasive multi-modal system has been developed for in vivo mouse retina imaging. It is configured for simultaneously detecting green and red fluorescent protein signals with scanning laser ophthalmoscopy (SLO) back-scattered light from the SLO illumination beam, and depth information about different retinal layers by means of Optical Coherence Tomography (OCT). Simultaneous assessment of retinal characteristics with different modalities can provide a wealth of information about the structural and functional changes in the retinal neural tissue and chorio-retinal vasculature in vivo. Additionally, simultaneous acquisition of multiple channels facilitates analysis of the data of different modalities by automatic temporal and structural co-registration. As an example of the instrument's performance we imaged the retina of a mouse with constitutive expression of GFP in microglia cells (Cx3cr1GFP/+), and which also expressed the red fluorescent protein mCherry in Müller glial cells by means of adeno-associated virus delivery (AAV2) of an mCherry cDNA driven by the GFAP (glial fibrillary acid protein) promoter.

Nanoparticles for multimodal in vivo imaging in nanomedicine

PubMed Central

Key, Jaehong; Leary, James F

2014-01-01

While nanoparticles are usually designed for targeted drug delivery, they can also simultaneously provide diagnostic information by a variety of in vivo imaging methods. These diagnostic capabilities make use of specific properties of nanoparticle core materials. Near-infrared fluorescent probes provide optical detection of cells targeted by real-time nanoparticle-distribution studies within the organ compartments of live, anesthetized animals. By combining different imaging modalities, we can start with deep-body imaging by magnetic resonance imaging or computed tomography, and by using optical imaging, get down to the resolution required for real-time fluorescence-guided surgery. PMID:24511229

Targeted detection of murine colonic dysplasia in vivo with flexible multispectral scanning fiber endoscopy

NASA Astrophysics Data System (ADS)

Joshi, Bishnu P.; Miller, Sharon J.; Lee, Cameron; Gustad, Adam; Seibel, Eric J.; Wang, Thomas D.

2012-02-01

We demonstrate a multi-spectral scanning fiber endoscope (SFE) that collects fluorescence images in vivo from three target peptides that bind specifically to murine colonic adenomas. This ultrathin endoscope was demonstrated in a genetically engineered mouse model of spontaneous colorectal adenomas based on somatic Apc (adenomatous polyposis coli) gene inactivation. The SFE delivers excitation at 440, 532, 635 nm with <2 mW per channel. The target 7-mer peptides were conjugated to visible organic dyes, including 7-Diethylaminocoumarin-3-carboxylic acid (DEAC) (λex=432 nm, λem=472 nm), 5-Carboxytetramethylrhodamine (5-TAMRA) (λex=535 nm, λem=568 nm), and CF-633 (λex=633 nm, λem=650 nm). Target peptides were first validated using techniques of pfu counting, flow cytometry and previously established methods of fluorescence endoscopy. Peptides were applied individually or in combination and detected with fluorescence imaging. The ability to image multiple channels of fluorescence concurrently was successful for all three channels in vitro, while two channels were resolved simultaneously in vivo. Selective binding of the peptide was evident to adenomas and not to adjacent normal-appearing mucosa. Multispectral wide-field fluorescence detection using the SFE is achievable, and this technology has potential to advance early cancer detection and image-guided therapy in human patients by simultaneously visualizing multiple over expressed molecular targets unique to dysplasia.

3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation

PubMed Central

Choi, Heejin; Tzeranis, Dimitrios S.; Cha, Jae Won; Clémenceau, Philippe; de Jong, Sander J. G.; van Geest, Lambertus K.; Moon, Joong Ho; Yannas, Ioannis V.; So, Peter T. C.

2012-01-01

Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude. PMID:23187477

An LED light source and novel fluorophore combinations improve fluorescence laparoscopic detection of metastatic pancreatic cancer in orthotopic mouse models.

PubMed

Metildi, Cristina A; Kaushal, Sharmeela; Lee, Claudia; Hardamon, Chanae R; Snyder, Cynthia S; Luiken, George A; Talamini, Mark A; Hoffman, Robert M; Bouvet, Michael

2012-06-01

The aim of this study was to improve fluorescence laparoscopy of pancreatic cancer in an orthotopic mouse model with the use of a light-emitting diode (LED) light source and optimal fluorophore combinations. Human pancreatic cancer models were established with fluorescent FG-RFP, MiaPaca2-GFP, BxPC-3-RFP, and BxPC-3 cancer cells implanted in 6-week-old female athymic mice. Two weeks postimplantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source or a Stryker X8000 xenon light source 24 hours after tail-vein injection of CEA antibodies conjugated with Alexa 488 or Alexa 555. Cancer lesions were detected and localized under each light mode. Intravital images were also obtained with the OV-100 Olympus and Maestro CRI Small Animal Imaging Systems, serving as a positive control. Tumors were collected for histologic analysis. Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor deposits in the peritoneal cavity. The simultaneous use of different fluorophores (Alexa 488 and Alexa 555), conjugated to antibodies, brightened the fluorescence signal, enhancing detection of submillimeter lesions without compromising background illumination. Adjustments to the LED light source permitted simultaneous detection of tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence. Using an LED light source with adjustments to the red, blue, and green wavelengths, it is possible to simultaneously identify tumor metastases expressing fluorescent proteins of different wavelengths, which greatly enhanced the signal without compromising background illumination. Development of this fluorescence laparoscopy technology for clinical use can improve staging and resection of pancreatic cancer. Copyright © 2012 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

RAMTaB: Robust Alignment of Multi-Tag Bioimages

PubMed Central

Raza, Shan-e-Ahmed; Humayun, Ahmad; Abouna, Sylvie; Nattkemper, Tim W.; Epstein, David B. A.; Khan, Michael; Rajpoot, Nasir M.

2012-01-01

Background In recent years, new microscopic imaging techniques have evolved to allow us to visualize several different proteins (or other biomolecules) in a visual field. Analysis of protein co-localization becomes viable because molecules can interact only when they are located close to each other. We present a novel approach to align images in a multi-tag fluorescence image stack. The proposed approach is applicable to multi-tag bioimaging systems which (a) acquire fluorescence images by sequential staining and (b) simultaneously capture a phase contrast image corresponding to each of the fluorescence images. To the best of our knowledge, there is no existing method in the literature, which addresses simultaneous registration of multi-tag bioimages and selection of the reference image in order to maximize the overall overlap between the images. Methodology/Principal Findings We employ a block-based method for registration, which yields a confidence measure to indicate the accuracy of our registration results. We derive a shift metric in order to select the Reference Image with Maximal Overlap (RIMO), in turn minimizing the total amount of non-overlapping signal for a given number of tags. Experimental results show that the Robust Alignment of Multi-Tag Bioimages (RAMTaB) framework is robust to variations in contrast and illumination, yields sub-pixel accuracy, and successfully selects the reference image resulting in maximum overlap. The registration results are also shown to significantly improve any follow-up protein co-localization studies. Conclusions For the discovery of protein complexes and of functional protein networks within a cell, alignment of the tag images in a multi-tag fluorescence image stack is a key pre-processing step. The proposed framework is shown to produce accurate alignment results on both real and synthetic data. Our future work will use the aligned multi-channel fluorescence image data for normal and diseased tissue specimens to analyze molecular co-expression patterns and functional protein networks. PMID:22363510

Imaging workflow and calibration for CT-guided time-domain fluorescence tomography

PubMed Central

Tichauer, Kenneth M.; Holt, Robert W.; El-Ghussein, Fadi; Zhu, Qun; Dehghani, Hamid; Leblond, Frederic; Pogue, Brian W.

2011-01-01

In this study, several key optimization steps are outlined for a non-contact, time-correlated single photon counting small animal optical tomography system, using simultaneous collection of both fluorescence and transmittance data. The system is presented for time-domain image reconstruction in vivo, illustrating the sensitivity from single photon counting and the calibration steps needed to accurately process the data. In particular, laser time- and amplitude-referencing, detector and filter calibrations, and collection of a suitable instrument response function are all presented in the context of time-domain fluorescence tomography and a fully automated workflow is described. Preliminary phantom time-domain reconstructed images demonstrate the fidelity of the workflow for fluorescence tomography based on signal from multiple time gates. PMID:22076264

Monte Carlo based method for fluorescence tomographic imaging with lifetime multiplexing using time gates

PubMed Central

Chen, Jin; Venugopal, Vivek; Intes, Xavier

2011-01-01

Time-resolved fluorescence optical tomography allows 3-dimensional localization of multiple fluorophores based on lifetime contrast while providing a unique data set for improved resolution. However, to employ the full fluorescence time measurements, a light propagation model that accurately simulates weakly diffused and multiple scattered photons is required. In this article, we derive a computationally efficient Monte Carlo based method to compute time-gated fluorescence Jacobians for the simultaneous imaging of two fluorophores with lifetime contrast. The Monte Carlo based formulation is validated on a synthetic murine model simulating the uptake in the kidneys of two distinct fluorophores with lifetime contrast. Experimentally, the method is validated using capillaries filled with 2.5nmol of ICG and IRDye™800CW respectively embedded in a diffuse media mimicking the average optical properties of mice. Combining multiple time gates in one inverse problem allows the simultaneous reconstruction of multiple fluorophores with increased resolution and minimal crosstalk using the proposed formulation. PMID:21483610

Sentinel lymph node biopsy under fluorescent indocyanin green guidance: Initial experience.

PubMed

Aydoğan, Fatih; Arıkan, Akif Enes; Aytaç, Erman; Velidedeoğlu, Mehmet; Yılmaz, Mehmet Halit; Sager, Muhammet Sait; Çelik, Varol; Uras, Cihan

2016-01-01

Sentinel lymph node biopsy can be applied by using either blue dye or radionuclide method or both in breast cancer. Fluorescent imaging with indocyanine green is a new defined method. This study evaluates the applicability of sentinel lymph node biopsy via fluorescent indocyanine green. IC-VIEW (Pulsion Medical Systems AG, Munich, Germany) infrared visualization system was used for imaging. Two mL of indocyanine green was injected to visualize sentinel lymph nodes. After injection, subcutaneous lymphatics were traced and sentinel lymph nodes were found with simultaneous imaging. Sentinel lymph nodes were excised under fluorescent light guidance, and excised lymph nodes were examined histopathologically. Patients with sentinel lymph node metastases underwent axillary dissection. Four patients with sentinel lymph node biopsy due to breast cancer were included in the study. Sentinel lymph nodes were visualized with indocyanine green in all patients. The median number of excised sentinel lymph node was 2 (2-3). Two patients with lymph node metastasis underwent axillary dissection. No metastasis was detected in lymph nodes other than the sentinel nodes in patients with axillary dissection. There was no complication during and after the operation related to the method. According to our limited experience, sentinel lymph node biopsy under fluorescent indocyanine green guidance, which has an advantage of simultaneous visualization, is technically feasible.

Guiding synchrotron X-ray diffraction by multimodal video-rate protein crystal imaging

DOE PAGES

Newman, Justin A.; Zhang, Shijie; Sullivan, Shane Z.; ...

2016-05-16

Synchronous digitization, in which an optical sensor is probed synchronously with the firing of an ultrafast laser, was integrated into an optical imaging station for macromolecular crystal positioning prior to synchrotron X-ray diffraction. Using the synchronous digitization instrument, second-harmonic generation, two-photon-excited fluorescence and bright field by laser transmittance were all acquired simultaneously with perfect image registry at up to video-rate (15 frames s –1). A simple change in the incident wavelength enabled simultaneous imaging by two-photon-excited ultraviolet fluorescence, one-photon-excited visible fluorescence and laser transmittance. Development of an analytical model for the signal-to-noise enhancement afforded by synchronous digitization suggests a 15.6-foldmore » improvement over previous photon-counting techniques. This improvement in turn allowed acquisition on nearly an order of magnitude more pixels than the preceding generation of instrumentation and reductions of well over an order of magnitude in image acquisition times. These improvements have allowed detection of protein crystals on the order of 1 µm in thickness under cryogenic conditions in the beamline. Lastly, these capabilities are well suited to support serial crystallography of crystals approaching 1 µm or less in dimension.« less

Guiding synchrotron X-ray diffraction by multimodal video-rate protein crystal imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Newman, Justin A.; Zhang, Shijie; Sullivan, Shane Z.

Synchronous digitization, in which an optical sensor is probed synchronously with the firing of an ultrafast laser, was integrated into an optical imaging station for macromolecular crystal positioning prior to synchrotron X-ray diffraction. Using the synchronous digitization instrument, second-harmonic generation, two-photon-excited fluorescence and bright field by laser transmittance were all acquired simultaneously with perfect image registry at up to video-rate (15 frames s –1). A simple change in the incident wavelength enabled simultaneous imaging by two-photon-excited ultraviolet fluorescence, one-photon-excited visible fluorescence and laser transmittance. Development of an analytical model for the signal-to-noise enhancement afforded by synchronous digitization suggests a 15.6-foldmore » improvement over previous photon-counting techniques. This improvement in turn allowed acquisition on nearly an order of magnitude more pixels than the preceding generation of instrumentation and reductions of well over an order of magnitude in image acquisition times. These improvements have allowed detection of protein crystals on the order of 1 µm in thickness under cryogenic conditions in the beamline. Lastly, these capabilities are well suited to support serial crystallography of crystals approaching 1 µm or less in dimension.« less

Guiding synchrotron X-ray diffraction by multimodal video-rate protein crystal imaging

PubMed Central

Newman, Justin A.; Zhang, Shijie; Sullivan, Shane Z.; Dow, Ximeng Y.; Becker, Michael; Sheedlo, Michael J.; Stepanov, Sergey; Carlsen, Mark S.; Everly, R. Michael; Das, Chittaranjan; Fischetti, Robert F.; Simpson, Garth J.

2016-01-01

Synchronous digitization, in which an optical sensor is probed synchronously with the firing of an ultrafast laser, was integrated into an optical imaging station for macromolecular crystal positioning prior to synchrotron X-ray diffraction. Using the synchronous digitization instrument, second-harmonic generation, two-photon-excited fluorescence and bright field by laser transmittance were all acquired simultaneously with perfect image registry at up to video-rate (15 frames s−1). A simple change in the incident wavelength enabled simultaneous imaging by two-photon-excited ultraviolet fluorescence, one-photon-excited visible fluorescence and laser transmittance. Development of an analytical model for the signal-to-noise enhancement afforded by synchronous digitization suggests a 15.6-fold improvement over previous photon-counting techniques. This improvement in turn allowed acquisition on nearly an order of magnitude more pixels than the preceding generation of instrumentation and reductions of well over an order of magnitude in image acquisition times. These improvements have allowed detection of protein crystals on the order of 1 µm in thickness under cryogenic conditions in the beamline. These capabilities are well suited to support serial crystallography of crystals approaching 1 µm or less in dimension. PMID:27359145

Stereoscopic Integrated Imaging Goggles for Multimodal Intraoperative Image Guidance

PubMed Central

Mela, Christopher A.; Patterson, Carrie; Thompson, William K.; Papay, Francis; Liu, Yang

2015-01-01

We have developed novel stereoscopic wearable multimodal intraoperative imaging and display systems entitled Integrated Imaging Goggles for guiding surgeries. The prototype systems offer real time stereoscopic fluorescence imaging and color reflectance imaging capacity, along with in vivo handheld microscopy and ultrasound imaging. With the Integrated Imaging Goggle, both wide-field fluorescence imaging and in vivo microscopy are provided. The real time ultrasound images can also be presented in the goggle display. Furthermore, real time goggle-to-goggle stereoscopic video sharing is demonstrated, which can greatly facilitate telemedicine. In this paper, the prototype systems are described, characterized and tested in surgeries in biological tissues ex vivo. We have found that the system can detect fluorescent targets with as low as 60 nM indocyanine green and can resolve structures down to 0.25 mm with large FOV stereoscopic imaging. The system has successfully guided simulated cancer surgeries in chicken. The Integrated Imaging Goggle is novel in 4 aspects: it is (a) the first wearable stereoscopic wide-field intraoperative fluorescence imaging and display system, (b) the first wearable system offering both large FOV and microscopic imaging simultaneously, (c) the first wearable system that offers both ultrasound imaging and fluorescence imaging capacities, and (d) the first demonstration of goggle-to-goggle communication to share stereoscopic views for medical guidance. PMID:26529249

Neutron, fluorescence, and optical imaging: An in situ combination of complementary techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagner, D.; Egelhaaf, S. U.; Hermes, H. E.

2015-09-15

An apparatus which enables the simultaneous combination of three complementary imaging techniques, optical imaging, fluorescence imaging, and neutron radiography, is presented. While each individual technique can provide information on certain aspects of the sample and their time evolution, a combination of the three techniques in one setup provides a more complete and consistent data set. The setup can be used in transmission and reflection modes and thus with optically transparent as well as opaque samples. Its capabilities are illustrated with two examples. A polymer hydrogel represents a transparent sample and the diffusion of fluorescent particles into and through this polymermore » matrix is followed. In reflection mode, the absorption of solvent by a nile red-functionalized mesoporous silica powder and the corresponding change in fluorescent signal are studied.« less

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera

NASA Astrophysics Data System (ADS)

Cruz Perez, Carlos; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera.

PubMed

Perez, Carlos Cruz; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

A multi-imaging approach to study the root–soil interface

PubMed Central

Rudolph-Mohr, Nicole; Vontobel, Peter; Oswald, Sascha E.

2014-01-01

Background and Aims Dynamic processes occurring at the soil–root interface crucially influence soil physical, chemical and biological properties at a local scale around the roots, and are technically challenging to capture in situ. This study presents a novel multi-imaging approach combining fluorescence and neutron radiography that is able to simultaneously monitor root growth, water content distribution, root respiration and root exudation. Methods Germinated seeds of white lupins (Lupinus albus) were planted in boron-free glass rhizotrons. After 11 d, the rhizotrons were wetted from the bottom and time series of fluorescence and neutron images were taken during the subsequent day and night cycles for 13 d. The following day (i.e. 25 d after planting) the rhizotrons were again wetted from the bottom and the measurements were repeated. Fluorescence sensor foils were attached to the inner sides of the glass and measurements of oxygen and pH were made on the basis of fluorescence intensity. The experimental set-up allowed for simultaneous fluorescence imaging and neutron radiography. Key Results The interrelated patterns of root growth and distribution in the soil, root respiration, exudation and water uptake could all be studied non-destructively and at high temporal and spatial resolution. The older parts of the root system with greater root-length density were associated with fast decreases of water content and rapid changes in oxygen concentration. pH values around the roots located in areas with low soil water content were significantly lower than the rest of the root system. Conclusions The results suggest that the combined imaging set-up developed here, incorporating fluorescence intensity measurements, is able to map important biogeochemical parameters in the soil around living plants with a spatial resolution that is sufficiently high enough to relate the patterns observed to the root system. PMID:25344936

Simultaneous multicolor detection system of the single-molecular microbial antigen by total internal reflection fluorescence microscopy with fluorescent nanocrystal quantum dots

NASA Astrophysics Data System (ADS)

Hoshino, Akiyoshi; Fujioka, Kouki; Yamamoto, Mayu; Manabe, Noriyoshi; Yasuhara, Masato; Suzuki, Kazuo; Yamamoto, Kenji

2005-11-01

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystals QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

Investigation of burn effect on skin using simultaneous Raman-Brillouin spectroscopy, and fluorescence microspectroscopy

NASA Astrophysics Data System (ADS)

Coker, Zachary; Meng, Zhaokai; Troyanova-Wood, Maria; Traverso, Andrew; Ballmann, Charles; Petrov, Georgi; Ibey, Bennett L.; Yakovlev, Vladislav

2017-02-01

Burns are thermal injuries that can completely damage or at least compromise the protective function of skin, and affect the ability of tissues to manage moisture. Burn-damaged tissues exhibit lower elasticity than healthy tissues, due to significantly reduced water concentrations and plasma retention. Current methods for determining burn intensity are limited to visual inspection, and potential hospital x-ray examination. We present a unique confocal microscope capable of measuring Raman and Brillouin spectra simultaneously, with concurrent fluorescence investigation from a single spatial location, and demonstrate application by investigating and characterizing the properties of burn-afflicted tissue on chicken skin model. Raman and Brillouin scattering offer complementary information about a material's chemical and mechanical structure, while fluorescence can serve as a useful diagnostic indicator and imaging tool. The developed instrument has the potential for very diverse analytical applications in basic biomedical science and biomedical diagnostics and imaging.

Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic β-cells.

PubMed

Elliott, Amicia D; Bedard, Noah; Ustione, Alessandro; Baird, Michelle A; Davidson, Michael W; Tkaczyk, Tomasz; Piston, David W

2017-01-01

Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS's capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic β-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic β-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.

Integrated image presentation of transmission and fluorescent X-ray CT using synchrotron radiation

NASA Astrophysics Data System (ADS)

Zeniya, T.; Takeda, T.; Yu, Q.; Hasegawa, Y.; Hyodo, K.; Yuasa, T.; Hiranaka, Y.; Itai, Y.; Akatsuka, T.

2001-07-01

We have developed a computed tomography (CT) system with synchrotron radiation (SR) to detect fluorescent X-rays and transmitted X-rays simultaneously. Both SR transmission X-ray CT (SR-TXCT) and SR fluorescent X-ray CT (SR-FXCT) can describe cross-sectional images with high spatial and contrast resolutions as compared to conventional CT. TXCT gives morphological information and FXCT gives functional information of organs. So, superposed display system for SR-FXCT and SR-TXCT images has been developed for clinical diagnosis with higher reliability. Preliminary experiment with brain phantom was carried out and the superposition of both images was performed. The superposed SR-CT image gave us both functional and morphological information easily with high reliability, thus demonstrating the usefulness of this system.

New method of acne disease fluorescent diagnostics in natural and fluorescent light and treatment control

NASA Astrophysics Data System (ADS)

Karimova, L. N.; Berezin, A. N.; Shevchik, S. A.; Kharnas, S. S.; Kusmin, S. G.; Loschenov, V. B.

2005-08-01

In the given research the new method of fluorescent diagnostics (FD) and photodynamic therapy (PDT) control of acne disease is submitted. Method is based on simultaneous diagnostics in natural and fluorescent light. PDT was based on using 5-ALA (5- aminolevulinic acid) preparation and 600-730 nanometers radiation. If the examined site of a skin possessed a high endogenous porphyrin fluorescence level, PDT was carried out without 5-ALA. For FD and treatment control a dot spectroscopy and the fluorescent imaging of the affected skin were used.

A multimodal imaging platform with integrated simultaneous photoacoustic microscopy, optical coherence tomography, optical Doppler tomography and fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dadkhah, Arash; Zhou, Jun; Yeasmin, Nusrat; Jiao, Shuliang

2018-02-01

Various optical imaging modalities with different optical contrast mechanisms have been developed over the past years. Although most of these imaging techniques are being used in many biomedical applications and researches, integration of these techniques will allow researchers to reach the full potential of these technologies. Nevertheless, combining different imaging techniques is always challenging due to the difference in optical and hardware requirements for different imaging systems. Here, we developed a multimodal optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissue in micrometer scale. This imaging system integrates photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and fluorescence microscopy in one platform. Optical-resolution PAM (OR-PAM) provides absorption-based imaging of biological tissues. Spectral domain OCT is able to provide structural information based on the scattering property of biological sample with no need for exogenous contrast agents. In addition, ODT is a functional extension of OCT with the capability of measurement and visualization of blood flow based on the Doppler effect. Fluorescence microscopy allows to reveal molecular information of biological tissue using autofluoresce or exogenous fluorophores. In-vivo as well as ex-vivo imaging studies demonstrated the capability of our multimodal imaging system to provide comprehensive microscopic information on biological tissues. Integrating all the aforementioned imaging modalities for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the near future.

Fluorescent magnetic hybrid nanoprobe for multimodal bioimaging

PubMed Central

Bright, Vanessa

2011-01-01

A fluorescent magnetic hybrid imaging nanoprobe (HINP) was fabricated by conjugation of superparamagnetic Fe3O4 nanoparticles and visible light-emitting (~600 nm) fluorescent CdTe/CdS quantum dots (QDs). The assembly strategy used the covalent linking of the oxidized dextran shell of magnetic particles to the glutathione ligands of QDs. Synthesized HINP formed stable water-soluble colloidal dispersions. The structure and properties of the particles were characterized by transmission electron and atomic force microscopy, energy dispersive X-ray analysis and inductively coupled plasma optical emission spectroscopy, dynamic light scattering analysis, optical absorption and photoluminescence spectroscopy, and fluorescent imaging. The luminescence imaging region of the nanoprobe was extended to the near-infrared (NIR) (~800 nm) by conjugation of superparamagnetic nanoparticles with synthesized CdHgTe/CdS QDs. Cadmium, mercury based QDs in HINP can be easily replaced by novel water soluble glutathione stabilized AgInS2/ZnS QDs to present a new class of cadmium-free multimodal imaging agents. Observed NIR photoluminescence of fluorescent magnetic nanocomposites supports their use for bioimaging. The developed HINP provides dual-imaging channels for simultaneous optical and magnetic resonance imaging. PMID:21597146

Recent advances in near-infrared fluorescence-guided imaging surgery using indocyanine green.

PubMed

Namikawa, Tsutomu; Sato, Takayuki; Hanazaki, Kazuhiro

2015-12-01

Near-infrared (NIR) fluorescence imaging has better tissue penetration, allowing for the effective rejection of excitation light and detection deep inside organs. Indocyanine green (ICG) generates NIR fluorescence after illumination by an NIR ray, enabling real-time intraoperative visualization of superficial lymphatic channels and vessels transcutaneously. The HyperEye Medical System (HEMS) can simultaneously detect NIR rays under room light to provide color imaging, which enables visualization under bright light. Thus, NIR fluorescence imaging using ICG can provide for excellent diagnostic accuracy in detecting sentinel lymph nodes in cancer and microvascular circulation in various ischemic diseases, to assist us with intraoperative decision making. Including HEMS in this system could further improve the sentinel lymph node mapping and intraoperative identification of blood supply in reconstructive organs and ischemic diseases, making it more attractive than conventional imaging. Moreover, the development of new laparoscopic imaging systems equipped with NIR will allow fluorescence-guided surgery in a minimally invasive setting. Future directions, including the conjugation of NIR fluorophores to target specific cancer markers might be realistic technology with diagnostic and therapeutic benefits.

Mirrored pyramidal wells for simultaneous multiple vantage point microscopy.

PubMed

Seale, K T; Reiserer, R S; Markov, D A; Ges, I A; Wright, C; Janetopoulos, C; Wikswo, J P

2008-10-01

We report a novel method for obtaining simultaneous images from multiple vantage points of a microscopic specimen using size-matched microscopic mirrors created from anisotropically etched silicon. The resulting pyramidal wells enable bright-field and fluorescent side-view images, and when combined with z-sectioning, provide additional information for 3D reconstructions of the specimen. We have demonstrated the 3D localization and tracking over time of the centrosome of a live Dictyostelium discoideum. The simultaneous acquisition of images from multiple perspectives also provides a five-fold increase in the theoretical collection efficiency of emitted photons, a property which may be useful for low-light imaging modalities such as bioluminescence, or low abundance surface-marker labelling.

Compact fluorescence and white-light imaging system for intraoperative visualization of nerves

NASA Astrophysics Data System (ADS)

Gray, Dan; Kim, Evgenia; Cotero, Victoria; Staudinger, Paul; Yazdanfar, Siavash; tan Hehir, Cristina

2012-02-01

Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

A compact fluorescence and white light imaging system for intraoperative visualization of nerves

NASA Astrophysics Data System (ADS)

Gray, Dan; Kim, Evgenia; Cotero, Victoria; Staudinger, Paul; Yazdanfar, Siavash; Tan Hehir, Cristina

2012-03-01

Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

An integrated instrumental setup for the combination of atomic force microscopy with optical spectroscopy.

PubMed

Owen, R J; Heyes, C D; Knebel, D; Röcker, C; Nienhaus, G U

2006-07-01

In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution. (c) 2006 Wiley Periodicals, Inc.

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

PubMed

Zhao, Ming; Li, Yu; Peng, Leilei

2014-05-05

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein–DNA complexes

PubMed Central

Ebenstein, Yuval; Gassman, Natalie; Kim, Soohong; Weiss, Shimon

2011-01-01

Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA. PMID:19452448

X-ray ptychographic and fluorescence microscopy of frozen-hydrated cells using continuous scanning

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2017-03-27

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. Here, by working with cells that have been rapidly frozen without the use of chemicalmore » fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2015-02-24

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and similar to 90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. This combined approach offers a way to study the role of trace elements in their structural context.« less

Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability.

PubMed

Hirabayashi, Kazuhisa; Hanaoka, Kenjiro; Egawa, Takahiro; Kobayashi, Chiaki; Takahashi, Shodai; Komatsu, Toru; Ueno, Tasuku; Terai, Takuya; Ikegaya, Yuji; Nagano, Tetsuo; Urano, Yasuteru

2016-10-01

Fluorescence imaging of calcium ions (Ca(2+)) has become an essential technique for investigation of signaling pathways involving Ca(2+) as a second messenger. But, Ca(2+) signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca(2+) and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca(2+) that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca(2+) concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca(2+) red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

New developments in clinical CARS

NASA Astrophysics Data System (ADS)

Weinigel, Martin; Breunig, Hans Georg; Kellner-Höfer, Marcel; Bückle, Rainer; Darvin, Maxim; Lademann, Juergen; König, Karsten

2013-02-01

We combined two-photon fluorescence and coherent anti-Stokes Raman scattering (CARS) imaging in a clinical hybrid multiphoton tomograph for in vivo imaging of human skin. The clinically approved TPEF/CARS system provides simultaneous imaging of endogenous fluorophores and non-fluorescent lipids. The Stokes laser for the two-beam configuration of CARS is based on spectral broadening of femtosecond laser pulses in a photonic crystal fiber (PCF). We report on the highly flexible medical TPEF/CARS tomograph MPTflex®-CARS with an articulated arm and first in vivo measurements on human skin.

Single-Molecule Fluorescence In Situ Hybridization (FISH) of Circular RNA CDR1as.

PubMed

Kocks, Christine; Boltengagen, Anastasiya; Piwecka, Monika; Rybak-Wolf, Agnieszka; Rajewsky, Nikolaus

2018-01-01

Individual mRNA molecules can be imaged in fixed cells by hybridization with multiple, singly labeled oligonucleotide probes, followed by computational identification of fluorescent signals. This approach, called single-molecule RNA fluorescence in situ hybridization (smRNA FISH), allows subcellular localization and absolute quantification of RNA molecules in individual cells. Here, we describe a simple smRNA FISH protocol for two-color imaging of a circular RNA, CDR1as, simultaneously with an unrelated messenger RNA. The protocol can be adapted to circRNAs that coexist with overlapping, noncircular mRNA isoforms produced from the same genetic locus.

Hybrid system for in vivo real-time planar fluorescence and volumetric optoacoustic imaging

NASA Astrophysics Data System (ADS)

Chen, Zhenyue; Deán-Ben, Xosé Luís.; Gottschalk, Sven; Razansky, Daniel

2018-02-01

Fluorescence imaging is widely employed in all fields of cell and molecular biology due to its high sensitivity, high contrast and ease of implementation. However, the low spatial resolution and lack of depth information, especially in strongly-scattering samples, restrict its applicability for deep-tissue imaging applications. On the other hand, optoacoustic imaging is known to deliver a unique set of capabilities such as high spatial and temporal resolution in three dimensions, deep penetration and spectrally-enriched imaging contrast. Since fluorescent substances can generate contrast in both modalities, simultaneous fluorescence and optoacoustic readings can provide new capabilities for functional and molecular imaging of living organisms. Optoacoustic images can further serve as valuable anatomical references based on endogenous hemoglobin contrast. Herein, we propose a hybrid system for in vivo real-time planar fluorescence and volumetric optoacoustic tomography, both operating in reflection mode, which synergistically combines the advantages of stand-alone systems. Validation of the spatial resolution and sensitivity of the system were first carried out in tissue mimicking phantoms while in vivo imaging was further demonstrated by tracking perfusion of an optical contrast agent in a mouse brain in the hybrid imaging mode. Experimental results show that the proposed system effectively exploits the contrast mechanisms of both imaging modalities, making it especially useful for accurate monitoring of fluorescence-based signal dynamics in highly scattering samples.

Versatile Polymer Nanoparticles as Two-Photon-Triggered Photosensitizers for Simultaneous Cellular, Deep-Tissue Imaging, and Photodynamic Therapy.

PubMed

Guo, Liang; Ge, Jiechao; Liu, Qian; Jia, Qingyan; Zhang, Hongyan; Liu, Weimin; Niu, Guangle; Liu, Sha; Gong, Jianru; Hackbarth, Steffen; Wang, Pengfei

2017-06-01

Clinical applications of current photodynamic therapy (PDT) photosensitizers (PSs) are often limited by their absorption in the UV-vis range that possesses limited tissue penetration ability, leading to ineffective therapeutic response for deep-seated tumors. Alternatively, two-photon excited PS (TPE-PS) using NIR light triggered is one the most promising candidates for PDT improvement. Herein, multimodal polymer nanoparticles (PNPs) from polythiophene derivative as two-photon fluorescence imaging as well as two-photon-excited PDT agent are developed. The prepared PNPs exhibit excellent water dispersibility, high photostability and pH stability, strong fluorescence brightness, and low dark toxicity. More importantly, the PNPs also possess other outstanding features including: (1) the high 1 O 2 quantum yield; (2) the strong two-photon-induced fluorescence and efficient 1 O 2 generation; (3) the specific accumulation in lysosomes of HeLa cells; and (4) the imaging detection depth up to 2100 µm in the mock tissue under two-photon. The multifunctional PNPs are promising candidates as TPE-PDT agent for simultaneous cellular, deep-tissue imaging, and highly efficient in vivo PDT of cancer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multimodal second harmonic generation and two photon fluorescence imaging of microdomain calcium contraction coupling in single cardiomyocytes

NASA Astrophysics Data System (ADS)

Chan, James; Awasthi, Samir; Izu, Leighton; Mao, Ziliang; Jian, Zhong; Landas, Trevor; Lerner, Aaron; Shimkunas, Rafael; Woldeyesus, Rahwa; Bossuyt, Julie; Wood, Brittani; Chen, Yi-Je; Matthews, Dennis; Lieu, Deborah; Chiamvimonvat, Nipavan; Lam, Kit; Chen-Izu, Ye

2016-11-01

The objective of this study was to develop a method for simultaneously measuring the calcium and contraction dynamics of single, live cardiomyocytes at high spatial resolutions. Such measurements are important to investigate local calcium release and the mechanical response at the sarcomere level (i.e. the basic unit of contraction), which have important implications in cardiac dysfunction and arrhythmias in conditions such as hypertension, atrial fibrillation, and myocardial infarction. Here, we describe a multimodal second harmonic generation (SHG) and two photon fluorescence (2PF) microscopy technique that is used to simultaneously measure subsarcomere calcium and contraction events at high spatial and temporal resolutions. The method takes advantage of the label-free nature of SHG for imaging the sarcomeres and the high spatial colocalization of the SHG signal and the fluorescence signal excited from calcium indicators. This microscope was used to measure calcium sparks and waves and associated contractions in subcellular microdomains, leading to the generation of subcellular strain. We anticipate this new imaging tool will play an important role in studying mechanical stress-induced heart disease.

Imaging of size-dependent uptake and identification of novel pathways in mouse Peyer's patches using fluorescent organosilica particles.

PubMed

Awaad, Aziz; Nakamura, Michihiro; Ishimura, Kazunori

2012-07-01

We investigated size-dependent uptake of fluorescent thiol-organosilica particles by Peyer's patches (PPs). We performed an oral single-particle administration (95, 130, 200, 340, 695 and 1050 nm) and a simultaneous dual-particle administration using 2 kinds of particles. Histological imaging and quantitative analysis revealed that particles taken up by the PP subepithelial dome were size dependent, and there was an optimal size range for higher uptake. Quantitative analysis of simultaneous dual-particle administration revealed that the percentage of fluorescence areas for 95, 130, 200, 340, 695 and 1050 nm with respect to 110 nm area was 124.0, 89.1, 73.8, 20.2, 9.2 and 0.5%, respectively. Additionally, imaging using fluorescent thiol-organosilica particles could detect 2 novel pathways through mouse PP epithelium: the transcellular pathway and the paracellular pathway. The uptake of nanoparticles based on an optimal size range and 2 novel pathways could indicate a new approach for vaccine delivery and nanomedicine development. Studying various sizes of fluorescent organosilica particles and their uptake in Peyer's patches, this team of authors determined the optimal size range of administration. Two novel pathways through mouse Peyer's patch epithelium were detected, i.e., the transcellular pathway and the paracellular pathway. This observation may have important applications in future vaccine delivery and nano-drug delivery. Copyright © 2012 Elsevier Inc. All rights reserved.

Sentinel lymph node biopsy under fluorescent indocyanin green guidance: Initial experience

PubMed Central

Aydoğan, Fatih; Arıkan, Akif Enes; Aytaç, Erman; Velidedeoğlu, Mehmet; Yılmaz, Mehmet Halit; Sager, Muhammet Sait; Çelik, Varol; Uras, Cihan

2016-01-01

Objective: Sentinel lymph node biopsy can be applied by using either blue dye or radionuclide method or both in breast cancer. Fluorescent imaging with indocyanine green is a new defined method. This study evaluates the applicability of sentinel lymph node biopsy via fluorescent indocyanine green. Material and Methods: IC-VIEW (Pulsion Medical Systems AG, Munich, Germany) infrared visualization system was used for imaging. Two mL of indocyanine green was injected to visualize sentinel lymph nodes. After injection, subcutaneous lymphatics were traced and sentinel lymph nodes were found with simultaneous imaging. Sentinel lymph nodes were excised under fluorescent light guidance, and excised lymph nodes were examined histopathologically. Patients with sentinel lymph node metastases underwent axillary dissection. Results: Four patients with sentinel lymph node biopsy due to breast cancer were included in the study. Sentinel lymph nodes were visualized with indocyanine green in all patients. The median number of excised sentinel lymph node was 2 (2–3). Two patients with lymph node metastasis underwent axillary dissection. No metastasis was detected in lymph nodes other than the sentinel nodes in patients with axillary dissection. There was no complication during and after the operation related to the method. Conclusion: According to our limited experience, sentinel lymph node biopsy under fluorescent indocyanine green guidance, which has an advantage of simultaneous visualization, is technically feasible. PMID:26985159

A turn-on fluorescent probe for simultaneous sensing of cysteine/homocysteine and hydrogen sulfide and its bioimaging applications.

PubMed

Chen, Fengzao; Han, Deman; Gao, Yuan; Liu, Heng; Wang, Shengfu; Zhou, Fangyu; Li, Kaibin; Zhang, Siqi; Shao, Wujun; He, Yanling

2018-09-01

Hydrogen sulfide and biothiol molecules such as Cys, Hcy, and GSH play important roles in biological systems. Exploiting a probe to simultaneously detect and distinguish them is quite important. In this work, a versatile fluorescent probe that can simultaneously detect and discriminate Cys/Hcy and H 2 S is reported. The probe easily prepared by the Knoevenagel condensation of cyanoacetylindole with chlorinated phenyl-propenal possessed three potential sites that could react with biothiols and H 2 S. This probe also exhibited rapidity, high selectivity, and sensitivity for Cys/Hcy and H 2 S with distinct optical signal changes. The probe was able to display obvious fluorescence enhancement at 480 nm for Cys/Hcy and unique absorbance enhancement at 500 nm for H 2 S. We also demonstrated that the probe can be successfully applied to image Cys in MCF-7 cells suing a confocal fluorescence microscope. Copyright © 2018 Elsevier B.V. All rights reserved.

Simultaneous visualization of water and hydrogen peroxide vapor using two-photon laser-induced fluorescence and photofragmentation laser-induced fluorescence.

PubMed

Larsson, Kajsa; Johansson, Olof; Aldén, Marcus; Bood, Joakim

2014-01-01

A concept based on a combination of photofragmentation laser-induced fluorescence (PF-LIF) and two-photon laser-induced fluorescence (LIF) is for the first time demonstrated for simultaneous detection of hydrogen peroxide (H2O2) and water (H2O) vapor. Water detection is based on two-photon excitation by an injection-locked krypton fluoride (KrF) excimer laser (248.28 nm), which induces broadband fluorescence (400-500 nm) from water. The same laser simultaneously photodissociates H2O2, whereupon the generated OH fragments are probed by LIF after a time delay of typically 50 ns, by a frequency-doubled dye laser (281.91 nm). Experiments in six different H2O2/H2O mixtures of known compositions show that both signals are linearly dependent on respective species concentration. For the H2O2 detection there is a minor interfering signal contribution from OH fragments created by two-photon photodissociation of H2O. Since the PF-LIF signal yield from H2O2 is found to be at least ∼24,000 times higher than the PF-LIF signal yield from H2O at room temperature, this interference is negligible for most H2O/H2O2 mixtures of practical interest. Simultaneous single-shot imaging of both species was demonstrated in a slightly turbulent flow. For single-shot imaging the minimum detectable H2O2 and H2O concentration is 10 ppm and 0.5%, respectively. The proposed measurement concept could be a valuable asset in several areas, for example, in atmospheric and combustion science and research on vapor-phase H2O2 sterilization in the pharmaceutical and aseptic food-packaging industries.

Fluorescence laminar optical tomography for brain imaging: system implementation and performance evaluation.

PubMed

Azimipour, Mehdi; Sheikhzadeh, Mahya; Baumgartner, Ryan; Cullen, Patrick K; Helmstetter, Fred J; Chang, Woo-Jin; Pashaie, Ramin

2017-01-01

We present our effort in implementing a fluorescence laminar optical tomography scanner which is specifically designed for noninvasive three-dimensional imaging of fluorescence proteins in the brains of small rodents. A laser beam, after passing through a cylindrical lens, scans the brain tissue from the surface while the emission signal is captured by the epi-fluorescence optics and is recorded using an electron multiplication CCD sensor. Image reconstruction algorithms are developed based on Monte Carlo simulation to model light–tissue interaction and generate the sensitivity matrices. To solve the inverse problem, we used the iterative simultaneous algebraic reconstruction technique. The performance of the developed system was evaluated by imaging microfabricated silicon microchannels embedded inside a substrate with optical properties close to the brain as a tissue phantom and ultimately by scanning brain tissue in vivo. Details of the hardware design and reconstruction algorithms are discussed and several experimental results are presented. The developed system can specifically facilitate neuroscience experiments where fluorescence imaging and molecular genetic methods are used to study the dynamics of the brain circuitries.

Clinical multiphoton and CARS microscopy

NASA Astrophysics Data System (ADS)

Breunig, H. G.; Weinigel, M.; Darvin, M. E.; Lademann, J.; König, K.

2012-03-01

We report on clinical CARS imaging of human skin in vivo with the certified hybrid multiphoton tomograph CARSDermaInspect. The CARS-DermaInspect provides simultaneous imaging of non-fluorescent intradermal lipid and water as well as imaging of two-photon excited fluorescence from intrinsic molecules. Two different excitation schemes for CARS imaging have been realized: In the first setup, a combination of fs oscillator and optical parametric oscillator provided fs-CARS pump and Stokes pulses, respectively. In the second setup a fs oscillator was combined with a photonic crystal fiber which provided a broadband spectrum. A spectral range out of the broadband-spectrum was selected and used for CARS excitation in combination with the residual fs-oscillator output. In both setups, in addition to CARS, single-beam excitation was used for imaging of two-photon excited fluorescence and second harmonic generation signals. Both CARS-excitation systems were successfully used for imaging of lipids inside the skin in vivo.

Multispectral Live-Cell Imaging.

PubMed

Cohen, Sarah; Valm, Alex M; Lippincott-Schwartz, Jennifer

2018-06-01

Fluorescent proteins and vital dyes are invaluable tools for studying dynamic processes within living cells. However, the ability to distinguish more than a few different fluorescent reporters in a single sample is limited by the spectral overlap of available fluorophores. Here, we present a protocol for imaging live cells labeled with six fluorophores simultaneously. A confocal microscope with a spectral detector is used to acquire images, and linear unmixing algorithms are applied to identify the fluorophores present in each pixel of the image. We describe the application of this method to visualize the dynamics of six different organelles, and to quantify the contacts between organelles. However, this method can be used to image any molecule amenable to tagging with a fluorescent probe. Thus, multispectral live-cell imaging is a powerful tool for systems-level analysis of cellular organization and dynamics. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

A CMOS image sensor with stacked photodiodes for lensless observation system of digital enzyme-linked immunosorbent assay

NASA Astrophysics Data System (ADS)

Takehara, Hironari; Miyazawa, Kazuya; Noda, Toshihiko; Sasagawa, Kiyotaka; Tokuda, Takashi; Kim, Soo Hyeon; Iino, Ryota; Noji, Hiroyuki; Ohta, Jun

2014-01-01

A CMOS image sensor with stacked photodiodes was fabricated using 0.18 µm mixed signal CMOS process technology. Two photodiodes were stacked at the same position of each pixel of the CMOS image sensor. The stacked photodiodes consist of shallow high-concentration N-type layer (N+), P-type well (PW), deep N-type well (DNW), and P-type substrate (P-sub). PW and P-sub were shorted to ground. By monitoring the voltage of N+ and DNW individually, we can observe two monochromatic colors simultaneously without using any color filters. The CMOS image sensor is suitable for fluorescence imaging, especially contact imaging such as a lensless observation system of digital enzyme-linked immunosorbent assay (ELISA). Since the fluorescence increases with time in digital ELISA, it is possible to observe fluorescence accurately by calculating the difference from the initial relation between the pixel values for both photodiodes.

Mirrored pyramidal wells for simultaneous multiple vantage point microscopy

PubMed Central

Seale, K.T.; Reiserer, R.S.; Markov, D.A.; Ges, I.A.; Wright, C.; Janetopoulos, C.; Wikswo, J.P.

2013-01-01

Summary We report a novel method for obtaining simultaneous images from multiple vantage points of a microscopic specimen using size-matched microscopic mirrors created from anisotropically etched silicon. The resulting pyramidal wells enable bright-field and fluorescent side-view images, and when combined with z-sectioning, provide additional information for 3D reconstructions of the specimen. We have demonstrated the 3D localization and tracking over time of the centrosome of a live Dictyostelium discoideum. The simultaneous acquisition of images from multiple perspectives also provides a five-fold increase in the theoretical collection efficiency of emitted photons, a property which may be useful for low-light imaging modalities such as bioluminescence, or low abundance surface-marker labelling. PMID:19017196

Soot Precursor Material: Spatial Location via Simultaneous LIF-LII Imaging and Characterization via TEM

NASA Technical Reports Server (NTRS)

VanderWal, Randall L.

1996-01-01

The chemical and physical transformation between gaseous fuel pyrolysis products and solid carbonaceous soot represents a critical step in soot formation. In this paper, simultaneous two-dimensional LIF-LII (laser-induced fluorescence - laser-induced incandescence) images identify the spatial location where the earliest identifiable chemical and physical transformation of material towards solid carbonaceous soot occurs along the axial streamline in a normal diffusion flame. The identification of the individual LIF and LII signals is achieved by examining both the excitation wavelength dependence and characteristic temporal decay of each signal. Spatially precise thermophoretic sampling measurements are guided by the LIF-LII images with characterization of the sampled material accomplished via both bright and dark field TEM. Both bright and dark field TEM measurements support the observed changes in photophysical properties which account for conversion of fluorescence to incandescence as fuel pyrolysis products evolve towards solid carbonaceous soot.

General route to multifunctional uniform yolk/mesoporous silica shell nanocapsules: a platform for simultaneous cancer-targeted imaging and magnetically guided drug delivery.

PubMed

Zhang, Lingyu; Wang, Tingting; Yang, Lei; Liu, Cong; Wang, Chungang; Liu, Haiyan; Wang, Y Andrew; Su, Zhongmin

2012-09-24

Hollow mesoporous SiO(2) (mSiO(2)) nanostructures with movable nanoparticles (NPs) as cores, so-called yolk-shell nanocapsules (NCs), have attracted great research interest. However, a highly efficient, simple and general way to produce yolk-mSiO(2) shell NCs with tunable functional cores and shell compositions is still a great challenge. A facile, general and reproducible strategy has been developed for fabricating discrete, monodisperse and highly uniform yolk-shell NCs under mild conditions, composed of mSiO(2) shells and diverse functional NP cores with different compositions and shapes. These NPs can be Fe(3)O(4) NPs, gold nanorods (GNRs), and rare-earth upconversion NRs, endowing the yolk-mSiO(2) shell NCs with magnetic, plasmonic, and upconversion fluorescent properties. In addition, multifunctional yolk-shell NCs with tunable interior hollow spaces and mSiO(2) shell thickness can be precisely controlled. More importantly, fluorescent-magnetic-biotargeting multifunctional polyethyleneimine (PEI)-modified fluorescent Fe(3)O(4)@mSiO(2) yolk-shell nanobioprobes as an example for simultaneous targeted fluorescence imaging and magnetically guided drug delivery to liver cancer cells is also demonstrated. This synthetic approach can be easily extended to the fabrication of multifunctional yolk@mSiO(2) shell nanostructures that encapsulate various functional movable NP cores, which construct a potential platform for the simultaneous targeted delivery of drug/gene/DNA/siRNA and bio-imaging. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous recording of fluorescence and electrical signals by photometric patch electrode in deep brain regions in vivo

PubMed Central

Hirai, Yasuharu; Nishino, Eri

2015-01-01

Despite its widespread use, high-resolution imaging with multiphoton microscopy to record neuronal signals in vivo is limited to the surface of brain tissue because of limited light penetration. Moreover, most imaging studies do not simultaneously record electrical neural activity, which is, however, crucial to understanding brain function. Accordingly, we developed a photometric patch electrode (PME) to overcome the depth limitation of optical measurements and also enable the simultaneous recording of neural electrical responses in deep brain regions. The PME recoding system uses a patch electrode to excite a fluorescent dye and to measure the fluorescence signal as a light guide, to record electrical signal, and to apply chemicals to the recorded cells locally. The optical signal was analyzed by either a spectrometer of high light sensitivity or a photomultiplier tube depending on the kinetics of the responses. We used the PME in Oregon Green BAPTA-1 AM-loaded avian auditory nuclei in vivo to monitor calcium signals and electrical responses. We demonstrated distinct response patterns in three different nuclei of the ascending auditory pathway. On acoustic stimulation, a robust calcium fluorescence response occurred in auditory cortex (field L) neurons that outlasted the electrical response. In the auditory midbrain (inferior colliculus), both responses were transient. In the brain-stem cochlear nucleus magnocellularis, calcium response seemed to be effectively suppressed by the activity of metabotropic glutamate receptors. In conclusion, the PME provides a powerful tool to study brain function in vivo at a tissue depth inaccessible to conventional imaging devices. PMID:25761950

Simultaneous recording of fluorescence and electrical signals by photometric patch electrode in deep brain regions in vivo.

PubMed

Hirai, Yasuharu; Nishino, Eri; Ohmori, Harunori

2015-06-01

Despite its widespread use, high-resolution imaging with multiphoton microscopy to record neuronal signals in vivo is limited to the surface of brain tissue because of limited light penetration. Moreover, most imaging studies do not simultaneously record electrical neural activity, which is, however, crucial to understanding brain function. Accordingly, we developed a photometric patch electrode (PME) to overcome the depth limitation of optical measurements and also enable the simultaneous recording of neural electrical responses in deep brain regions. The PME recoding system uses a patch electrode to excite a fluorescent dye and to measure the fluorescence signal as a light guide, to record electrical signal, and to apply chemicals to the recorded cells locally. The optical signal was analyzed by either a spectrometer of high light sensitivity or a photomultiplier tube depending on the kinetics of the responses. We used the PME in Oregon Green BAPTA-1 AM-loaded avian auditory nuclei in vivo to monitor calcium signals and electrical responses. We demonstrated distinct response patterns in three different nuclei of the ascending auditory pathway. On acoustic stimulation, a robust calcium fluorescence response occurred in auditory cortex (field L) neurons that outlasted the electrical response. In the auditory midbrain (inferior colliculus), both responses were transient. In the brain-stem cochlear nucleus magnocellularis, calcium response seemed to be effectively suppressed by the activity of metabotropic glutamate receptors. In conclusion, the PME provides a powerful tool to study brain function in vivo at a tissue depth inaccessible to conventional imaging devices. Copyright © 2015 the American Physiological Society.

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging

PubMed Central

Zhao, Ming; Li, Yu; Peng, Leilei

2014-01-01

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community. PMID:24921725

In vivo bioluminescence imaging of cell differentiation in biomaterials: a platform for scaffold development.

PubMed

Bagó, Juli R; Aguilar, Elisabeth; Alieva, Maria; Soler-Botija, Carolina; Vila, Olaia F; Claros, Silvia; Andrades, José A; Becerra, José; Rubio, Nuria; Blanco, Jerónimo

2013-03-01

In vivo testing is a mandatory last step in scaffold development. Agile longitudinal noninvasive real-time monitoring of stem cell behavior in biomaterials implanted in live animals should facilitate the development of scaffolds for tissue engineering. We report on a noninvasive bioluminescence imaging (BLI) procedure for simultaneous monitoring of changes in the expression of multiple genes to evaluate scaffold performance in vivo. Adipose tissue-derived stromal mensenchymal cells were dually labeled with Renilla red fluorescent protein and firefly green fluorescent protein chimeric reporters regulated by cytomegalovirus and tissue-specific promoters, respectively. Labeled cells were induced to differentiate in vitro and in vivo, by seeding in demineralized bone matrices (DBMs) and monitored by BLI. Imaging results were validated by RT-polymerase chain reaction and histological procedures. The proposed approach improves molecular imaging and measurement of changes in gene expression of cells implanted in live animals. This procedure, applicable to the simultaneous analysis of multiple genes from cells seeded in DBMs, should facilitate engineering of scaffolds for tissue repair.

In Vivo Bioluminescence Imaging of Cell Differentiation in Biomaterials: A Platform for Scaffold Development

PubMed Central

Bagó, Juli R.; Aguilar, Elisabeth; Alieva, Maria; Soler-Botija, Carolina; Vila, Olaia F.; Claros, Silvia; Andrades, José A.; Becerra, José; Rubio, Nuria

2013-01-01

In vivo testing is a mandatory last step in scaffold development. Agile longitudinal noninvasive real-time monitoring of stem cell behavior in biomaterials implanted in live animals should facilitate the development of scaffolds for tissue engineering. We report on a noninvasive bioluminescence imaging (BLI) procedure for simultaneous monitoring of changes in the expression of multiple genes to evaluate scaffold performance in vivo. Adipose tissue-derived stromal mensenchymal cells were dually labeled with Renilla red fluorescent protein and firefly green fluorescent protein chimeric reporters regulated by cytomegalovirus and tissue-specific promoters, respectively. Labeled cells were induced to differentiate in vitro and in vivo, by seeding in demineralized bone matrices (DBMs) and monitored by BLI. Imaging results were validated by RT-polymerase chain reaction and histological procedures. The proposed approach improves molecular imaging and measurement of changes in gene expression of cells implanted in live animals. This procedure, applicable to the simultaneous analysis of multiple genes from cells seeded in DBMs, should facilitate engineering of scaffolds for tissue repair. PMID:23013334

Visualisation of blood and lymphatic vessels with increasing exposure time of the detector

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalchenko, V V; Kuznetsov, Yu L; Meglinski, I V

2013-07-31

We describe the laser speckle contrast method for simultaneous noninvasive imaging of blood and lymphatic vessels of living organisms, based on increasing detector exposure time. In contrast to standard methods of fluorescent angiography, this technique of vascular bed imaging and lymphatic and blood vessel demarcation does not employ toxic fluorescent markers. The method is particularly promising with respect to the physiology of the cardiovascular system under in vivo conditions. (laser applications in biology and medicine)

Compressive hyperspectral time-resolved wide-field fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Pian, Qi; Yao, Ruoyang; Sinsuebphon, Nattawut; Intes, Xavier

2017-07-01

Spectrally resolved fluorescence lifetime imaging and spatial multiplexing have offered information content and collection-efficiency boosts in microscopy, but efficient implementations for macroscopic applications are still lacking. An imaging platform based on time-resolved structured light and hyperspectral single-pixel detection has been developed to perform quantitative macroscopic fluorescence lifetime imaging (MFLI) over a large field of view (FOV) and multiple spectral bands simultaneously. The system makes use of three digital micromirror device (DMD)-based spatial light modulators (SLMs) to generate spatial optical bases and reconstruct N by N images over 16 spectral channels with a time-resolved capability (∼40 ps temporal resolution) using fewer than N2 optical measurements. We demonstrate the potential of this new imaging platform by quantitatively imaging near-infrared (NIR) Förster resonance energy transfer (FRET) both in vitro and in vivo. The technique is well suited for quantitative hyperspectral lifetime imaging with a high sensitivity and paves the way for many important biomedical applications.

Designed Er(3+)-singly doped NaYF4 with double excitation bands for simultaneous deep macroscopic and microscopic upconverting bioimaging.

PubMed

Wen, Xuanyuan; Wang, Baoju; Wu, Ruitao; Li, Nana; He, Sailing; Zhan, Qiuqiang

2016-06-01

Simultaneous deep macroscopic imaging and microscopic imaging is in urgent demand, but is challenging to achieve experimentally due to the lack of proper fluorescent probes. Herein, we have designed and successfully synthesized simplex Er(3+)-doped upconversion nanoparticles (UCNPs) with double excitation bands for simultaneous deep macroscopic and microscopic imaging. The material structure and the excitation wavelength of Er(3+)-singly doped UCNPs were further optimized to enhance the upconversion emission efficiency. After optimization, we found that NaYF4:30%Er(3+)@NaYF4:2%Er(3+) could simultaneously achieve efficient two-photon excitation (2PE) macroscopic tissue imaging and three-photon excitation (3PE) deep microscopic when excited by 808 nm continuous wave (CW) and 1480 nm CW lasers, respectively. In vitro cell imaging and in vivo imaging have also been implemented to demonstrate the feasibility and potential of the proposed simplex Er(3+)-doped UCNPs as bioprobe.

Pyridine Based Fluorescence Probe: Simultaneous Detection and Removal of Arsenate from Real Samples with Living Cell Imaging Properties.

PubMed

Nandi, Sandip; Sahana, Animesh; Sarkar, Bidisha; Mukhopadhyay, Subhra Kanti; Das, Debasis

2015-09-01

Pyridine based fluorescence probe, DFPPIC and its functionalized Merrifield polymer has been synthesized, characterized and used as an arsenate selective fluorescence sensor. Arsenate induced fluorescence enhancement is attributed to inter-molecular H-bonding assisted CHEF process. The detection limit for arsenate is 0.001 μM, much below the WHO recommended tolerance level in drinking water. DFPPIC can detect intracellular arsenate in drinking water of Purbasthali, West Bengal, India efficiently. Graphical Abstract DFPPIC and its Merrifield conjugate polymer are used for selective determination and removal of arsenate from real drinking water samples of Purbasthali, a highly arsenic contaminated region of West Bengal, India. DFPPIC is very promising to imaging arsenate in living cells.

In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Gray, Daniel C.; Merigan, William; Wolfing, Jessica I.; Gee, Bernard P.; Porter, Jason; Dubra, Alfredo; Twietmeyer, Ted H.; Ahamd, Kamran; Tumbar, Remy; Reinholz, Fred; Williams, David R.

2006-08-01

The ability to resolve single cells noninvasively in the living retina has important applications for the study of normal retina, diseased retina, and the efficacy of therapies for retinal disease. We describe a new instrument for high-resolution, in vivo imaging of the mammalian retina that combines the benefits of confocal detection, adaptive optics, multispectral, and fluorescence imaging. The instrument is capable of imaging single ganglion cells and their axons through retrograde transport in ganglion cells of fluorescent dyes injected into the monkey lateral geniculate nucleus (LGN). In addition, we demonstrate a method involving simultaneous imaging in two spectral bands that allows the integration of very weak signals across many frames despite inter-frame movement of the eye. With this method, we are also able to resolve the smallest retinal capillaries in fluorescein angiography and the mosaic of retinal pigment epithelium (RPE) cells with lipofuscin autofluorescence.

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images.

PubMed

Zhang, Zhiqing; Kuzmin, Nikolay V; Groot, Marie Louise; de Munck, Jan C

2018-01-01

Third harmonic generation (THG) microscopy is a label-free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all-nuclei-highlighted fluorescence images, acquired simultaneously from the same tissue area. For quantitative comparison of a substantial pair of images, we present here a segmentation workflow that is applicable for both THG and fluorescence images, with a precision of 91.3 % and 95.8 % achieved respectively. We find that the correspondence between the main features of the two imaging modalities amounts to 88.9 %, providing quantitative evidence of the interpretation of dark holes as brain cells. Moreover, 80 % bright objects in THG images overlap with nuclei highlighted in the fluorescence images, and they are 2 times smaller than the dark holes, showing that cells of different morphologies can be recognized in THG images. We expect that the described quantitative comparison is applicable to other types of brain tissue and with more specific staining experiments for cell type identification. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Development of a Surgical Navigation System with Beam Split and Fusion of the Visible and Near-Infrared Fluorescence].

PubMed

Yang, Xiaofeng; Wu, Wei; Wang, Guoan

2015-04-01

This paper presents a surgical optical navigation system with non-invasive, real-time, and positioning characteristics for open surgical procedure. The design was based on the principle of near-infrared fluorescence molecular imaging. The in vivo fluorescence excitation technology, multi-channel spectral camera technology and image fusion software technology were used. Visible and near-infrared light ring LED excitation source, multi-channel band pass filters, spectral camera 2 CCD optical sensor technology and computer systems were integrated, and, as a result, a new surgical optical navigation system was successfully developed. When the near-infrared fluorescence was injected, the system could display anatomical images of the tissue surface and near-infrared fluorescent functional images of surgical field simultaneously. The system can identify the lymphatic vessels, lymph node, tumor edge which doctor cannot find out with naked eye intra-operatively. Our research will guide effectively the surgeon to remove the tumor tissue to improve significantly the success rate of surgery. The technologies have obtained a national patent, with patent No. ZI. 2011 1 0292374. 1.

Practical three color live cell imaging by widefield microscopy

PubMed Central

Xia, Jianrun; Kim, Song Hon H.; Macmillan, Susan

2006-01-01

Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope. PMID:16909160

Patch-based anisotropic diffusion scheme for fluorescence diffuse optical tomography--part 2: image reconstruction.

PubMed

Correia, Teresa; Koch, Maximilian; Ale, Angelique; Ntziachristos, Vasilis; Arridge, Simon

2016-02-21

Fluorescence diffuse optical tomography (fDOT) provides 3D images of fluorescence distributions in biological tissue, which represent molecular and cellular processes. The image reconstruction problem is highly ill-posed and requires regularisation techniques to stabilise and find meaningful solutions. Quadratic regularisation tends to either oversmooth or generate very noisy reconstructions, depending on the regularisation strength. Edge preserving methods, such as anisotropic diffusion regularisation (AD), can preserve important features in the fluorescence image and smooth out noise. However, AD has limited ability to distinguish an edge from noise. We propose a patch-based anisotropic diffusion regularisation (PAD), where regularisation strength is determined by a weighted average according to the similarity between patches around voxels within a search window, instead of a simple local neighbourhood strategy. However, this method has higher computational complexity and, hence, we wavelet compress the patches (PAD-WT) to speed it up, while simultaneously taking advantage of the denoising properties of wavelet thresholding. Furthermore, structural information can be incorporated into the image reconstruction with PAD-WT to improve image quality and resolution. In this case, the weights used to average voxels in the image are calculated using the structural image, instead of the fluorescence image. The regularisation strength depends on both structural and fluorescence images, which guarantees that the method can preserve fluorescence information even when it is not structurally visible in the anatomical images. In part 1, we tested the method using a denoising problem. Here, we use simulated and in vivo mouse fDOT data to assess the algorithm performance. Our results show that the proposed PAD-WT method provides high quality and noise free images, superior to those obtained using AD.

Multimodal flexible cystoscopy for creating co-registered panoramas of the bladder urothelium

NASA Astrophysics Data System (ADS)

Seibel, Eric J.; Soper, Timothy D.; Burkhardt, Matthew R.; Porter, Michael P.; Yoon, W. Jong

2012-02-01

Bladder cancer is the most expensive cancer to treat due to the high rate of recurrence. Though white light cystoscopy is the gold standard for bladder cancer surveillance, the advent of fluorescence biomarkers provides an opportunity to improve sensitivity for early detection and reduced recurrence resulting from more accurate excision. Ideally, fluorescence information could be combined with standard reflectance images to provide multimodal views of the bladder wall. The scanning fiber endoscope (SFE) of 1.2mm in diameter is able to acquire wide-field multimodal video from a bladder phantom with fluorescence cancer "hot-spots". The SFE generates images by scanning red, green, and blue (RGB) laser light and detects the backscatter signal for reflectance video of 500-line resolution at 30 frames per second. We imaged a bladder phantom with painted vessels and mimicked fluorescent lesions by applying green fluorescent microspheres to the surface. By eliminating the green laser illumination, simultaneous reflectance and fluorescence images can be acquired at the same field of view, resolution, and frame rate. Moreover, the multimodal SFE is combined with a robotic steering mechanism and image stitching software as part of a fully automated bladder surveillance system. Using this system, the SFE can be reliably articulated over the entire 360° bladder surface. Acquired images can then be stitched into a multimodal 3D panorama of the bladder using software developed in our laboratory. In each panorama, the fluorescence images are exactly co-registered with RGB reflectance.

Synthesizing and binding dual-mode poly (lactic-co-glycolic acid) (PLGA) nanobubbles for cancer targeting and imaging.

PubMed

Xu, Jeff S; Huang, Jiwei; Qin, Ruogu; Hinkle, George H; Povoski, Stephen P; Martin, Edward W; Xu, Ronald X

2010-03-01

Accurate assessment of tumor boundaries and recognition of occult disease are important oncologic principles in cancer surgeries. However, existing imaging modalities are not optimized for intraoperative cancer imaging applications. We developed a nanobubble (NB) contrast agent for cancer targeting and dual-mode imaging using optical and ultrasound (US) modalities. The contrast agent was fabricated by encapsulating the Texas Red dye in poly (lactic-co-glycolic acid) (PLGA) NBs and conjugating NBs with cancer-targeting ligands. Both one-step and three-step cancer-targeting strategies were tested on the LS174T human colon cancer cell line. For the one-step process, NBs were conjugated with the humanized HuCC49 Delta C(H)2 antibody to target the over-expressed TAG-72 antigen. For the three-step process, cancer cells were targeted by successive application of the biotinylated HuCC49 Delta C(H)2 antibody, streptavidin, and the biotinylated NBs. Both one-step and three-step processes successfully targeted the cancer cells with high binding affinity. NB-assisted dual-mode imaging was demonstrated on a gelatin phantom that embedded multiple tumor simulators at different NB concentrations. Simultaneous fluorescence and US images were acquired for these tumor simulators and linear correlations were observed between the fluorescence/US intensities and the NB concentrations. Our research demonstrated the technical feasibility of using the dual-mode NB contrast agent for cancer targeting and simultaneous fluorescence/US imaging. (c) 2009 Elsevier Ltd. All rights reserved.

Simultaneous fluorometry and phosphorometry of Langendorff perfused rat heart: ex vivo animal studies

NASA Astrophysics Data System (ADS)

Ranji, Mahsa; Jaggard, Dwight L.; Apreleva, Sofia V.; Vinogradov, Sergei A.; Chance, Britton

2006-10-01

Fluorescence imaging of intrinsic fluorophores of tissue is a powerful method to assess metabolic changes at the cellular and intracellular levels. At the same time, exogenous phosphorescent probes can be used to accurately measure intravascular tissue oxygenation. Heart failure is the leading cause of death in America. A rat heart can potentially model the human heart to study failures or other abnormalities optically. We report simultaneous fluorescence and phosphorescence measurements performed on a rat heart. We have used two different optical systems to acquire fluorescence signals of flavoprotein and nicotinamide adenine dinucleotide—the two intrinsic fluorophores of mitochondria—and the phosphorescence signal of an intravascular oxygen probe to extract intracellular and intravascular metabolism loads, respectively.

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography).

PubMed

Siegel, Nisan; Storrie, Brian; Bruce, Marc; Brooker, Gary

2015-02-07

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called "CINCH". An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution.

“Optical communication with brain cells by means of an implanted duplex micro-device with optogenetics and Ca2+ fluoroimaging”

PubMed Central

Kobayashi, Takuma; Haruta, Makito; Sasagawa, Kiyotaka; Matsumata, Miho; Eizumi, Kawori; Kitsumoto, Chikara; Motoyama, Mayumi; Maezawa, Yasuyo; Ohta, Yasumi; Noda, Toshihiko; Tokuda, Takashi; Ishikawa, Yasuyuki; Ohta, Jun

2016-01-01

To better understand the brain function based on neural activity, a minimally invasive analysis technology in a freely moving animal is necessary. Such technology would provide new knowledge in neuroscience and contribute to regenerative medical techniques and prosthetics care. An application that combines optogenetics for voluntarily stimulating nerves, imaging to visualize neural activity, and a wearable micro-instrument for implantation into the brain could meet the abovementioned demand. To this end, a micro-device that can be applied to the brain less invasively and a system for controlling the device has been newly developed in this study. Since the novel implantable device has dual LEDs and a CMOS image sensor, photostimulation and fluorescence imaging can be performed simultaneously. The device enables bidirectional communication with the brain by means of light. In the present study, the device was evaluated in an in vitro experiment using a new on-chip 3D neuroculture with an extracellular matrix gel and an in vivo experiment involving regenerative medical transplantation and gene delivery to the brain by using both photosensitive channel and fluorescent Ca2+ indicator. The device succeeded in activating cells locally by selective photostimulation, and the physiological Ca2+ dynamics of neural cells were visualized simultaneously by fluorescence imaging. PMID:26878910

“Optical communication with brain cells by means of an implanted duplex micro-device with optogenetics and Ca2+ fluoroimaging”

NASA Astrophysics Data System (ADS)

Kobayashi, Takuma; Haruta, Makito; Sasagawa, Kiyotaka; Matsumata, Miho; Eizumi, Kawori; Kitsumoto, Chikara; Motoyama, Mayumi; Maezawa, Yasuyo; Ohta, Yasumi; Noda, Toshihiko; Tokuda, Takashi; Ishikawa, Yasuyuki; Ohta, Jun

2016-02-01

To better understand the brain function based on neural activity, a minimally invasive analysis technology in a freely moving animal is necessary. Such technology would provide new knowledge in neuroscience and contribute to regenerative medical techniques and prosthetics care. An application that combines optogenetics for voluntarily stimulating nerves, imaging to visualize neural activity, and a wearable micro-instrument for implantation into the brain could meet the abovementioned demand. To this end, a micro-device that can be applied to the brain less invasively and a system for controlling the device has been newly developed in this study. Since the novel implantable device has dual LEDs and a CMOS image sensor, photostimulation and fluorescence imaging can be performed simultaneously. The device enables bidirectional communication with the brain by means of light. In the present study, the device was evaluated in an in vitro experiment using a new on-chip 3D neuroculture with an extracellular matrix gel and an in vivo experiment involving regenerative medical transplantation and gene delivery to the brain by using both photosensitive channel and fluorescent Ca2+ indicator. The device succeeded in activating cells locally by selective photostimulation, and the physiological Ca2+ dynamics of neural cells were visualized simultaneously by fluorescence imaging.

Multispectral analog-mean-delay fluorescence lifetime imaging combined with optical coherence tomography

PubMed Central

Nam, Hyeong Soo; Kang, Woo Jae; Lee, Min Woo; Song, Joon Woo; Kim, Jin Won; Oh, Wang-Yuhl; Yoo, Hongki

2018-01-01

The pathophysiological progression of chronic diseases, including atherosclerosis and cancer, is closely related to compositional changes in biological tissues containing endogenous fluorophores such as collagen, elastin, and NADH, which exhibit strong autofluorescence under ultraviolet excitation. Fluorescence lifetime imaging (FLIm) provides robust detection of the compositional changes by measuring fluorescence lifetime, which is an inherent property of a fluorophore. In this paper, we present a dual-modality system combining a multispectral analog-mean-delay (AMD) FLIm and a high-speed swept-source optical coherence tomography (OCT) to simultaneously visualize the cross-sectional morphology and biochemical compositional information of a biological tissue. Experiments using standard fluorescent solutions showed that the fluorescence lifetime could be measured with a precision of less than 40 psec using the multispectral AMD-FLIm without averaging. In addition, we performed ex vivo imaging on rabbit iliac normal-looking and atherosclerotic specimens to demonstrate the feasibility of the combined FLIm-OCT system for atherosclerosis imaging. We expect that the combined FLIm-OCT will be a promising next-generation imaging technique for diagnosing atherosclerosis and cancer due to the advantages of the proposed label-free high-precision multispectral lifetime measurement. PMID:29675330

Enabling Histopathological Annotations on Immunofluorescent Images through Virtualization of Hematoxylin and Eosin

PubMed Central

Lahiani, Amal; Klaiman, Eldad; Grimm, Oliver

2018-01-01

Context: Medical diagnosis and clinical decisions rely heavily on the histopathological evaluation of tissue samples, especially in oncology. Historically, classical histopathology has been the gold standard for tissue evaluation and assessment by pathologists. The most widely and commonly used dyes in histopathology are hematoxylin and eosin (H&E) as most malignancies diagnosis is largely based on this protocol. H&E staining has been used for more than a century to identify tissue characteristics and structures morphologies that are needed for tumor diagnosis. In many cases, as tissue is scarce in clinical studies, fluorescence imaging is necessary to allow staining of the same specimen with multiple biomarkers simultaneously. Since fluorescence imaging is a relatively new technology in the pathology landscape, histopathologists are not used to or trained in annotating or interpreting these images. Aims, Settings and Design: To allow pathologists to annotate these images without the need for additional training, we designed an algorithm for the conversion of fluorescence images to brightfield H&E images. Subjects and Methods: In this algorithm, we use fluorescent nuclei staining to reproduce the hematoxylin information and natural tissue autofluorescence to reproduce the eosin information avoiding the necessity to specifically stain the proteins or intracellular structures with an additional fluorescence stain. Statistical Analysis Used: Our method is based on optimizing a transform function from fluorescence to H&E images using least mean square optimization. Results: It results in high quality virtual H&E digital images that can easily and efficiently be analyzed by pathologists. We validated our results with pathologists by making them annotate tumor in real and virtual H&E whole slide images and we obtained promising results. Conclusions: Hence, we provide a solution that enables pathologists to assess tissue and annotate specific structures based on multiplexed fluorescence images. PMID:29531846

Enabling Histopathological Annotations on Immunofluorescent Images through Virtualization of Hematoxylin and Eosin.

PubMed

Lahiani, Amal; Klaiman, Eldad; Grimm, Oliver

2018-01-01

Medical diagnosis and clinical decisions rely heavily on the histopathological evaluation of tissue samples, especially in oncology. Historically, classical histopathology has been the gold standard for tissue evaluation and assessment by pathologists. The most widely and commonly used dyes in histopathology are hematoxylin and eosin (H&E) as most malignancies diagnosis is largely based on this protocol. H&E staining has been used for more than a century to identify tissue characteristics and structures morphologies that are needed for tumor diagnosis. In many cases, as tissue is scarce in clinical studies, fluorescence imaging is necessary to allow staining of the same specimen with multiple biomarkers simultaneously. Since fluorescence imaging is a relatively new technology in the pathology landscape, histopathologists are not used to or trained in annotating or interpreting these images. To allow pathologists to annotate these images without the need for additional training, we designed an algorithm for the conversion of fluorescence images to brightfield H&E images. In this algorithm, we use fluorescent nuclei staining to reproduce the hematoxylin information and natural tissue autofluorescence to reproduce the eosin information avoiding the necessity to specifically stain the proteins or intracellular structures with an additional fluorescence stain. Our method is based on optimizing a transform function from fluorescence to H&E images using least mean square optimization. It results in high quality virtual H&E digital images that can easily and efficiently be analyzed by pathologists. We validated our results with pathologists by making them annotate tumor in real and virtual H&E whole slide images and we obtained promising results. Hence, we provide a solution that enables pathologists to assess tissue and annotate specific structures based on multiplexed fluorescence images.

Design and validation of a near-infrared fluorescence endoscope for detection of early esophageal malignancy using a targeted imaging probe

NASA Astrophysics Data System (ADS)

Waterhouse, Dale J.; Joseph, James; Neves, Andre A.; di Pietro, Massimiliano; Brindle, Kevin M.; Fitzgerald, Rebecca C.; Bohndiek, Sarah E.

2016-03-01

Barrett's esophagus is a condition that predisposes patients to esophageal cancer. Early detection of cancer in these patients can be curative, but is confounded by a lack of contrast in white light endoscopy (WLE). Application of fluorescently-labeled lectins to the esophagus during endoscopy can more accurately delineate dysplasia emerging within Barrett's than WLE1, but strong tissue autofluorescence has limited sensitivity and dynamic range of this approach. To overcome this challenge, we synthesized a near-infrared (NIR) fluorescent lectin and have constructed a clinically translatable endoscope for simultaneous WLE and NIR imaging. An imaging fiber bundle, shielded from patient contact using a disposable catheter, relays collected light into an optical path that splits the WL reflectance and NIR emission onto two cameras for simultaneous video-rate recording. The captured images are co-registered and the honeycomb artifact arising from the fiber bundle is removed using interpolation between image points derived from individual fibers. A minimum detectable concentration of 110 nM was determined using a dilution series of IRDye800CW-lectin in black well plates. We have demonstrated the ability to use our endoscope to distinguish between different tissue types in ex vivo mouse stomachs. Future work using human ex vivo tissue specimens will determine safe illumination limits and sensitivity for dysplasia and adenocarcinoma in Barrett's esophagus, prior to commencing clinical trials.

Design and characterization of a handheld multimodal imaging device for the assessment of oral epithelial lesions

NASA Astrophysics Data System (ADS)

Higgins, Laura M.; Pierce, Mark C.

2014-08-01

A compact handpiece combining high resolution fluorescence (HRF) imaging with optical coherence tomography (OCT) was developed to provide real-time assessment of oral lesions. This multimodal imaging device simultaneously captures coregistered en face images with subcellular detail alongside cross-sectional images of tissue microstructure. The HRF imaging acquires a 712×594 μm2 field-of-view at the sample with a spatial resolution of 3.5 μm. The OCT images were acquired to a depth of 1.5 mm with axial and lateral resolutions of 9.3 and 8.0 μm, respectively. HRF and OCT images are simultaneously displayed at 25 fps. The handheld device was used to image a healthy volunteer, demonstrating the potential for in vivo assessment of the epithelial surface for dysplastic and neoplastic changes at the cellular level, while simultaneously evaluating submucosal involvement. We anticipate potential applications in real-time assessment of oral lesions for improved surveillance and surgical guidance.

Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations

PubMed Central

Mlodzianoski, Michael J.; Curthoys, Nikki M.; Gunewardene, Mudalige S.; Carter, Sean; Hess, Samuel T.

2016-01-01

Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample. PMID:27002724

Simultaneous confocal fluorescence microscopy and optical coherence tomography for drug distribution and tissue integrity assessment

NASA Astrophysics Data System (ADS)

Rinehart, Matthew T.; LaCroix, Jeffrey; Henderson, Marcus; Katz, David; Wax, Adam

2011-03-01

The effectiveness of microbicidal gels, topical products developed to prevent infection by sexually transmitted diseases including HIV/AIDS, is governed by extent of gel coverage, pharmacokinetics of active pharmaceutical ingredients (APIs), and integrity of vaginal epithelium. While biopsies provide localized information about drug delivery and tissue structure, in vivo measurements are preferable in providing objective data on API and gel coating distribution as well as tissue integrity. We are developing a system combining confocal fluorescence microscopy with optical coherence tomography (OCT) to simultaneously measure local concentrations and diffusion coefficients of APIs during transport from microbicidal gels into tissue, while assessing tissue integrity. The confocal module acquires 2-D images of fluorescent APIs multiple times per second allowing analysis of lateral diffusion kinetics. The custom Fourier domain OCT module has a maximum a-scan rate of 54 kHz and provides depth-resolved tissue integrity information coregistered with the confocal fluorescence measurements. The combined system is validated by imaging phantoms with a surrogate fluorophore. Time-resolved API concentration measured at fixed depths is analyzed for diffusion kinetics. This multimodal system will eventually be implemented in vivo for objective evaluation of microbicide product performance.

ReagentTF: a rapid and versatile optical clearing method for biological imaging(Conference Presentation)

NASA Astrophysics Data System (ADS)

Yu, Tingting; Zhu, Jingtan; Li, Yusha; Qi, Yisong; Xu, Jianyi; Gong, Hui; Luo, Qingming; Zhu, Dan

2017-02-01

The emergence of various optical clearing methods provides a great potential for imaging deep inside tissues by combining with multiple-labelling and microscopic imaging techniques. They were generally developed for specific imaging demand thus presented some non-negligible limitations such as long incubation time, tissue deformation, fluorescence quenching, incompatibility with immunostaining or lipophilic tracers. In this study, we developed a rapid and versatile clearing method, termed ReagentTF, for deep imaging of various fluorescent samples. This method can not only efficiently clear embryos, neonatal whole-brains and adult thick brain sections by simple immersion in aqueous mixtures with minimal volume change, but also can preserve fluorescence of various fluorescent proteins and simultaneously be compatible with immunostaining and lipophilic neuronal dyes. We demonstrate the effectiveness of this method in reconstructing the cell distributions of mouse hippocampus, visualizing the neural projection from CA1 (Cornu Ammonis 1) to HDB (nucleus of the horizontal limb of the diagonal band), and observing the growth of forelimb plexus in whole-mount embryos. These results suggest that ReagentTF is useful for large-volume imaging and will be an option for the deep imaging of biological tissues.

Imaging intracellular protein dynamics by spinning disk confocal microscopy

PubMed Central

Stehbens, Samantha; Pemble, Hayley; Murrow, Lindsay; Wittmann, Torsten

2012-01-01

The palette of fluorescent proteins has grown exponentially over the last decade, and as a result live imaging of cells expressing fluorescently tagged proteins is becoming more and more main stream. Spinning disk confocal microscopy (SDC) is a high speed optical sectioning technique, and a method of choice to observe and analyze intracellular fluorescent protein dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low noise scientific grade cooled charged-coupled device (CCD) cameras, and can achieve frame rates of up 1000 frames per second. In this chapter we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy, and provide a rationale for specific design choices. We also give guidelines how other imaging techniques such as total internal reflection (TIRF) microscopy or spatially controlled photoactivation can be coupled with SDC imaging, and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction. PMID:22264541

Hyperspectral fluorescence imaging with multi wavelength LED excitation

NASA Astrophysics Data System (ADS)

Luthman, A. Siri; Dumitru, Sebastian; Quirós-Gonzalez, Isabel; Bohndiek, Sarah E.

2016-04-01

Hyperspectral imaging (HSI) can combine morphological and molecular information, yielding potential for real-time and high throughput multiplexed fluorescent contrast agent imaging. Multiplexed readout from targets, such as cell surface receptors overexpressed in cancer cells, could improve both sensitivity and specificity of tumor identification. There remains, however, a need for compact and cost effective implementations of the technology. We have implemented a low-cost wide-field multiplexed fluorescence imaging system, which combines LED excitation at 590, 655 and 740 nm with a compact commercial solid state HSI system operating in the range 600 - 1000 nm. A key challenge for using reflectance-based HSI is the separation of contrast agent fluorescence from the reflectance of the excitation light. Here, we illustrate how it is possible to address this challenge in software, using two offline reflectance removal methods, prior to least-squares spectral unmixing. We made a quantitative comparison of the methods using data acquired from dilutions of contrast agents prepared in well-plates. We then established the capability of our HSI system for non-invasive in vivo fluorescence imaging in small animals using the optimal reflectance removal method. The HSI presented here enables quantitative unmixing of at least four fluorescent contrast agents (Alexa Fluor 610, 647, 700 and 750) simultaneously in living mice. A successful unmixing of the four fluorescent contrast agents was possible both using the pure contrast agents and with mixtures. The system could in principle also be applied to imaging of ex vivo tissue or intraoperative imaging in a clinical setting. These data suggest a promising approach for developing clinical applications of HSI based on multiplexed fluorescence contrast agent imaging.

Noninvasive measurement of pharmacokinetics by near-infrared fluorescence imaging in the eye of mice

NASA Astrophysics Data System (ADS)

Dobosz, Michael; Strobel, Steffen; Stubenrauch, Kay-Gunnar; Osl, Franz; Scheuer, Werner

2014-01-01

Purpose: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. Procedures: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(-alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). Results: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F( fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). Conclusions: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

Enhanced visualization of the bile duct via parallel white light and indocyanine green fluorescence laparoscopic imaging

NASA Astrophysics Data System (ADS)

Demos, Stavros G.; Urayama, Shiro

2014-03-01

Despite best efforts, bile duct injury during laparoscopic cholecystectomy is a major potential complication. Precise detection method of extrahepatic bile duct during laparoscopic procedures would minimize the risk of injury. Towards this goal, we have developed a compact imaging instrumentation designed to enable simultaneous acquisition of conventional white color and NIR fluorescence endoscopic/laparoscopic imaging using ICG as contrast agent. The capabilities of this system, which offers optimized sensitivity and functionality, are demonstrated for the detection of the bile duct in an animal model. This design could also provide a low-cost real-time surgical navigation capability to enhance the efficacy of a variety of other image-guided minimally invasive procedures.

Laser-Excited Luminescent Tracers for Planar Concentration Measurements in Gaseous Jets

NASA Astrophysics Data System (ADS)

Lozano, Antonio

Tracers currently used in planar laser-induced fluorescence concentration measurements are not ideal for some experimental conditions, e.g., non-reacting turbulent gaseous flows at standard temperature and pressure. In this work, a number of chemicals have been evaluated, through consideration of their physical and photophysical properties, for use as luminescent concentration markers in turbulent gaseous flows. Two selected substances, biacetyl and acetone, have been studied in more detail. Acetone PLIF concentration images have been acquired in a non-reacting air jet, and the results have been compared to similar images obtained seeding with biacetyl. Acetone has proven to be a superior tracer when imaging fluorescence emission. Acetone has also been used as a fuel marker in hydrogen and methane diffusion flames. This single -laser technique enables simultaneous recording of the acetone and OH fluorescence emissions, as well as Mie scattering from ambient air dust particles. Acetone-sensitized, collisionally-induced biacetyl phosphorescence has been used to visualize molecular mixing in gaseous flows. Initial attempts to produce quantitative results with this method through simultaneous imaging of acetone fluorescence and collisionally-induced biacetyl emission, are described. Using laser-induced biacetyl phosphorescence imaging, a data set of cross-cut concentration images has been acquired in a nitrogen coflowing jet (Re = 5,000). The images have been statistically analyzed. Very simple models of the instantaneous concentration profile have been compared to the experimental data. Of all the tested models, a paraboloid has resulted to be the best approximation to the instantaneous 2-D profile. Finally, an experiment to study jet mixing in crossflow using acetone PLIF imaging has been designed. The flow facility has been constructed, and preliminary images obtained with a high quantum efficiency, thinned CCD detector have revealed the presence of jet structures inside the wake region that appear to be dependent on the jet/crossflow velocity ratio.

Three-dimensional fluorescence-enhanced optical tomography using a hand-held probe based imaging system

PubMed Central

Ge, Jiajia; Zhu, Banghe; Regalado, Steven; Godavarty, Anuradha

2008-01-01

Hand-held based optical imaging systems are a recent development towards diagnostic imaging of breast cancer. To date, all the hand-held based optical imagers are used to perform only surface mapping and target localization, but are not capable of demonstrating tomographic imaging. Herein, a novel hand-held probe based optical imager is developed towards three-dimensional (3-D) optical tomography studies. The unique features of this optical imager, which primarily consists of a hand-held probe and an intensified charge coupled device detector, are its ability to; (i) image large tissue areas (5×10 sq. cm) in a single scan, (ii) perform simultaneous multiple point illumination and collection, thus reducing the overall imaging time; and (iii) adapt to varying tissue curvatures, from a flexible probe head design. Experimental studies are performed in the frequency domain on large slab phantoms (∼650 ml) using fluorescence target(s) under perfect uptake (1:0) contrast ratios, and varying target depths (1–2 cm) and X-Y locations. The effect of implementing simultaneous over sequential multiple point illumination towards 3-D tomography is experimentally demonstrated. The feasibility of 3-D optical tomography studies has been demonstrated for the first time using a hand-held based optical imager. Preliminary fluorescence-enhanced optical tomography studies are able to reconstruct 0.45 ml target(s) located at different target depths (1–2 cm). However, the depth recovery was limited as the actual target depth increased, since only reflectance measurements were acquired. Extensive tomography studies are currently carried out to determine the resolution and performance limits of the imager on flat and curved phantoms. PMID:18697559

Three-dimensional fluorescence-enhanced optical tomography using a hand-held probe based imaging system.

PubMed

Ge, Jiajia; Zhu, Banghe; Regalado, Steven; Godavarty, Anuradha

2008-07-01

Hand-held based optical imaging systems are a recent development towards diagnostic imaging of breast cancer. To date, all the hand-held based optical imagers are used to perform only surface mapping and target localization, but are not capable of demonstrating tomographic imaging. Herein, a novel hand-held probe based optical imager is developed towards three-dimensional (3-D) optical tomography studies. The unique features of this optical imager, which primarily consists of a hand-held probe and an intensified charge coupled device detector, are its ability to; (i) image large tissue areas (5 x 10 sq. cm) in a single scan, (ii) perform simultaneous multiple point illumination and collection, thus reducing the overall imaging time; and (iii) adapt to varying tissue curvatures, from a flexible probe head design. Experimental studies are performed in the frequency domain on large slab phantoms (approximately 650 ml) using fluorescence target(s) under perfect uptake (1:0) contrast ratios, and varying target depths (1-2 cm) and X-Y locations. The effect of implementing simultaneous over sequential multiple point illumination towards 3-D tomography is experimentally demonstrated. The feasibility of 3-D optical tomography studies has been demonstrated for the first time using a hand-held based optical imager. Preliminary fluorescence-enhanced optical tomography studies are able to reconstruct 0.45 ml target(s) located at different target depths (1-2 cm). However, the depth recovery was limited as the actual target depth increased, since only reflectance measurements were acquired. Extensive tomography studies are currently carried out to determine the resolution and performance limits of the imager on flat and curved phantoms.

Quantifying cancer cell receptors with paired-agent fluorescent imaging: a novel method to account for tissue optical property effects

NASA Astrophysics Data System (ADS)

Sadeghipour, Negar; Davis, Scott C.; Tichauer, Kenneth M.

2018-02-01

Dynamic fluorescence imaging approaches can be used to estimate the concentration of cell surface receptors in vivo. Kinetic models are used to generate the final estimation by taking the targeted imaging agent concentration as a function of time. However, tissue absorption and scattering properties cause the final readout signal to be on a different scale than the real fluorescent agent concentration. In paired-agent imaging approaches, simultaneous injection of a suitable control imaging agent with a targeted one can account for non-specific uptake and retention of the targeted agent. Additionally, the signal from the control agent can be a normalizing factor to correct for tissue optical property differences. In this study, the kinetic model used for paired-agent imaging analysis (i.e., simplified reference tissue model) is modified and tested in simulation and experimental data in a way that accounts for the scaling correction within the kinetic model fit to the data to ultimately extract an estimate of the targeted biomarker concentration.

Instrumentation for simultaneous kinetic imaging of multiple fluorophores in single living cells

NASA Astrophysics Data System (ADS)

Morris, Stephen J.; Beatty, Diane M.; Welling, Larry W.; Wiegmann, Thomas B.

1991-05-01

Low-light fluorescence video microscopy has established itself as an excellent method for investigations of cell dynamics. There is a growing interest in resolving multiple images of 'ratio' fluorophores like indo or BCECF or the emission from multiple dyes placed in the same cell system. For rapid kinetic studies, the problems of photodynamic damage and photobleaching on one hand and the need for good spatial and temporal resolution on the other, press the resolution of the instrumentation. Rapid resolution of multiple probes at multiple wavelengths presents a third set of problems of exciting the probes and appropriately imaging the emitted light. The authors have designed a new real-time low-light fluorescence video microscope for capturing intensified images of up to four dyes contained in the same cell system. These can be two dual-emission wavelength 'ratio' dyes or multiple dyes. The optics allow simultaneous excitation of up to four fluorophores and the real-time (30 frames/second) capture of four separate fluorescence emission images. Each emission wavelength is imaged simultaneously by one of four cameras, then digitized and appropriately combined at standard video frame rates to be stored at high resolution on tape or video disk for further off-line correction and analysis. The design has no moving parts in its optical train, which overcomes a number of technical difficulties encountered in filter wheel or mechanical shutter designs for multiple imaging. The instrument can be assembled form off-the-shelf components. Coupled to compatible image processing software utilizing PC-AT computers, it can be realized for relatively low cost. Two examples of simultaneous multi-parameter imaging are presented. Synchronous observations of calcium and pH distribution in kidney epithelial cells, loaded with both indo-1 and SNARF-1TM, show that both are altered in response to ionomycin treatment; however, the kinetics for the two changes are quite different. Intracellular calcium increases rapidly when the bath Ca2+ is raised. The pH remains stable for several seconds, then suddenly collapses. The second example concerns fusion of human red blood cells (RBC) to fibroblasts expressing influenza hemagglutinin. Movement of soluble and membrane-bound dyes follow different kinetics, depending upon the molecular weight of the soluble dye. Furthermore, the swelling of the RBC occurs after the onset of fusion, and therefore cannot provide the driving force.

Bioconjugated Quantum Dots for In Vivo Molecular and Cellular Imaging

PubMed Central

Smith, Andrew M.; Duan, Hongwei; Mohs, Aaron M.; Nie, Shuming

2008-01-01

Semiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometer scale, and are emerging as a new class of fluorescent labels for biology and medicine. In comparison with organic dyes and fluorescent proteins, they have unique optical and electronic properties, with size-tunable light emission, superior signal brightness, resistance to photobleaching, and broad absorption spectra for simultaneous excitation of multiple fluorescence colors. QDs also provide a versatile nanoscale scaffold for designing multifunctional nanoparticles with both imaging and therapeutic functions. When linked with targeting ligands such as antibodies, peptides or small molecules, QDs can be used to target tumor biomarkers as well as tumor vasculatures with high affinity and specificity. Here we discuss the synthesis and development of state-of-the-art QD probes and their use for molecular and cellular imaging. We also examine key issues for in vivo imaging and therapy, such as nanoparticle biodistribution, pharmacokinetics, and toxicology. PMID:18495291

Ultrafast Method for the Analysis of Fluorescence Lifetime Imaging Microscopy Data Based on the Laguerre Expansion Technique

PubMed Central

Jo, Javier A.; Fang, Qiyin; Marcu, Laura

2007-01-01

We report a new deconvolution method for fluorescence lifetime imaging microscopy (FLIM) based on the Laguerre expansion technique. The performance of this method was tested on synthetic and real FLIM images. The following interesting properties of this technique were demonstrated. 1) The fluorescence intensity decay can be estimated simultaneously for all pixels, without a priori assumption of the decay functional form. 2) The computation speed is extremely fast, performing at least two orders of magnitude faster than current algorithms. 3) The estimated maps of Laguerre expansion coefficients provide a new domain for representing FLIM information. 4) The number of images required for the analysis is relatively small, allowing reduction of the acquisition time. These findings indicate that the developed Laguerre expansion technique for FLIM analysis represents a robust and extremely fast deconvolution method that enables practical applications of FLIM in medicine, biology, biochemistry, and chemistry. PMID:19444338

Single cell systems biology by super-resolution imaging and combinatorial labeling

PubMed Central

Lubeck, Eric; Cai, Long

2012-01-01

Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral separability of fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using Fluorescence in situ Hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured the mRNA levels of 32 genes simultaneously in single S. cerevisiae cells. These experiments demonstrate that combinatorial labeling and super-resolution imaging of single cells provides a natural approach to bring systems biology into single cells. PMID:22660740

Simultaneous optical coherence tomography and lipofuscin autofluorescence imaging of the retina with a single broadband light source at 480nm.

PubMed

Jiang, Minshan; Liu, Tan; Liu, Xiaojing; Jiao, Shuliang

2014-12-01

We accomplished spectral domain optical coherence tomography and auto-fluorescence microscopy for imaging the retina with a single broadband light source centered at 480 nm. This technique is able to provide simultaneous structural imaging and lipofuscin molecular contrast of the retina. Since the two imaging modalities are provided by the same group of photons, their images are intrinsically registered. To test the capabilities of the technique we periodically imaged the retinas of the same rats for four weeks. The images successfully demonstrated lipofuscin accumulation in the retinal pigment epithelium with aging. The experimental results showed that the dual-modal imaging system can be a potentially powerful tool in the study of age-related degenerative retinal diseases.

Rotational multispectral fluorescence lifetime imaging and intravascular ultrasound: bimodal system for intravascular applications

PubMed Central

Ma, Dinglong; Bec, Julien; Yankelevich, Diego R.; Gorpas, Dimitris; Fatakdawala, Hussain; Marcu, Laura

2014-01-01

Abstract. We report the development and validation of a hybrid intravascular diagnostic system combining multispectral fluorescence lifetime imaging (FLIm) and intravascular ultrasound (IVUS) for cardiovascular imaging applications. A prototype FLIm system based on fluorescence pulse sampling technique providing information on artery biochemical composition was integrated with a commercial IVUS system providing information on artery morphology. A customized 3-Fr bimodal catheter combining a rotational side-view fiberoptic and a 40-MHz IVUS transducer was constructed for sequential helical scanning (rotation and pullback) of tubular structures. Validation of this bimodal approach was conducted in pig heart coronary arteries. Spatial resolution, fluorescence detection efficiency, pulse broadening effect, and lifetime measurement variability of the FLIm system were systematically evaluated. Current results show that this system is capable of temporarily resolving the fluorescence emission simultaneously in multiple spectral channels in a single pullback sequence. Accurate measurements of fluorescence decay characteristics from arterial segments can be obtained rapidly (e.g., 20 mm in 5 s), and accurate co-registration of fluorescence and ultrasound features can be achieved. The current finding demonstrates the compatibility of FLIm instrumentation with in vivo clinical investigations and its potential to complement conventional IVUS during catheterization procedures. PMID:24898604

Artifact mitigation of ptychography integrated with on-the-fly scanning probe microscopy

DOE PAGES

Huang, Xiaojing; Yan, Hanfei; Ge, Mingyuan; ...

2017-07-11

In this paper, we report our experiences with conducting ptychography simultaneously with the X-ray fluorescence measurement using the on-the-fly mode for efficient multi-modality imaging. We demonstrate that the periodic artifact inherent to the raster scan pattern can be mitigated using a sufficiently fine scan step size to provide an overlap ratio of >70%. This allows us to obtain transmitted phase contrast images with enhanced spatial resolution from ptychography while maintaining the fluorescence imaging with continuous-motion scans on pixelated grids. Lastly, this capability will greatly improve the competence and throughput of scanning probe X-ray microscopy.

Fluorescence-Raman Dual Modal Endoscopic System for Multiplexed Molecular Diagnostics

NASA Astrophysics Data System (ADS)

Jeong, Sinyoung; Kim, Yong-Il; Kang, Homan; Kim, Gunsung; Cha, Myeong Geun; Chang, Hyejin; Jung, Kyung Oh; Kim, Young-Hwa; Jun, Bong-Hyun; Hwang, Do Won; Lee, Yun-Sang; Youn, Hyewon; Lee, Yoon-Sik; Kang, Keon Wook; Lee, Dong Soo; Jeong, Dae Hong

2015-03-01

Optical endoscopic imaging, which was recently equipped with bioluminescence, fluorescence, and Raman scattering, allows minimally invasive real-time detection of pathologies on the surface of hollow organs. To characterize pathologic lesions in a multiplexed way, we developed a dual modal fluorescence-Raman endomicroscopic system (FRES), which used fluorescence and surface-enhanced Raman scattering nanoprobes (F-SERS dots). Real-time, in vivo, and multiple target detection of a specific cancer was successful, based on the fast imaging capability of fluorescence signals and the multiplex capability of simultaneously detected SERS signals using an optical fiber bundle for intraoperative endoscopic system. Human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) on the breast cancer xenografts in a mouse orthotopic model were successfully detected in a multiplexed way, illustrating the potential of FRES as a molecular diagnostic instrument that enables real-time tumor characterization of receptors during routine endoscopic procedures.

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography)

PubMed Central

Siegel, Nisan; Storrie, Brian; Bruce, Marc

2016-01-01

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called “CINCH”. An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution. PMID:26839443

On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements.

PubMed

Macháň, Radek; Foo, Yong Hwee; Wohland, Thorsten

2016-07-12

Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are widely used methods to determine diffusion coefficients. However, they often do not yield the same results. With the advent of camera-based imaging FCS, which measures the diffusion coefficient in each pixel of an image, and proper bleaching corrections, it is now possible to measure the diffusion coefficient by FRAP and FCS in the exact same images. We thus performed simultaneous FCS and FRAP measurements on supported lipid bilayers and live cell membranes to test how far the two methods differ in their results and whether the methodological differences, in particular the high bleach intensity in FRAP, the bleach corrections, and the fitting procedures in the two methods explain observed differences. Overall, we find that the FRAP bleach intensity does not measurably influence the diffusion in the samples, but that bleach correction and fitting introduce large uncertainties in FRAP. We confirm our results by simulations. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A study of MRI-guided diffuse fluorescence molecular tomography for monitoring PDT effects in pancreas cancer

NASA Astrophysics Data System (ADS)

Samkoe, Kimberley S.; Davis, Scott C.; Srinivasan, Subhadra; O'Hara, Julia A.; Hasan, Tayyaba; Pogue, Brian W.

2009-06-01

Over the last several decades little progress has been made in the therapy and treatment monitoring of pancreas adenocarcinoma, a devastating and aggressive form of cancer that has a 5-year patient survival rate of 3%. Currently, investigations for the use of interstitial Verteporfin photodynamic therapy (PDT) are being undertaken in both orthotopic xenograft mouse models and in human clinical trials. In the mouse models, magnetic resonance (MR) imaging has been used as a measure of surrogate response to Verteporfin PDT; however, MR imaging alone lacks the molecular information required to assess the metabolic function and growth rates of the tumor immediately after treatment. We propose the implementation of MR-guided fluorescence tomography in conjunction with a fluorescently labeled (IR-Dye 800 CW, LI-COR) epidermal growth factor (EGF) as a molecular measure of surrogate response. To demonstrate the effectiveness of MR-guided diffuse fluorescence tomography for molecular imaging, we have used the AsPC-1 (+EGFR) human pancreatic adenocarcinoma in an orthotopic mouse model. EGF IRDye 800CW was injected 48 hours prior to imaging. MR image sequences were collected simultaneously with the fluorescence data using a MR-coupled diffuse optical tomography system. Image reconstruction was performed multiple times with varying abdominal organ segmentation in order to obtain a optimal tomographic image. It is shown that diffuse fluorescence tomography of the orthotopic pancreas model is feasible, with consideration of confounding fluorescence signals from the multiple organs and tissues surrounding the pancreas. MR-guided diffuse fluorescence tomography will be used to monitor EGF response after photodynamic therapy. Additionally, it provide the opportunity to individualize subsequent therapies based on response to PDT as well as to evaluate the success of combination therapies, such as PDT with chemotherapy, antibody therapy or even radiation.

Simultaneous tracking of drug molecules and carriers using aptamer-functionalized fluorescent superstable gold nanorod-carbon nanocapsules during thermo-chemotherapy

NASA Astrophysics Data System (ADS)

Wang, Xue-Wei; Gao, Wei; Fan, Huanhuan; Ding, Ding; Lai, Xiao-Fang; Zou, Yu-Xiu; Chen, Long; Chen, Zhuo; Tan, Weihong

2016-04-01

Controlling and monitoring the drug delivery process is critical to its intended therapeutic function. Many nanocarrier systems for drug delivery have been successfully developed. However, biocompatibility, stability, and simultaneously tracing drugs and nanocarriers present significant limitations. Herein, we have fabricated a multifunctional nanocomposite by coating the gold nanorod (AuNR) with a biocompatible, superstable and fluorescent carbon layer, obtaining the AuNR@carbon core-shell nanocapsule. In this system, the carbon shell, originally obtained in aqueous glucose solutions and, therefore, biocompatible in physiological environments, could be simply loaded with cell-specific aptamers and therapeutic molecules through π-π interactions, a useful tool for cancer-targeted cellular imaging and therapy. Moreover, such a stable and intrinsic fluorescence effect of the AuNR@carbon enabled simultaneous tracking of released therapeutic molecules and nanocarriers under thermo-chemotherapy. The AuNR@carbons had high surface areas and stable shells, as well as unique optical and photothermal properties, making them promising nanostructures for biomedical applications.Controlling and monitoring the drug delivery process is critical to its intended therapeutic function. Many nanocarrier systems for drug delivery have been successfully developed. However, biocompatibility, stability, and simultaneously tracing drugs and nanocarriers present significant limitations. Herein, we have fabricated a multifunctional nanocomposite by coating the gold nanorod (AuNR) with a biocompatible, superstable and fluorescent carbon layer, obtaining the AuNR@carbon core-shell nanocapsule. In this system, the carbon shell, originally obtained in aqueous glucose solutions and, therefore, biocompatible in physiological environments, could be simply loaded with cell-specific aptamers and therapeutic molecules through π-π interactions, a useful tool for cancer-targeted cellular imaging and therapy. Moreover, such a stable and intrinsic fluorescence effect of the AuNR@carbon enabled simultaneous tracking of released therapeutic molecules and nanocarriers under thermo-chemotherapy. The AuNR@carbons had high surface areas and stable shells, as well as unique optical and photothermal properties, making them promising nanostructures for biomedical applications. Electronic supplementary information (ESI) available: Experimental details and characterization data for all new compounds. See DOI: 10.1039/c6nr00369a

Multi-channel medical imaging system

DOEpatents

Frangioni, John V

2013-12-31

A medical imaging system provides simultaneous rendering of visible light and fluorescent images. The system may employ dyes in a small-molecule form that remain in the subject's blood stream for several minutes, allowing real-time imaging of the subject's circulatory system superimposed upon a conventional, visible light image of the subject. The system may provide an excitation light source to excite the fluorescent substance and a visible light source for general illumination within the same optical guide used to capture images. The system may be configured for use in open surgical procedures by providing an operating area that is closed to ambient light. The systems described herein provide two or more diagnostic imaging channels for capture of multiple, concurrent diagnostic images and may be used where a visible light image may be usefully supplemented by two or more images that are independently marked for functional interest.

Multi-channel medical imaging system

DOEpatents

Frangioni, John V.

2016-05-03

A medical imaging system provides simultaneous rendering of visible light and fluorescent images. The system may employ dyes in a small-molecule form that remain in a subject's blood stream for several minutes, allowing real-time imaging of the subject's circulatory system superimposed upon a conventional, visible light image of the subject. The system may provide an excitation light source to excite the fluorescent substance and a visible light source for general illumination within the same optical guide used to capture images. The system may be configured for use in open surgical procedures by providing an operating area that is closed to ambient light. The systems described herein provide two or more diagnostic imaging channels for capture of multiple, concurrent diagnostic images and may be used where a visible light image may be usefully supplemented by two or more images that are independently marked for functional interest.

Two-photon fluorescence imaging and bimodal phototherapy of epidermal cancer cells with biocompatible self-assembled polymer nanoparticles.

PubMed

Kandoth, Noufal; Kirejev, Vladimir; Monti, Sandra; Gref, Ruxandra; Ericson, Marica B; Sortino, Salvatore

2014-05-12

We have developed herein an engineered polymer-based nanoplatform showing the convergence of two-photon fluorescence imaging and bimodal phototherapeutic activity in a single nanostructure. It was achieved through the appropriate choice of three different components: a β-cyclodextrin-based polymer acting as a suitable carrier, a zinc phthalocyanine emitting red fluorescence simultaneously as being a singlet oxygen ((1)O2) photosensitizer, and a tailored nitroaniline derivative, functioning as a nitric oxide (NO) photodonor. The self-assembly of these components results in photoactivable nanoparticles, approximately 35 nm in diameter, coencapsulating a multifunctional cargo, which can be delivered to carcinoma cells. The combination of steady-state and time-resolved spectroscopic and photochemical techniques shows that the two photoresponsive guests do not interfere with each other while being enclosed in their supramolecular container and can thus be operated in parallel under control of light stimuli. Specifically, two-photon fluorescence microscopy allows mapping of the nanoassembly, here applied to epidermal cancer cells. By detecting the red emission from the phthalocyanine fluorophore it was also possible to investigate the tissue distribution after topical delivery onto human skin ex vivo. Irradiation of the nanoassembly with visible light triggers the simultaneous delivery of cytotoxic (1)O2 and NO, resulting in an amplified cell photomortality due to a combinatory effect of the two cytotoxic agents. The potential of dual therapeutic photodynamic action and two-photon fluorescence imaging capability in a single nanostructure make this system an appealing candidate for further studies in biomedical research.

Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

PubMed

Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

2017-07-01

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

Dual-channel (green and red) fluorescence microendoscope with subcellular resolution

NASA Astrophysics Data System (ADS)

de Paula D'Almeida, Camila; Fortunato, Thereza Cury; Teixeira Rosa, Ramon Gabriel; Romano, Renan Arnon; Moriyama, Lilian Tan; Pratavieira, Sebastião.

2018-02-01

Usually, tissue images at cellular level need biopsies to be done. Considering this, diagnostic devices, such as microendoscopes, have been developed with the purpose of do not be invasive. This study goal is the development of a dual-channel microendoscope, using two fluorescent labels: proflavine and protoporphyrin IX (PpIX), both approved by Food and Drug Administration. This system, with the potential to perform a microscopic diagnosis and to monitor a photodynamic therapy (PDT) session, uses a halogen lamp and an image fiber bundle to perform subcellular image. Proflavine fluorescence indicates the nuclei of the cell, which is the reference for PpIX localization on image tissue. Preliminary results indicate the efficacy of this optical technique to detect abnormal tissues and to improve the PDT dosimetry. This was the first time, up to our knowledge, that PpIX fluorescence was microscopically observed in vivo, in real time, combined to other fluorescent marker (Proflavine), which allowed to simultaneously observe the spatial localization of the PpIX in the mucosal tissue. We believe this system is very promising tool to monitor PDT in mucosa as it happens. Further experiments have to be performed in order to validate the system for PDT monitoring.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Junjing; Vine, David J.; Chen, Si

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. Here, by working with cells that have been rapidly frozen without the use of chemicalmore » fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.« less

Towards automated early cancer detection: Non-invasive, fluorescence-based approaches for quantitative assessment of cells and tissue to identify pre-cancers

NASA Astrophysics Data System (ADS)

Levitt, Jonathan Michael

Cancer is the second leading cause of death globally, second only to heart disease. As in many diseases, patient survival is directly related to how early lesions are detected. Using conventional screening methods, the early changes associated with cancer, which occur on the microscopic scale, can easily go overlooked. Due to the inherent drawbacks of conventional techniques we present non-invasive, optically based methods to acquire high resolution images from live samples and assess cellular function associated with the onset of disease. Specifically, we acquired fluorescence images from NADH and FAD to quantify morphology and metabolic activity. We first conducted studies to monitor monolayers of keratinocytes in response to apoptosis which has been shown to be disrupted during cancer progression. We found that as keratinocytes undergo apoptosis there are populations of mitochondria that exhibit a higher metabolic activity that become progressively confined to a gradually smaller perinuclear region. To further assess the changes associated with early cancer growth we developed automated methods to rapidly quantify fluorescence images and extract morphological and metabolic information from life tissue. In this study, we simultaneously quantified mitochondrial organization, metabolic activity, nuclear size distribution, and the localization of the structural protein keratin, to differentiate between normal and pre-cancerous engineered tissues. We found the degree mitochondrial organization, as determined from the fractal derived Hurst parameter, was well correlated to level of cellular differentiation. We also found that the metabolic activity in the pre-cancerous cells was greater and more consistent throughout tissue depths in comparison to normal tissue. Keratin localization, also quantified from the fluorescence images, we found it to be confined to the uppermost layers of normal tissue while it was more evenly distributed in the precancerous tissues. To allow for evaluation of the early cancerous changes in vivo, we developed video-rate confocal reflectance/multi-photon fluorescence microscope as a clinical prototype. This device was specifically designed to rapidly acquire and assess non-invasively acquire fluorescence images using the automated methods we have developed. We have demonstrated the ability of this microscope to simultaneously acquire fluorescence, confocal reflectance, and second-harmonic generation images as well as assess blood flow in vivo.

Integrated micro-endoscopy system for simultaneous fluorescence and optical-resolution photoacoustic imaging.

PubMed

Shao, Peng; Shi, Wei; Hajireza, Parsin; Zemp, Roger J

2012-07-01

We present a new integrated micro-endoscopy system combining label-free, fiber-based, real-time C-scan optical-resolution photoacoustic microscopy (F-OR-PAM) and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the F-OR-PAM sub-system is able to reach a resolution of ∼7  μm. The fluorescence subsystem, which does not require any mechanical scanning, consists of a 447.5-nm-centered diode laser as the light source, an objective lens, and a CCD camera. Proflavine is used as the fluorescent contrast agent by topical application. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single-mode fibers. The absorption of proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural information given by F-OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping visualize angiogenesis and the effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Integrated micro-endoscopy system for simultaneous fluorescence and optical-resolution photoacoustic imaging

NASA Astrophysics Data System (ADS)

Shao, Peng; Shi, Wei; Hajireza, Parsin; Zemp, Roger J.

2012-07-01

We present a new integrated micro-endoscopy system combining label-free, fiber-based, real-time C-scan optical-resolution photoacoustic microscopy (F-OR-PAM) and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the F-OR-PAM sub-system is able to reach a resolution of ~7 μm. The fluorescence subsystem, which does not require any mechanical scanning, consists of a 447.5-nm-centered diode laser as the light source, an objective lens, and a CCD camera. Proflavine is used as the fluorescent contrast agent by topical application. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single-mode fibers. The absorption of proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural information given by F-OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping visualize angiogenesis and the effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Clinical application of photodynamic medicine technology using light-emitting fluorescence imaging based on a specialized luminous source.

PubMed

Namikawa, Tsutomu; Fujisawa, Kazune; Munekage, Eri; Iwabu, Jun; Uemura, Sunao; Tsujii, Shigehiro; Maeda, Hiromichi; Kitagawa, Hiroyuki; Fukuhara, Hideo; Inoue, Keiji; Sato, Takayuki; Kobayashi, Michiya; Hanazaki, Kazuhiro

2018-04-04

The natural amino acid 5-aminolevulinic acid (ALA) is a protoporphyrin IX (PpIX) precursor and a new-generation photosensitive substance that accumulates specifically in cancer cells. When indocyanine green (ICG) is irradiated with near-infrared (NIR) light, it shifts to a higher energy state and emits infrared light with a longer wavelength than the irradiated NIR light. Photodynamic diagnosis (PDD) using ALA and ICG-based NIR fluorescence imaging has emerged as a new diagnostic technique. Specifically, in laparoscopic examinations for serosa-invading advanced gastric cancer, peritoneal metastases could be detected by ALA-PDD, but not by conventional visible-light imaging. The HyperEye Medical System (HEMS) can visualize ICG fluorescence as color images simultaneously projected with visible light in real time. This ICG fluorescence method is widely applicable, including for intraoperative identification of sentinel lymph nodes, visualization of blood vessels in organ resection, and blood flow evaluation during surgery. Fluorescence navigation by ALA-PDD and NIR using ICG imaging provides good visualization and detection of the target lesions that is not possible with the naked eye. We propose that this technique should be used in fundamental research on the relationship among cellular dynamics, metabolic enzymes, and tumor tissues, and to evaluate clinical efficacy and safety in multicenter cooperative clinical trials.

Snapshot 3D tracking of insulin granules in live cells

NASA Astrophysics Data System (ADS)

Wang, Xiaolei; Huang, Xiang; Gdor, Itay; Daddysman, Matthew; Yi, Hannah; Selewa, Alan; Haunold, Theresa; Hereld, Mark; Scherer, Norbert F.

2018-02-01

Rapid and accurate volumetric imaging remains a challenge, yet has the potential to enhance understanding of cell function. We developed and used a multifocal microscope (MFM) for 3D snapshot imaging to allow 3D tracking of insulin granules labeled with mCherry in MIN6 cells. MFM employs a special diffractive optical element (DOE) to simultaneously image multiple focal planes. This simultaneous acquisition of information determines the 3D location of single objects at a speed only limited by the array detector's frame rate. We validated the accuracy of MFM imaging/tracking with fluorescence beads; the 3D positions and trajectories of single fluorescence beads can be determined accurately over a wide range of spatial and temporal scales. The 3D positions and trajectories of single insulin granules in a 3.2um deep volume were determined with imaging processing that combines 3D decovolution, shift correction, and finally tracking using the Imaris software package. We find that the motion of the granules is superdiffusive, but less so in 3D than 2D for cells grown on coverslip surfaces, suggesting an anisotropy in the cytoskeleton (e.g. microtubules and action).

Semiconductor Quantum Dots for Bioimaging and Biodiagnostic Applications

NASA Astrophysics Data System (ADS)

Kairdolf, Brad A.; Smith, Andrew M.; Stokes, Todd H.; Wang, May D.; Young, Andrew N.; Nie, Shuming

2013-06-01

Semiconductor quantum dots (QDs) are light-emitting particles on the nanometer scale that have emerged as a new class of fluorescent labels for chemical analysis, molecular imaging, and biomedical diagnostics. Compared with traditional fluorescent probes, QDs have unique optical and electronic properties such as size-tunable light emission, narrow and symmetric emission spectra, and broad absorption spectra that enable the simultaneous excitation of multiple fluorescence colors. QDs are also considerably brighter and more resistant to photobleaching than are organic dyes and fluorescent proteins. These properties are well suited for dynamic imaging at the single-molecule level and for multiplexed biomedical diagnostics at ultrahigh sensitivity. Here, we discuss the fundamental properties of QDs; the development of next-generation QDs; and their applications in bioanalytical chemistry, dynamic cellular imaging, and medical diagnostics. For in vivo and clinical imaging, the potential toxicity of QDs remains a major concern. However, the toxic nature of cadmium-containing QDs is no longer a factor for in vitro diagnostics, so the use of multicolor QDs for molecular diagnostics and pathology is probably the most important and clinically relevant application for semiconductor QDs in the immediate future.

Semiconductor quantum dots for bioimaging and biodiagnostic applications.

PubMed

Kairdolf, Brad A; Smith, Andrew M; Stokes, Todd H; Wang, May D; Young, Andrew N; Nie, Shuming

2013-01-01

Semiconductor quantum dots (QDs) are light-emitting particles on the nanometer scale that have emerged as a new class of fluorescent labels for chemical analysis, molecular imaging, and biomedical diagnostics. Compared with traditional fluorescent probes, QDs have unique optical and electronic properties such as size-tunable light emission, narrow and symmetric emission spectra, and broad absorption spectra that enable the simultaneous excitation of multiple fluorescence colors. QDs are also considerably brighter and more resistant to photobleaching than are organic dyes and fluorescent proteins. These properties are well suited for dynamic imaging at the single-molecule level and for multiplexed biomedical diagnostics at ultrahigh sensitivity. Here, we discuss the fundamental properties of QDs; the development of next-generation QDs; and their applications in bioanalytical chemistry, dynamic cellular imaging, and medical diagnostics. For in vivo and clinical imaging, the potential toxicity of QDs remains a major concern. However, the toxic nature of cadmium-containing QDs is no longer a factor for in vitro diagnostics, so the use of multicolor QDs for molecular diagnostics and pathology is probably the most important and clinically relevant application for semiconductor QDs in the immediate future.

Semiconductor Quantum Dots for Bioimaging and Biodiagnostic Applications

PubMed Central

Kairdolf, Brad A.; Smith, Andrew M.; Stokes, Todd H.; Wang, May D.; Young, Andrew N.; Nie, Shuming

2013-01-01

Semiconductor quantum dots (QDs) are light-emitting particles on the nanometer scale that have emerged as a new class of fluorescent labels for chemical analysis, molecular imaging, and biomedical diagnostics. Compared with traditional fluorescent probes, QDs have unique optical and electronic properties such as size-tunable light emission, narrow and symmetric emission spectra, and broad absorption spectra that enable the simultaneous excitation of multiple fluorescence colors. QDs are also considerably brighter and more resistant to photobleaching than are organic dyes and fluorescent proteins. These properties are well suited for dynamic imaging at the single-molecule level and for multiplexed biomedical diagnostics at ultrahigh sensitivity. Here, we discuss the fundamental properties of QDs; the development of next-generation QDs; and their applications in bioanalytical chemistry, dynamic cellular imaging, and medical diagnostics. For in vivo and clinical imaging, the potential toxicity of QDs remains a major concern. However, the toxic nature of cadmium-containing QDs is no longer a factor for in vitro diagnostics, so the use of multicolor QDs for molecular diagnostics and pathology is probably the most important and clinically relevant application for semiconductor QDs in the immediate future. PMID:23527547

The Application of Fluorescent Quantum Dots to Confocal, Multiphoton, and Electron Microscopic Imaging

PubMed Central

Deerinck, Thomas J.

2009-01-01

Fluorescent quantum dots are emerging as an important tool for imaging cells and tissues, and their unique optical and physical properties have captured the attention of the research community. The most common types of commercially available quantum dots consist of a nanocrystalline semiconductor core composed of cadmium selenide with a zinc sulfide capping layer and an outer polymer layer to facilitate conjugation to targeting biomolecules such as immunoglobulins. They exhibit high fluorescent quantum yields and have large absorption cross-sections, possess excellent photostability, and can be synthesized so that their narrow-band fluorescence emission can occur in a wide spectrum of colors. These properties make them excellent candidates for serving as multiplexing molecular beacons using a variety of imaging modalities including highly correlated microscopies. Whereas much attention has been focused on quantum-dot applications for live-cell imaging, we have sought to characterize and exploit their utility for enabling simultaneous multiprotein immunolabeling in fixed cells and tissues. Considerations for their application to immunolabeling for correlated light and electron microscopic analysis are discussed. PMID:18337229

Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy.

PubMed

Byrne, Gerard D; Vllasaliu, Driton; Falcone, Franco H; Somekh, Michael G; Stolnik, Snjezana

2015-11-02

In this work we utilize the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labeled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF coimaging enables live visualization of the process of colloidal particle interaction with the labeled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm in diameter model polystyrene colloid associates with clathrin, prior to and during its cellular internalization. This association is not apparent with larger, 1 μm in diameter colloids, indicating an upper particle size limit for clathrin-mediated endocytosis.

Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy

PubMed Central

Chan, Jacky C. K.; Diebold, Eric D.; Buckley, Brandon W.; Mao, Sien; Akbari, Najva; Jalali, Bahram

2014-01-01

Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat’s-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures. PMID:25574449

Parallel detection experiment of fluorescence confocal microscopy using DMD.

PubMed

Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

2016-05-01

Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

A portable fluorescence microscopic imaging system for cholecystectomy

NASA Astrophysics Data System (ADS)

Ye, Jian; Yang, Chaoyu; Gan, Qi; Ma, Rong; Zhang, Zeshu; Chang, Shufang; Shao, Pengfei; Zhang, Shiwu; Liu, Chenhai; Xu, Ronald

2016-03-01

In this paper we proposed a portable fluorescence microscopic imaging system to prevent iatrogenic biliary injuries from occurring during cholecystectomy due to misidentification of the cystic structures. The system consisted of a light source module, a CMOS camera, a Raspberry Pi computer and a 5 inch HDMI LCD. Specifically, the light source module was composed of 690 nm and 850 nm LEDs, allowing the CMOS camera to simultaneously acquire both fluorescence and background images. The system was controlled by Raspberry Pi using Python programming with the OpenCV library under Linux. We chose Indocyanine green(ICG) as a fluorescent contrast agent and then tested fluorescence intensities of the ICG aqueous solution at different concentration levels by our fluorescence microscopic system compared with the commercial Xenogen IVIS system. The spatial resolution of the proposed fluorescence microscopic imaging system was measured by a 1951 USAF resolution target and the dynamic response was evaluated quantitatively with an automatic displacement platform. Finally, we verified the technical feasibility of the proposed system in mouse models of bile duct, performing both correct and incorrect gallbladder resection. Our experiments showed that the proposed system can provide clear visualization of the confluence between the cystic duct and common bile duct or common hepatic duct, suggesting that this is a potential method for guiding cholecystectomy. The proposed portable system only cost a total of $300, potentially promoting its use in resource-limited settings.

In vivo magnetic resonance and fluorescence dual imaging of tumor sites by using dye-doped silica-coated iron oxide nanoparticles

NASA Astrophysics Data System (ADS)

Jang, Haeyun; Lee, Chaedong; Nam, Gi-Eun; Quan, Bo; Choi, Hyuck Jae; Yoo, Jung Sun; Piao, Yuanzhe

2016-02-01

The difficulty in delineating tumor is a major obstacle for better outcomes in cancer treatment of patients. The use of single-imaging modality is often limited by inadequate sensitivity and resolution. Here, we present the synthesis and the use of monodisperse iron oxide nanoparticles coated with fluorescent silica nano-shells for fluorescence and magnetic resonance dual imaging of tumor. The as-synthesized core-shell nanoparticles were designed to improve the accuracy of diagnosis via simultaneous tumor imaging with dual imaging modalities by a single injection of contrast agent. The iron oxide nanocrystals ( 11 nm) were coated with Rhodamine B isothiocyanate-doped silica shells via reverse microemulsion method. Then, the core-shell nanoparticles ( 54 nm) were analyzed to confirm their size distribution by transmission electron microscopy and dynamic laser scattering. Photoluminescence spectroscopy was used to characterize the fluorescent property of the dye-doped silica shell-coated nanoparticles. The cellular compatibility of the as-prepared nanoparticles was confirmed by a trypan blue dye exclusion assay and the potential as a dual-imaging contrast agent was verified by in vivo fluorescence and magnetic resonance imaging. The experimental results show that the uniform-sized core-shell nanoparticles are highly water dispersible and the cellular toxicity of the nanoparticles is negligible. In vivo fluorescence imaging demonstrates the capability of the developed nanoparticles to selectively target tumors by the enhanced permeability and retention effects and ex vivo tissue analysis was corroborated this. Through in vitro phantom test, the core/shell nanoparticles showed a T2 relaxation time comparable to Feridex® with smaller size, indicating that the as-made nanoparticles are suitable for imaging tumor. This new dual-modality-nanoparticle approach has promised for enabling more accurate tumor imaging.

Improving diffuse optical tomography with structural a priori from fluorescence diffuse optical tomography

NASA Astrophysics Data System (ADS)

Ma, Wenjuan; Gao, Feng; Duan, Linjing; Zhu, Qingzhen; Wang, Xin; Zhang, Wei; Wu, Linhui; Yi, Xi; Zhao, Huijuan

2012-03-01

We obtain absorption and scattering reconstructed images by incorporating a priori information of target location obtained from fluorescence diffuse optical tomography (FDOT) into the diffuse optical tomography (DOT). The main disadvantage of DOT lies in the low spatial resolution resulting from highly scattering nature of tissue in the near-infrared (NIR), but one can use it to monitor hemoglobin concentration and oxygen saturation simultaneously, as well as several other cheomphores such as water, lipids, and cytochrome-c-oxidase. Up to date, extensive effort has been made to integrate DOT with other imaging modalities such as MRI, CT, to obtain accurate optical property maps of the tissue. However, the experimental apparatus is intricate. In this study, DOT image reconstruction algorithm that incorporates a prior structural information provided by FDOT is investigated in an attempt to optimize recovery of a simulated optical property distribution. By use of a specifically designed multi-channel time-correlated single photon counting system, the proposed scheme in a transmission mode is experimentally validated to achieve simultaneous reconstruction of the fluorescent yield, lifetime, absorption and scattering coefficient. The experimental results demonstrate that the quantitative recovery of the tumor optical properties has doubled and the spatial resolution improves as well by applying the new improved method.

A portable array biosensor for food safety

NASA Astrophysics Data System (ADS)

Golden, Joel P.; Ngundi, Miriam M.; Shriver-Lake, Lisa C.; Taitt, Chris R.; Ligler, Frances S.

2004-11-01

An array biosensor developed for simultaneous analysis of multiple samples has been utilized to develop assays for toxins and pathogens in a variety of foods. The biochemical component of the multi-analyte biosensor consists of a patterned array of biological recognition elements immobilized on the surface of a planar waveguide. A fluorescence assay is performed on the patterned surface, yielding an array of fluorescent spots, the locations of which are used to identify what analyte is present. Signal transduction is accomplished by means of a diode laser for fluorescence excitation, optical filters and a CCD camera for image capture. A laptop computer controls the miniaturized fluidics system and image capture. Results for four mycotoxin competition assays in buffer and food samples are presented.

Design and implementation of a dual-wavelength intrinsic fluorescence camera system

NASA Astrophysics Data System (ADS)

Ortega-Martinez, Antonio; Musacchia, Joseph J.; Gutierrez-Herrera, Enoch; Wang, Ying; Franco, Walfre

2017-03-01

Intrinsic UV fluorescence imaging is a technique that permits the observation of spatial differences in emitted fluorescence. It relies on the fluorescence produced by the innate fluorophores in the sample, and thus can be used for marker-less in-vivo assessment of tissue. It has been studied as a tool for the study of the skin, specifically for the classification of lesions, the delimitation of lesion borders and the study of wound healing, among others. In its most basic setup, a sample is excited with a narrow-band UV light source and the resulting fluorescence is imaged with a UV sensitive camera filtered to the emission wavelength of interest. By carefully selecting the excitation/emission pair, we can observe changes in fluorescence associated with physiological processes. One of the main drawbacks of this simple setup is the inability to observe more than a single excitation/emission pair at the same time, as some phenomena are better studied when two or more different pairs are studied simultaneously. In this work, we describe the design and the hardware and software implementation of a dual wavelength portable UV fluorescence imaging system. Its main components are an UV camera, a dual wavelength UV LED illuminator (295 and 345 nm) and two different emission filters (345 and 390 nm) that can be swapped by a mechanical filter wheel. The system is operated using a laptop computer and custom software that performs basic pre-processing to improve the image. The system was designed to allow us to image fluorescent peaks of tryptophan and collagen cross links in order to study wound healing progression.

Fast widefield techniques for fluorescence and phase endomicroscopy

NASA Astrophysics Data System (ADS)

Ford, Tim N.

Endomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast wide-field imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated.

Magnetic resonance-coupled fluorescence tomography scanner for molecular imaging of tissue

NASA Astrophysics Data System (ADS)

Davis, Scott C.; Pogue, Brian W.; Springett, Roger; Leussler, Christoph; Mazurkewitz, Peter; Tuttle, Stephen B.; Gibbs-Strauss, Summer L.; Jiang, Shudong S.; Dehghani, Hamid; Paulsen, Keith D.

2008-06-01

A multichannel spectrally resolved optical tomography system to image molecular targets in small animals from within a clinical MRI is described. Long source/detector fibers operate in contact mode and couple light from the tissue surface in the magnet bore to 16 spectrometers, each containing two optical gratings optimized for the near infrared wavelength range. High sensitivity, cooled charge coupled devices connected to each spectrograph provide detection of the spectrally resolved signal, with exposure times that are automated for acquisition at each fiber. The design allows spectral fitting of the remission light, thereby separating the fluorescence signal from the nonspecific background, which improves the accuracy and sensitivity when imaging low fluorophore concentrations. Images of fluorescence yield are recovered using a nonlinear reconstruction approach based on the diffusion approximation of photon propagation in tissue. The tissue morphology derived from the MR images serves as an imaging template to guide the optical reconstruction algorithm. Sensitivity studies show that recovered values of indocyanine green fluorescence yield are linear to concentrations of 1nM in a 70mm diameter homogeneous phantom, and detection is feasible to near 10pM. Phantom data also demonstrate imaging capabilities of imperfect fluorophore uptake in tissue volumes of clinically relevant sizes. A unique rodent MR coil provides optical fiber access for simultaneous optical and MR data acquisition of small animals. A pilot murine study using an orthotopic glioma tumor model demonstrates optical-MRI imaging of an epidermal growth factor receptor targeted fluorescent probe in vivo.

Three-dimensional refractive index and fluorescence tomography using structured illumination (Conference Presentation)

NASA Astrophysics Data System (ADS)

Park, GwangSik; Shin, SeungWoo; Kim, Kyoohyun; Park, YongKeun

2017-02-01

Optical diffraction tomography (ODT) has been an emerging optical technique for label-free imaging of three-dimensional (3-D) refractive index (RI) distribution of biological samples. ODT employs interferometric microscopy for measuring multiple holograms of samples with various incident angles, from which the Fourier diffraction theorem reconstructs the 3-D RI distribution of samples from retrieved complex optical fields. Since the RI value is linearly proportional to the protein concentration of biological samples where the proportional coefficient is called as refractive index increment (RII), reconstructed 3-D RI tomograms provide precise structural and biochemical information of individual biological samples. Because most proteins have similar RII value, however, ODT has limited molecular specificity, especially for imaging eukaryotic cells having various types of proteins and subcellular organelles. Here, we present an ODT system combined with structured illumination microscopy which can measure the 3-D RI distribution of biological samples as well as 3-D super-resolution fluorescent images in the same optical setup. A digital micromirror device (DMD) controls the incident angle of the illumination beam for tomogram reconstruction, and the same DMD modulates the structured illumination pattern of the excitation beam for super-resolution fluorescent imaging. We first validate the proposed method for simultaneous optical diffraction tomographic imaging and super-resolution fluorescent imaging of fluorescent beads. The proposed method is also exploited for various biological samples.

Label-free imaging of brain and brain tumor specimens with combined two-photon excited fluorescence and second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Jiang, Liwei; Wang, Xingfu; Wu, Zanyi; Du, Huiping; Wang, Shu; Li, Lianhuang; Fang, Na; Lin, Peihua; Chen, Jianxin; Kang, Dezhi; Zhuo, Shuangmu

2017-10-01

Label-free imaging techniques are gaining acceptance within the medical imaging field, including brain imaging, because they have the potential to be applied to intraoperative in situ identifications of pathological conditions. In this paper, we describe the use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopy in combination for the label-free detection of brain and brain tumor specimens; gliomas. Two independently detecting channels were chosen to subsequently collect TPEF/SHG signals from the specimen to increase TPEF/SHG image contrasts. Our results indicate that the combined TPEF/SHG microscopic techniques can provide similar rat brain structural information and produce a similar resolution like conventional H&E staining in neuropathology; including meninges, cerebral cortex, white-matter structure corpus callosum, choroid plexus, hippocampus, striatum, and cerebellar cortex. It can simultaneously detect infiltrating human brain tumor cells, the extracellular matrix collagen fiber of connective stroma within brain vessels and collagen depostion in tumor microenvironments. The nuclear-to-cytoplasmic ratio and collagen content can be extracted as quantitative indicators for differentiating brain gliomas from healthy brain tissues. With the development of two-photon fiberscopes and microendoscope probes and their clinical applications, the combined TPEF and SHG microcopy may become an important multimodal, nonlinear optical imaging approach for real-time intraoperative histological diagnostics of residual brain tumors. These occur in various brain regions during ongoing surgeries through the method of simultaneously identifying tumor cells, and the change of tumor microenvironments, without the need for the removal biopsies and without the need for tissue labelling or fluorescent markers.

Visualizing photosynthesis through processing of chlorophyll fluorescence images

NASA Astrophysics Data System (ADS)

Daley, Paul F.; Ball, J. Timothy; Berry, Joseph A.; Patzke, Juergen; Raschke, Klaus E.

1990-05-01

Measurements of terrestrial plant photosynthesis frequently exploit sensing of gas exchange from leaves enclosed in gas-tight, climate controlled chambers. These methods are typically slow, and do not resolve variation in photosynthesis below the whole leaf level. A photosynthesis visualization technique is presented that uses images of leaves employing light from chlorophyll (Chl) fluorescence. Images of Chl fluorescence from whole leaves undergoing steady-state photosynthesis, photosynthesis induction, or response to stress agents were digitized during light flashes that saturated photochemical reactions. Use of saturating flashes permitted deconvolution of photochemical energy use from biochemical quenching mechanisms (qN) that dissipate excess excitation energy, otherwise damaging to the light harvesting apparatus. Combination of the digital image frames of variable fluorescence with reference frames obtained from the same leaves when dark-adapted permitted derivation of frames in which grey scale represented the magnitude of qN. Simultaneous measurements with gas-exchange apparatus provided data for non-linear calibration filters for subsequent rendering of grey-scale "images" of photosynthesis. In several experiments significant non-homogeneity of photosynthetic activity was observed following treatment with growth hormones, or shifts in light or humidity, and following infection by virus. The technique provides a rapid, non-invasive probe for stress physiology and plant disease detection.

Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope

NASA Astrophysics Data System (ADS)

Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

2016-03-01

Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.

Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope.

PubMed

Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

2016-03-01

Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.

Motion corrected photoacoustic difference imaging of fluorescent contrast agents

NASA Astrophysics Data System (ADS)

Märk, Julia; Wagener, Asja; Pönick, Sarah; Grötzinger, Carsten; Zhang, Edward; Laufer, Jan

2016-03-01

In fluorophores, such as exogenous dyes and genetically expressed proteins, the excited state lifetime can be modulated using pump-probe excitation at wavelengths corresponding to the absorption and fluorescence spectra. Simultaneous pump-probe pulses induce stimulated emission (SE) which, in turn, modulates the thermalized energy, and hence the photoacoustic (PA) signal amplitude. For time-delayed pulses, by contrast, SE is suppressed. Since this is not observed in endogenous chromophores, the location of the fluorophore can be determined by subtracting images acquired using simultaneous and time-delayed pump-probe excitation. This simple experimental approach exploits a fluorophorespecific contrast mechanism, and has the potential to enable deep-tissue molecular imaging at fluences below the MPE. In this study, some of the challenges to its in vivo implementation are addressed. First, the PA signal amplitude generated in fluorophores in vivo is often much smaller than that in blood. Second, tissue motion can give rise to artifacts that correspond to endogenous chromophores in the difference image. This would not allow the unambiguous detection of fluorophores. A method to suppress motion artifacts based on fast switching between simultaneous and time-delayed pump-probe excitation was developed. This enables the acquisition of PA signals using the two excitation modes with minimal time delay (20 ms), thus minimizing the effects of tissue motion. The feasibility of this method is demonstrated by visualizing a fluorophore (Atto680) in tissue phantoms, which were moved during the image acquisition to mimic tissue motion.

Time-resolved fluorescence monitoring of cholesterol in peripheral blood mononuclear cells

NASA Astrophysics Data System (ADS)

Martinakova, Z.; Horilova, J.; Lajdova, I.; Marcek Chorvatova, A.

2014-12-01

Precise evaluation of intracellular cholesterol distribution is crucial for improving diagnostics of diseased states associated with cholesterol alteration. Time-resolved fluorescence techniques are tested for non-invasive investigation of cholesterol in living cells. Fluorescent probe NBD attached to cholesterol was employed to evaluate cholesterol distribution in peripheral blood mononuclear cells (PBMC) isolated from the human blood. Fluorescence Lifetime Imaging Microscopy (FLIM) was successfully applied to simultaneously monitor the spatial distribution and the timeresolved characteristics of the NBD-cholesterol fluorescence in PBMC. Gathered data are the first step in the development of a new perspective non-invasive diagnostic method for evaluation of cholesterol modifications in diseases associated with disorders of lipid metabolism.

High-Resolution Detector For X-Ray Diffraction

NASA Technical Reports Server (NTRS)

Carter, Daniel C.; Withrow, William K.; Pusey, Marc L.; Yost, Vaughn H.

1988-01-01

Proposed x-ray-sensitive imaging detector offers superior spatial resolution, counting-rate capacity, and dynamic range. Instrument based on laser-stimulated luminescence and reusable x-ray-sensitive film. Detector scans x-ray film line by line. Extracts latent image in film and simultaneously erases film for reuse. Used primarily for protein crystallography. Principle adapted to imaging detectors for electron microscopy and fluorescence spectroscopy and general use in astronomy, engineering, and medicine.

In-vivo multi-nonlinear optical imaging of a living cell using a supercontinuum light source generated from a photonic crystal fiber

NASA Astrophysics Data System (ADS)

Kano, Hideaki; Hamaguchi, Hiro-O.

2006-04-01

A supercontinuum light source generated with a femtosecond Ti:Sapphire oscillator has been used to obtain both vibrational and two-photon excitation fluorescence (TPEF) images of a living cell simultaneously at different wavelengths. Owing to an ultrabroadband spectral profile of the supercontinuum, multiple vibrational resonances have been detected through coherent anti-Stokes Raman scattering (CARS) process. In addition to the multiplex CARS process, multiple electronic states can be excited due to the broadband electronic two-photon excitation using the supercontinuum, giving rise to a two-photon excitation fluorescence (TPEF) signal. Using a living yeast cell whose nucleus is labeled by green fluorescent protein (GFP), we have succeeded in visualizing organelles such as mitochondria, septum, and nucleus through the CARS and the TPEF processes. The supercontinuum enables us to perform unique multi-nonlinear optical imaging through two different nonlinear optical processes.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

PubMed Central

Gulati, Srishti; Cao, Vania Y.; Otte, Stephani

2017-01-01

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms. PMID:28654056

Design and daytime performance of laser-induced fluorescence spectrum lidar for simultaneous detection of multiple components, dissolved organic matter, phycocyanin, and chlorophyll in river water.

PubMed

Saito, Yasunori; Kakuda, Kei; Yokoyama, Mizuho; Kubota, Tomoki; Tomida, Takayuki; Park, Ho-Dong

2016-08-20

In this work, we developed mobile laser-induced fluorescence spectrum (LIFS) lidar based on preliminary experiments on the excitation emission matrix of a water sample and a method for reducing solar background light using the synchronous detection technique. The combination of a UV short-pulse laser (355 nm, 6 ns) for fluorescence excitation with a 10-100 ns short-time synchronous detection using a gated image-intensified multi-channel CCD of the fluorescence made the LIFS lidar operation possible even in daytime. The LIFS lidar with this construction demonstrated the potential of natural river/lake water quality monitoring at the Tenryu River/Lake Suwa. Three main components in the fluorescence data of the water, dissolved organic matter, phycocyanin, and chlorophyll, were extracted by spectral analysis using the standard spectral functions of these components. Their concentrations were estimated by adapting experimentally calibrated data. Results of long-term field observations using our LIFS lidar from 2010 to 2012 show the necessity of simultaneous multi-component detection to understand the natural water environment.

Simultaneous fast scanning XRF, dark field, phase-, and absorption contrast tomography

NASA Astrophysics Data System (ADS)

Medjoubi, Kadda; Bonissent, Alain; Leclercq, Nicolas; Langlois, Florent; Mercère, Pascal; Somogyi, Andrea

2013-09-01

Scanning hard X-ray nanoprobe imaging provides a unique tool for probing specimens with high sensitivity and large penetration depth. Moreover, the combination of complementary techniques such as X-ray fluorescence, absorption, phase contrast and dark field imaging gives complete quantitative information on the sample structure, composition and chemistry. The multi-technique "FLYSCAN" data acquisition scheme developed at Synchrotron SOLEIL permits to perform fast continuous scanning imaging and as such makes scanning tomography techniques feasible in a time-frame well-adapted to typical user experiments. Here we present the recent results of simultaneous fast scanning multi-technique tomography performed at Soleil. This fast scanning scheme will be implemented at the Nanoscopium beamline for large field of view 2D and 3D multimodal imaging.

Simultaneous acquisition of trajectory and fluorescence lifetime of moving single particles

NASA Astrophysics Data System (ADS)

Wu, Qianqian; Qi, Jing; Lin, Danying; Yan, Wei; Hu, Rui; Peng, Xiao; Qu, Junle

2017-02-01

Fluorescence lifetime imaging (FLIM) has been a powerful tool in life science because it can reveal the interactions of an excited fluorescent molecule and its environment. The combination with two-photon excitation (TPE) and timecorrelated single photon counting (TCSPC) provides it the ability of optical sectioning, high time resolution and detection efficiency. In previous work, we have introduced a two-dimensional acousto-optic deflector (AOD) into TCSPC-based FLIM to achieve fast and flexible FLIM. In this work, we combined the AOD-FLIM system with a single particle tracking (SPT) setup and algorithm and developed an SPT-FLIM system. Using the system, we acquired the trajectory and fluorescence lifetime of a moving particle simultaneously and reconstructed a life-time-marked pseudocolored trajectory, which might reflect dynamic interaction between the moving particle and its local environment along its motion trail. The results indicated the potential of the technique for studying the interaction between specific moving biological macromolecules and the ambient micro-environment in live cells.

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

PubMed Central

Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Effting, Andries P. J.; Voorneveld, Philip W.; Lucas, Miriam S.; Hardwick, James C.; Wepf, Roger A.; Kruit, Pieter; Hoogenboom, Jacob P.

2013-01-01

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown. PMID:23409024

Multiplexed 3D FRET imaging in deep tissue of live embryos

PubMed Central

Zhao, Ming; Wan, Xiaoyang; Li, Yu; Zhou, Weibin; Peng, Leilei

2015-01-01

Current deep tissue microscopy techniques are mostly restricted to intensity mapping of fluorophores, which significantly limit their applications in investigating biochemical processes in vivo. We present a deep tissue multiplexed functional imaging method that probes multiple Förster resonant energy transfer (FRET) sensors in live embryos with high spatial resolution. The method simultaneously images fluorescence lifetimes in 3D with multiple excitation lasers. Through quantitative analysis of triple-channel intensity and lifetime images, we demonstrated that Ca2+ and cAMP levels of live embryos expressing dual FRET sensors can be monitored simultaneously at microscopic resolution. The method is compatible with a broad range of FRET sensors currently available for probing various cellular biochemical functions. It opens the door to imaging complex cellular circuitries in whole live organisms. PMID:26387920

Solvent and solute ingress into hydrogels resolved by a combination of imaging techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagner, D.; Burbach, J.; Egelhaaf, S. U.

2016-05-28

Using simultaneous neutron, fluorescence, and optical brightfield transmission imaging, the diffusion of solvent, fluorescent dyes, and macromolecules into a crosslinked polyacrylamide hydrogel was investigated. This novel combination of different imaging techniques enables us to distinguish the movements of the solvent and fluorescent molecules. Additionally, the swelling or deswelling of the hydrogels can be monitored. From the sequence of images, dye and solvent concentrations were extracted spatially and temporally resolved. Diffusion equations and different boundary conditions, represented by different models, were used to quantitatively analyze the temporal evolution of these concentration profiles and to determine the diffusion coefficients of solvent andmore » solutes. Solute size and network properties were varied and their effect was investigated. Increasing the crosslinking ratio or partially drying the hydrogel was found to hinder solute diffusion due to the reduced pore size. By contrast, solvent diffusion seemed to be slightly faster if the hydrogel was only partially swollen and hence solvent uptake enhanced.« less

Elemental mapping and microimaging by x-ray capillary optics.

PubMed

Hampai, D; Dabagov, S B; Cappuccio, G; Longoni, A; Frizzi, T; Cibin, G; Guglielmotti, V; Sala, M

2008-12-01

Recently, many experiments have highlighted the advantage of using polycapillary optics for x-ray fluorescence studies. We have developed a special confocal scheme for micro x-ray fluorescence measurements that enables us to obtain not only elemental mapping of the sample but also simultaneously its own x-ray imaging. We have designed the prototype of a compact x-ray spectrometer characterized by a spatial resolution of less than 100 microm for fluorescence and less than 10 microm for imaging. A couple of polycapillary lenses in a confocal configuration together with a silicon drift detector allow elemental studies of extended samples (approximately 3 mm) to be performed, while a CCD camera makes it possible to record an image of the same samples with 6 microm spatial resolution, which is limited only by the pixel size of the camera. By inserting a compound refractive lens between the sample and the CCD camera, we hope to develop an x-ray microscope for more enlarged images of the samples under test.

MultiFocus Polarization Microscope (MF-PolScope) for 3D polarization imaging of up to 25 focal planes simultaneously

PubMed Central

Abrahamsson, Sara; McQuilken, Molly; Mehta, Shalin B.; Verma, Amitabh; Larsch, Johannes; Ilic, Rob; Heintzmann, Rainer; Bargmann, Cornelia I.; Gladfelter, Amy S.; Oldenbourg, Rudolf

2015-01-01

We have developed an imaging system for 3D time-lapse polarization microscopy of living biological samples. Polarization imaging reveals the position, alignment and orientation of submicroscopic features in label-free as well as fluorescently labeled specimens. Optical anisotropies are calculated from a series of images where the sample is illuminated by light of different polarization states. Due to the number of images necessary to collect both multiple polarization states and multiple focal planes, 3D polarization imaging is most often prohibitively slow. Our MF-PolScope system employs multifocus optics to form an instantaneous 3D image of up to 25 simultaneous focal-planes. We describe this optical system and show examples of 3D multi-focus polarization imaging of biological samples, including a protein assembly study in budding yeast cells. PMID:25837112

Non-rigid multi-frame registration of cell nuclei in live cell fluorescence microscopy image data.

PubMed

Tektonidis, Marco; Kim, Il-Han; Chen, Yi-Chun M; Eils, Roland; Spector, David L; Rohr, Karl

2015-01-01

The analysis of the motion of subcellular particles in live cell microscopy images is essential for understanding biological processes within cells. For accurate quantification of the particle motion, compensation of the motion and deformation of the cell nucleus is required. We introduce a non-rigid multi-frame registration approach for live cell fluorescence microscopy image data. Compared to existing approaches using pairwise registration, our approach exploits information from multiple consecutive images simultaneously to improve the registration accuracy. We present three intensity-based variants of the multi-frame registration approach and we investigate two different temporal weighting schemes. The approach has been successfully applied to synthetic and live cell microscopy image sequences, and an experimental comparison with non-rigid pairwise registration has been carried out. Copyright © 2014 Elsevier B.V. All rights reserved.

Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

PubMed Central

Peddie, Christopher J.; Blight, Ken; Wilson, Emma; Melia, Charlotte; Marrison, Jo; Carzaniga, Raffaella; Domart, Marie-Charlotte; O׳Toole, Peter; Larijani, Banafshe; Collinson, Lucy M.

2014-01-01

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. PMID:24637200

NIR-to-visible upconversion nanoparticles for fluorescent labeling and targeted delivery of siRNA

NASA Astrophysics Data System (ADS)

Jiang, Shan; Zhang, Yong; Lim, Kian Meng; Sim, Eugene K. W.; Ye, Lei

2009-04-01

Near-infrared (NIR)-to-visible upconversion fluorescent nanoparticles were synthesized and used for imaging and targeted delivery of small interfering RNA (siRNA) to cancer cells. Silica-coated NaYF4 upconversion nanoparticles (UCNs) co-doped with lanthanide ions (Yb/Er) were synthesized. Folic acid and anti-Her2 antibody conjugated UCNs were used to fluorescently label the folate receptors of HT-29 cells and Her2 receptors of SK-BR-3 cells, respectively. The intracellular uptake of the folic acid and antibody conjugated UCNs was visualized using a confocal fluorescence microscope equipped with an NIR laser. siRNA was attached to anti-Her2 antibody conjugated UCNs and the delivery of these nanoparticles to SK-BR-3 cells was studied. Meanwhile, a luciferase assay was established to confirm the gene silencing effect of siRNA. Upconversion nanoparticles can serve as a fluorescent probe and delivery system for simultaneous imaging and delivery of biological molecules.

Laser-Induced Photofragmentation Fluorescence Imaging of Alkali Compounds in Flames.

PubMed

Leffler, Tomas; Brackmann, Christian; Aldén, Marcus; Li, Zhongshan

2017-06-01

Laser-induced photofragmentation fluorescence has been investigated for the imaging of alkali compounds in premixed laminar methane-air flames. An ArF excimer laser, providing pulses of wavelength 193 nm, was used to photodissociate KCl, KOH, and NaCl molecules in the post-flame region and fluorescence from the excited atomic alkali fragment was detected. Fluorescence emission spectra showed distinct lines of the alkali atoms allowing for efficient background filtering. Temperature data from Rayleigh scattering measurements together with simulations of potassium chemistry presented in literature allowed for conclusions on the relative contributions of potassium species KOH and KCl to the detected signal. Experimental approaches for separate measurements of these components are discussed. Signal power dependence and calculated fractions of dissociated molecules indicate the saturation of the photolysis process, independent on absorption cross-section, under the experimental conditions. Quantitative KCl concentrations up to 30 parts per million (ppm) were evaluated from the fluorescence data and showed good agreement with results from ultraviolet absorption measurements. Detection limits for KCl photofragmentation fluorescence imaging of 0.5 and 1.0 ppm were determined for averaged and single-shot data, respectively. Moreover, simultaneous imaging of KCl and NaCl was demonstrated using a stereoscope with filters. The results indicate that the photofragmentation method can be employed for detailed studies of alkali chemistry in laboratory flames for validation of chemical kinetic mechanisms crucial for efficient biomass fuel utilization.

Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector

PubMed Central

Kodaira, Satoshi; Konishi, Teruaki; Kobayashi, Alisa; Maeda, Takeshi; Ahmad, Tengku Ahbrizal Farizal Tengku; Yang, Gen; Akselrod, Mark S.; Furusawa, Yoshiya; Uchihori, Yukio

2015-01-01

Abstract The geometric locations of ion traversals in mammalian cells constitute important information in the study of heavy ion-induced biological effect. Single ion traversal through a cellular nucleus produces complex and massive DNA damage at a nanometer level, leading to cell inactivation, mutations and transformation. We present a novel approach that uses a fluorescent nuclear track detector (FNTD) for the simultaneous detection of the geometrical images of ion traversals and DNA damage in single cells using confocal microscopy. HT1080 or HT1080–53BP1-GFP cells were cultured on the surface of a FNTD and exposed to 5.1-MeV/n neon ions. The positions of the ion traversals were obtained as fluorescent images of a FNTD. Localized DNA damage in cells was identified as fluorescent spots of γ-H2AX or 53BP1-GFP. These track images and images of damaged DNA were obtained in a short time using a confocal laser scanning microscope. The geometrical distribution of DNA damage indicated by fluorescent γ-H2AX spots in fixed cells or fluorescent 53BP1-GFP spots in living cells was found to correlate well with the distribution of the ion traversals. This method will be useful for evaluating the number of ion hits on individual cells, not only for micro-beam but also for random-beam experiments. PMID:25324538

In vivo molecular imaging of colorectal cancer using quantum dots targeted to vascular endothelial growth factor receptor 2 and optical coherence tomography/laser-induced fluorescence dual-modality imaging

NASA Astrophysics Data System (ADS)

Carbary-Ganz, Jordan L.; Welge, Weston A.; Barton, Jennifer K.; Utzinger, Urs

2015-09-01

Optical coherence tomography/laser induced fluorescence (OCT/LIF) dual-modality imaging allows for minimally invasive, nondestructive endoscopic visualization of colorectal cancer in mice. This technology enables simultaneous longitudinal tracking of morphological (OCT) and biochemical (fluorescence) changes as colorectal cancer develops, compared to current methods of colorectal cancer screening in humans that rely on morphological changes alone. We have shown that QDot655 targeted to vascular endothelial growth factor receptor 2 (QD655-VEGFR2) can be applied to the colon of carcinogen-treated mice and provides significantly increased contrast between the diseased and undiseased tissue with high sensitivity and specificity ex vivo. QD655-VEGFR2 was used in a longitudinal in vivo study to investigate the ability to correlate fluorescence signal to tumor development. QD655-VEGFR2 was applied to the colon of azoxymethane (AOM-) or saline-treated control mice in vivo via lavage. OCT/LIF images of the distal colon were taken at five consecutive time points every three weeks after the final AOM injection. Difficulties in fully flushing unbound contrast agent from the colon led to variable background signal; however, a spatial correlation was found between tumors identified in OCT images, and high fluorescence intensity of the QD655 signal, demonstrating the ability to detect VEGFR2 expressing tumors in vivo.

Photo-multiplier Tube Based Hybrid MRI and Frequency Domain Fluorescence Tomography System for Small Animal Imaging

PubMed Central

Lin, Y; Ghijsen, M T; Gao, H; Liu, N; Nalcioglu, O; Gulsen, G

2014-01-01

Fluorescence tomography (FT) is a promising molecular imaging technique that can spatially resolve both fluorophore concentration and lifetime parameters. However, recovered fluorophore parameters highly depend on the size and depth of the object due to the ill-posedness of the FT inverse problem. Structural a priori information from another high spatial resolution imaging modality has been demonstrated to significantly improve FT reconstruction accuracy. In this study, we have constructed a combined magnetic resonance imaging (MRI) and FT system for small animal imaging. A photo-multiplier tube (PMT) is used as the detector to acquire frequency domain FT measurements. This is the first MR-compatible time-resolved FT system that can reconstruct both fluorescence concentration and lifetime maps simultaneously. The performance of the hybrid system is evaluated with phantom studies. Two different fluorophores, Indocyanine Green (ICG) and 3-3′ Diethylthiatricarbocyanine Iodide (DTTCI), which have similar excitation and emission spectra but different lifetimes, are utilized. The fluorescence concentration and lifetime maps are both reconstructed with and without the structural a priori information obtained from MRI for comparison. We show that the hybrid system can accurately recover both fluorescence intensity and lifetime within 10% error for two 4.2 mm-diameter cylindrical objects embedded in a 38 mm-diameter cylindrical phantom when MRI structural a priori information is utilized. PMID:21753235

Blood flow speed of the gastric conduit assessed by indocyanine green fluorescence

PubMed Central

Koyanagi, Kazuo; Ozawa, Soji; Oguma, Junya; Kazuno, Akihito; Yamazaki, Yasushi; Ninomiya, Yamato; Ochiai, Hiroki; Tachimori, Yuji

2016-01-01

Abstract Anastomotic leakage is considered as an independent risk factor for postoperative mortality after esophagectomy, and an insufficient blood flow in the reconstructed conduit may be a risk factor of anastomotic leakage. We investigated the clinical significance of blood flow visualization by indocyanine green (ICG) fluorescence in the gastric conduit as a means of predicting the leakage of esophagogastric anastomosis after esophagectomy. Forty patients who underwent an esophagectomy with gastric conduit reconstruction were prospectively investigated. ICG fluorescence imaging of the gastric conduit was detected by a near-infrared camera system during esophagectomy and correlated with clinical parameters or surgical outcomes. In 25 patients, the flow speed of ICG fluorescence in the gastric conduit wall was simultaneous with that of the greater curvature vessels (simultaneous group), whereas in 15 patients this was slower than that of the greater curvature vessels (delayed group). The reduced speed of ICG fluorescence stream in the gastric conduit wall was associated with intraoperative blood loss (P = 0.008). Although anastomotic leakage was not found in the simultaneous group, it occurred in 7 patients of the delayed group (P < 0.001). A flow speed of ICG fluorescence in the gastric conduit wall of 1.76 cm/s or less was determined by a receiver operating characteristic (ROC) curve, identified as a significant independent predictor of anastomotic leakage after esophagectomy (P = 0.004). This preliminary study demonstrates that intraoperative evaluation of blood flow speed by ICG fluorescence in the gastric conduit wall is a useful means to predict the risk of anastomotic leakage after esophagectomy. PMID:27472732

Oxygen sensing PLIM together with FLIM of intrinsic cellular fluorophores for metabolic mapping

NASA Astrophysics Data System (ADS)

Kalinina, Sviatlana; Schaefer, Patrick; Breymayer, Jasmin; Bisinger, Dominik; Chakrabortty, Sabyasachi; Rueck, Angelika

2018-02-01

Otical imaging techniques based on time correlated single photon counting (TCSPC) has found wide applications in medicine and biology. Non-invasive and information-rich fluorescence lifetime imaging microscopy (FLIM) is successfully used for monitoring fluorescent intrinsic metabolic coenzymes as NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) and FAD+ (flavin adenine dinucleotide) in living cells and tissues. The ratio between proteinbound and free coenzymes gives an information about the balance between oxidative phosphorylation and glycolysis in the cells. The changes of the ratio reflects major cellular disorders. A correlation exists between metabolic activity, redox ratio and fluorescence lifetime during stem cell differentiation, neurodegenerative diseases, and carcinogenesis. A multichannel FLIM detection system was designed for monitoring the redox state of NAD(P)H and FAD+ and other intrinsic fluorophores as protoporphyrin IX. In addition, the unique upgrade is useful to perform FLIM and PLIM (phosphorescence lifetime imaging microscopy) simultaneously. PLIM is a promising method to investigate oxygen sensing in biomedical samples. In detail, the oxygen-dependent quenching of phosphorescence of some compounds as transition metal complexes enables measuring of oxygen partial pressure (pO2). Using a two-channel FLIM/PLIM system we monitored intrinsic pO2 by PLIM simultaneously with NAD(P)H by FLIM providing complex metabolic and redox imaging of living cells. Physico-chemical properties of oxygen sensitive probes define certain parameters including their localisation. We present results of some ruthenium based complexes including those specifically bound to mitochondria.

Live imaging of dense-core vesicles in primary cultured hippocampal neurons.

PubMed

Kwinter, David M; Silverman, Michael A; Kwinter, David; Michael, Silverman

2009-05-29

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Multi-scale fluorescence imaging of bacterial infections in animal models

NASA Astrophysics Data System (ADS)

Bixler, Joel N.; Kong, Ying; Cirillo, Jeffrey D.; Maitland, Kristen C.

2013-03-01

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), currently affects roughly one-third of the world's population. Drug resistant strains of Mtb decrease the effectiveness of current therapeutics and demand the development of new antimicrobial therapies. In addition, the current vaccine, Bacille Calmette Guérin (BCG), has variable efficacy for disease prevention in different populations. Animal studies are often limited by the need to sacrifice at discrete time points for pathology and tissue homogenization, which greatly reduces spatial and temporal resolution. Optical imaging offers the potential for a minimally-invasive solution to imaging on a macroscopic and microscopic scale, allowing for high resolution study of infection. We have integrated a fluorescence microendoscope into a whole-animal optical imaging system, allowing for simultaneous microscopic and macroscopic imaging of tdTomato expressing BCG in vivo. A 535 nm LED was collimated and launched into a 10,000 element fiber bundle with an outer diameter of 0.66 mm. The fiber bundle can be inserted through an intra-tracheal catheter into the lung of a mouse. Fluorescence emission can either be (1) collected by the bundle and imaged onto the surface of a CCD camera for localized detection or (2) the fluorescence can be imaged by the whole animal imaging system providing macroscopic information. Results from internal localized excitation and external whole body detection indicate the potential for imaging bacterial infections down to 100 colony forming units. This novel imaging technique has the potential to allow for functional studies, enhancing the ability to assess new therapeutic agents.

A Red-Emitting, Multidimensional Sensor for the Simultaneous Cellular Imaging of Biothiols and Phosphate Ions †

PubMed Central

Herrero-Foncubierta, Pilar; Cuerva, Juan M.; Miguel, Delia

2018-01-01

The development of new fluorescent probes for cellular imaging is currently a very active field because of the large potential in understanding cell physiology, especially targeting anomalous behaviours due to disease. In particular, red-emitting dyes are keenly sought, as the light in this spectral region presents lower interferences and a deeper depth of penetration in tissues. In this work, we have synthesized a red-emitting, dual probe for the multiplexed intracellular detection of biothiols and phosphate ions. We have prepared a fluorogenic construct involving a silicon-substituted fluorescein for red emission. The fluorogenic reaction is selectively started by the presence of biothiols. In addition, the released fluorescent moiety undergoes an excited-state proton transfer reaction promoted by the presence of phosphate ions, which modulates its fluorescence lifetime, τ, with the total phosphate concentration. Therefore, in a multidimensional approach, the intracellular levels of biothiols and phosphate can be detected simultaneously using a single fluorophore and with spectral clearing of cell autofluorescence interferences. We have applied this concept to different cell lines, including photoreceptor cells, whose levels of biothiols are importantly altered by light irradiation and other oxidants. PMID:29315248

Mass spectrometric imaging of red fluorescent protein in breast tumor xenografts.

PubMed

Chughtai, Kamila; Jiang, Lu; Post, Harm; Winnard, Paul T; Greenwood, Tiffany R; Raman, Venu; Bhujwalla, Zaver M; Heeren, Ron M A; Glunde, Kristine

2013-05-01

Mass spectrometric imaging (MSI) in combination with electrospray mass spectrometry (ESI-MS) is a powerful technique for visualization and identification of a variety of different biomolecules directly from thin tissue sections. As commonly used tools for molecular reporting, fluorescent proteins are molecular reporter tools that have enabled the elucidation of a multitude of biological pathways and processes. To combine these two approaches, we have performed targeted MS analysis and MALDI-MSI visualization of a tandem dimer (td)Tomato red fluorescent protein, which was expressed exclusively in the hypoxic regions of a breast tumor xenograft model. For the first time, a fluorescent protein has been visualized by both optical microscopy and MALDI-MSI. Visualization of tdTomato by MALDI-MSI directly from breast tumor tissue sections will allow us to simultaneously detect and subsequently identify novel molecules present in hypoxic regions of the tumor. MS and MALDI-MSI of fluorescent proteins, as exemplified in our study, is useful for studies in which the advantages of MS and MSI will benefit from the combination with molecular approaches that use fluorescent proteins as reporters.

Visible continuum pulses based on enhanced dispersive wave generation for endogenous fluorescence imaging.

PubMed

Cui, Quan; Chen, Zhongyun; Liu, Qian; Zhang, Zhihong; Luo, Qingming; Fu, Ling

2017-09-01

In this study, we demonstrate endogenous fluorescence imaging using visible continuum pulses based on 100-fs Ti:sapphire oscillator and a nonlinear photonic crystal fiber. Broadband (500-700 nm) and high-power (150 mW) continuum pulses are generated through enhanced dispersive wave generation by pumping femtosecond pulses at the anomalous dispersion region near zero-dispersion wavelength of high-nonlinear photonic crystal fibers. We also minimize the continuum pulse width by determining the proper fiber length. The visible-wavelength two-photon microscopy produces NADH and tryptophan images of mice tissues simultaneously. Our 500-700 nm continuum pulses support extending nonlinear microscopy to visible wavelength range that is inaccessible to 100-fs Ti:sapphire oscillators and other applications requiring visible laser pulses.

Fluorescence imaging and spectroscopy of ALA-induced protoporphyrin IX preferentially accumulated in tumor tissue

NASA Astrophysics Data System (ADS)

Stepp, Herbert G.; Baumgartner, Reinhold; Beyer, Wolfgang; Knuechel, Ruth; Koerner, T. O.; Kriegmair, M.; Rick, Kai; Steinbach, Pia; Hofstetter, Alfons G.

1995-12-01

In a clinical pilot study performed on 104 patients suffering from bladder cancer it could be shown that intravesical instillation of a solution of 5-aminolevulinic acid (5-ALA) induces a tumorselective accumulation of Protoporphyrin IX (PPIX). Malignant lesions could be detected with a sensitivity of 97% and a specificity of 67%. The Kr+-laser as excitation light source could successfully be replaced by a filtered short arc Xe-lamp. Its emission wavelength band (375 nm - 440 nm) leads to an efficiency of 58% for PPIX- excitation compared to the laser. Two-hundred-sixty mW of output power at the distal end of a slightly modified cystoscope could be obtained. This is sufficient for recording fluorescence images with a target integrating color CCD-camera. Red fluorescence and blue remitted light are displayed simultaneously. Standard white light observation is possible with the same instrumentation. Pharmacokinetic measurements were performed on 18 patients after different routes of 5-ALA application (oral, inhalation and intravesical instillation). PPIX-fluorescence measurements were made on the skin and on the blood plasma. Pharmacokinetic of 5-ALA could be performed on blood plasma. Endoscopical florescence spectroscopy showed the high fluorescence contrast between tumor and normal tissue with a mean value of 10.7. Forthcoming clinical multicenter studies require an objective measure of the fluorescence intensity. Monte Carlo computer simulations showed that artifacts due to observation geometry and varying absorption can largely be reduced by ratioing fluorescence (red channel of camera) to remission (blue channel). Real time image ratioing provides false color images with a reliable fluorescence information.

High-speed fluorescent thermal imaging of quench propagation in high temperature superconductor tapes

NASA Astrophysics Data System (ADS)

Gyuráki, Roland; Sirois, Frédéric; Grilli, Francesco

2018-07-01

Fluorescent microthermographic imaging, a method using rare-earth fluorescent coatings with temperature dependent light emission, was used for quench investigation in high temperature superconductors (HTS). A fluorophore was embedded in a polymer matrix and used as a coating on top of an HTS tape, while being excited with UV light and recorded with a high-speed camera. Simultaneously, the tape was pulsed with high amplitude, short duration DC current, and brought to quench with the help of a localised defect. The Joule heating during a quench influences the fluorescent light intensity emitted from the coating, and by recording the local variations in this intensity over time, the heating of the tape can be visualised and the developed temperatures can be calculated. In this paper, the fluorophore europium tris[3-(trifluoromethylhydroxymethylene)-(+)-camphorate] (EuTFC) provided sufficient temperature sensitivity and a usable temperature range from 77-260 K. With the help of 2500 image captures per second, the normal zone development was imaged in a 20 μm copper stabilised HTS tape held in a liquid nitrogen bath, and using a calibration curve, the temperatures reached during the quench have been calculated.

Experimental investigation of a HOPG crystal fan for x-ray fluorescence molecular imaging

NASA Astrophysics Data System (ADS)

Rosentreter, Tanja; Müller, Bernhard; Schlattl, Helmut; Hoeschen, Christoph

2017-03-01

Imaging x-ray fluorescence generally generates a conflict between the best image quality or highest sensitivity and lowest possible radiation dose. Consequently many experimental studies investigating the feasibility of this molecular imaging method, deal with either monochromatic x-ray sources that are not practical in clinical environment or accept high x-ray doses in order to maintain the advantage of high sensitivity and producing high quality images. In this work we present a x-ray fluorescence imaging setup using a HOPG crystal fan construction consisting of a Bragg reflecting analyzer array together with a scatter reducing radial collimator. This method allows for the use of polychromatic x-ray tubes that are in general easily accessible in contrast to monochromatic x-ray sources such as synchrotron facilities. Moreover this energy-selecting device minimizes the amount of Compton scattered photons while simultaneously increasing the fluorescence signal yield, thus significantly reducing the signal to noise ratio. The aim is to show the feasibility of this approach by measuring the Bragg reflected Kα fluorescence signal of an object containing an iodine solution using a large area detector with moderate energy resolution. Contemplating the anisotropic energy distribution of background scattered x-rays we compare the detection sensitivity, applying two different detector angular configurations. Our results show that even for large area detectors with limited energy resolution, iodine concentrations of 0.12 % can be detected. However, the potentially large scan times and therefore high radiation dose need to be decreased in further investigations.

Fluorescence Imaging In Vivo at Wavelengths beyond 1500 nm.

PubMed

Diao, Shuo; Blackburn, Jeffrey L; Hong, Guosong; Antaris, Alexander L; Chang, Junlei; Wu, Justin Z; Zhang, Bo; Cheng, Kai; Kuo, Calvin J; Dai, Hongjie

2015-12-01

Compared to imaging in the visible and near-infrared regions below 900 nm, imaging in the second near-infrared window (NIR-II, 1000-1700 nm) is a promising method for deep-tissue high-resolution optical imaging in vivo mainly owing to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single-walled carbon nanotubes with large diameters were used for in vivo fluorescence imaging in the long-wavelength NIR region (1500-1700 nm, NIR-IIb). With this imaging agent, 3-4 μm wide capillary blood vessels at a depth of about 3 mm could be resolved. Meanwhile, the blood-flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR-IIb tumor imaging of a live mouse was explored. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700 nm for high-performance in vivo optical imaging. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimizing pulse compressibility in completely all-fibered Ytterbium chirped pulse amplifiers for in vivo two photon laser scanning microscopy

PubMed Central

Fernández, A.; Grüner-Nielsen, L.; Andreana, M.; Stadler, M.; Kirchberger, S.; Sturtzel, C.; Distel, M.; Zhu, L.; Kautek, W.; Leitgeb, R.; Baltuska, A.; Jespersen, K.; Verhoef, A.

2017-01-01

A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy. PMID:28856032

Optimizing pulse compressibility in completely all-fibered Ytterbium chirped pulse amplifiers for in vivo two photon laser scanning microscopy.

PubMed

Fernández, A; Grüner-Nielsen, L; Andreana, M; Stadler, M; Kirchberger, S; Sturtzel, C; Distel, M; Zhu, L; Kautek, W; Leitgeb, R; Baltuska, A; Jespersen, K; Verhoef, A

2017-08-01

A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy.

Expanding Imaging Capabilities for Microfluidics: Applicability of Darkfield Internal Reflection Illumination (DIRI) to Observations in Microfluidics

PubMed Central

Kawano, Yoshihiro; Otsuka, Chino; Sanzo, James; Higgins, Christopher; Nirei, Tatsuo; Schilling, Tobias; Ishikawa, Takuji

2015-01-01

Microfluidics is used increasingly for engineering and biomedical applications due to recent advances in microfabrication technologies. Visualization of bubbles, tracer particles, and cells in a microfluidic device is important for designing a device and analyzing results. However, with conventional methods, it is difficult to observe the channel geometry and such particles simultaneously. To overcome this limitation, we developed a Darkfield Internal Reflection Illumination (DIRI) system that improved the drawbacks of a conventional darkfield illuminator. This study was performed to investigate its utility in the field of microfluidics. The results showed that the developed system could clearly visualize both microbubbles and the channel wall by utilizing brightfield and DIRI illumination simultaneously. The methodology is useful not only for static phenomena, such as clogging, but also for dynamic phenomena, such as the detection of bubbles flowing in a channel. The system was also applied to simultaneous fluorescence and DIRI imaging. Fluorescent tracer beads and channel walls were observed clearly, which may be an advantage for future microparticle image velocimetry (μPIV) analysis, especially near a wall. Two types of cell stained with different colors, and the channel wall, can be recognized using the combined confocal and DIRI system. Whole-slide imaging was also conducted successfully using this system. The tiling function significantly expands the observing area of microfluidics. The developed system will be useful for a wide variety of engineering and biomedical applications for the growing field of microfluidics. PMID:25748425

Expanding imaging capabilities for microfluidics: applicability of darkfield internal reflection illumination (DIRI) to observations in microfluidics.

PubMed

Kawano, Yoshihiro; Otsuka, Chino; Sanzo, James; Higgins, Christopher; Nirei, Tatsuo; Schilling, Tobias; Ishikawa, Takuji

2015-01-01

Microfluidics is used increasingly for engineering and biomedical applications due to recent advances in microfabrication technologies. Visualization of bubbles, tracer particles, and cells in a microfluidic device is important for designing a device and analyzing results. However, with conventional methods, it is difficult to observe the channel geometry and such particles simultaneously. To overcome this limitation, we developed a Darkfield Internal Reflection Illumination (DIRI) system that improved the drawbacks of a conventional darkfield illuminator. This study was performed to investigate its utility in the field of microfluidics. The results showed that the developed system could clearly visualize both microbubbles and the channel wall by utilizing brightfield and DIRI illumination simultaneously. The methodology is useful not only for static phenomena, such as clogging, but also for dynamic phenomena, such as the detection of bubbles flowing in a channel. The system was also applied to simultaneous fluorescence and DIRI imaging. Fluorescent tracer beads and channel walls were observed clearly, which may be an advantage for future microparticle image velocimetry (μPIV) analysis, especially near a wall. Two types of cell stained with different colors, and the channel wall, can be recognized using the combined confocal and DIRI system. Whole-slide imaging was also conducted successfully using this system. The tiling function significantly expands the observing area of microfluidics. The developed system will be useful for a wide variety of engineering and biomedical applications for the growing field of microfluidics.

In vivo wide-field multispectral scanning laser ophthalmoscopy–optical coherence tomography mouse retinal imager: longitudinal imaging of ganglion cells, microglia, and Müller glia, and mapping of the mouse retinal and choroidal vasculature

PubMed Central

Zhang, Pengfei; Zam, Azhar; Jian, Yifan; Wang, Xinlei; Li, Yuanpei; Lam, Kit S.; Burns, Marie E.; Sarunic, Marinko V.; Pugh, Edward N.; Zawadzki, Robert J.

2015-01-01

Abstract. Scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) provide complementary views of the retina, with the former collecting fluorescence data with good lateral but relatively low-axial resolution, and the latter collecting label-free backscattering data with comparable lateral but much higher axial resolution. To take maximal advantage of the information of both modalities in mouse retinal imaging, we have constructed a compact, four-channel, wide-field (∼50  deg) system that simultaneously acquires and automatically coregisters three channels of confocal SLO and Fourier domain OCT data. The scanner control system allows “zoomed” imaging of a region of interest identified in a wide-field image, providing efficient digital sampling and localization of cellular resolution features in longitudinal imaging of individual mice. The SLO is equipped with a “flip-in” spectrometer that enables spectral “fingerprinting” of fluorochromes. Segmentation of retina layers and en face display facilitate spatial comparison of OCT data with SLO fluorescence patterns. We demonstrate that the system can be used to image an individual retinal ganglion cell over many months, to simultaneously image microglia and Müller glia expressing different fluorochromes, to characterize the distinctive spatial distributions and clearance times of circulating fluorochromes with different molecular sizes, and to produce unequivocal images of the heretofore uncharacterized mouse choroidal vasculature. PMID:26677070

A practical implementation of multi-frequency widefield frequency-domain FLIM

PubMed Central

Chen, Hongtao

2013-01-01

Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime, especially in kinetic studies in biomedical researches. However, the small range of modulation frequencies available in commercial instruments makes this technique limited in its applications. Here we describe a practical implementation of multi-frequency widefield FD-FLIM using a pulsed supercontinuum laser and a direct digital synthesizer. In this instrument we use a pulse to modulate the image intensifier rather than the more conventional sine wave modulation. This allows parallel multi-frequency FLIM measurement using the Fast Fourier Transform and the cross-correlation technique, which permits precise and simultaneous isolation of individual frequencies. In addition, the pulse modulation at the cathode of image intensifier restored the loss of optical resolution caused by the defocusing effect when the voltage at the cathode is sinusoidally modulated. Furthermore, in our implementation of this technique, data can be graphically analyzed by the phasor method while data are acquired, which allows easy fit-free lifetime analysis of FLIM images. Here our measurements of standard fluorescent samples and a Föster resonance energy transfer pair demonstrate that the widefield multi-frequency FLIM system is a valuable and simple tool in fluorescence imaging studies. PMID:23296945

Compact 3D printed module for fluorescence and label-free imaging using evanescent excitation

NASA Astrophysics Data System (ADS)

Pandey, Vikas; Gupta, Shalini; Elangovan, Ravikrishnan

2018-01-01

Total internal reflection fluorescence (TIRF) microscopy is widely used for selective excitation and high-resolution imaging of fluorophores, and more recently label-free nanosized objects, with high vertical confinement near a liquid-solid interface. Traditionally, high numerical aperture objectives (>1.4) are used to simultaneously generate evanescent waves and collect fluorescence emission signals which limits their use to small area imaging (<0.1 mm2). Objective-based TIRFs are also expensive as they require dichroic mirrors and efficient notch filters to prevent specular reflection within the objective lenses. We have developed a compact 3D module called cTIRF that can generate evanescent waves in microscope glass slides via a planar waveguide illumination. The module can be attached as a fixture to any existing optical microscope, converting it into a TIRF and enabling high signal-to-noise ratio (SNR) fluorescence imaging using any magnification objective. As the incidence optics is perpendicular to the detector, label-free evanescent scattering-based imaging of submicron objects can also be performed without using emission filters. SNR is significantly enhanced in this case as compared to cTIRF alone, as seen through our model experiments performed on latex beads and mammalian cells. Extreme flexibility and the low cost of our approach makes it scalable for limited resource settings.

Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels.

PubMed

Yang, Liju; Li, Yanbin

2006-03-01

In this study, we explored the use of semiconductor quantum dots (QDs) as fluorescence labels in immunoassays for simultaneous detection of two species of foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. QDs with different sizes can be excited with a single wavelength of light, resulting in different emission peaks that can be measured simultaneously. Highly fluorescent semiconductor quantum dots with different emission wavelengths (525 nm and 705 nm) were conjugated to anti-E. coli O157 and anti-Salmonella antibodies, respectively. Target bacteria were separated from samples by using specific antibody coated magnetic beads. The bead-cell complexes reacted with QD-antibody conjugates to form bead-cell-QD complexes. Fluorescent microscopic images of QD labeled E. coli and Salmonella cells demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of bacterial cells, indicating that the conjugated QD molecules still retain their effective fluorescence, while the conjugated antibody molecules remain active and are able to recognize their specific target bacteria in a complex mixture. The intensities of fluorescence emission peaks at 525 nm and 705 nm of the final complexes were measured for quantitative detection of E. coli O157:H7 and S. Typhimurium simultaneously. The fluorescence intensity (FI) as a function of cell number (N) was found for Salmonella and E. coli, respectively. The regression models can be expressed as: FI = 60.6 log N- 250.9 with R(2) = 0.97 for S. Typhimurium, and FI = 77.8 log N- 245.2 with R(2) = 0.91 for E. coli O157:H7 in the range of cell numbers from 10(4) to 10(7) cfu ml(-1). The detection limit of this method was 10(4) cfu ml(-1). The detection could be completed within 2 hours. The principle of this method could be extended to detect multiple species of bacteria (3-4 species) simultaneously, depending on the availability of each type of QD-antibody conjugates with a unique emission peak and the antibody coated magnetic beads specific to each species of bacteria.

Synthesis of novel taspine diphenyl derivatives as fluorescence probes and inhibitors of breast cancer cell proliferation.

PubMed

He, Huaizhen; Zhan, Yingzhuan; Zhang, Yanmin; Zhang, Jie; He, Langchong

2012-01-01

Two novel taspine diphenyl derivatives (Ta-dD) were designed and synthesized by introducing different coumarin fluorescent groups into the basic structure of Ta-dD. The main advantage of these two compounds is that they can be used as fluorescence probes and inhibitors simultaneously. In the present study, the fluorescent properties of the probes were measured and their inhibition of four breast cancer cell lines was tested. Different concentrations of the fluorescence probe were added to MCF-7 breast cancer cells for fluorescence imaging analysis under normal conditions. The results suggested that both of the new compounds have not only fluorescence but also the ability to inhibit effects on different breast cancer cell lines, which indicates their possible further use as dual functional fluorescence probes in tracer analysis. Copyright © 2011 John Wiley & Sons, Ltd.

Multimodal digital color imaging system for facial skin lesion analysis

NASA Astrophysics Data System (ADS)

Bae, Youngwoo; Lee, Youn-Heum; Jung, Byungjo

2008-02-01

In dermatology, various digital imaging modalities have been used as an important tool to quantitatively evaluate the treatment effect of skin lesions. Cross-polarization color image was used to evaluate skin chromophores (melanin and hemoglobin) information and parallel-polarization image to evaluate skin texture information. In addition, UV-A induced fluorescent image has been widely used to evaluate various skin conditions such as sebum, keratosis, sun damages, and vitiligo. In order to maximize the evaluation efficacy of various skin lesions, it is necessary to integrate various imaging modalities into an imaging system. In this study, we propose a multimodal digital color imaging system, which provides four different digital color images of standard color image, parallel and cross-polarization color image, and UV-A induced fluorescent color image. Herein, we describe the imaging system and present the examples of image analysis. By analyzing the color information and morphological features of facial skin lesions, we are able to comparably and simultaneously evaluate various skin lesions. In conclusion, we are sure that the multimodal color imaging system can be utilized as an important assistant tool in dermatology.

Optical Mapping of Membrane Potential and Epicardial Deformation in Beating Hearts.

PubMed

Zhang, Hanyu; Iijima, Kenichi; Huang, Jian; Walcott, Gregory P; Rogers, Jack M

2016-07-26

Cardiac optical mapping uses potentiometric fluorescent dyes to image membrane potential (Vm). An important limitation of conventional optical mapping is that contraction is usually arrested pharmacologically to prevent motion artifacts from obscuring Vm signals. However, these agents may alter electrophysiology, and by abolishing contraction, also prevent optical mapping from being used to study coupling between electrical and mechanical function. Here, we present a method to simultaneously map Vm and epicardial contraction in the beating heart. Isolated perfused swine hearts were stained with di-4-ANEPPS and fiducial markers were glued to the epicardium for motion tracking. The heart was imaged at 750 Hz with a video camera. Fluorescence was excited with cyan or blue LEDs on alternating camera frames, thus providing a 375-Hz effective sampling rate. Marker tracking enabled the pixel(s) imaging any epicardial site within the marked region to be identified in each camera frame. Cyan- and blue-elicited fluorescence have different sensitivities to Vm, but other signal features, primarily motion artifacts, are common. Thus, taking the ratio of fluorescence emitted by a motion-tracked epicardial site in adjacent frames removes artifacts, leaving Vm (excitation ratiometry). Reconstructed Vm signals were validated by comparison to monophasic action potentials and to conventional optical mapping signals. Binocular imaging with additional video cameras enabled marker motion to be tracked in three dimensions. From these data, epicardial deformation during the cardiac cycle was quantified by computing finite strain fields. We show that the method can simultaneously map Vm and strain in a left-sided working heart preparation and can image changes in both electrical and mechanical function 5 min after the induction of regional ischemia. By allowing high-resolution optical mapping in the absence of electromechanical uncoupling agents, the method relieves a long-standing limitation of optical mapping and has potential to enhance new studies in coupled cardiac electromechanics. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Morphology and Topography of Retinal Pericytes in the Living Mouse Retina Using In Vivo Adaptive Optics Imaging and Ex Vivo Characterization

PubMed Central

Schallek, Jesse; Geng, Ying; Nguyen, HoanVu; Williams, David R.

2013-01-01

Purpose. To noninvasively image retinal pericytes in the living eye and characterize NG2-positive cell topography and morphology in the adult mouse retina. Methods. Transgenic mice expressing fluorescent pericytes (NG2, DsRed) were imaged using a two-channel, adaptive optics scanning laser ophthalmoscope (AOSLO). One channel imaged vascular perfusion with near infrared light. A second channel simultaneously imaged fluorescent retinal pericytes. Mice were also imaged using wide-field ophthalmoscopy. To confirm in vivo imaging, five eyes were enucleated and imaged in flat mount with conventional fluorescent microscopy. Cell topography was quantified relative to the optic disc. Results. We observed strong DsRed fluorescence from NG2-positive cells. AOSLO revealed fluorescent vascular mural cells enveloping all vessels in the living retina. Cells were stellate on larger venules, and showed banded morphology on arterioles. NG2-positive cells indicative of pericytes were found on the smallest capillaries of the retinal circulation. Wide-field SLO enabled quick assessment of NG2-positive distribution, but provided insufficient resolution for cell counts. Ex vivo microscopy showed relatively even topography of NG2-positive capillary pericytes at eccentricities more than 0.3 mm from the optic disc (515 ± 94 cells/mm2 of retinal area). Conclusions. We provide the first high-resolution images of retinal pericytes in the living animal. Subcellular resolution enabled morphological identification of NG2-positive cells on capillaries showing classic features and topography of retinal pericytes. This report provides foundational basis for future studies that will track and quantify pericyte topography, morphology, and function in the living retina over time, especially in the progression of microvascular disease. PMID:24150762

Two-wavelength single laser CH and CH(4) imaging in a lifted turbulent diffusion flame.

PubMed

Namazian, M; Schmitt, R L; Long, M B

1988-09-01

A new technique has been developed which allows simultaneous 2-D mapping of CH and CH 4 in a turbulent methane flame. A flashlamp-pumped dye laser using two back mirrors produces output at 431.5 and 444 nm simultaneously. The 431.5-nm line is used to excite the (0, 0) band of the A(2)Delta-X(2)Pi system of CH, and the fluorescence of the (0, 1) transition is observed at 489 nm. Coincidentally, the spontaneous Raman scattering from CH(4) also occurs near 489 nm for a 431.5-nm excitation. To separate the CH(4) and CH contributions, the 444-nm line is used to produce a spontaneous Raman signal from CH(4) that is spectrally separated from the CH fluorescence. Subtraction of the signals generated by the 431.5- and 444-nm wavelength beams yields separate measurements of CH(4) and CH. Raman-scattered light records the instantaneous distribution of the fuel, and simultaneously the CH fluorescence indicates the location of the flame zone. The resulting composite images provide important insight on the interrelationship between fuel-air mixing and subsequent combustion.M. Namazian is with Altex Technologies Corporation, 109 Via De Tesoros, Los Gatos, California 95030; R. L. Schmitt is with Sandia National Laboratories, Combustion Research Facility, Livermore, California 94550; and M. B. Long is with Yale University, Department of Mechanical Engineering, New Haven, Connecticut 06520.

Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy

PubMed Central

Wu, Yicong; Chandris, Panagiotis; Winter, Peter W.; Kim, Edward Y.; Jaumouillé, Valentin; Kumar, Abhishek; Guo, Min; Leung, Jacqueline M.; Smith, Corey; Rey-Suarez, Ivan; Liu, Huafeng; Waterman, Clare M.; Ramamurthi, Kumaran S.; La Riviere, Patrick J.; Shroff, Hari

2016-01-01

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence. PMID:27761486

Advances in Fluorescence Sensing Systems for the Remote Assessment of Nitrogen Supply in Field Corn

NASA Technical Reports Server (NTRS)

Corp, L. A.; Chappelle, E. W.; McMurtrey, J. E.; Daughtry, C. S. T.; Kim, M. S.

2000-01-01

The studies described herein were conducted to better define changes in fluorescence properties of leaves from field grown corn (Zea mays L.) as they relate to varying levels of nitrogen (N) fertilization. This research was directed toward: 1) providing a remote non-destructive sensing technique to aid in the determination of optimal rates of N fertilization in corn crops and, 2) defining parameters for further development of fluorescence instrumentation to be operated remotely at field canopy levels. Fluorescence imaging bands centered in the blue (450 nm), green (525 nm), red (680 nm), and far-red (740 nm) and ratios of these bands were compared with the following plant parameters: rates of photosynthesis, N:C ratio, pigment concentrations, and grain yields. Both the fluorescence and physiological measures exhibited similar curvilinear responses to N fertilization level while significant linear correlations were obtained among fluorescence bands and band ratios to certain physiological measures of plant productivity. The red / blue, red / green, far-red / blue, far-red /green fluorescence ratios are well suited for remote observation and provided high correlations to grain yield, LAI, N:C, and chlorophyll contents. The results from this investigation indicate that fluorescence technology could aid in the determination of N fertilization requirements for corn. This discussion will also address design concepts and preliminary field trials of a mobile field-based Laser Induced Fluorescence Imaging System (LIFIS) capable of simultaneously acquiring images of four fluorescence emission bands from areas of plant canopies equaling 1 sq m and greater without interference of ambient solar radiation.

Tumor acidity-activatable TAT targeted nanomedicine for enlarged fluorescence/magnetic resonance imaging-guided photodynamic therapy.

PubMed

Gao, Meng; Fan, Feng; Li, Dongdong; Yu, Yue; Mao, Kuirong; Sun, Tianmeng; Qian, Haisheng; Tao, Wei; Yang, Xianzhu

2017-07-01

Nanoparticles simultaneously integrated the photosensitizers and diagnostic agents represent an emerging approach for imaging-guided photodynamic therapy (PDT). However, the diagnostic sensitivity and therapeutic efficacy of nanoparticles as well as the heterogeneity of tumors pose tremendous challenges for clinical imaging-guided PDT treatment. Herein, a polymeric nanoparticle with tumor acidity (pH e )-activatable TAT targeting ligand that encapsulates the photosensitizer chlorin e6 (Ce6) and chelates contrast agent Gd 3+ is successfully developed for fluorescence/magnetic resonance (MR) dual-model imaging-guided precision PDT. We show clear evidence that the resulting nanoparticle DA TAT-NP [its TAT lysine residues' amines was modified by 2,3-dimethylmaleic anhydride (DA)] efficiently avoids the rapid clearance by reticuloendothelial system (RES) by masking of the TAT peptide, resulting in the significantly prolonged circulation time in the blood. Once accumulating in the tumor tissues, DA TAT-NP is reactivated by tumor acidity to promote cellular uptake, resulting in enlarged fluorescence/MR imaging signal intensity and elevated in vivo PDT therapeutic effect. This concept provides new avenues to design tumor acidity-activatable targeted nanoparticles for imaging-guided cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Statistical Deconvolution for Superresolution Fluorescence Microscopy

PubMed Central

Mukamel, Eran A.; Babcock, Hazen; Zhuang, Xiaowei

2012-01-01

Superresolution microscopy techniques based on the sequential activation of fluorophores can achieve image resolution of ∼10 nm but require a sparse distribution of simultaneously activated fluorophores in the field of view. Image analysis procedures for this approach typically discard data from crowded molecules with overlapping images, wasting valuable image information that is only partly degraded by overlap. A data analysis method that exploits all available fluorescence data, regardless of overlap, could increase the number of molecules processed per frame and thereby accelerate superresolution imaging speed, enabling the study of fast, dynamic biological processes. Here, we present a computational method, referred to as deconvolution-STORM (deconSTORM), which uses iterative image deconvolution in place of single- or multiemitter localization to estimate the sample. DeconSTORM approximates the maximum likelihood sample estimate under a realistic statistical model of fluorescence microscopy movies comprising numerous frames. The model incorporates Poisson-distributed photon-detection noise, the sparse spatial distribution of activated fluorophores, and temporal correlations between consecutive movie frames arising from intermittent fluorophore activation. We first quantitatively validated this approach with simulated fluorescence data and showed that deconSTORM accurately estimates superresolution images even at high densities of activated fluorophores where analysis by single- or multiemitter localization methods fails. We then applied the method to experimental data of cellular structures and demonstrated that deconSTORM enables an approximately fivefold or greater increase in imaging speed by allowing a higher density of activated fluorophores/frame. PMID:22677393

Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

PubMed Central

Warren, Sean C.; Margineanu, Anca; Alibhai, Dominic; Kelly, Douglas J.; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Katan, Matilda

2013-01-01

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment. PMID:23940626

Visualization of hydrogen injection in a scramjet engine by simultaneous PLIF imaging and laser holographic imaging

NASA Technical Reports Server (NTRS)

Anderson, Robert C.; Trucco, Richard E.; Rubin, L. F.; Swain, D. M.

1992-01-01

Flowfield characterization has been accomplished for several fuel injector configurations using simultaneous planar laser induced fluorescence (PLIF) and laser holographic imaging (LHI). The experiments were carried out in the GASL-NASA HYPULSE real gas expansion tube facility, a pulsed facility with steady test times of about 350 microsec. The tests were done at simulated Mach numbers 13.5 and 17. The focus of this paper is on the measurement technologies used and their application in a research facility. The HYPULSE facility, the models used for the experiments, and the setup for the LHI and PLIF measurements are described. Measurement challenges and solutions are discussed. Results are presented for experiments with several fuel injector configurations and several equivalence ratios.

Carbon nanotube-mediated siRNA delivery for gene silencing in cancer cells

NASA Astrophysics Data System (ADS)

Hong, Tu; Guo, Honglian; Xu, Yaqiong

2011-10-01

Small interfering RNA (siRNA) is potentially a promising tool in influencing gene expression with a high degree of target specificity. However, its poor intracellular uptake, instability in vivo, and non-specific immune stimulations impeded its effect in clinical applications. In this study, carbon nanotubes (CNTs) functionalized with two types of phospholipid-polyethylene glycol (PEG) have shown capabilities to stabilize siRNA in cell culture medium during the transfection and efficiently deliver siRNA into neuroblastoma and breast cancer cells. Moreover, the intrinsic optical properties of CNTs have been investigated through absorption and fluorescence measurements. We have found that the directly-functionalized groups play an important role on the fluorescence imaging of functionalized CNTs. The unique fluorescence imaging and high delivery efficiency make CNTs a promising material to deliver drugs and evaluate the treatment effect simultaneously.

A dynamic system with digital lock-in-photon-counting for pharmacokinetic diffuse fluorescence tomography

NASA Astrophysics Data System (ADS)

Yin, Guoyan; Zhang, Limin; Zhang, Yanqi; Liu, Han; Du, Wenwen; Ma, Wenjuan; Zhao, Huijuan; Gao, Feng

2018-02-01

Pharmacokinetic diffuse fluorescence tomography (DFT) can describe the metabolic processes of fluorescent agents in biomedical tissue and provide helpful information for tumor differentiation. In this paper, a dynamic DFT system was developed by employing digital lock-in-photon-counting with square wave modulation, which predominates in ultra-high sensitivity and measurement parallelism. In this system, 16 frequency-encoded laser diodes (LDs) driven by self-designed light source system were distributed evenly in the imaging plane and irradiated simultaneously. Meanwhile, 16 detection fibers collected emission light in parallel by the digital lock-in-photon-counting module. The fundamental performances of the proposed system were assessed with phantom experiments in terms of stability, linearity, anti-crosstalk as well as images reconstruction. The results validated the availability of the proposed dynamic DFT system.

Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow.

PubMed

Wang, Sheng; Ding, Miao; Xue, Boxin; Hou, Yingping; Sun, Yujie

2018-05-18

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

Single Probe for Imaging and Biosensing of pH, Cu(2+) Ions, and pH/Cu(2+) in Live Cells with Ratiometric Fluorescence Signals.

PubMed

Han, Yingying; Ding, Changqin; Zhou, Jie; Tian, Yang

2015-01-01

It is very essential to disentangle the complicated inter-relationship between pH and Cu in the signal transduction and homeostasis. To this end, reporters that can display distinct signals to pH and Cu are highly valuable. Unfortunately, there is still no report on the development of biosensors that can simultaneously respond to pH and Cu(2+), to the best of our knowledge. In this work, we developed a single fluorescent probe, AuNC@FITC@DEAC (AuNC, gold cluster; FITC, fluorescein isothiocyanate; DEAC, 7-diethylaminocoumarin-3-carboxylic acid), for biosensing of pH, Cu(2+), and pH/Cu(2+) with different ratiometric fluorescent signals. First, 2,2',2″-(2,2',2″-nitrilotris(ethane-2,1-diyl)tris((pyridin-2-yl-methyl)azanediyl))triethanethiol (TPAASH) was designed for specific recognition of Cu(2+), as well as for organic ligand to synthesize fluorescent AuNCs. Then, pH-sensitive molecule, FITC emitting at 518 nm, and inner reference molecule, DEAC with emission peak at 472 nm, were simultaneously conjugated on the surface of AuNCs emitting at 722 nm, thus, constructing a single fluorescent probe, AuNC@FITC@DEAC, to sensing pH, Cu(2+), and pH/Cu(2+) excited by 405 nm light. The developed probe exhibited high selectivity and accuracy for independent determination of pH and Cu(2+) against reactive oxygen species (ROS), other metal ions, amino acids, and even copper-containing proteins. The AuNC-based inorganic-organic probe with good cell-permeability and high biocompatibility was eventually applied in monitoring both pH and Cu(2+) and in understanding the interplaying roles of Cu(2+) and pH in live cells by ratiometric multicolor fluorescent imaging.

Simultaneous Spectral Temporal Adaptive Raman Spectrometer - SSTARS

NASA Technical Reports Server (NTRS)

Blacksberg, Jordana

2010-01-01

Raman spectroscopy is a prime candidate for the next generation of planetary instruments, as it addresses the primary goal of mineralogical analysis, which is structure and composition. However, large fluorescence return from many mineral samples under visible light excitation can render Raman spectra unattainable. Using the described approach, Raman and fluorescence, which occur on different time scales, can be simultaneously obtained from mineral samples using a compact instrument in a planetary environment. This new approach is taken based on the use of time-resolved spectroscopy for removing the fluorescence background from Raman spectra in the laboratory. In the SSTARS instrument, a visible excitation source (a green, pulsed laser) is used to generate Raman and fluorescence signals in a mineral sample. A spectral notch filter eliminates the directly reflected beam. A grating then disperses the signal spectrally, and a streak camera provides temporal resolution. The output of the streak camera is imaged on the CCD (charge-coupled device), and the data are read out electronically. By adjusting the sweep speed of the streak camera, anywhere from picoseconds to milliseconds, it is possible to resolve Raman spectra from numerous fluorescence spectra in the same sample. The key features of SSTARS include a compact streak tube capable of picosecond time resolution for collection of simultaneous spectral and temporal information, adaptive streak tube electronics that can rapidly change from one sweep rate to another over ranges of picoseconds to milliseconds, enabling collection of both Raman and fluorescence signatures versus time and wavelength, and Synchroscan integration that allows for a compact, low-power laser without compromising ultimate sensitivity.

Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-Tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic-Targeting and Imaging.

PubMed

Sun, Lu; Xie, Shuping; Qi, Jing; Liu, Ergang; Liu, Di; Liu, Quan; Chen, Sunhui; He, Huining; Yang, Victor C

2017-11-15

Matrix metalloproteinases (MMPs) activatable imaging probe has been explored for tumor detection. However, activation of the probe is mainly done in the extracellular space without intracellular uptake of the probe for more sensitivity. Although cell-penetrating peptides (CPPs) have been demonstrated to enable intracellular delivery of the imaging probe, they nevertheless encounter off-target delivery of the cargos to normal tissues. Herein, we have developed a dual MMP-2-activatable and tumor cell-permeable magnetic nanoprobe to simultaneously achieve selective and intracellular tumor imaging. This novel imaging probe was constructed by self-assembling a hexahistidine-tagged (His-tagged) fluorescent fusion protein chimera and nickel ferrite nanoparticles via a chelation mechanism. The His-tagged fluorescent protein chimera consisted of a red fluorescent protein mCherry that acted as the fluorophore, the low-molecular-weight protamine peptide as the CPP, and the MMP-2 cleavage sequence fused with the hexahistidine tag, whereas the nickel ferrite nanoparticles functioned as the His-tagged protein binder and also the fluorescent quencher. Both in vitro and in vivo results revealed that this imaging probe would not only remain nonpermeable to normal tissues, thereby offsetting the nonselective cellular uptake, but was also suppressed of fluorescent signals during magnetic tumor-targeting in the circulation, primarily because of the masking of the CPP activity and quenching of the fluorophore by the associated NiFe 2 O 4 nanoparticles. However, these properties were recovered or "turned on" by the action of tumor-associated MMP-2 stimuli, leading to cell penetration of the nanoprobes as well as fluorescence restoration and visualization within the tumor cells. In this regard, the presented tumor-activatable and cell-permeable system deems to be an appealing platform to achieve selective tumor imaging and intracellular protein delivery. Its impact is therefore significant, far-reaching, and wide-spread.

Simultaneous measurement of liposome extravasation and content release in tumors.

PubMed

Wu, N Z; Braun, R D; Gaber, M H; Lin, G M; Ong, E T; Shan, S; Papahadjopoulos, D; Dewhirst, M W

1997-03-01

The success of liposome-based drug delivery systems for tumor targeting relies on maximum extravasation of liposomes into tumor interstitium, as well as optimal release of contents from the liposomes once within the tumor Liposome extravasation and content release are two separate processes that can be individually or jointly manipulated so a method is needed to monitor these two processes independently and simultaneously. In this report, we describe a method to measure liposome extravasation and content release in tumor tissues growing in a rat skinfold window chamber preparation. Mixtures of liposomes containing either doxorubicin or calcein, both of which are fluorescent, and liposomes surface-labeled with rhodamine were injected intravenously. Fluorescent, light intensities in a tumor region in two fluorescent channels were measured using an image-processing system. Light intensities of plasma from blood samples were also measured using this system. These measurements were used to calculate the amounts of liposomes and released contents in both plasma and tumor interstitium. The calculations were based on the fact that the liposome surface labels and contents emit fluorescent light at different wavelengths and when encapsulated, the contents fluorescence is self-quenched. The model included equations to account for fluorescent light "cross-contamination" by the two fluorochromes as well as equations relating the measured fluorescent light intensities to the amounts of liposomes and released contents. This method was applied to three situations in which liposome extravasation and content release were manipulated in different, predictable ways. Our results indicate that this method can perform simultaneous independent and quantitative measurements of liposome extravasation and content release. This method can potentially be used to study drug delivery of other carrier systems in vivo.

A multiplexed fluorescent assay for independent second-messenger systems: decoding GPCR activation in living cells.

PubMed

Tewson, Paul H; Quinn, Anne Marie; Hughes, Thomas E

2013-08-01

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein-coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca(2+)). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca(2+) sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

Mapping Diffusion in a Living Cell via the Phasor Approach

PubMed Central

Ranjit, Suman; Lanzano, Luca; Gratton, Enrico

2014-01-01

Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created. PMID:25517145

1-Million droplet array with wide-field fluorescence imaging for digital PCR.

PubMed

Hatch, Andrew C; Fisher, Jeffrey S; Tovar, Armando R; Hsieh, Albert T; Lin, Robert; Pentoney, Stephen L; Yang, David L; Lee, Abraham P

2011-11-21

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.

Improving confocal microscopy with solid-state semiconductor excitation sources

NASA Astrophysics Data System (ADS)

Sivers, Nelson L.

To efficiently excite the fluorescent dyes used in imaging biological samples with a confocal microscope, the wavelengths of the exciting laser must be near the fluorochrome absorption peak. However, this causes imaging problems when the fluorochrome absorption and emission spectra overlap significantly, i.e. have small Stokes shifts, which is the case for most fluorochromes that emit in the red to infrared. As a result, the reflected laser excitation cannot be distinguished from the information-containing fluorescence signal. However, cryogenically cooling the exciting laser diode enabled the laser emission wavelengths to be tuned to shorter wavelengths, decreasing the interference between the laser and the fluorochrome's fluorescence. This reduced the amount of reflected laser light in the confocal image. However, the cooled laser diode's shorter wavelength signal resulted in slightly less efficient fluorochrome excitation. Spectrophotometric analysis showed that as the laser diodes were cooled, their output power increased, which more than compensated for the lower fluorochrome excitation and resulted in significantly more intense fluorescence. Thus, by tuning the laser diode emission wavelengths away from the fluorescence signal, less reflected laser light and more fluorescence information reached the detector, creating images with better signal to noise ratios. Additionally, new, high, luminous flux, light-emitting diodes (LEDs) are now powerful enough to create confocal fluorescence signals comparable to those produced by the traditional laser excitation sources in fluorescence confocal microscopes. The broader LED spectral response effectively excited the fluorochrome, yet was spectrally limited enough for standard filter sets to separate the LED excitation from the fluorochrome fluorescence signal. Spectrophotometric analysis of the excitation and fluorescence spectra of several fluorochromes showed that high-powered, LED-induced fluorescence contained the same spectral information and could be more intense than that produced by lasers. An alternative, LED-based, confocal microscope is proposed in this thesis that would be capable of exciting multiple fluorochromes in a single specimen, producing images of several distinct cellular components simultaneously. The inexpensive, LED-based, confocal microscope would require lower peak excitation intensities to produce fluorescence signals equal to those produced by laser excitation, reducing cellular damage and slowing fluorochrome photobleaching.

Array biosensor: recent developments

NASA Astrophysics Data System (ADS)

Golden, Joel P.; Rowe-Taitt, Chris A.; Feldstein, Mark J.; Ligler, Frances S.

1999-05-01

A fluorescence-based immunosensor has been developed for simultaneous analyses of multiple samples for 1 to 6 different antigens. A patterned array of recognition antibodies immobilized on the surface of a planar waveguide is used to 'capture' analyte present in samples. Bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. A new design for a fluidics distribution system is shown, as well as results from assays for physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D- dimer, a marker of sepsis and thrombotic disorders.

Spectroscopic imaging system for high-throughput viability assessment of ovarian spheroids or microdissected tumor tissues (MDTs) in a microfluidic chip

NASA Astrophysics Data System (ADS)

St-Georges-Robillard, A.; Masse, M.; Kendall-Dupont, J.; Strupler, M.; Patra, B.; Jermyn, M.; Mes-Masson, A.-M.; Leblond, F.; Gervais, T.

2016-02-01

There is a growing effort in the biomicrosystems community to develop a personalized treatment response assay for cancer patients using primary cells, patient-derived spheroids, or live tissues on-chip. Recently, our group has developed a technique to cut tumors in 350 μm diameter microtissues and keep them alive on-chip, enabling multiplexed in vitro drug assays on primary tumor tissue. Two-photon microscopy, confocal microscopy and flow cytometry are the current standard to assay tissue chemosensitivity on-chip. While these techniques provide microscopic and molecular information, they are not adapted for high-throughput analysis of microtissues. We present a spectroscopic imaging system that allows rapid quantitative measurements of multiple fluorescent viability markers simultaneously by using a liquid crystal tunable filter to record fluorescence and transmittance spectra. As a proof of concept, 24 spheroids composed of ovarian cancer cell line OV90 were formed in a microfluidic chip, stained with two live cell markers (CellTrackerTM Green and Orange), and imaged. Fluorescence images acquired were normalized to the acquisition time and gain of the camera, dark noise was removed, spectral calibration was applied, and spatial uniformity was corrected. Spectral un-mixing was applied to separate each fluorophore's contribution. We have demonstrated that rapid and simultaneous viability measurements on multiple spheroids can be achieved, which will have a significant impact on the prediction of a tumor's response to multiple treatment options. This technique may be applied as well in drug discovery to assess the potential of a drug candidate directly on human primary tissue.

Time-resolved autofluorescence imaging of human donor retina tissue from donors with significant extramacular drusen.

PubMed

Schweitzer, Dietrich; Gaillard, Elizabeth R; Dillon, James; Mullins, Robert F; Russell, Stephen; Hoffmann, Birgit; Peters, Sven; Hammer, Martin; Biskup, Christoph

2012-06-08

Time and spectrally resolved measurements of autofluorescence have the potential to monitor metabolism at the cellular level. Fluorophores that emit with the same fluorescence intensity can be discriminated from each other by decay time of fluorescence intensity after pulsed excitation. We performed time-resolved autofluorescence measurements on fundus samples from a donor with significant extramacular drusen. Tissue sections from two human donors were prepared and imaged with a laser scanning microscope. The sample was excited with a titanium-sapphire laser, which was tuned to 860 nm, and frequency doubled by a BBO crystal to 430 nm. The repetition rate was 76 MHz and the pulse width was 170 femtoseconds (fs). The time-resolved autofluorescence was recorded simultaneously in 16 spectral channels (445-605 nm) and bi-exponentially fitted. RPE can be discriminated clearly from Bruch's membrane, drusen, and choroidal connective tissue by fluorescence lifetime. In RPE, bright fluorescence of lipofuscin could be detected with a maximum at 510 nm and extending beyond 600 nm. The lifetime was 385 ps. Different types of drusen were found. Most of them did not contain lipofuscin and exhibited a weak fluorescence, with a maximum at 470 nm. The lifetime was 1785 picoseconds (ps). Also, brightly emitting lesions, presumably representing basal laminar deposits, with fluorescence lifetimes longer than those recorded in RPE could be detected. The demonstrated differentiation of fluorescent structures by their fluorescence decay time is important for interpretation of in vivo measurements by the new fluorescence lifetime imaging (FLIM) ophthalmoscopy on healthy subjects as well as on patients.

A colorimetric and fluorescent probe for detecting intracellular biothiols.

PubMed

Chen, Chunyang; Liu, Wei; Xu, Cong; Liu, Weisheng

2016-11-15

A new rapid and highly sensitive coumarin-based probe (probe 1) has been designed and synthesized for detecting intracellular thiols. Probe 1 was prepared by a 4-step procedure as a latent fluorescence probe to achieve high sensitivity and fluorescence turn-on response toward cysteine and homocysteine over GSH and other various natural amino acids under physiological conditions. Owing to specific cyclization between thiols and aldehyde group, probe 1 displayed a highly selectivity toward cysteine and homocysteine. Above all, probe 1 was successfully used for fluorescence imaging of biothiols in Hela cells, and quantitative determination had been achieved within a certain range. Then specific fluorescence imaging of mice organ tissues was obtained for proving the permeability of probe 1. Simultaneously, the viability was measured to be more than 80%, which shows probe 1 can be a rapid and biocompatible probe for biothiols in cells. Furthermore, the measurement of thiols detection in 5 kinds of animal serum showed that probe 1 can be used in determination of biothiols in blood. Copyright © 2016 Elsevier B.V. All rights reserved.

Self-phase modulation and two-photon absorption imaging of cells and active neurons

NASA Astrophysics Data System (ADS)

Fischer, Martin C.; Liu, Henry; Piletic, Ivan R.; Ye, Tong; Yasuda, Ryohei; Warren, Warren S.

2007-02-01

Even though multi-photon fluorescence microscopy offers higher resolution and better penetration depth than traditional fluorescence microscopy, its use is restricted to the detection of molecules that fluoresce. Two-photon absorption (TPA) imaging can provide contrast in non-fluorescent molecules while retaining the high resolution and sectioning capabilities of nonlinear imaging modalities. In the long-wavelength water window, tissue TPA is dominated by the endogenous molecules melanin and hemoglobin with an almost complete absence of endogenous two-photon fluorescence. A complementary nonlinear contrast mechanism is self-phase modulation (SPM), which can provide intrinsic signatures that can depend on local tissue anisotropy, chemical environment, or other structural properties. We have developed a spectral hole refilling measurement technique for TPA and SPM measurements using shaped ultrafast laser pulses. Here we report on a microscopy setup to simultaneously acquire 3D, high-resolution TPA and SPM images. We have acquired data in mounted B16 melanoma cells with very modest laser power levels. We will also discuss the possible application of this measurement technique to neuronal imaging. Since SPM is sensitive to material structure we can expect SPM properties of neurons to change during neuronal firing. Using our hole-refilling technique we have now demonstrated strong novel intrinsic nonlinear signatures of neuronal activation in a hippocampal brain slice. The observed changes in nonlinear signal upon collective activation were up to factors of two, unlike other intrinsic optical signal changes on the percent level. These results show that TPA and SPM imaging can provide important novel functional contrast in tissue using very modest power levels suitable for in vivo applications.

Development of fast parallel multi-technique scanning X-ray imaging at Synchrotron Soleil

NASA Astrophysics Data System (ADS)

Medjoubi, K.; Leclercq, N.; Langlois, F.; Buteau, A.; Lé, S.; Poirier, S.; Mercère, P.; Kewish, C. M.; Somogyi, A.

2013-10-01

A fast multimodal scanning X-ray imaging scheme is prototyped at Soleil Synchrotron. It permits the simultaneous acquisition of complementary information on the sample structure, composition and chemistry by measuring transmission, differential phase contrast, small-angle scattering, and X-ray fluorescence by dedicated detectors with ms dwell time per pixel. The results of the proof of principle experiments are presented in this paper.

Intravital multiphoton fluorescence imaging and optical manipulation of spinal cord in mice, using a compact fiber laser system.

PubMed

Oshima, Yusuke; Horiuch, Hideki; Honkura, Naoki; Hikita, Atsuhiko; Ogata, Tadanori; Miura, Hiromasa; Imamura, Takeshi

2014-09-01

Near-infrared ultrafast lasers are widely used for multiphoton excited fluorescence microscopy in living animals. Ti:Sapphire lasers are typically used for multiphoton excitation, but their emission wavelength is restricted below 1,000 nm. The aim of this study is to evaluate the performance of a compact Ytterbium-(Yb-) fiber laser at 1,045 nm for multiphoton excited fluorescence microscopy in spinal cord injury. In this study, we employed a custom-designed microscopy system with a compact Yb-fiber laser and evaluated the performance of this system in in vivo imaging of brain cortex and spinal cord in YFP-H transgenic mice. For in vivo imaging of brain cortex, sharp images of basal dendrites, and pyramidal cells expressing EYFP were successfully captured using the Yb-fiber laser in our microscopy system. We also performed in vivo imaging of axon fibers of spinal cord in the transgenic mice. The obtained images were almost as sharp as those obtained using a conventional ultrafast laser system. In addition, laser ablation and multi-color imaging could be performed simultaneously using the Yb-fiber laser. The high-peak pulse Yb-fiber laser is potentially useful for multimodal bioimaging methods based on a multiphoton excited fluorescence microscopy system that incorporates laser ablation techniques. Our results suggest that microscopy systems of this type could be utilized in studies of neuroscience and clinical use in diagnostics and therapeutic tool for spinal cord injury in the future. © 2014 Wiley Periodicals, Inc.

High-Resolution Ultrasound-Switchable Fluorescence Imaging in Centimeter-Deep Tissue Phantoms with High Signal-To-Noise Ratio and High Sensitivity via Novel Contrast Agents

PubMed Central

Cheng, Bingbing; Bandi, Venugopal; Wei, Ming-Yuan; Pei, Yanbo; D’Souza, Francis; Nguyen, Kytai T.; Hong, Yi; Yuan, Baohong

2016-01-01

For many years, investigators have sought after high-resolution fluorescence imaging in centimeter-deep tissue because many interesting in vivo phenomena—such as the presence of immune system cells, tumor angiogenesis, and metastasis—may be located deep in tissue. Previously, we developed a new imaging technique to achieve high spatial resolution in sub-centimeter deep tissue phantoms named continuous-wave ultrasound-switchable fluorescence (CW-USF). The principle is to use a focused ultrasound wave to externally and locally switch on and off the fluorophore emission from a small volume (close to ultrasound focal volume). By making improvements in three aspects of this technique: excellent near-infrared USF contrast agents, a sensitive frequency-domain USF imaging system, and an effective signal processing algorithm, for the first time this study has achieved high spatial resolution (~ 900 μm) in 3-centimeter-deep tissue phantoms with high signal-to-noise ratio (SNR) and high sensitivity (3.4 picomoles of fluorophore in a volume of 68 nanoliters can be detected). We have achieved these results in both tissue-mimic phantoms and porcine muscle tissues. We have also demonstrated multi-color USF to image and distinguish two fluorophores with different wavelengths, which might be very useful for simultaneously imaging of multiple targets and observing their interactions in the future. This work has opened the door for future studies of high-resolution centimeter-deep tissue fluorescence imaging. PMID:27829050

High-Resolution Ultrasound-Switchable Fluorescence Imaging in Centimeter-Deep Tissue Phantoms with High Signal-To-Noise Ratio and High Sensitivity via Novel Contrast Agents.

PubMed

Cheng, Bingbing; Bandi, Venugopal; Wei, Ming-Yuan; Pei, Yanbo; D'Souza, Francis; Nguyen, Kytai T; Hong, Yi; Yuan, Baohong

2016-01-01

For many years, investigators have sought after high-resolution fluorescence imaging in centimeter-deep tissue because many interesting in vivo phenomena-such as the presence of immune system cells, tumor angiogenesis, and metastasis-may be located deep in tissue. Previously, we developed a new imaging technique to achieve high spatial resolution in sub-centimeter deep tissue phantoms named continuous-wave ultrasound-switchable fluorescence (CW-USF). The principle is to use a focused ultrasound wave to externally and locally switch on and off the fluorophore emission from a small volume (close to ultrasound focal volume). By making improvements in three aspects of this technique: excellent near-infrared USF contrast agents, a sensitive frequency-domain USF imaging system, and an effective signal processing algorithm, for the first time this study has achieved high spatial resolution (~ 900 μm) in 3-centimeter-deep tissue phantoms with high signal-to-noise ratio (SNR) and high sensitivity (3.4 picomoles of fluorophore in a volume of 68 nanoliters can be detected). We have achieved these results in both tissue-mimic phantoms and porcine muscle tissues. We have also demonstrated multi-color USF to image and distinguish two fluorophores with different wavelengths, which might be very useful for simultaneously imaging of multiple targets and observing their interactions in the future. This work has opened the door for future studies of high-resolution centimeter-deep tissue fluorescence imaging.

Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging.

PubMed

Valm, Alex M; Mark Welch, Jessica L; Rieken, Christopher W; Hasegawa, Yuko; Sogin, Mitchell L; Oldenbourg, Rudolf; Dewhirst, Floyd E; Borisy, Gary G

2011-03-08

Microbes in nature frequently function as members of complex multitaxon communities, but the structural organization of these communities at the micrometer level is poorly understood because of limitations in labeling and imaging technology. We report here a combinatorial labeling strategy coupled with spectral image acquisition and analysis that greatly expands the number of fluorescent signatures distinguishable in a single image. As an imaging proof of principle, we first demonstrated visualization of Escherichia coli labeled by fluorescence in situ hybridization (FISH) with 28 different binary combinations of eight fluorophores. As a biological proof of principle, we then applied this Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH) strategy using genus- and family-specific probes to visualize simultaneously and differentiate 15 different phylotypes in an artificial mixture of laboratory-grown microbes. We then illustrated the utility of our method for the structural analysis of a natural microbial community, namely, human dental plaque, a microbial biofilm. We demonstrate that 15 taxa in the plaque community can be imaged simultaneously and analyzed and that this community was dominated by early colonizers, including species of Streptococcus, Prevotella, Actinomyces, and Veillonella. Proximity analysis was used to determine the frequency of inter- and intrataxon cell-to-cell associations which revealed statistically significant intertaxon pairings. Cells of the genera Prevotella and Actinomyces showed the most interspecies associations, suggesting a central role for these genera in establishing and maintaining biofilm complexity. The results provide an initial systems-level structural analysis of biofilm organization.

A Planar-Fluorescence Imaging Technique for Studying Droplet-Turbulence Interactions in Vaporizing Sprays

NASA Technical Reports Server (NTRS)

Santavicca, Dom A.; Coy, E.

1990-01-01

Droplet turbulence interactions directly affect the vaporization and dispersion of droplets in liquid sprays and therefore play a major role in fuel oxidizer mixing in liquid fueled combustion systems. Proper characterization of droplet turbulence interactions in vaporizing sprays require measurement of droplet size velocity and size temperature correlations. A planar, fluorescence imaging technique is described which is being developed for simultaneously measuring the size, velocity, and temperature of individual droplets in vaporizing sprays. Preliminary droplet size velocity correlation measurements made with this technique are presented. These measurements are also compared to and show very good agreement with measurements made in the same spray using a phase Doppler particle analyzer.

Two-dimensional imaging of molecular hydrogen in H2-air diffusion flames using two-photon laser-induced fluorescence

NASA Technical Reports Server (NTRS)

Lempert, W.; Kumar, V.; Glesk, I.; Miles, R.; Diskin, G.

1991-01-01

The use of a tunable ArF laser at 193.26 nm to record simultaneous single-laser-shot, planar images of molecular hydrogen and hot oxygen in a turbulent H2-air diffusion flame. Excitation spectra of fuel and oxidant-rich flame zones confirm a partial overlap of the two-photon H2 and single-photon O2 Schumann-Runge absorption bands. UV Rayleigh scattering images of flame structure and estimated detection limits for the H2 two-photon imaging are also presented.

Near-infrared fluorescent probes in cancer imaging and therapy: an emerging field

PubMed Central

Yi, Xiaomin; Wang, Fuli; Qin, Weijun; Yang, Xiaojian; Yuan, Jianlin

2014-01-01

Near-infrared fluorescence (NIRF) imaging is an attractive modality for early cancer detection with high sensitivity and multi-detection capability. Due to convenient modification by conjugating with moieties of interests, NIRF probes are ideal candidates for cancer targeted imaging. Additionally, the combinatory application of NIRF imaging and other imaging modalities that can delineate anatomical structures extends fluorometric determination of biomedical information. Moreover, nanoparticles loaded with NIRF dyes and anticancer agents contribute to the synergistic management of cancer, which integrates the advantage of imaging and therapeutic functions to achieve the ultimate goal of simultaneous diagnosis and treatment. Appropriate probe design with targeting moieties can retain the original properties of NIRF and pharmacokinetics. In recent years, great efforts have been made to develop new NIRF probes with better photostability and strong fluorescence emission, leading to the discovery of numerous novel NIRF probes with fine photophysical properties. Some of these probes exhibit tumoricidal activities upon light radiation, which holds great promise in photothermal therapy, photodynamic therapy, and photoimmunotherapy. This review aims to provide a timely and concise update on emerging NIRF dyes and multifunctional agents. Their potential uses as agents for cancer specific imaging, lymph node mapping, and therapeutics are included. Recent advances of NIRF dyes in clinical use are also summarized. PMID:24648733

3-D photoacoustic and pulse echo imaging of prostate tumor progression in the mouse window chamber

NASA Astrophysics Data System (ADS)

Bauer, Daniel R.; Olafsson, Ragnar; Montilla, Leonardo G.; Witte, Russell S.

2011-02-01

Understanding the tumor microenvironment is critical to characterizing how cancers operate and predicting their response to treatment. We describe a novel, high-resolution coregistered photoacoustic (PA) and pulse echo (PE) ultrasound system used to image the tumor microenvironment. Compared to traditional optical systems, the platform provides complementary contrast and important depth information. Three mice are implanted with a dorsal skin flap window chamber and injected with PC-3 prostate tumor cells transfected with green fluorescent protein. The ensuing tumor invasion is mapped during three weeks or more using simultaneous PA and PE imaging at 25 MHz, combined with optical and fluorescent techniques. Pulse echo imaging provides details of tumor structure and the surrounding environment with 100-μm3 resolution. Tumor size increases dramatically with an average volumetric growth rate of 5.35 mm3/day, correlating well with 2-D fluorescent imaging (R = 0.97, p < 0.01). Photoacoustic imaging is able to track the underlying vascular network and identify hemorrhaging, while PA spectroscopy helps classify blood vessels according to their optical absorption spectrum, suggesting variation in blood oxygen saturation. Photoacoustic and PE imaging are safe, translational modalities that provide enhanced depth resolution and complementary contrast to track the tumor microenvironment, evaluate new cancer therapies, and develop molecular contrast agents in vivo.

Near-infrared fluorescent probes in cancer imaging and therapy: an emerging field.

PubMed

Yi, Xiaomin; Wang, Fuli; Qin, Weijun; Yang, Xiaojian; Yuan, Jianlin

2014-01-01

Near-infrared fluorescence (NIRF) imaging is an attractive modality for early cancer detection with high sensitivity and multi-detection capability. Due to convenient modification by conjugating with moieties of interests, NIRF probes are ideal candidates for cancer targeted imaging. Additionally, the combinatory application of NIRF imaging and other imaging modalities that can delineate anatomical structures extends fluorometric determination of biomedical information. Moreover, nanoparticles loaded with NIRF dyes and anticancer agents contribute to the synergistic management of cancer, which integrates the advantage of imaging and therapeutic functions to achieve the ultimate goal of simultaneous diagnosis and treatment. Appropriate probe design with targeting moieties can retain the original properties of NIRF and pharmacokinetics. In recent years, great efforts have been made to develop new NIRF probes with better photostability and strong fluorescence emission, leading to the discovery of numerous novel NIRF probes with fine photophysical properties. Some of these probes exhibit tumoricidal activities upon light radiation, which holds great promise in photothermal therapy, photodynamic therapy, and photoimmunotherapy. This review aims to provide a timely and concise update on emerging NIRF dyes and multifunctional agents. Their potential uses as agents for cancer specific imaging, lymph node mapping, and therapeutics are included. Recent advances of NIRF dyes in clinical use are also summarized.

Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

PubMed

Cooper, Mark S; Szeto, Daniel P; Sommers-Herivel, Greg; Topczewski, Jacek; Solnica-Krezel, Lila; Kang, Hee-Chol; Johnson, Iain; Kimelman, David

2005-02-01

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Copyright 2004 Wiley-Liss, Inc.

Label-free NIR reflectance imaging as a complimentary tool for two-photon fluorescence microscopy: multimodal investigation of stroke (Conference Presentation)

NASA Astrophysics Data System (ADS)

Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S.

2016-03-01

Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.

Development and Applications of Laminar Optical Tomography for In Vivo Imaging

NASA Astrophysics Data System (ADS)

Burgess, Sean A.

Laminar optical tomography (LOT) is an optical imaging technique capable of making depth-resolved measurements of absorption and fluorescence contrast in scattering tissue. LOT was first demonstrated in 2004 by Hillman et al [1]. The technique combines a non-contact laser scanning geometry, similar to a low magnification confocal microscope, with the imaging principles of diffuse optical tomography (DOT). This thesis describes the development and application of a second generation LOT system, which acquires both fluorescence and multi-wavelength measurements simultaneously and is better suited for in vivo measurements. Chapter 1 begins by reviewing the interactions of light with tissue that form the foundation of optical imaging. A range of related optical imaging techniques and the basic principles of LOT imaging are then described. In Chapter 2, the development of the new LOT imaging system is described including the implementation of a series of interfaces to allow clinical imaging. System performance is then evaluated on a range of imaging phantoms. Chapter 3 describes two in vivo imaging applications explored using the second generation LOT system, first in a clinical setting where skin lesions were imaged, and then in a laboratory setting where LOT imaging was performed on exposed rat cortex. The final chapter provides a brief summary and describes future directions for LOT. LOT has the potential to find applications in medical diagnostics, surgical guidance, and in-situ monitoring owing to its sensitivity to absorption and fluorescence contrast as well as its ability to provide depth sensitive measures. Optical techniques can characterize blood volume and oxygenation, two important biological parameters, through measurements at different wavelengths. Fluorescence measurements, either from autofluorescence or fluorescent dyes, have shown promise for identifying and analyzing lesions in various epithelial tissues including skin [2, 3], colon [4], esophagus [5, 6], oral mucosa [7, 8], and cervix [9]. The desire to capture these types of measurements with LOT motivated much of the work presented here.

Lateral resolution testing of a novel developed confocal microscopic imaging system

NASA Astrophysics Data System (ADS)

Zhang, Xin; Zhang, Yunhai; Chang, Jian; Huang, Wei; Xue, Xiaojun; Xiao, Yun

2015-10-01

Laser scanning confocal microscope has been widely used in biology, medicine and material science owing to its advantages of high resolution and tomographic imaging. Based on a set of confirmatory experiments and system design, a novel confocal microscopic imaging system is developed. The system is composed of a conventional fluorescence microscope and a confocal scanning unit. In the scanning unit a laser beam coupling module provides four different wavelengths 405nm 488nm 561nm and 638nm which can excite a variety of dyes. The system works in spot-to-spot scanning mode with a two-dimensional galvanometer. A 50 microns pinhole is used to guarantee that stray light is blocked and only the fluorescence signal from the focal point can be received . The three-channel spectral splitter is used to perform fluorescence imaging at three different working wavelengths simultaneously. The rat kidney tissue slice is imaged using the developed confocal microscopic imaging system. Nucleues labeled by DAPI and kidney spherule curved pipe labeled by Alexa Fluor 488 can be imaged clearly and respectively, realizing the distinction between the different components of mouse kidney tissue. The three-dimensional tomographic imaging of mouse kidney tissue is reconstructed by several two-dimensional images obtained in different depths. At last the resolution of the confocal microscopic imaging system is tested quantitatively. The experimental result shows that the system can achieve lateral resolution priority to 230nm.

High sensitivity optical molecular imaging system

NASA Astrophysics Data System (ADS)

An, Yu; Yuan, Gao; Huang, Chao; Jiang, Shixin; Zhang, Peng; Wang, Kun; Tian, Jie

2018-02-01

Optical Molecular Imaging (OMI) has the advantages of high sensitivity, low cost and ease of use. By labeling the regions of interest with fluorescent or bioluminescence probes, OMI can noninvasively obtain the distribution of the probes in vivo, which play the key role in cancer research, pharmacokinetics and other biological studies. In preclinical and clinical application, the image depth, resolution and sensitivity are the key factors for researchers to use OMI. In this paper, we report a high sensitivity optical molecular imaging system developed by our group, which can improve the imaging depth in phantom to nearly 5cm, high resolution at 2cm depth, and high image sensitivity. To validate the performance of the system, special designed phantom experiments and weak light detection experiment were implemented. The results shows that cooperated with high performance electron-multiplying charge coupled device (EMCCD) camera, precision design of light path system and high efficient image techniques, our OMI system can simultaneously collect the light-emitted signals generated by fluorescence molecular imaging, bioluminescence imaging, Cherenkov luminance and other optical imaging modality, and observe the internal distribution of light-emitting agents fast and accurately.

NIR-Cyanine Dye Linker: a Promising Candidate for Isochronic Fluorescence Imaging in Molecular Cancer Diagnostics and Therapy Monitoring.

PubMed

Komljenovic, Dorde; Wiessler, Manfred; Waldeck, Waldemar; Ehemann, Volker; Pipkorn, Ruediger; Schrenk, Hans-Hermann; Debus, Jürgen; Braun, Klaus

2016-01-01

Personalized anti-cancer medicine is boosted by the recent development of molecular diagnostics and molecularly targeted drugs requiring rapid and efficient ligation routes. Here, we present a novel approach to synthetize a conjugate able to act simultaneously as an imaging and as a chemotherapeutic agent by coupling functional peptides employing solid phase peptide synthesis technologies. Development and the first synthesis of a fluorescent dye with similarity in the polymethine part of the Cy7 molecule whose indolenine-N residues were substituted with a propylene linker are described. Methylating agent temozolomide is functionalized with a tetrazine as a diene component whereas Cy7-cell penetrating peptide conjugate acts as a dienophilic reaction partner for the inverse Diels-Alder click chemistry-mediated ligation route yielding a theranostic conjugate, 3-mercapto-propionic-cyclohexenyl-Cy7-bis-temozolomide-bromide-cell penetrating peptide. Synthesis route described here may facilitate targeted delivery of the therapeutic compound to achieve sufficient local concentrations at the target site or tissue. Its versatility allows a choice of adequate imaging tags applicable in e.g. PET, SPECT, CT, near-infrared imaging, and therapeutic substances including cytotoxic agents. Imaging tags and therapeutics may be simultaneously bound to the conjugate applying click chemistry. Theranostic compound presented here offers a solid basis for a further improvement of cancer management in a precise, patient-specific manner.

NIR-Cyanine Dye Linker: a Promising Candidate for Isochronic Fluorescence Imaging in Molecular Cancer Diagnostics and Therapy Monitoring

PubMed Central

Komljenovic, Dorde; Wiessler, Manfred; Waldeck, Waldemar; Ehemann, Volker; Pipkorn, Ruediger; Schrenk, Hans-Hermann; Debus, Jürgen; Braun, Klaus

2016-01-01

Personalized anti-cancer medicine is boosted by the recent development of molecular diagnostics and molecularly targeted drugs requiring rapid and efficient ligation routes. Here, we present a novel approach to synthetize a conjugate able to act simultaneously as an imaging and as a chemotherapeutic agent by coupling functional peptides employing solid phase peptide synthesis technologies. Development and the first synthesis of a fluorescent dye with similarity in the polymethine part of the Cy7 molecule whose indolenine-N residues were substituted with a propylene linker are described. Methylating agent temozolomide is functionalized with a tetrazine as a diene component whereas Cy7-cell penetrating peptide conjugate acts as a dienophilic reaction partner for the inverse Diels-Alder click chemistry-mediated ligation route yielding a theranostic conjugate, 3-mercapto-propionic-cyclohexenyl-Cy7-bis-temozolomide-bromide-cell penetrating peptide. Synthesis route described here may facilitate targeted delivery of the therapeutic compound to achieve sufficient local concentrations at the target site or tissue. Its versatility allows a choice of adequate imaging tags applicable in e.g. PET, SPECT, CT, near-infrared imaging, and therapeutic substances including cytotoxic agents. Imaging tags and therapeutics may be simultaneously bound to the conjugate applying click chemistry. Theranostic compound presented here offers a solid basis for a further improvement of cancer management in a precise, patient-specific manner. PMID:26722379

A novel multiwavelength fluorescence image-guided surgery imaging system

NASA Astrophysics Data System (ADS)

Volpi, D.; Tullis, I. D. C.; Laios, A.; Pathiraja, P. N. J.; Haldar, K.; Ahmed, A. A.; Vojnovic, B.

2014-02-01

We describe the development and performance analysis of two clinical near-infrared fluorescence image-guided surgery (FIGS) devices that aim to overcome some of the limitations of current FIGS systems. The devices operate in a widefield-imaging mode and can work (1) in conjunction with a laparoscope, during minimally invasive surgery, and (2) as a hand-held, open surgery imaging system. In both cases, narrow-band excitation light, delivered at multiple wavelengths, is efficiently combined with white reflectance light. Light is delivered to ~100 cm2 surgical field at 1-2 mW/cm2 for white light and 3-7 mW/cm2 (depending on wavelength) of red - near infrared excitation, at a typical working distance of 350 mm for the hand-held device and 100 mm for the laparoscope. A single, sensitive, miniaturized color camera collects both fluorescence and white reflectance light. The use of a single imager eliminates image alignment and software overlay complexity. A novel filtering and illumination arrangement allows simultaneous detection of white reflectance and fluorescence emission from multiple dyes in real-time. We will present both fluorescence detection sensitivity modeling and practical performance data. We have demonstrated the efficiency and the advantages of the devices both pre-clinically and during live surgery on humans. Both the hand-held and the laparoscopic systems have proved to be reliable and beneficial in an ongoing clinical trial involving sentinel lymph node detection in gynecological cancers. We will show preliminary results using two clinically approved dyes, Methylene blue and indocyanine green. We anticipate that this technology can be integrated and routinely used in a larger variety of surgical procedures.

Optically trapped atomic resonant devices for narrow linewidth spectral imaging

NASA Astrophysics Data System (ADS)

Qian, Lipeng

This thesis focuses on the development of atomic resonant devices for spectroscopic applications. The primary emphasis is on the imaging properties of optically thick atomic resonant fluorescent filters and their applications. In addition, this thesis presents a new concept for producing very narrow linewidth light as from an atomic vapor lamp pumped by a nanosecond pulse system. This research was motivated by application for missile warning system, and presents an innovative approach to a wide angle, ultra narrow linewidth imaging filter using a potassium vapor cell. The approach is to image onto and collect the fluorescent photons emitted from the surface of an optically thick potassium vapor cell, generating a 2 GHz pass-band imaging filter. This linewidth is narrow enough to fall within a Fraunhefer dark zone in the solar spectrum, thus make the detection solar blind. Experiments are conducted to measure the absorption line shape of the potassium resonant filter, the quantum efficiency of the fluorescent behavior, and the resolution of the fluorescent image. Fluorescent images with different spatial frequency components are analyzed by using a discrete Fourier transform, and the imaging capability of the fluorescent filter is described by its Modulation Transfer Function. For the detection of radiation that is spectrally broader than the linewidth of the potassium imaging filter, the fluorescent image is seen to be blurred by diffuse fluorescence from the slightly off resonant photons. To correct this, an ultra-thin potassium imaging filter is developed and characterized. The imaging property of the ultra-thin potassium imaging cell is tested with a potassium seeded flame, yielding a resolution image of ˜ 20 lines per mm. The physics behind the atomic resonant fluorescent filter is radiation trapping. The diffusion process of the resonant photons trapped in the atomic vapor is theoretically described in this thesis. A Monte Carlo method is used to simulate the absorption and fluorescence. The optimum resolution of the fluorescent image is predicted by simulation. Radiation trapping is also shown to be useful for the generation of ultra-narrow linewidth light from an atomic vapor flash lamp. A 2 nanosecond, high voltage pulse is used to excite low pressure mercury vapor mixed with noble gases, producing high intensity emission at the mercury resonant line at 253.7 nm. With a nanosecond pumping time and high electrical current, the radiation intensity of the mercury discharge is increased significantly compared to a normal glow discharge lamp, while simultaneously suppressing the formation of an arc discharge. By avoiding the arc discharge, discrete spectral lines of mercury were kept at narrow bandwidth. Due to radiation trapping, the emission linewidth from the nanosecond mercury lamp decreases with time and produces ultra-narrow linewidth emission 100 ns after of the excitation, this linewidth is verified by absorption measurements through low pressure mercury absorption filter. The lamp is used along with mercury absorption filters for spectroscopic applications, including Filtered Rayleigh Scattering with different CO2 pressures and Raman scattering from methanol.

Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells.

PubMed

Norcross, Stevie; Trull, Keelan J; Snaider, Jordan; Doan, Sara; Tat, Kiet; Huang, Libai; Tantama, Mathew

2017-11-22

Reactive oxygen species (ROS) mediate both intercellular and intraorganellar signaling, and ROS propagate oxidative stress between cellular compartments such as mitochondria and the cytosol. Each cellular compartment contains its own sources of ROS as well as antioxidant mechanisms, which contribute to dynamic fluctuations in ROS levels that occur during signaling, metabolism, and stress. However, the coupling of redox dynamics between cellular compartments has not been well studied because of the lack of available sensors to simultaneously measure more than one subcellular compartment in the same cell. Currently, the redox-sensitive green fluorescent protein, roGFP, has been used extensively to study compartment-specific redox dynamics because it provides a quantitative ratiometric readout and it is amenable to subcellular targeting as a genetically encoded sensor. Here, we report a new family of genetically encoded fluorescent protein sensors that extend the fluorescence emission of roGFP via Förster-type resonance energy transfer to an acceptor red fluorescent protein for dual-color live-cell microscopy. We characterize the redox and optical properties of the sensor proteins, and we demonstrate that they can be used to simultaneously measure cytosolic and mitochondrial ROS in living cells. Furthermore, we use these sensors to reveal cell-to-cell heterogeneity in redox coupling between the cytosol and mitochondria when neuroblastoma cells are exposed to reductive and metabolic stresses.

Indocyanine green fluorescence in second near-infrared (NIR-II) window

PubMed Central

Bhavane, Rohan; Ghaghada, Ketan B.; Vasudevan, Sanjeev A.; Kaay, Alexander; Annapragada, Ananth

2017-01-01

Indocyanine green (ICG), a FDA approved near infrared (NIR) fluorescent agent, is used in the clinic for a variety of applications including lymphangiography, intra-operative lymph node identification, tumor imaging, superficial vascular imaging, and marking ischemic tissues. These applications operate in the so-called “NIR-I” window (700–900 nm). Recently, imaging in the “NIR-II” window (1000–1700 nm) has attracted attention since, at longer wavelengths, photon absorption, and scattering effects by tissue components are reduced, making it possible to image deeper into the underlying tissue. Agents for NIR-II imaging are, however, still in pre-clinical development. In this study, we investigated ICG as a NIR-II dye. The absorbance and NIR-II fluorescence emission of ICG were measured in different media (PBS, plasma and ethanol) for a range of ICG concentrations. In vitro and in vivo testing were performed using a custom-built spectral NIR assembly to facilitate simultaneous imaging in NIR-I and NIR-II window. In vitro studies using ICG were performed using capillary tubes (as a simulation of blood vessels) embedded in Intralipid solution and tissue phantoms to evaluate depth of tissue penetration in NIR-I and NIR-II window. In vivo imaging using ICG was performed in nude mice to evaluate vascular visualization in the hind limb in the NIR-I and II windows. Contrast-to-noise ratios (CNR) were calculated for comparison of image quality in NIR-I and NIR-II window. ICG exhibited significant fluorescence emission in the NIR-II window and this emission (similar to the absorption profile) is substantially affected by the environment of the ICG molecules. In vivo imaging further confirmed the utility of ICG as a fluorescent dye in the NIR-II domain, with the CNR values being ~2 times those in the NIR-I window. The availability of an FDA approved imaging agent could accelerate the clinical translation of NIR-II imaging technology. PMID:29121078

Differential phase contrast with a segmented detector in a scanning X-ray microprobe

PubMed Central

Hornberger, B.; de Jonge, M. D.; Feser, M.; Holl, P.; Holzner, C.; Jacobsen, C.; Legnini, D.; Paterson, D.; Rehak, P.; Strüder, L.; Vogt, S.

2008-01-01

Scanning X-ray microprobes are unique tools for the nanoscale investigation of specimens from the life, environmental, materials and other fields of sciences. Typically they utilize absorption and fluorescence as contrast mechanisms. Phase contrast is a complementary technique that can provide strong contrast with reduced radiation dose for weakly absorbing structures in the multi-keV range. In this paper the development of a segmented charge-integrating silicon detector which provides simultaneous absorption and differential phase contrast is reported. The detector can be used together with a fluorescence detector for the simultaneous acquisition of transmission and fluorescence data. It can be used over a wide range of photon energies, photon rates and exposure times at third-generation synchrotron radiation sources, and is currently operating at two beamlines at the Advanced Photon Source. Images obtained at around 2 keV and 10 keV demonstrate the superiority of phase contrast over absorption for specimens composed of light elements. PMID:18552427

In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

PubMed

Christiansen, Eric M; Yang, Samuel J; Ando, D Michael; Javaherian, Ashkan; Skibinski, Gaia; Lipnick, Scott; Mount, Elliot; O'Neil, Alison; Shah, Kevan; Lee, Alicia K; Goyal, Piyush; Fedus, William; Poplin, Ryan; Esteva, Andre; Berndl, Marc; Rubin, Lee L; Nelson, Philip; Finkbeiner, Steven

2018-04-19

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire. Copyright © 2018 Elsevier Inc. All rights reserved.

Modification of measurement methods for evaluation of tissue-engineered cartilage function and biochemical properties using nanosecond pulsed laser

NASA Astrophysics Data System (ADS)

Ishihara, Miya; Sato, Masato; Kutsuna, Toshiharu; Ishihara, Masayuki; Mochida, Joji; Kikuchi, Makoto

2008-02-01

There is a demand in the field of regenerative medicine for measurement technology that enables determination of functions and components of engineered tissue. To meet this demand, we developed a method for extracellular matrix characterization using time-resolved autofluorescence spectroscopy, which enabled simultaneous measurements with mechanical properties using relaxation of laser-induced stress wave. In this study, in addition to time-resolved fluorescent spectroscopy, hyperspectral sensor, which enables to capture both spectral and spatial information, was used for evaluation of biochemical characterization of tissue-engineered cartilage. Hyperspectral imaging system provides spectral resolution of 1.2 nm and image rate of 100 images/sec. The imaging system consisted of the hyperspectral sensor, a scanner for x-y plane imaging, magnifying optics and Xenon lamp for transmmissive lighting. Cellular imaging using the hyperspectral image system has been achieved by improvement in spatial resolution up to 9 micrometer. The spectroscopic cellular imaging could be observed using cultured chondrocytes as sample. At early stage of culture, the hyperspectral imaging offered information about cellular function associated with endogeneous fluorescent biomolecules.

Combined planar imaging of schlieren photography with OH-LIPF and spontaneous OH-emission in a 2-D valveless pulse combustor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishino, Yojiro; Hasegawa, Tatsuya; Yamaguchi, Shigeki

1999-07-01

Using a novel optical system, simultaneous imaging of schlieren photography and laser induced predissociation fluorescence of OH radicals (OH-LIPF) have been carried out to examine combustion processes and flame structure in a two-dimensional valveless pulse combustor. Simultaneous imaging of schlieren photographs and spontaneous OH-emission have also been made, in order to obtain information on the behavior of the flame front during a cycle of pulsation. The pulse combustor used in this experiment consists of a combustion chamber of a volume of 125 cm{sup 3} and a tailpipe of a length of 976 mm, which is followed by an automobile muffler.more » The fuel used is commercial grade gaseous propane.« less

3D visualization of additive occlusion and tunable full-spectrum fluorescence in calcite

PubMed Central

Green, David C.; Ihli, Johannes; Thornton, Paul D.; Holden, Mark A.; Marzec, Bartosz; Kim, Yi-Yeoun; Kulak, Alex N.; Levenstein, Mark A.; Tang, Chiu; Lynch, Christophe; Webb, Stephen E. D.; Tynan, Christopher J.; Meldrum, Fiona C.

2016-01-01

From biomineralization to synthesis, organic additives provide an effective means of controlling crystallization processes. There is growing evidence that these additives are often occluded within the crystal lattice. This promises an elegant means of creating nanocomposites and tuning physical properties. Here we use the incorporation of sulfonated fluorescent dyes to gain new understanding of additive occlusion in calcite (CaCO3), and to link morphological changes to occlusion mechanisms. We demonstrate that these additives are incorporated within specific zones, as defined by the growth conditions, and show how occlusion can govern changes in crystal shape. Fluorescence spectroscopy and lifetime imaging microscopy also show that the dyes experience unique local environments within different zones. Our strategy is then extended to simultaneously incorporate mixtures of dyes, whose fluorescence cascade creates calcite nanoparticles that fluoresce white. This offers a simple strategy for generating biocompatible and stable fluorescent nanoparticles whose output can be tuned as required. PMID:27857076

Magneto-Fluorescent Core-Shell Supernanoparticles

PubMed Central

Chen, Ou; Riedemann, Lars; Etoc, Fred; Herrmann, Hendrik; Coppey, Mathieu; Barch, Mariya; Farrar, Christian T.; Zhao, Jing; Bruns, Oliver T.; Wei, He; Guo, Peng; Cui, Jian; Jensen, Russ; Chen, Yue; Harris, Daniel K.; Cordero, Jose M.; Wang, Zhongwu; Jasanoff, Alan; Fukumura, Dai; Reimer, Rudolph; Dahan, Maxime; Jain, Rakesh K.; Bawendi, Moungi G.

2014-01-01

Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magneto-fluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface, and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close packed magnetic nanoparticle “core” which is fully surrounded by a “shell” of fluorescent quantum dots. A thin layer of silica-coating provides high colloidal stability and biocompatiblity and a versatile surface functionality. We demonstrate that after surface pegylation, these silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe. PMID:25298155

Imaging of Neuronal Activity in Awake Mice by Measurements of Flavoprotein Autofluorescence Corrected for Cerebral Blood Flow.

PubMed

Takahashi, Manami; Urushihata, Takuya; Takuwa, Hiroyuki; Sakata, Kazumi; Takado, Yuhei; Shimizu, Eiji; Suhara, Tetsuya; Higuchi, Makoto; Ito, Hiroshi

2017-01-01

Green fluorescence imaging (e.g., flavoprotein autofluorescence imaging, FAI) can be used to measure neuronal activity and oxygen metabolism in living brains without expressing fluorescence proteins. It is useful for understanding the mechanism of various brain functions and their abnormalities in age-related brain diseases. However, hemoglobin in cerebral blood vessels absorbs green fluorescence, hampering accurate assessments of brain function in animal models with cerebral blood vessel dysfunctions and subsequent cerebral blood flow (CBF) alterations. In the present study, we developed a new method to correct FAI signals for hemoglobin-dependent green fluorescence reductions by simultaneous measurements of green fluorescence and intrinsic optical signals. Intrinsic optical imaging enabled evaluations of light absorption and scatters by hemoglobin, which could then be applied to corrections of green fluorescence intensities. Using this method, enhanced flavoprotein autofluorescence by sensory stimuli was successfully detected in the brains of awake mice, despite increases of CBF, and hemoglobin interference. Moreover, flavoprotein autofluorescence could be properly quantified in a resting state and during sensory stimulation by a CO 2 inhalation challenge, which modified vascular responses without overtly affecting neuronal activities. The flavoprotein autofluorescence signal data obtained here were in good agreement with the previous findings from a condition with drug-induced blockade of cerebral vasodilation, justifying the current assaying methodology. Application of this technology to studies on animal models of brain diseases with possible changes of CBF, including age-related neurological disorders, would provide better understanding of the mechanisms of neurovascular coupling in pathological circumstances.

Novel techniques with multiphoton microscopy: Deep-brain imaging with microprisms, neurometabolism of epilepsy, and counterfeit paper money detection

NASA Astrophysics Data System (ADS)

Chia, Thomas H.

Multiphoton microscopy is a laser-scanning fluorescence imaging method with extraordinary potential. We describe three innovative multiphoton microscopy techniques across various disciplines. Traditional in vivo fluorescence microscopy of the mammalian brain has a limited penetration depth (<400 microm). We present a method of imaging 1 mm deep into mouse neocortex by using a glass microprism to relay the excitation and emission light. This technique enables simultaneous imaging of multiple cortical layers, including layer V, at an angle typical of slice preparations. At high-magnification imaging using an objective with 1-mm of coverglass correction, resolution was sufficient to resolve dendritic spines on layer V GFP neurons. Functional imaging of blood flow at various neocortical depths is also presented, allowing for quantification of red blood cell flux and velocity. Multiphoton fluorescence lifetime imaging (FLIM) of NADH reveals information on neurometabolism. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic coenzyme, has a lifetime dependent on enzymatic binding. A novel NADH FLIM algorithm is presented that produces images showing spatially distinct NADH fluorescence lifetimes in mammalian brain slices. This program provides advantages over traditional FLIM processing of multi-component lifetime data. We applied this technique to a GFP-GFAP pilocarpine mouse model of temporal lobe epilepsy. Results indicated significant changes in the neurometabolism of astrocytes and neuropil in the cell and dendritic layers of the hippocampus when compared to control tissue. Data obtained with NADH FLIM were subsequently interpreted based on the abnormal activity reported in epileptic tissue. Genuine U.S. Federal Reserve Notes have a consistent, two-component intrinsic fluorescence lifetime. This allows for detection of counterfeit paper money because of its significant differences in fluorescence lifetime when compared to genuine paper money. We used scanning multiphoton laser excitation to sample a ˜4 mm2 region from 54 genuine Reserve Notes. Three types of counterfeit samples were tested. Four out of the nine counterfeit samples fit to a one-component decay. Five out of nine counterfeit samples fit to a two-component model, but are identified as counterfeit due to significant deviations in the longer lifetime component compared to genuine bills.

Multimodal molecular 3D imaging for the tumoral volumetric distribution assessment of folate-based biosensors.

PubMed

Ramírez-Nava, Gerardo J; Santos-Cuevas, Clara L; Chairez, Isaac; Aranda-Lara, Liliana

2017-12-01

The aim of this study was to characterize the in vivo volumetric distribution of three folate-based biosensors by different imaging modalities (X-ray, fluorescence, Cerenkov luminescence, and radioisotopic imaging) through the development of a tridimensional image reconstruction algorithm. The preclinical and multimodal Xtreme imaging system, with a Multimodal Animal Rotation System (MARS), was used to acquire bidimensional images, which were processed to obtain the tridimensional reconstruction. Images of mice at different times (biosensor distribution) were simultaneously obtained from the four imaging modalities. The filtered back projection and inverse Radon transformation were used as main image-processing techniques. The algorithm developed in Matlab was able to calculate the volumetric profiles of 99m Tc-Folate-Bombesin (radioisotopic image), 177 Lu-Folate-Bombesin (Cerenkov image), and FolateRSense™ 680 (fluorescence image) in tumors and kidneys of mice, and no significant differences were detected in the volumetric quantifications among measurement techniques. The imaging tridimensional reconstruction algorithm can be easily extrapolated to different 2D acquisition-type images. This characteristic flexibility of the algorithm developed in this study is a remarkable advantage in comparison to similar reconstruction methods.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manohar, N; Cho, S; Reynoso, F

Purpose: To make benchtop x-ray fluorescence computed tomography (XFCT) practical for routine preclinical imaging tasks with gold nanoparticles (GNPs) by deploying, integrating, and characterizing a dedicated high-performance x-ray source and addition of simultaneous micro-CT functionality. Methods: Considerable research effort is currently under way to develop a polychromatic benchtop cone-beam XFCT system capable of imaging GNPs by stimulation and detection of gold K-shell x-ray fluorescence (XRF) photons. Recently, an ad hoc high-power x-ray source was incorporated and used to image the biodistribution of GNPs within a mouse, postmortem. In the current work, a dedicated x-ray source system featuring a liquid-cooled tungsten-targetmore » x-ray tube (max 160 kVp, ∼3 kW power) was deployed. The source was operated at 125 kVp, 24 mA. The tube’s compact dimensions allowed greater flexibility for optimizing both the irradiation and detection geometries. Incident x-rays were shaped by a conical collimator and filtered by 2 mm of tin. A compact “OEM” cadmium-telluride x-ray detector was implemented for detecting XRF/scatter spectra. Additionally, a flat panel detector was installed to allow simultaneous transmission CT imaging. The performance of the system was characterized by determining the detection limit (10-second acquisition time) for inserts filled with water/GNPs at various concentrations (0 and 0.010–1.0 wt%) and embedded in a small-animal-sized phantom. The phantom was loaded with 0.5, 0.3, and 0.1 wt% inserts and imaged using XFCT and simultaneous micro-CT. Results: An unprecedented detection limit of 0.030 wt% was experimentally demonstrated, with a 33% reduction in acquisition time. The reconstructed XFCT image accurately localized the imaging inserts. Micro-CT imaging did not provide enough contrast to distinguish imaging inserts from the phantom under the current conditions. Conclusion: The system is immediately capable of in vivo preclinical XFCT imaging with GNPs. Micro-CT imaging will require optimization of irradiation parameters to improve contrast. Supported by NIH/NCI grant R01CA155446; This investigation was supported by NIH/NCI grant R01CA155446.« less

Spectral unmixing of multi-color tissue specific in vivo fluorescence in mice

NASA Astrophysics Data System (ADS)

Zacharakis, Giannis; Favicchio, Rosy; Garofalakis, Anikitos; Psycharakis, Stylianos; Mamalaki, Clio; Ripoll, Jorge

2007-07-01

Fluorescence Molecular Tomography (FMT) has emerged as a powerful tool for monitoring biological functions in vivo in small animals. It provides the means to determine volumetric images of fluorescent protein concentration by applying the principles of diffuse optical tomography. Using different probes tagged to different proteins or cells, different biological functions and pathways can be simultaneously imaged in the same subject. In this work we present a spectral unmixing algorithm capable of separating signal from different probes when combined with the tomographic imaging modality. We show results of two-color imaging when the algorithm is applied to separate fluorescence activity originating from phantoms containing two different fluorophores, namely CFSE and SNARF, with well separated emission spectra, as well as Dsred- and GFP-fused cells in F5-b10 transgenic mice in vivo. The same algorithm can furthermore be applied to tissue-specific spectroscopy data. Spectral analysis of a variety of organs from control, DsRed and GFP F5/B10 transgenic mice showed that fluorophore detection by optical systems is highly tissue-dependent. Spectral data collected from different organs can provide useful insight into experimental parameter optimisation (choice of filters, fluorophores, excitation wavelengths) and spectral unmixing can be applied to measure the tissue-dependency, thereby taking into account localized fluorophore efficiency. Summed up, tissue spectral unmixing can be used as criteria in choosing the most appropriate tissue targets as well as fluorescent markers for specific applications.

Fluorescence-based surface magnifying chromoendoscopy and optical coherence tomography endoscope

NASA Astrophysics Data System (ADS)

Wall, R. Andrew; Barton, Jennifer K.

2012-02-01

A side-viewing, 2 mm diameter, surface magnifying chromoendoscopy (SMC)-optical coherence tomography (OCT) endoscope has been designed for simultaneous, non-destructive surface fluorescence visualization and cross-sectional imaging. We apply this endoscope to in vivo examination of mouse colon. A 30,000 element fiber bundle is combined with single mode fibers. The distal optics consist of a gradient-index lens and spacer to provide a magnification of 1 at a working distance of 1.58 mm in air, necessary to image the sample through a 0.23 mm thick outer glass envelope, and an aluminized right-angle prism fixed to the distal end of the GRIN lens assembly. The resulting 1:1 imaging system is capable of 3.9 μm lateral and 2.3 μm axial resolution in the OCT channel, and 125 lp/mm resolution across a 0.70 mm field of view in the SMC channel. The endoscope can perform high contrast crypt visualization, molecular imaging, and cross-sectional imaging of colon microstructure.

ICG-enhanced imaging of arthritis with an integrated Optical Imaging/X-ray System

PubMed Central

Meier, Reinhard; Krug, Christian; Golovko, Daniel; Boddington, Sophie; Piontek, Guido; Rudelius, Martina; Sutton, Elizabeth J.; Baur-Melnyk, Andrea; Jones, Ella F.; Daldrup-Link, Heike E.

2010-01-01

Background Optical Imaging (OI) is a promising technique that is quick, inexpensive and, in combination with Indocyanine Green (ICG), an FDA-approved fluorescent dye, could provide early detection of rheumatoid arthritis. Objective The purpose of this study was to evaluate a combined X-ray/OI imaging system for ICG-enhanced detection of arthritic joints in a rat model of antigen induced arthritis. Methods Arthritis of the knee and ankle joints was induced in six Harlan rats with peptidoglycan polysaccharide polymers (PGPS). Three rats served as non-treated controls. Optical imaging of the knee and ankle joints was done with an integrated OI/X-ray system before and up to 24h post intravenous injection (p.i.) of 10mg/kg ICG. The fluorescence signal intensities of arthritic and normal joints were compared for significant differences using generalized estimation equation models. Specimen of knee and ankle joints were further processed and evaluated by histology. Results ICG provided a significant increase in fluorescence signal of arthritic joints compared to baseline values immediately after administration (p<0.05). The fluorescence signal of arthritic joints was significantly higher compared to the non-arthritic control joints at 1 - 720 min p.i. (p<0.05). Fusion of ICG-enhanced OI and X-rays allowed for anatomical co-registration of the inflamed tissue with the associated joint. H&E stains confirmed marked synovial inflammation of arthritic joints and absence of inflammation in control joints. Conclusion ICG-enhanced OI is a clinically applicable tool for detection of arthritic tissue. Using relatively high doses of ICG, a long term fluorescence enhancement of arthritic joints can be achieved. This may facilitate simultaneous evaluations of multiple joints in a clinical setting. Fusion of ICG-OI scans with X-ray imaging increases anatomical resolution. PMID:20506388

Quantitative Cell Cycle Analysis Based on an Endogenous All-in-One Reporter for Cell Tracking and Classification.

PubMed

Zerjatke, Thomas; Gak, Igor A; Kirova, Dilyana; Fuhrmann, Markus; Daniel, Katrin; Gonciarz, Magdalena; Müller, Doris; Glauche, Ingmar; Mansfeld, Jörg

2017-05-30

Cell cycle kinetics are crucial to cell fate decisions. Although live imaging has provided extensive insights into this relationship at the single-cell level, the limited number of fluorescent markers that can be used in a single experiment has hindered efforts to link the dynamics of individual proteins responsible for decision making directly to cell cycle progression. Here, we present fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA) as an all-in-one cell cycle reporter that allows simultaneous analysis of cell cycle progression, including the transition into quiescence, and the dynamics of individual fate determinants. We also provide an image analysis pipeline for automated segmentation, tracking, and classification of all cell cycle phases. Combining the all-in-one reporter with labeled endogenous cyclin D1 and p21 as prime examples of cell-cycle-regulated fate determinants, we show how cell cycle and quantitative protein dynamics can be simultaneously extracted to gain insights into G1 phase regulation and responses to perturbations. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

PubMed

Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

2017-08-01

Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes

PubMed Central

Sun, Minjie; Sun, Bin; Liu, Yun; Shen, Qun-Dong; Jiang, Shaojun

2016-01-01

Rapid growth in biological applications of nanomaterials brings about pressing needs for exploring nanomaterial-cell interactions. Cationic blue-emissive and anionic green-emissive conjugated polymers are applied as dual-color fluorescence probes to the surface of negatively charged magnetic nanoparticles through sequentially electrostatic adsorption. These conjugated polymers have large extinction coefficients and high fluorescence quantum yield (82% for PFN and 62% for ThPFS). Thereby, one can visualize trace amount (2.7 μg/mL) of fluorescence-labeled nanoparticles within cancer cells by confocal laser scanning microscopy. Fluorescence labeling by the conjugated polymers is also validated for quantitative determination of the internalized nanoparticles in each individual cell by flow cytometry analysis. Extensive overlap of blue and green fluorescence signals in the cytoplasm indicates that both conjugated polymer probes tightly bind to the surface of the nanoparticles during cellular internalization. The highly charged and fluorescence-labeled nanoparticles non-specifically bind to the cell membranes, followed by cellular uptake through endocytosis. The nanoparticles form aggregates inside endosomes, which yields a punctuated staining pattern. Cellular internalization of the nanoparticles is dependent on the dosage and time. Uptake efficiency can be enhanced three-fold by application of an external magnetic field. The nanoparticles are low cytotoxicity and suitable for simultaneously noninvasive fluorescence and magnetic resonance imaging application. PMID:26931282

Multiple excitation nano-spot generation and confocal detection for far-field microscopy.

PubMed

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy

NASA Astrophysics Data System (ADS)

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Simultaneous Visualization of Hydrogen Peroxide and Water Concentrations Using Photofragmentation Laser-Induced Fluorescence.

PubMed

Larsson, Kajsa; Aldén, Marcus; Bood, Joakim

2017-09-01

A concept based on photofragmentation laser-induced fluorescence (PFLIF) is for the first time demonstrated for simultaneous detection of hydrogen peroxide (H 2 O 2 ) and water (H 2 O) vapor in various mixtures containing the two constituents in a bath of argon gas. A photolysis laser pulse at 248 nm dissociates H 2 O 2 into OH fragments, whereupon a probe pulse, delayed 100 ns and tuned to an absorption line in the A 2 Σ + (v = 1) ← X 2 Π(v = 0) band of OH near 282 nm, induces fluorescence. The total OH fluorescence reflects the H 2 O 2 concentration, while its spectral shape is utilized to determine the H 2 O concentration via a model predicting the ratio between the fluorescence intensities of the A 2 Σ + (v = 1) → X 2 Π(v = 1) and the A 2 Σ + (v = 0) → X 2 Π(v = 0) bands. The H 2 O detection scheme requires that the bath gas has a collisional cross-section with OH(A) that is significantly lower than that of H 2 O, which is the case for argon. Spectrally dispersed OH fluorescence spectra were recorded for five different H 2 O 2 /H 2 O/Ar mixtures; the H 2 O 2 concentration in the range of 30-500 ppm and the H 2 O concentration in the range of 0-3%. Fluorescence intensity ratios predicted by the model for these mixtures agree very well with corresponding experimental data, which thus validates the model. The concept was also demonstrated for two-dimensional imaging, using two intensified charge-coupled device (CCD) cameras for signal detection. Water content was here sensed through the different temporal characteristics of the two fluorescence bands by triggering the two cameras so that one captures the total OH fluorescence while the other one captures only the early part, which mainly stems from A 2 Σ + (v = 1) → X 2 Π(v = 1) fluorescence. Hence, the H 2 O 2 concentration is reflected by the image of the camera recording the total OH fluorescence, whereas H 2 O concentration is extracted from the ratio between the two camera images. Quantification of the concentrations was carried out based on calibration measurements performed in known mixtures of H 2 O 2 (30-500 ppm) and H 2 O (0-3%) in bulk argon. The detection limits for single-shot imaging are estimated to be 20 ppm for H 2 O 2 and 0.05% for H 2 O. The authors believe that the concept provides a valuable asset in, for example, pharmaceutical or aseptic food packaging applications, where H 2 O 2 /H 2 O vapor is routinely used for sterilization.

Fully integrated optical coherence tomography, ultrasound, and indocyanine green based fluorescence tri-modality system for intravascular imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Li, Yan; Jing, Joseph C.; Qu, Yueqiao; Miao, Yusi; Ma, Teng; Yu, Mingyue; Zhou, Qifa; Chen, Zhongping

2017-02-01

The rupture of atherosclerotic plaques is the leading cause of acute coronary events, so accurate assessment of plaque is critical. A large lipid pool, thin fibrous cap, and inflammatory reaction are the crucial characteristics for identifying vulnerable plaques. In our study, a tri-modality imaging system for intravascular imaging was designed and implemented. The tri-modality imaging system with a 1-mm probe diameter is able to simultaneously acquire optical coherence tomography (OCT), intravascular ultrasound (IVUS), and fluorescence imaging. Moreover, for fluorescence imaging, we used the FDA-approved indocyanine green (ICG) dye as the contrast agent to target lipid-loaded macrophages. Firstly, IVUS is used as the first step for identifying plaque since IVUS enables the visualization of the layered structures of the artery wall. Due to low soft-tissue contrast, IVUS only provides initial identification of the lipid plaque. Then OCT is used for differentiating fibrosis and lipid pool based on its relatively higher soft tissue contrast and high sensitivity/specificity. Last, fluorescence imaging is used for identifying inflammatory reaction to further confirm whether the plaque is vulnerable or not. Ex vivo experiment of a male New Zealand white rabbit aorta was performed to validate the performance of our tri-modality system. H and E histology results of the rabbit aorta were also presented to check assessment accuracy. The miniature tri-modality probe, together with the use of ICG dye suggest that the system is of great potential for providing a more accurate assessment of vulnerable plaques in clinical applications.

Review of fluorescence guided surgery systems: identification of key performance capabilities beyond indocyanine green imaging

PubMed Central

DSouza, Alisha V.; Lin, Huiyun; Henderson, Eric R.; Samkoe, Kimberley S.; Pogue, Brian W.

2016-01-01

Abstract. There is growing interest in using fluorescence imaging instruments to guide surgery, and the leading options for open-field imaging are reviewed here. While the clinical fluorescence-guided surgery (FGS) field has been focused predominantly on indocyanine green (ICG) imaging, there is accelerated development of more specific molecular tracers. These agents should help advance new indications for which FGS presents a paradigm shift in how molecular information is provided for resection decisions. There has been a steady growth in commercially marketed FGS systems, each with their own differentiated performance characteristics and specifications. A set of desirable criteria is presented to guide the evaluation of instruments, including: (i) real-time overlay of white-light and fluorescence images, (ii) operation within ambient room lighting, (iii) nanomolar-level sensitivity, (iv) quantitative capabilities, (v) simultaneous multiple fluorophore imaging, and (vi) ergonomic utility for open surgery. In this review, United States Food and Drug Administration 510(k) cleared commercial systems and some leading premarket FGS research systems were evaluated to illustrate the continual increase in this performance feature base. Generally, the systems designed for ICG-only imaging have sufficient sensitivity to ICG, but a fraction of the other desired features listed above, with both lower sensitivity and dynamic range. In comparison, the emerging research systems targeted for use with molecular agents have unique capabilities that will be essential for successful clinical imaging studies with low-concentration agents or where superior rejection of ambient light is needed. There is no perfect imaging system, but the feature differences among them are important differentiators in their utility, as outlined in the data and tables here. PMID:27533438

Review of fluorescence guided surgery systems: identification of key performance capabilities beyond indocyanine green imaging

NASA Astrophysics Data System (ADS)

DSouza, Alisha V.; Lin, Huiyun; Henderson, Eric R.; Samkoe, Kimberley S.; Pogue, Brian W.

2016-08-01

There is growing interest in using fluorescence imaging instruments to guide surgery, and the leading options for open-field imaging are reviewed here. While the clinical fluorescence-guided surgery (FGS) field has been focused predominantly on indocyanine green (ICG) imaging, there is accelerated development of more specific molecular tracers. These agents should help advance new indications for which FGS presents a paradigm shift in how molecular information is provided for resection decisions. There has been a steady growth in commercially marketed FGS systems, each with their own differentiated performance characteristics and specifications. A set of desirable criteria is presented to guide the evaluation of instruments, including: (i) real-time overlay of white-light and fluorescence images, (ii) operation within ambient room lighting, (iii) nanomolar-level sensitivity, (iv) quantitative capabilities, (v) simultaneous multiple fluorophore imaging, and (vi) ergonomic utility for open surgery. In this review, United States Food and Drug Administration 510(k) cleared commercial systems and some leading premarket FGS research systems were evaluated to illustrate the continual increase in this performance feature base. Generally, the systems designed for ICG-only imaging have sufficient sensitivity to ICG, but a fraction of the other desired features listed above, with both lower sensitivity and dynamic range. In comparison, the emerging research systems targeted for use with molecular agents have unique capabilities that will be essential for successful clinical imaging studies with low-concentration agents or where superior rejection of ambient light is needed. There is no perfect imaging system, but the feature differences among them are important differentiators in their utility, as outlined in the data and tables here.

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

PubMed

Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H G; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

2016-06-02

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

Molecular profiling of single cancer cells and clinical tissue specimens with semiconductor quantum dots

PubMed Central

Xing, Yun; Smith, Andrew M; Agrawal, Amit; Ruan, Gang; Nie, Shuming

2006-01-01

Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens. PMID:17722280

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

PubMed Central

Malide, Daniela; Métais, Jean-Yves; Dunbar, Cynthia E.

2014-01-01

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner. PMID:25145579

In vivo cell tracking and quantification method in adult zebrafish

NASA Astrophysics Data System (ADS)

Zhang, Li; Alt, Clemens; Li, Pulin; White, Richard M.; Zon, Leonard I.; Wei, Xunbin; Lin, Charles P.

2012-03-01

Zebrafish have become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provide unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems. In this paper we introduce an optical method for simultaneous visualization and cell quantification, which combines the laser scanning confocal microscopy (LSCM) and the in vivo flow cytometry (IVFC). The system is designed specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish casper, under physiological conditions in the same fish over time. The confocal imaging part in this system serves the dual purposes of imaging fish tissue microstructure and a 3D navigation tool to locate a suitable vessel for circulating cell counting. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously. We demonstrate initial testing of this novel instrument by imaging vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed transgenic casper fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. Circulating fluorescent cell incidents were recorded and counted repeatedly over time and in different types of vessels. Great application opportunities in cancer and stem cell researches are discussed.

Automatic neutron dosimetry system based on fluorescent nuclear track detector technology.

PubMed

Akselrod, M S; Fomenko, V V; Bartz, J A; Haslett, T L

2014-10-01

For the first time, the authors are describing an automatic fluorescent nuclear track detector (FNTD) reader for neutron dosimetry. FNTD is a luminescent integrating type of detector made of aluminium oxide crystals that does not require electronics or batteries during irradiation. Non-destructive optical readout of the detector is performed using a confocal laser scanning fluorescence imaging with near-diffraction limited resolution. The fully automatic table-top reader allows one to load up to 216 detectors on a tray, read their engraved IDs using a CCD camera and optical character recognition, scan and process simultaneously two types of images in fluorescent and reflected laser light contrast to eliminate false-positive tracks related to surface and volume crystal imperfections. The FNTD dosimetry system allows one to measure neutron doses from 0.1 mSv to 20 Sv and covers neutron energies from thermal to 20 MeV. The reader is characterised by a robust, compact optical design, fast data processing electronics and user-friendly software. © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy.

PubMed

Dardikman, Gili; Nygate, Yoav N; Barnea, Itay; Turko, Nir A; Singh, Gyanendra; Javidi, Barham; Shaked, Natan T

2018-03-01

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.

In vivo imaging of the dynamics of different variants of EGFR in glioblastomas.

PubMed

Shah, Khalid

2011-01-01

A number of altered pathways in cancer cells depend on growth factor receptors. The amplification/alteration of the epidermal growth factor receptor (EGFR) has been shown to play a significant role in enhancing tumor burden in a number of tumors, including malignant glioblastomas (GBM). To dissect the role of EGFR expression in tumor progression in mouse models of cancer and ultimately evaluate targeted therapies, it is necessary to visualize the dynamics of EGFR in real time in vivo. Non-invasive imaging based on quantitative and qualitative changes in light emission by fluorescent and bioluminescent markers offers a huge potential to facilitate drug development. Multiple approaches could be used to follow a molecular target or pathway with the fusion of a bioluminescent-fluorescent marker. This unit describes a protocol for simultaneously imaging EGFR activity and progression of GBM in a mouse model. Human glioma cells transduced with lentiviral vectors bearing different combinations of fluorescent and bioluminescent proteins either fused to EGFR or expressed alone can be grown as monolayers and maintained over several passages. The unit begins with a method for transducing glioma cells with lentiviral vectors for stable expression of these fluorescent and bioluminescent markers in vitro, followed by transplantation of engineered glioma cells in mice, and, finally, sequential bioluminescent imaging of EGFR expression and GBM progression in mice. The protocol details characterization of engineered glioma cells in culture, surgical preparation, craniotomy, cell implantation, animal recovery, and imaging procedures to study kinetics of EGFR expression and GBM progression.

Simultaneous imaging of temporal changes of NF-κB activity and viable tumor cells in Huh7/NF-κB-tk-luc2/rfp tumor-bearing mice.

PubMed

Wang, Wei-Hsun; Chiang, I-Tsang; Liu, Yu-Chang; Hsu, Fei-Ting; Chen, Hong-Wen; Chen, Chuan-Lin; Lee, Yi-Jang; Lin, Wuu-Jyh; Hwang, Jeng-Jong

2013-01-01

Few studies have reported that the effect of sorafenib on advanced human hepatocellular carcinoma (HCC) is taking place via the inhibition of NF-κB signal transduction. Here we constructed a human HCC Huh7 stable clone with NF-κB-responsive element to drive dual reporter genes, herpes simplex virus thymidine kinase (tk) and firefly luciferase (luc2), and co-transfected with a third red fluorescent protein (rfp) gene, renamed as Huh7/NF-κB-tk-luc2/rfp cells, and combined with bioluminescent imaging (BLI) and red fluorescent protein imaging (RFPI) to monitor the effect of sorafenib on NF-κB activation and tumor inhibition. The results show that sorafenib could suppress the NF-κB-DNA binding activity, and the expression of downstream effector proteins. Notably, the relative photon fluxes obtained from RFPI and BLI, which represent the viable tumor cells and cells with NF-κB activation, decreased after sorafenib treatment by 50 to 65%, and 87.5 to >90%, respectively, suggesting that NF-κB activation is suppressed in viable HCC cells by sorafenib. Simultaneous molecular imaging of the temporal change of NF-κB activity and of viable cells in the same Huh7/NF-κB-tk-luc2/rfp tumors of the animal may reflect the real status of NF-κB activity and the viable tumor cells at the time of imaging.

Quantitative imaging of gold nanoparticle distribution in a tumor-bearing mouse using benchtop x-ray fluorescence computed tomography

PubMed Central

Manohar, Nivedh; Reynoso, Francisco J.; Diagaradjane, Parmeswaran; Krishnan, Sunil; Cho, Sang Hyun

2016-01-01

X-ray fluorescence computed tomography (XFCT) is a technique that can identify, quantify, and locate elements within objects by detecting x-ray fluorescence (characteristic x-rays) stimulated by an excitation source, typically derived from a synchrotron. However, the use of a synchrotron limits practicality and accessibility of XFCT for routine biomedical imaging applications. Therefore, we have developed the ability to perform XFCT on a benchtop setting with ordinary polychromatic x-ray sources. Here, we report our postmortem study that demonstrates the use of benchtop XFCT to accurately image the distribution of gold nanoparticles (GNPs) injected into a tumor-bearing mouse. The distribution of GNPs as determined by benchtop XFCT was validated using inductively coupled plasma mass spectrometry. This investigation shows drastically enhanced sensitivity and specificity of GNP detection and quantification with benchtop XFCT, up to two orders of magnitude better than conventional x-ray CT. The results also reaffirm the unique capabilities of benchtop XFCT for simultaneous determination of the spatial distribution and concentration of nonradioactive metallic probes, such as GNPs, within the context of small animal imaging. Overall, this investigation identifies a clear path toward in vivo molecular imaging using benchtop XFCT techniques in conjunction with GNPs and other metallic probes. PMID:26912068

CT/FMT dual-model imaging of breast cancer based on peptide-lipid nanoparticles

NASA Astrophysics Data System (ADS)

Xu, Guoqiang; Lin, Qiaoya; Lian, Lichao; Qian, Yuan; Lu, Lisen; Zhang, Zhihong

2016-03-01

Breast cancer is one of the most harmful cancers in human. Its early diagnosis is expected to improve the patients' survival rate. X-ray computed tomography (CT) has been widely used in tumor detection for obtaining three-dimentional information. Fluorescence Molecular Tomography (FMT) imaging combined with near-infrared fluorescent dyes provides a powerful tool for the acquisition of molecular biodistribution information in deep tissues. Thus, the combination of CT and FMT imaging modalities allows us to better differentiate diseased tissues from normal tissues. Here we developed a tumor-targeting nanoparticle for dual-modality imaging based on a biocompatible HDL-mimicking peptide-phospholipid scaffold (HPPS) nanocarrier. By incorporation of CT contrast agents (iodinated oil) and far-infrared fluorescent dyes (DiR-BOA) into the hydrophobic core of HPPS, we obtained the FMT and CT signals simultaneously. Increased accumulation of the nanoparticles in the tumor lesions was achieved through the effect of the tumor-targeting peptide on the surface of nanoparticle. It resulted in excellent contrast between lesions and normal tissues. Together, the abilities to sensitively separate the lesions from adjacent normal tissues with the aid of a FMT/CT dual-model imaging approach make the targeting nanoparticles a useful tool for the diagnostics of breast cancer.

Preliminary study on X-ray fluorescence computed tomography imaging of gold nanoparticles: Acceleration of data acquisition by multiple pinholes scheme

NASA Astrophysics Data System (ADS)

Sasaya, Tenta; Sunaguchi, Naoki; Seo, Seung-Jum; Hyodo, Kazuyuki; Zeniya, Tsutomu; Kim, Jong-Ki; Yuasa, Tetsuya

2018-04-01

Gold nanoparticles (GNPs) have recently attracted attention in nanomedicine as novel contrast agents for cancer imaging. A decisive tomographic imaging technique has not yet been established to depict the 3-D distribution of GNPs in an object. An imaging technique known as pinhole-based X-ray fluorescence computed tomography (XFCT) is a promising method that can be used to reconstruct the distribution of GNPs from the X-ray fluorescence emitted by GNPs. We address the acceleration of data acquisition in pinhole-based XFCT for preclinical use using a multiple pinhole scheme. In this scheme, multiple projections are simultaneously acquired through a multi-pinhole collimator with a 2-D detector and full-field volumetric beam to enhance the signal-to-noise ratio of the projections; this enables fast data acquisition. To demonstrate the efficacy of this method, we performed an imaging experiment using a physical phantom with an actual multi-pinhole XFCT system that was constructed using the beamline AR-NE7A at KEK. The preliminary study showed that the multi-pinhole XFCT achieved a data acquisition time of 20 min at a theoretical detection limit of approximately 0.1 Au mg/ml and at a spatial resolution of 0.4 mm.

Stochastic Simulation of Dopamine Neuromodulation for Implementation of Fluorescent Neurochemical Probes in the Striatal Extracellular Space.

PubMed

Beyene, Abraham G; McFarlane, Ian R; Pinals, Rebecca L; Landry, Markita P

2017-10-18

Imaging the dynamic behavior of neuromodulatory neurotransmitters in the extracelluar space that arise from individual quantal release events would constitute a major advance in neurochemical imaging. Spatial and temporal resolution of these highly stochastic neuromodulatory events requires concurrent advances in the chemical development of optical nanosensors selective for neuromodulators in concert with advances in imaging methodologies to capture millisecond neurotransmitter release. Herein, we develop and implement a stochastic model to describe dopamine dynamics in the extracellular space (ECS) of the brain dorsal striatum to guide the design and implementation of fluorescent neurochemical probes that record neurotransmitter dynamics in the ECS. Our model is developed from first-principles and simulates release, diffusion, and reuptake of dopamine in a 3D simulation volume of striatal tissue. We find that in vivo imaging of neuromodulation requires simultaneous optimization of dopamine nanosensor reversibility and sensitivity: dopamine imaging in the striatum or nucleus accumbens requires nanosensors with an optimal dopamine dissociation constant (K d ) of 1 μM, whereas K d s above 10 μM are required for dopamine imaging in the prefrontal cortex. Furthermore, as a result of the probabilistic nature of dopamine terminal activity in the striatum, our model reveals that imaging frame rates of 20 Hz are optimal for recording temporally resolved dopamine release events. Our work provides a modeling platform to probe how complex neuromodulatory processes can be studied with fluorescent nanosensors and enables direct evaluation of nanosensor chemistry and imaging hardware parameters. Our stochastic model is generic for evaluating fluorescent neurotransmission probes, and is broadly applicable to the design of other neurotransmitter fluorophores and their optimization for implementation in vivo.

Thrombin-activatable fluorescent peptide incorporated gold nanoparticles for dual optical/computed tomography thrombus imaging.

PubMed

Kwon, Sung-Pil; Jeon, Sangmin; Lee, Sung-Hoon; Yoon, Hong Yeol; Ryu, Ju Hee; Choi, Dayil; Kim, Jeong-Yeon; Kim, Jiwon; Park, Jae Hyung; Kim, Dong-Eog; Kwon, Ick Chan; Kim, Kwangmeyung; Ahn, Cheol-Hee

2018-01-01

Thrombosis is an important pathophysiologic phenomenon in various cardiovascular diseases, which can lead to oxygen deprivation and infarction of tissues by generation of a thrombus. Thus, direct thrombus imaging can provide beneficial in diagnosis and therapy of thrombosis. Herein, we developed thrombin-activatable fluorescent peptide (TAP) incorporated silica-coated gold nanoparticles (TAP-SiO 2 @AuNPs) for direct imaging of thrombus by dual near-infrared fluorescence (NIRF) and micro-computed tomography (micro-CT) imaging, wherein TAP molecules were used as targeted thrombin-activatable peptide probes for thrombin-specific NIRF imaging. The freshly prepared TAP-SiO 2 @AuNPs had an average diameter of 39.8 ± 2.55 nm and they showed the quenched NIRF signal in aqueous condition, due to the excellent quenching effect of TAP molecules on the silica-gold nanoparticle surface. However, 30.31-fold higher NIRF intensity was rapidly recovered in the presence of thrombin in vitro, due to the thrombin-specific cleavage of quenched TAP molecules on the gold particle surface. Furthermore, TAP-SiO 2 @AuNPs were successfully accumulated in thrombus by their particle size-dependent capturing property, and they presented a potential X-ray absorption property in a dose-dependent manner. Finally, thrombotic lesion was clearly distinguished from peripheral tissues by dual NIRF/micro-CT imaging after intravenous injection of TAP-SiO 2 @AuNPs in the in situ thrombotic mouse model, simultaneously. This study showed that thrombin-activatable fluorescent peptide incorporated silica-coated gold nanoparticles can be potentially used as a dual imaging probe for direct thrombus imaging and therapy in clinical applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

A gold nanocluster-based fluorescent probe for simultaneous pH and temperature sensing and its application to cellular imaging and logic gates.

PubMed

Wu, Yun-Tse; Shanmugam, Chandirasekar; Tseng, Wei-Bin; Hiseh, Ming-Mu; Tseng, Wei-Lung

2016-06-07

Metal nanocluster-based nanomaterials for the simultaneous determination of temperature and pH variations in micro-environments are still a challenge. In this study, we develop a dual-emission fluorescent probe consisting of bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) and fluorescein-5-isothiocyanate (FITC) as temperature- and pH-responsive fluorescence signals. Under single wavelength excitation the FITC/BSA-AuNCs exhibited well-separated dual emission bands at 525 and 670 nm. When FITC was used as a reference fluorophore, FITC/BSA-AuNCs showed a good linear response over the temperature range 1-71 °C and offered temperature-independent spectral shifts, temperature accuracy, activation energy, and reusability. The possible mechanism for high temperature-induced fluorescence quenching of FITC/BSA-AuNCs could be attributed to a weakening of the Au-S bond, thereby lowering the charge transfer from BSA to AuNCs. Additionally, the pH- and temperature-responsive properties of FITC/BSA-AuNCs allow simultaneous temperature sensing from 21 to 41 °C (at intervals of 5 °C) and pH from 6.0 to 8.0 (at intervals of 0.5 pH unit), facilitating the construction of two-input AND logic gates. Three-input AND logic gates were also designed using temperature, pH, and trypsin as inputs. The practicality of using FITC/BSA-AuNCs to determine the temperature and pH changes in HeLa cells is also validated.

In vivo multi-modality photoacoustic and pulse echo tracking of prostate tumor growth using a window chamber

NASA Astrophysics Data System (ADS)

Bauer, Daniel R.; Olafsson, Ragnar; Montilla, Leonardo G.; Witte, Russell S.

2010-02-01

Understanding the tumor microenvironment is critical to characterizing how cancers operate and predicting how they will eventually respond to treatment. The mouse window chamber model is an excellent tool for cancer research, because it enables high resolution tumor imaging and cross-validation using multiple modalities. We describe a novel multimodality imaging system that incorporates three dimensional (3D) photoacoustics with pulse echo ultrasound for imaging the tumor microenvironment and tracking tissue growth in mice. Three mice were implanted with a dorsal skin flap window chamber. PC-3 prostate tumor cells, expressing green fluorescent protein (GFP), were injected into the skin. The ensuing tumor invasion was mapped using photoacoustic and pulse echo imaging, as well as optical and fluorescent imaging for comparison and cross validation. The photoacoustic imaging and spectroscopy system, consisting of a tunable (680-1000nm) pulsed laser and 25 MHz ultrasound transducer, revealed near infrared absorbing regions, primarily blood vessels. Pulse echo images, obtained simultaneously, provided details of the tumor microstructure and growth with 100-μm3 resolution. The tumor size in all three mice increased between three and five fold during 3+ weeks of imaging. Results were consistent with the optical and fluorescent images. Photoacoustic imaging revealed detailed maps of the tumor vasculature, whereas photoacoustic spectroscopy identified regions of oxygenated and deoxygenated blood vessels. The 3D photoacoustic and pulse echo imaging system provided complementary information to track the tumor microenvironment, evaluate new cancer therapies, and develop molecular imaging agents in vivo. Finally, these safe and noninvasive techniques are potentially applicable for human cancer imaging.

Image restoration for three-dimensional fluorescence microscopy using an orthonormal basis for efficient representation of depth-variant point-spread functions

PubMed Central

Patwary, Nurmohammed; Preza, Chrysanthe

2015-01-01

A depth-variant (DV) image restoration algorithm for wide field fluorescence microscopy, using an orthonormal basis decomposition of DV point-spread functions (PSFs), is investigated in this study. The efficient PSF representation is based on a previously developed principal component analysis (PCA), which is computationally intensive. We present an approach developed to reduce the number of DV PSFs required for the PCA computation, thereby making the PCA-based approach computationally tractable for thick samples. Restoration results from both synthetic and experimental images show consistency and that the proposed algorithm addresses efficiently depth-induced aberration using a small number of principal components. Comparison of the PCA-based algorithm with a previously-developed strata-based DV restoration algorithm demonstrates that the proposed method improves performance by 50% in terms of accuracy and simultaneously reduces the processing time by 64% using comparable computational resources. PMID:26504634

Multi-color localization microscopy of fixed cells as a promising tool to study organization of bacterial cytoskeleton

NASA Astrophysics Data System (ADS)

Vedyaykin, A. D.; Gorbunov, V. V.; Sabantsev, A. V.; Polinovskaya, V. S.; Vishnyakov, I. E.; Melnikov, A. S.; Serdobintsev, P. Yu; Khodorkovskii, M. A.

2015-11-01

Localization microscopy allows visualization of biological structures with resolution well below the diffraction limit. Localization microscopy was used to study FtsZ organization in Escherichia coli previously in combination with fluorescent protein labeling, but the fact that fluorescent chimeric protein was unable to rescue temperature-sensitive ftsZ mutants suggests that obtained images may not represent native FtsZ structures faithfully. Indirect immunolabeling of FtsZ not only overcomes this problem, but also allows the use of the powerful visualization methods arsenal available for different structures in fixed cells. In this work we simultaneously obtained super-resolution images of FtsZ structures and diffraction-limited or super-resolution images of DNA and cell surface in E. coli, which allows for the study of the spatial arrangement of FtsZ structures with respect to the nucleoid positions and septum formation.

Fluorescence-based surface magnifying chromoendoscopy and optical coherence tomography endoscope

NASA Astrophysics Data System (ADS)

Wall, R. Andrew; Barton, Jennifer K.

2012-08-01

A side-viewing, 2.3-mm diameter, surface magnifying chromoendoscopy-optical coherence tomography (SMC-OCT) endoscope has been designed for simultaneous, nondestructive surface fluorescence visualization and cross-sectional imaging. We apply this endoscope to in vivo examination of the mouse colon. A 30,000 element fiber bundle is combined with single mode fibers, for SMC and OCT imaging, respectively. The distal optics consist of a gradient-index lens and spacer to provide a 1× magnification at a working distance of 1.58 mm in air, necessary to image the sample through a 0.23-mm thick outer glass envelope, and an aluminized right-angle prism fixed to the distal end of the gradient-index lens assembly. The resulting 1∶1 imaging system is capable of 3.9-μm lateral and 2.3-μm axial resolution in the OCT channel, and 125-lp/mm resolution across a 0.70-mm field of view in the SMC channel. The endoscope can perform high contrast crypt visualization, molecular imaging, and cross-sectional imaging of colon microstructure.

Fluorescence-based surface magnifying chromoendoscopy and optical coherence tomography endoscope

PubMed Central

Wall, R. Andrew

2012-01-01

Abstract. A side-viewing, 2.3-mm diameter, surface magnifying chromoendoscopy-optical coherence tomography (SMC-OCT) endoscope has been designed for simultaneous, nondestructive surface fluorescence visualization and cross-sectional imaging. We apply this endoscope to in vivo examination of the mouse colon. A 30,000 element fiber bundle is combined with single mode fibers, for SMC and OCT imaging, respectively. The distal optics consist of a gradient-index lens and spacer to provide a 1× magnification at a working distance of 1.58 mm in air, necessary to image the sample through a 0.23-mm thick outer glass envelope, and an aluminized right-angle prism fixed to the distal end of the gradient-index lens assembly. The resulting 1∶1 imaging system is capable of 3.9-µm lateral and 2.3-µm axial resolution in the OCT channel, and 125-lp/mm resolution across a 0.70-mm field of view in the SMC channel. The endoscope can perform high contrast crypt visualization, molecular imaging, and cross-sectional imaging of colon microstructure. PMID:23224190

Multicolor probe-based confocal laser endomicroscopy: a new world for in vivo and real-time cellular imaging

NASA Astrophysics Data System (ADS)

Vercauteren, Tom; Doussoux, François; Cazaux, Matthieu; Schmid, Guillaume; Linard, Nicolas; Durin, Marie-Amélie; Gharbi, Hédi; Lacombe, François

2013-03-01

Since its inception in the field of in vivo imaging, endomicroscopy through optical fiber bundles, or probe-based Confocal Laser Endomicroscopy (pCLE), has extensively proven the benefit of in situ and real-time examination of living tissues at the microscopic scale. By continuously increasing image quality, reducing invasiveness and improving system ergonomics, Mauna Kea Technologies has turned pCLE not only into an irreplaceable research instrument for small animal imaging, but also into an accurate clinical decision making tool with applications as diverse as gastrointestinal endoscopy, pulmonology and urology. The current implementation of pCLE relies on a single fluorescence spectral band making different sources of in vivo information challenging to distinguish. Extending the pCLE approach to multi-color endomicroscopy therefore appears as a natural plan. Coupling simultaneous multi-laser excitation with minimally invasive, microscopic resolution, thin and flexible optics, allows the fusion of complementary and valuable biological information, thus paving the way to a combination of morphological and functional imaging. This paper will detail the architecture of a new system, Cellvizio Dual Band, capable of video rate in vivo and in situ multi-spectral fluorescence imaging with a microscopic resolution. In its standard configuration, the system simultaneously operates at 488 and 660 nm, where it automatically performs the necessary spectral, photometric and geometric calibrations to provide unambiguously co-registered images in real-time. The main hardware and software features, including calibration procedures and sub-micron registration algorithms, will be presented as well as a panorama of its current applications, illustrated with recent results in the field of pre-clinical imaging.

Hard X-ray Ptychography: Making It Cool, Colorful and Fast

NASA Astrophysics Data System (ADS)

Deng, Junjing

Ptychography is a recently developed coherent imaging technique for extended objects, with a resolution not limited by the lens. Because X-rays have short wavelengths and high penetration ability, X-ray ptychography provides a powerful and unique tool for studying thick samples at high spatial resolution. We have advanced X-ray ptychography by making it cool, colorful, and fast. We make it cool by carrying out ptychography experiments at cryogenic conditions to image frozen-hydrated specimens. This largely removes the limitations of radiation damage on the achievable resolution, and allows one to obtain excellent preservation of structure and chemistry in biological specimens. We make it colorful by combining it with X-ray fluorescence measurements of chemical element distributions. In studies of biological specimens, this means that ptychography can reveal cellular ultrastructure at high contrast and at a resolution well beyond that of X-ray focusing optics, while X-ray fluorescence is used to simultaneously image the distribution of trace elements in cells (such as metals that play key roles in cell functions and which can be used in various disease therapeutic agents). Because X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular materials, this combined approach provides the unique tool to obtain simultaneous views of ultrastructure and elemental compositions of specimens. We make it fast by using continuous-scan (or "fly-scan") methods. Conventional ptychography is implemented in a move-settle-measure approach, which is slow due to the positioning overheads. To overcome this bottleneck, we have developed fly-scan ptychography that is able to speed up the data collection, and real time on-site data analysis can be achieved by using a parallelized reconstruction code. With these advances, we conducted combined cryo X-ray ptychography and fluorescence imaging at 5.2 keV in a more practical way using fly scan, well-preserved cryogenic samples and rapid reconstructions, and obtained images of a whole frozen-hydrated eukaryotic cell at 18 nm resolution which we believe to be the highest spatial resolution obtained in X-ray imaging of frozen-hydrated biological samples to date. After a successful demonstration of fly-scan 3D ptychography on a gold test sample, we also obtained fly-scan 3D ptychography and fluorescence data on frozen-hydrated cells with an imaging speedup of factor more than 7. Finally, we applied fly-scan X-ray ptychography on un-thinned integrated circuits (ICs) using 10 keV X-rays, and were able to see the circuit details within the thick IC chips with a high resolution of 11.6 nm. All of these achievements point the way toward high-speed X-ray imaging without lens-imposed resolution limit.

3D mesoscopic fluorescence tomography for imaging micro-distribution of antibody-photon absorber conjugates during near infrared photoimmunotherapy in vivo.

PubMed

Tang, Qinggong; Nagaya, Tadanobu; Liu, Yi; Horng, Hannah; Lin, Jonathan; Sato, Kazuhide; Kobayashi, Hisataka; Chen, Yu

2018-06-10

As a novel low-side-effect cancer therapy, photo-immunotherapy (PIT) is based on conjugating monoclonal antibody (mAb) with a near-infrared (NIR) phthalocyanine dye IRDye700DX (IR 700). IR700 is not only fluorescent to be used as an imaging agent, but also phototoxic. When illuminating with NIR light, PIT can induce highly-selective cancer cell death while leaving most of tumor blood vessels unharmed, leading to an effect termed super-enhanced permeability and retention (SUPR), which can significantly improve the effectiveness of anti-cancer drug. Currently, the therapeutic effects of PIT are monitored using 2D macroscopic fluorescence reflectance imager, which lacks the resolution and depth information to reveal the 3D distribution of mAb-IR700. In the study, we applied a multi-modal optical imaging approach including high-resolution optical coherence tomography (OCT) and high-sensitivity fluorescence laminar optical tomography (FLOT), to provide 3D tumor micro-structure and micro-distribution of mAb-IR700 in the tumor simultaneously during PIT in situ and in vivo. The multi-wavelength FLOT can also provide the blood vessels morphology of the tumor. Thus, the 3D FLOT reconstructed images allow us to evaluate the IR700 fluorescence distribution change with respect to the blood vessels and at different tumor locations/depths non-invasively, thereby enabling evaluation of the therapeutic effects in vivo and optimization of treatment regimens accordingly. The mAb-IR700 can access more tumor areas after PIT treatment, which can be explained by increased vascular permeability immediately after NIR-PIT. Two-photon microscopy was also used to record the mAb-IR700 on the tumor surface near the blood vessels to verify the results. Published by Elsevier B.V.

Neuronal network imaging in acute slices using Ca2+ sensitive bioluminescent reporter.

PubMed

Tricoire, Ludovic; Lambolez, Bertrand

2014-01-01

Genetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. Imaging of Ca(2+) indicators is widely used to record transient intracellular Ca(2+) increases associated with bioelectrical activity. The natural bioluminescent Ca(2+) sensor aequorin has been historically the first Ca(2+) indicator used to address biological questions. Aequorin imaging offers several advantages over fluorescent reporters: it is virtually devoid of background signal; it does not require light excitation and interferes little with intracellular processes. Genetically encoded sensors such as aequorin are commonly used in dissociated cultured cells; however it becomes more challenging to express them in differentiated intact specimen such as brain tissue. Here we describe a method to express a GFP-aequorin (GA) fusion protein in pyramidal cells of neocortical acute slices using recombinant Sindbis virus. This technique allows expressing GA in several hundreds of neurons on the same slice and to perform the bioluminescence recording of Ca(2+) transients in single neurons or multiple neurons simultaneously.

Cellular Oxygen and Nutrient Sensing in Microgravity Using Time-Resolved Fluorescence Microscopy

NASA Technical Reports Server (NTRS)

Szmacinski, Henryk

2003-01-01

Oxygen and nutrient sensing is fundamental to the understanding of cell growth and metabolism. This requires identification of optical probes and suitable detection technology without complex calibration procedures. Under this project Microcosm developed an experimental technique that allows for simultaneous imaging of intra- and inter-cellular events. The technique consists of frequency-domain Fluorescence Lifetime Imaging Microscopy (FLIM), a set of identified oxygen and pH probes, and methods for fabrication of microsensors. Specifications for electronic and optical components of FLIM instrumentation are provided. Hardware and software were developed for data acquisition and analysis. Principles, procedures, and representative images are demonstrated. Suitable lifetime sensitive oxygen, pH, and glucose probes for intra- and extra-cellular measurements of analyte concentrations have been identified and tested. Lifetime sensing and imaging have been performed using PBS buffer, culture media, and yeast cells as a model systems. Spectral specifications, calibration curves, and probes availability are also provided in the report.

Probing cellular uptake and tracking of differently shaped gelatin-coated gold nanoparticles inside of ovarian cancer cells by two-photon excited photoluminescence analyzed by fluorescence lifetime imaging (FLIM).

PubMed

Suarasan, Sorina; Licarete, Emilia; Astilean, Simion; Craciun, Ana-Maria

2018-06-01

Nowadays, the non-linear optical effect of two-photon excited (TPE) fluorescence has recently grown in interest in recent years over other optical imaging method, due to improved 3D spatial resolution, deep penetrability and less photodamage of living organism owing to the excitation in near-infrared region (NIR). In parallel, gold nanoparticles (AuNPs) have gain considerable attention for NIR TPE bio-imaging applications due to their appealing ability to generate strong intrinsic photoluminescence (PL). Here, we demonstrate the capability of differently shaped gelatin-coated AuNPs to perform as reliable label-free contrast agents for the non-invasive NIR imaging of NIH:OVCAR-3 ovary cancer cells via TPE Fluorescence Lifetime Imaging Microscopy (FLIM). Examination of the spectroscopic profile of the intrinsic signals exhibited by AuNPs inside cells confirm the plasmonic nature of the emitted PL, while the evaluation of time-dependent profile of the TPE PL signal under continuous irradiation indicates the photo-stability of the signal revealing simultaneously a photo-blinking behavior. Finally, we assess the dependence of the TPE PL signal on laser excitation power and wavelength in view of contributing to a better understanding of plasmonic TPE PL in biological media towards the improvement of TPE FLIM imaging applications based on AuNPs. Copyright © 2018 Elsevier B.V. All rights reserved.

Dual-color multiple-particle tracking at 50-nm localization and over 100-µm range in 3D with temporal focusing two-photon microscopy

PubMed Central

Ding, Yu; Li, Chunqiang

2016-01-01

Nanoscale particle tracking in three dimensions is crucial to directly observe dynamics of molecules and nanoparticles in living cells. Here we present a three-dimensional particle tracking method based on temporally focused two-photon excitation. Multiple particles are imaged at 30 frames/s in volume up to 180 × 180 × 100 µm3. The spatial localization precision can reach 50 nm. We demonstrate its capability of tracking fast swimming microbes at speed of ~200 µm/s. Two-photon dual-color tracking is achieved by simultaneously exciting two kinds of fluorescent beads at 800 nm to demonstrate its potential in molecular interaction studies. Our method provides a simple wide-field fluorescence imaging approach for deep multiple-particle tracking. PMID:27867724

In Vivo Fluorescence Imaging and Tracking of Circulating Cells and Therapeutic Nanoparticles

NASA Astrophysics Data System (ADS)

Markovic, Stacey

Noninvasive enumeration of rare circulating cells in small animals is of great importance in many areas of biomedical research, but most existing enumeration techniques involve drawing and enriching blood which is known to be problematic. Recently, small animal "in vivo flow cytometry" (IVFC) techniques have been developed, where cells flowing through small arterioles are counted continuously and noninvasively in vivo. However, higher sensitivity IVFC techniques are needed for studying low-abundance (<100/mL) circulating cells. To this end, we developed a macroscopic fluorescence imaging system and automated computer vision algorithm that allows in vivo detection, enumeration and tracking of circulating fluorescently labeled cells from multiple large blood vessels in the ear of a mouse. This technique ---"computer vision IVFC" (CV-IVFC) --- allows cell detection and enumeration at concentrations of 20 cells/mL. Performance of CV-IVFC was also characterized for low-contrast imaging scenarios, representing conditions of weak cell fluorescent labeling or high background tissue autofluorescence, and showed efficient tracking and enumeration of circulating cells with 50% sensitivity in contrast conditions degraded 2 orders of magnitude compared to in vivo testing supporting the potential utility of CV-IVFC in a range of biological models. Refinement of prior work in our lab of a separate rare-cell detection platform - "diffuse fluorescence flow cytometry" (DFFC) --- implemented a "frequency encoding" scheme by modulating two excitation lasers. Fluorescent light from both lasers can be simultaneously detected and split by frequency allowing for better discrimination of noise, sensitivity, and cell localization. The system design is described in detail and preliminary data is shown. Last, we developed a broad-field transmission fluorescence imaging system to observe nanoparticle (NP) diffusion in bulk biological tissue. Novel, implantable NP spacers allow controlled, long-term release of drugs. However, kinetics of NP (drug) diffusion over time is still poorly understood. Our imaging system allowed us to quantify diffusion of free dye and NPs of different sizes in vitro and in vivo. Subsequent analysis verified that there was continuous diffusion which could be controlled based on particle size. Continued use of this imaging system will aid optimization of NP spacers.

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images.

PubMed

Ferro, Anabela; Mestre, Tânia; Carneiro, Patrícia; Sahumbaiev, Ivan; Seruca, Raquel; Sanches, João M

2017-05-01

In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k=3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives. Importantly, this novel imaging approach is a non-disruptive method that allows an integrative and simultaneous quantitative analysis of molecular and morphological parameters, thus awarding the possibility of cell cycle profiling in cytological and histological samples.

Combined synchrotron X-ray tomography and X-ray powder diffraction using a fluorescing metal foil.

PubMed

Kappen, P; Arhatari, B D; Luu, M B; Balaur, E; Caradoc-Davies, T

2013-06-01

This study realizes the concept of simultaneous micro-X-ray computed tomography and X-ray powder diffraction using a synchrotron beamline. A thin zinc metal foil was placed in the primary, monochromatic synchrotron beam to generate a divergent wave to propagate through the samples of interest onto a CCD detector for tomographic imaging, thus removing the need for large beam illumination and high spatial resolution detection. Both low density materials (kapton tubing and a piece of plant) and higher density materials (Egyptian faience) were investigated, and elemental contrast was explored for the example of Cu and Ni meshes. The viability of parallel powder diffraction using the direct beam transmitted through the foil was demonstrated. The outcomes of this study enable further development of the technique towards in situ tomography∕diffraction studies combining micrometer and crystallographic length scales, and towards elemental contrast imaging and reconstruction methods using well defined fluorescence outputs from combinations of known fluorescence targets (elements).

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow.

PubMed

Barendrecht, Arjan D; Verhoef, Johan J F; Pignatelli, Silvia; Pasterkamp, Gerard; Heijnen, Harry F G; Maas, Coen

2017-07-10

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions. A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo

PubMed Central

Davis, Scott C.; Samkoe, Kimberley S.; Tichauer, Kenneth M.; Sexton, Kristian J.; Gunn, Jason R.; Deharvengt, Sophie J.; Hasan, Tayyaba; Pogue, Brian W.

2013-01-01

The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies. PMID:23671066

Biological applications of near-field scanning optical microscopy

NASA Astrophysics Data System (ADS)

Moers, Marco H. P.; Ruiter, A. G. T.; Jalocha, Alain; van Hulst, Niko F.; Kalle, W. H. J.; Wiegant, J. C. A. G.; Raap, A. K.

1995-09-01

Near-field Scanning Optical Microscopy (NSOM) is a true optical microscopic technique allowing fluorescence, absorption, reflection and polarization contrast with the additional advantage of nanometer lateral resolution, unlimited by diffraction and operation at ambient conditions. NSOM based on metal coated adiabatically tapered fibers, combined with shear force feedback and operated in illumination mode, has proven to be the most powerful NSOM arrangement, because of its true localization of the optical interaction, its various optical contrast possibilities and its sensitivity down to the single molecular level. In this paper applications of `aperture' NSOM to Fluorescence In Situ Hybridization of human metaphase chromosomes are presented, where the localized fluorescence allows to identify specific DNA sequences. All images are accompanied by the simultaneously acquired force image, enabling direct comparison of the optical contrast with the sample topography on nanometer scale, far beyond the diffraction limit. Thus the unique combination of high resolution, specific optical contrast and ambient operation offers many new direction possibilities in biological studies.

Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized epifluorescence laser scanning

NASA Astrophysics Data System (ADS)

Favicchio, Rosy; Zacharakis, Giannis; Oikonomaki, Katerina; Zacharopoulos, Athanasios; Mamalaki, Clio; Ripoll, Jorge

2012-07-01

Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.

Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering.

PubMed

Nuñez, Isaac; Matute, Tamara; Herrera, Roberto; Keymer, Juan; Marzullo, Timothy; Rudge, Timothy; Federici, Fernán

2017-01-01

The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential use in a high school environment to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for education, scientific research and bioengineering. All the resources developed here are available under open source licenses.

Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering

PubMed Central

Herrera, Roberto; Keymer, Juan; Marzullo, Timothy; Rudge, Timothy

2017-01-01

The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential use in a high school environment to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for education, scientific research and bioengineering. All the resources developed here are available under open source licenses. PMID:29140977

Dual-Mode SERS-Fluorescence Immunoassay Using Graphene Quantum Dot Labeling on One-Dimensional Aligned Magnetoplasmonic Nanoparticles.

PubMed

Zou, Fengming; Zhou, Hongjian; Tan, Tran Van; Kim, Jeonghyo; Koh, Kwangnak; Lee, Jaebeom

2015-06-10

A novel dual-mode immunoassay based on surface-enhanced Raman scattering (SERS) and fluorescence was designed using graphene quantum dot (GQD) labels to detect a tuberculosis (TB) antigen, CFP-10, via a newly developed sensing platform of linearly aligned magnetoplasmonic (MagPlas) nanoparticles (NPs). The GQDs were excellent bilabeling materials for simultaneous Raman scattering and photoluminescence (PL). The one-dimensional (1D) alignment of MagPlas NPs simplified the immunoassay process and enabled fast, enhanced signal transduction. With a sandwich-type immunoassay using dual-mode nanoprobes, both SERS signals and fluorescence images were recognized in a highly sensitive and selective manner with a detection limit of 0.0511 pg mL(-1).

Spatially resolved density and ionization measurements of shocked foams using x-ray fluorescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacDonald, M. J.; Keiter, P. A.; Montgomery, D. S.

2016-09-28

We present experiments at the Trident laser facility demonstrating the use of x-ray fluorescence (XRF) to simultaneously measure density, ionization state populations, and electron temperature in shocked foams. An imaging x-ray spectrometer obtained spatially resolved measurements of Ti K-α emission. Density profiles were measured from K-α intensity. Ti ionization state distributions and electron temperatures were inferred by fitting K-α spectra to spectra from CRETIN simulations. This work shows that XRF provides a powerful tool to complement other diagnostics to make equation of state measurements of shocked materials containing a suitable tracer element.

Simultaneous one-dimensional fluorescence lifetime measurements of OH and CO in premixed flames

NASA Astrophysics Data System (ADS)

Jonsson, Malin; Ehn, Andreas; Christensen, Moah; Aldén, Marcus; Bood, Joakim

2014-04-01

A method for simultaneous measurements of fluorescence lifetimes of two species along a line is described. The experimental setup is based on picosecond laser pulses from two tunable optical parametric generator/optical parametric amplifier systems together with a streak camera. With an appropriate optical time delay between the two laser pulses, whose wavelengths are tuned to excite two different species, laser-induced fluorescence can be both detected temporally and spatially resolved by the streak camera. Hence, our method enables one-dimensional imaging of fluorescence lifetimes of two species in the same streak camera recording. The concept is demonstrated for fluorescence lifetime measurements of CO and OH in a laminar methane/air flame on a Bunsen-type burner. Measurements were taken in flames with four different equivalence ratios, namely ϕ = 0.9, 1.0, 1.15, and 1.25. The measured one-dimensional lifetime profiles generally agree well with lifetimes calculated from quenching cross sections found in the literature and quencher concentrations predicted by the GRI 3.0 mechanism. For OH, there is a systematic deviation of approximately 30 % between calculated and measured lifetimes. It is found that this is mainly due to the adiabatic assumption regarding the flame and uncertainty in H2O quenching cross section. This emphasizes the strength of measuring the quenching rates rather than relying on models. The measurement concept might be useful for single-shot measurements of fluorescence lifetimes of several species pairs of vital importance in combustion processes, hence allowing fluorescence signals to be corrected for quenching and ultimately yield quantitative concentration profiles.

Quantum dots in imaging, drug delivery and sensor applications

PubMed Central

Matea, Cristian T; Mocan, Teodora; Tabaran, Flaviu; Pop, Teodora; Mosteanu, Ofelia; Puia, Cosmin; Iancu, Cornel; Mocan, Lucian

2017-01-01

Quantum dots (QDs), also known as nanoscale semiconductor crystals, are nanoparticles with unique optical and electronic properties such as bright and intensive fluorescence. Since most conventional organic label dyes do not offer the near-infrared (>650 nm) emission possibility, QDs, with their tunable optical properties, have gained a lot of interest. They possess characteristics such as good chemical and photo-stability, high quantum yield and size-tunable light emission. Different types of QDs can be excited with the same light wavelength, and their narrow emission bands can be detected simultaneously for multiple assays. There is an increasing interest in the development of nano-theranostics platforms for simultaneous sensing, imaging and therapy. QDs have great potential for such applications, with notable results already published in the fields of sensors, drug delivery and biomedical imaging. This review summarizes the latest developments available in literature regarding the use of QDs for medical applications. PMID:28814860

Simultaneous quadruple modal nonlinear optical imaging for gastric diseases diagnosis and characterization

NASA Astrophysics Data System (ADS)

Wang, Zi; Zheng, Wei; Lin, Jian; Huang, Zhiwei

2015-03-01

We report the development of a unique simultaneous quadruple-modal nonlinear optical microscopy (i.e., stimulated Raman scattering (SRS), second-harmonic generation (SHG), two-photon excitation fluorescence (TPEF), and third-harmonic generation (THG)) platform for characterization of the gastric diseases (i.e., gastritis, intestinal metaplasia (IM), intestinal type adenocarcinoma). SRS highlights the goblet cells found in IM. SHG images the distribution of collagen in lamina propria. Collagen is found to aggregate for intestinal type adenocarcinoma. TPEF reveals the cell morphology and can reflect the damage inside glands caused by the diseases. THG visualizes the nuclei with high spatial resolution, which facilitates the identification of neutrophils that are usually used as a feature of inflammation. This work shows that the co-registration of quadruple-modal images can be an effective means for diagnosis and characterization of gastric diseases at the cellular and molecular levels.

Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping

NASA Astrophysics Data System (ADS)

Bongiovanni, Marie N.; Godet, Julien; Horrocks, Mathew H.; Tosatto, Laura; Carr, Alexander R.; Wirthensohn, David C.; Ranasinghe, Rohan T.; Lee, Ji-Eun; Ponjavic, Aleks; Fritz, Joelle V.; Dobson, Christopher M.; Klenerman, David; Lee, Steven F.

2016-12-01

Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping

PubMed Central

Bongiovanni, Marie N.; Godet, Julien; Horrocks, Mathew H.; Tosatto, Laura; Carr, Alexander R.; Wirthensohn, David C.; Ranasinghe, Rohan T.; Lee, Ji-Eun; Ponjavic, Aleks; Fritz, Joelle V.; Dobson, Christopher M.; Klenerman, David; Lee, Steven F.

2016-01-01

Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane. PMID:27929085

Quantum dots in imaging, drug delivery and sensor applications.

PubMed

Matea, Cristian T; Mocan, Teodora; Tabaran, Flaviu; Pop, Teodora; Mosteanu, Ofelia; Puia, Cosmin; Iancu, Cornel; Mocan, Lucian

2017-01-01

Quantum dots (QDs), also known as nanoscale semiconductor crystals, are nanoparticles with unique optical and electronic properties such as bright and intensive fluorescence. Since most conventional organic label dyes do not offer the near-infrared (>650 nm) emission possibility, QDs, with their tunable optical properties, have gained a lot of interest. They possess characteristics such as good chemical and photo-stability, high quantum yield and size-tunable light emission. Different types of QDs can be excited with the same light wavelength, and their narrow emission bands can be detected simultaneously for multiple assays. There is an increasing interest in the development of nano-theranostics platforms for simultaneous sensing, imaging and therapy. QDs have great potential for such applications, with notable results already published in the fields of sensors, drug delivery and biomedical imaging. This review summarizes the latest developments available in literature regarding the use of QDs for medical applications.

An intraoperative spectroscopic imaging system for quantification of Protoporphyrin IX during glioma surgery (Conference Presentation)

NASA Astrophysics Data System (ADS)

Angulo-Rodríguez, Leticia M.; Laurence, Audrey; Jermyn, Michael; Sheehy, Guillaume; Sibai, Mira; Petrecca, Kevin; Roberts, David W.; Paulsen, Keith D.; Wilson, Brian C.; Leblond, Frédéric

2016-03-01

Cancer tissue often remains after brain tumor resection due to the inability to detect the full extent of cancer during surgery, particularly near tumor boundaries. Commercial systems are available for intra-operative real-time aminolevulenic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence imaging. These are standard white-light neurosurgical microscopes adapted with optical components for fluorescence excitation and detection. However, these instruments lack sensitivity and specificity, which limits the ability to detect low levels of PpIX and distinguish it from tissue auto-fluorescence. Current systems also cannot provide repeatable and un-biased quantitative fluorophore concentration values because of the unknown and highly variable light attenuation by tissue. We present a highly sensitive spectroscopic fluorescence imaging system that is seamlessly integrated onto a neurosurgical microscope. Hardware and software were developed to achieve through-microscope spatially-modulated illumination for 3D profilometry and to use this information to extract tissue optical properties to correct for the effects of tissue light attenuation. This gives pixel-by-pixel quantified fluorescence values and improves detection of low PpIX concentrations. This is achieved using a high-sensitivity Electron Multiplying Charge Coupled Device (EMCCD) with a Liquid Crystal Tunable Filter (LCTF) whereby spectral bands are acquired sequentially; and a snapshot camera system with simultaneous acquisition of all bands is used for profilometry and optical property recovery. Sensitivity and specificity to PpIX is demonstrated using brain tissue phantoms and intraoperative human data acquired in an on-going clinical study using PpIX fluorescence to guide glioma resection.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

PubMed Central

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-01-01

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

PubMed

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-05-07

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

Dual light-emitting diode-based multichannel microscopy for whole-slide multiplane, multispectral and phase imaging.

PubMed

Liao, Jun; Wang, Zhe; Zhang, Zibang; Bian, Zichao; Guo, Kaikai; Nambiar, Aparna; Jiang, Yutong; Jiang, Shaowei; Zhong, Jingang; Choma, Michael; Zheng, Guoan

2018-02-01

We report the development of a multichannel microscopy for whole-slide multiplane, multispectral and phase imaging. We use trinocular heads to split the beam path into 6 independent channels and employ a camera array for parallel data acquisition, achieving a maximum data throughput of approximately 1 gigapixel per second. To perform single-frame rapid autofocusing, we place 2 near-infrared light-emitting diodes (LEDs) at the back focal plane of the condenser lens to illuminate the sample from 2 different incident angles. A hot mirror is used to direct the near-infrared light to an autofocusing camera. For multiplane whole-slide imaging (WSI), we acquire 6 different focal planes of a thick specimen simultaneously. For multispectral WSI, we relay the 6 independent image planes to the same focal position and simultaneously acquire information at 6 spectral bands. For whole-slide phase imaging, we acquire images at 3 focal positions simultaneously and use the transport-of-intensity equation to recover the phase information. We also provide an open-source design to further increase the number of channels from 6 to 15. The reported platform provides a simple solution for multiplexed fluorescence imaging and multimodal WSI. Acquiring an instant focal stack without z-scanning may also enable fast 3-dimensional dynamic tracking of various biological samples. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expanded palette of Nano-lanterns for real-time multicolor luminescence imaging.

PubMed

Takai, Akira; Nakano, Masahiro; Saito, Kenta; Haruno, Remi; Watanabe, Tomonobu M; Ohyanagi, Tatsuya; Jin, Takashi; Okada, Yasushi; Nagai, Takeharu

2015-04-07

Fluorescence live imaging has become an essential methodology in modern cell biology. However, fluorescence requires excitation light, which can sometimes cause potential problems, such as autofluorescence, phototoxicity, and photobleaching. Furthermore, combined with recent optogenetic tools, the light illumination can trigger their unintended activation. Because luminescence imaging does not require excitation light, it is a good candidate as an alternative imaging modality to circumvent these problems. The application of luminescence imaging, however, has been limited by the two drawbacks of existing luminescent protein probes, such as luciferases: namely, low brightness and poor color variants. Here, we report the development of bright cyan and orange luminescent proteins by extending our previous development of the bright yellowish-green luminescent protein Nano-lantern. The color change and the enhancement of brightness were both achieved by bioluminescence resonance energy transfer (BRET) from enhanced Renilla luciferase to a fluorescent protein. The brightness of these cyan and orange Nano-lanterns was ∼20 times brighter than wild-type Renilla luciferase, which allowed us to perform multicolor live imaging of intracellular submicron structures. The rapid dynamics of endosomes and peroxisomes were visualized at around 1-s temporal resolution, and the slow dynamics of focal adhesions were continuously imaged for longer than a few hours without photobleaching or photodamage. In addition, we extended the application of these multicolor Nano-lanterns to simultaneous monitoring of multiple gene expression or Ca(2+) dynamics in different cellular compartments in a single cell.

Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue.

PubMed

Gaertner, Maria; Cimalla, Peter; Meissner, Sven; Kuebler, Wolfgang M; Koch, Edmund

2012-07-01

Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 μm line pair feature and has an approximately 11 μm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 μm (laterally), and 5.9 μm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation.

PubMed

MacNevin, Christopher J; Toutchkine, Alexei; Marston, Daniel J; Hsu, Chia-Wen; Tsygankov, Denis; Li, Li; Liu, Bei; Qi, Timothy; Nguyen, Dan-Vinh; Hahn, Klaus M

2016-03-02

Biosensors that report endogenous protein activity in vivo can be based on environment-sensing fluorescent dyes. The dyes can be attached to reagents that bind selectively to a specific conformation of the targeted protein, such that binding leads to a fluorescence change. Dyes that are sufficiently bright for use at low, nonperturbing intracellular concentrations typically undergo changes in intensity rather than the shifts in excitation or emission maxima that would enable precise quantitation through ratiometric imaging. We report here mero199, an environment-sensing dye that undergoes a 33 nm solvent-dependent shift in excitation. The dye was used to generate a ratiometric biosensor of Cdc42 (CRIB199) without the need for additional fluorophores. CRIB199 was used in the same cell with a FRET sensor of Rac1 activation to simultaneously observe Cdc42 and Rac1 activity in cellular protrusions, indicating that Rac1 but not Cdc42 activity was reduced during tail retraction, and specific protrusions had reduced Cdc42 activity. A novel program (EdgeProps) used to correlate localized activation with cell edge dynamics indicated that Rac1 was specifically reduced during retraction.

Fluorescent Heterodoped Nanotetrapods as Synergistically Enhancing Positive and Negative Magnetic Resonance Imaging Contrast Agents.

PubMed

Sharma, V K; Alipour, A; Soran-Erdem, Z; Kelestemur, Y; Aykut, Z G; Demir, H V

2016-05-18

In this work, we report Mn-Fe heterodoped ZnSe tetrapod nanocrystals (NCs) synthesized to synergistically enhance contrast in both T1- and T2-weighted magnetic resonance imaging (MRI). The proposed NCs were prepared using a customized heteroarchitecture such that the manganese (Mn) is confined in the core and iron (Fe) in the branches of the tetrapods. The elemental composition and profile of these NCs were studied using X-ray photoelectron spectroscopy, energy-dispersive X-ray spectroscopy, and inductively coupled plasma mass spectroscopy. Photoluminescence quantum yield of these heterodoped NCs in water is ∼30%. Magnetic measurements reveal the simultaneous presence of superparamagnetic and paramagnetic behavior in these NCs because of the coexistence of Mn(2+) and Fe(2+) dopants. Their potential as simultaneous positive and negative MRI contrast agents was demonstrated by relaxivity measurements and in vivo MRI. From the in vivo studies, we also found that these NCs (with a hydrodynamic diameter of 20 nm) are excreted from the body within 24 h after the injection. Therefore, these heterodoped tetrapods NCs, while being fluorescent and safe, hold great future as a synergistically enhancing dual-modal MRI contrast agent.

Simultaneous velocity and concentration measurements in the near field of a turbulent low-pressure jet by digital particle image velocimetry-planar laser-induced fluorescence

NASA Astrophysics Data System (ADS)

Borg, A.; Bolinder, J.; Fuchs, L.

The main purpose of this work is to develop a method for simultaneous measurement of velocity and passive scalar concentration by means of digital particle image velocimetry and planar laser-induced fluorescence. Details of the implementation of the method are given, and the technique is applied to measurements of concentration and velocity in the centre-plane of a liquid jet with a Reynolds number of 6,000. The measurements are compared with large eddy simulations. Mean velocities and concentrations, fluctuating velocities and concentrations, and correlation between fluctuating velocities and concentrations are analysed for the first six diameters downstream of the jet exit. The general agreement between measured and simulated results was found to be good, in particular for mean quantities. Mean profiles are also found to be in good agreement with other experimental work on jets reported in the literature. The ``whole-plane'' measurement method was found to be very useful for detailed comparisons of turbulent statistics with simulated data. The inadequacy of models for turbulent mass transport based on the standard gradient diffusion concept is demonstrated through the experimental data.

The utilization of a non-invasive fluorescence imaging system to follow clinical dermatological MAL-PDT

NASA Astrophysics Data System (ADS)

Tyrrell, Jessica; Campbell, Sandra; Curnow, Alison

2009-06-01

This study employed a commercially available, non-invasive, fluorescence imaging system (Dyaderm, Biocam, Germany), to measure protoporphyrin IX (PpIX) concentration at several different stages during clinical dermatological methyl aminolevulinate photodynamic therapy (MAL-PDT). We validated the system prior to use to ensure that the PpIX changes witnessed were accurate and not due to environmental or user induced artifacts. The system was then employed to acquire color (morphological) and fluorescent (physiological) images simultaneously during dermatological PDT. Clinical data was collected from a range of licensed dermatological conditions (actinic keratosis, Bowen's disease and superficial basal cell carcinoma) during initial and subsequent PDT treatment cycles. The initial clinical data indicated that each type of licensed lesion considered responded in a similar manner following the application of Metvix (Galderma, U.K.) and the subsequent light irradiation (Aktilite, Galderma, U.K.). Images acquired three hours after Metvix application showed a significant increase in PpIX concentration within the lesion (P < 0.05), whilst PpIX levels in the surrounding normal tissue remained unaltered. After irradiation, the PpIX concentration was significantly decreased and returned to a level similar to the initial concentration originally observed. Lesions that received subsequent treatment cycles accumulated significantly less PpIX (P < 0.05) prior to irradiation.

Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-10

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Combined optical resolution photoacoustic and fluorescence micro-endoscopy

NASA Astrophysics Data System (ADS)

Shao, Peng; Shi, Wei; Hajireza, Parsin; Zemp, Roger J.

2012-02-01

We present a new micro-endoscopy system combining real-time C-scan optical-resolution photoacoustic micro-endoscopy (OR-PAME), and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the OR-PAM sub-system is capable of imaging with a resolution of ~ 7μm. The fluorescence sub-system consists of a diode laser with 445 nm-centered emissions as the light source, an objective lens and a CCD camera. Proflavine, a FDA approved drug for human use, is used as the fluorescent contrast agent by topical application. The fluorescence system does not require any mechanical scanning. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single mode fibers. The absorption of Proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural and functional information given by OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping researchers and clinicians visualize angiogenesis, effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

PubMed Central

Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

2016-01-01

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

PubMed

Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Swann, Karl; Borri, Paola

2016-06-15

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. © 2016. Published by The Company of Biologists Ltd.

Automatic vision system for analysis of microscopic behavior of flow and transport in porous media

NASA Astrophysics Data System (ADS)

Rashidi, Mehdi; Dehmeshki, Jamshid; Dickenson, Eric; Daemi, M. Farhang

1997-10-01

This paper describes the development of a novel automated and efficient vision system to obtain velocity and concentration measurement within a porous medium. An aqueous fluid lace with a fluorescent dye to microspheres flows through a transparent, refractive-index-matched column packed with transparent crystals. For illumination purposes, a planar sheet of laser passes through the column as a CCD camera records all the laser illuminated planes. Detailed microscopic velocity and concentration fields have been computed within a 3D volume of the column. For measuring velocities, while the aqueous fluid, laced with fluorescent microspheres, flows through the transparent medium, a CCD camera records the motions of the fluorescing particles by a video cassette recorder. The recorded images are acquired automatically frame by frame and transferred to the computer for processing, by using a frame grabber an written relevant algorithms through an RS-232 interface. Since the grabbed image is poor in this stage, some preprocessings are used to enhance particles within images. Finally, these enhanced particles are monitored to calculate velocity vectors in the plane of the beam. For concentration measurements, while the aqueous fluid, laced with a fluorescent organic dye, flows through the transparent medium, a CCD camera sweeps back and forth across the column and records concentration slices on the planes illuminated by the laser beam traveling simultaneously with the camera. Subsequently, these recorded images are transferred to the computer for processing in similar fashion to the velocity measurement. In order to have a fully automatic vision system, several detailed image processing techniques are developed to match exact images that have different intensities values but the same topological characteristics. This results in normalized interstitial chemical concentrations as a function of time within the porous column.

An ultra-small, multi-point, and multi-color photo-detection system with high sensitivity and high dynamic range.

PubMed

Anazawa, Takashi; Yamazaki, Motohiro

2017-12-05

Although multi-point, multi-color fluorescence-detection systems are widely used in various sciences, they would find wider applications if they are miniaturized. Accordingly, an ultra-small, four-emission-point and four-color fluorescence-detection system was developed. Its size (space between emission points and a detection plane) is 15 × 10 × 12 mm, which is three-orders-of-magnitude smaller than that of a conventional system. Fluorescence from four emission points with an interval of 1 mm on the same plane was respectively collimated by four lenses and split into four color fluxes by four dichroic mirrors. Then, a total of sixteen parallel color fluxes were directly input into an image sensor and simultaneously detected. The emission-point plane and the detection plane (the image-sensor surface) were parallel and separated by a distance of only 12 mm. The developed system was applied to four-capillary array electrophoresis and successfully achieved Sanger DNA sequencing. Moreover, compared with a conventional system, the developed system had equivalent high fluorescence-detection sensitivity (lower detection limit of 17 pM dROX) and 1.6-orders-of-magnitude higher dynamic range (4.3 orders of magnitude).

Simultaneous Fluorescence Visualization of Endoplasmic Reticulum Superoxide Anion and Polarity in Myocardial Cells and Tissue.

PubMed

Xiao, Haibin; Wu, Chuanchen; Li, Ping; Tang, Bo

2018-05-15

Diabetic cardiomyopathy (DCM) is a critical complication of diabetes, the accurate pathogenesis of which remains elusive. It is widely accepted that endoplasmic reticulum (ER) stress and abnormal fluctuations of reactive oxygen species (ROS) are considered to be closely associated with progress of DCM. In addition, DCM-induced changes of myocardial tissue and ROS-derived oxidation of proteins will cause changes of the hydrophilic and hydrophobic domains and may further seriously alter the myocardial cell polarity. Thus, real-time detection of ROS and polarity in ER of live cells and in tissue will contribute to revealing the exact molecular mechanisms of DCM. In this article, we first present an ER-targetable fluorogenic probe termed ER-NAPC for sensitive and selective detection of superoxide anion (O 2 •- ). ER-NAPC can precisely target ER and visualize the increase of O 2 •- level in a live H9c2 cardiomyocyte cell during ER stress. Meanwhile, by combining ER-NAPC with a polarity-sensitive probe, ER-P, we accomplish the simultaneous fluorescence visualization of O 2 •- and polarity in ER of live cells and diabetic myocardial tissue. The dual-color fluorescence imaging results indicate that the O 2 •- level and polarity will synergistically rise during ER stress in live cells and diabetic myocardial tissue. The proposed dual-color imaging strategy may offer a proven methodology for studying coordinated variation of different parameters during ER stress oriented disease.

Protein recognition by a pattern-generating fluorescent molecular probe.

PubMed

Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M; Motiei, Leila; Margulies, David

2017-12-01

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor.

PubMed

Fan, Yichong; Makar, Merna; Wang, Michael X; Ai, Hui-Wang

2017-09-01

Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

Protein recognition by a pattern-generating fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M.; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M.; Motiei, Leila; Margulies, David

2017-12-01

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

PubMed Central

Tichauer, Kenneth M.; Holt, Robert W.; Samkoe, Kimberley S.; El-Ghussein, Fadi; Gunn, Jason R.; Jermyn, Michael; Dehghani, Hamid; Leblond, Frederic; Pogue, Brian W.

2012-01-01

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3. Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model. The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging. Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17. PMID:22847515

Computed tomography-guided time-domain diffuse fluorescence tomography in small animals for localization of cancer biomarkers.

PubMed

Tichauer, Kenneth M; Holt, Robert W; Samkoe, Kimberley S; El-Ghussein, Fadi; Gunn, Jason R; Jermyn, Michael; Dehghani, Hamid; Leblond, Frederic; Pogue, Brian W

2012-07-17

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals. Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity, while our group and a few others have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model. The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control, laser referencing, and co-registration with a small animal computed tomography (microCT) system. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging. Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat.

Imaging stem cell distribution, growth, migration, and differentiation in 3-D scaffolds for bone tissue engineering using mesoscopic fluorescence tomography.

PubMed

Tang, Qinggong; Piard, Charlotte; Lin, Jonathan; Nan, Kai; Guo, Ting; Caccamese, John; Fisher, John; Chen, Yu

2018-01-01

Regenerative medicine has emerged as an important discipline that aims to repair injury or replace damaged tissues or organs by introducing living cells or functioning tissues. Successful regenerative medicine strategies will likely depend upon a simultaneous optimization strategy for the design of biomaterials, cell-seeding methods, cell-biomaterial interactions, and molecular signaling within the engineered tissues. It remains a challenge to image three-dimensional (3-D) structures and functions of the cell-seeded scaffold in mesoscopic scale (>2 ∼ 3 mm). In this study, we utilized angled fluorescence laminar optical tomography (aFLOT), which allows depth-resolved molecular characterization of engineered tissues in 3-D to investigate cell viability, migration, and bone mineralization within bone tissue engineering scaffolds in situ. © 2017 Wiley Periodicals, Inc.

RBC micromotors carrying multiple cargos towards potential theranostic applications

NASA Astrophysics Data System (ADS)

Wu, Zhiguang; Esteban-Fernández de Ávila, Berta; Martín, Aída; Christianson, Caleb; Gao, Weiwei; Thamphiwatana, Soracha Kun; Escarpa, Alberto; He, Qiang; Zhang, Liangfang; Wang, Joseph

2015-08-01

Red blood cell (RBC)-based micromotors containing both therapeutic and diagnostic modalities are described as a means for potential theranostic applications. In this natural RBC-based multicargo-loaded micromotor system, quantum dots (QDs), anti-cancer drug doxorubicin (DOX), and magnetic nanoparticles (MNPs), were co-encapsulated into RBC micromotors. The fluorescent emission of both QDs and DOX provides direct visualization of their loading inside the RBC motors at two distinct wavelengths. The presence of MNPs within the RBCs allows for efficient magnetic guidance under ultrasound propulsion along with providing the potential for magnetic resonance imaging. The simultaneous encapsulation of the imaging nanoparticles and therapeutic payloads within the same RBC micromotor has a minimal effect upon its propulsion behavior. The ability of the RBC micromotors to transport imaging and therapeutic agents at high speed and spatial precision through a complex microchannel network is also demonstrated. Such ability to load and transport diagnostic imaging agents and therapeutic drugs within a single cell-based motor, in addition to a lower toxicity observed once the drug is encapsulated within the multicargo RBC motor, opens the door to the development of theranostic micromotors that may simultaneously treat and monitor diseases.Red blood cell (RBC)-based micromotors containing both therapeutic and diagnostic modalities are described as a means for potential theranostic applications. In this natural RBC-based multicargo-loaded micromotor system, quantum dots (QDs), anti-cancer drug doxorubicin (DOX), and magnetic nanoparticles (MNPs), were co-encapsulated into RBC micromotors. The fluorescent emission of both QDs and DOX provides direct visualization of their loading inside the RBC motors at two distinct wavelengths. The presence of MNPs within the RBCs allows for efficient magnetic guidance under ultrasound propulsion along with providing the potential for magnetic resonance imaging. The simultaneous encapsulation of the imaging nanoparticles and therapeutic payloads within the same RBC micromotor has a minimal effect upon its propulsion behavior. The ability of the RBC micromotors to transport imaging and therapeutic agents at high speed and spatial precision through a complex microchannel network is also demonstrated. Such ability to load and transport diagnostic imaging agents and therapeutic drugs within a single cell-based motor, in addition to a lower toxicity observed once the drug is encapsulated within the multicargo RBC motor, opens the door to the development of theranostic micromotors that may simultaneously treat and monitor diseases. Electronic supplementary information (ESI) available: Videos of the propulsion of the multicargo-loaded, RBC-based micromotors and more data are available in the ESI. See DOI: 10.1039/c5nr03730a

Comparison between two time-resolved approaches for prostate cancer diagnosis: high rate imager vs. photon counting system

NASA Astrophysics Data System (ADS)

Boutet, J.; Debourdeau, M.; Laidevant, A.; Hervé, L.; Dinten, J.-M.

2010-02-01

Finding a way to combine ultrasound and fluorescence optical imaging on an endorectal probe may improve early detection of prostate cancer. A trans-rectal probe adapted to fluorescence diffuse optical tomography measurements was developed by our team. This probe is based on a pulsed NIR laser source, an optical fiber network and a time-resolved detection system. A reconstruction algorithm was used to help locate and quantify fluorescent prostate tumors. In this study, two different kinds of time-resolved detectors are compared: High Rate Imaging system (HRI) and a photon counting system. The HRI is based on an intensified multichannel plate and a CCD Camera. The temporal resolution is obtained through a gating of the HRI. Despite a low temporal resolution (300ps), this system allows a simultaneous acquisition of the signal from a large number of detection fibers. In the photon counting setup, 4 photomultipliers are connected to a Time Correlated Single Photon Counting (TCSPC) board, providing a better temporal resolution (0.1 ps) at the expense of a limited number of detection fibers (4). At last, we show that the limited number of detection fibers of the photon counting setup is enough for a good localization and dramatically improves the overall acquisition time. The photon counting approach is then validated through the localization of fluorescent inclusions in a prostate-mimicking phantom.

Hyperspectral and chlorophyll fluorescence imaging to analyse the impact of Fusarium culmorum on the photosynthetic integrity of infected wheat ears.

PubMed

Bauriegel, Elke; Giebel, Antje; Herppich, Werner B

2011-01-01

Head blight on wheat, caused by Fusarium spp., is a serious problem for both farmers and food production due to the concomitant production of highly toxic mycotoxins in infected cereals. For selective mycotoxin analyses, information about the on-field status of infestation would be helpful. Early symptom detection directly on ears, together with the corresponding geographic position, would be important for selective harvesting. Hence, the capabilities of various digital imaging methods to detect head blight disease on winter wheat were tested. Time series of images of healthy and artificially Fusarium-infected ears were recorded with a laboratory hyperspectral imaging system (wavelength range: 400 nm to 1,000 nm). Disease-specific spectral signatures were evaluated with an imaging software. Applying the 'Spectral Angle Mapper' method, healthy and infected ear tissue could be clearly classified. Simultaneously, chlorophyll fluorescence imaging of healthy and infected ears, and visual rating of the severity of disease was performed. Between six and eleven days after artificial inoculation, photosynthetic efficiency of infected compared to healthy ears decreased. The severity of disease highly correlated with photosynthetic efficiency. Above an infection limit of 5% severity of disease, chlorophyll fluorescence imaging reliably recognised infected ears. With this technique, differentiation of the severity of disease was successful in steps of 10%. Depending on the quality of chosen regions of interests, hyperspectral imaging readily detects head blight 7 d after inoculation up to a severity of disease of 50%. After beginning of ripening, healthy and diseased ears were hardly distinguishable with the evaluated methods.

Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells

NASA Astrophysics Data System (ADS)

McGuinness, L. P.; Yan, Y.; Stacey, A.; Simpson, D. A.; Hall, L. T.; MacLaurin, D.; Prawer, S.; Mulvaney, P.; Wrachtrup, J.; Caruso, F.; Scholten, R. E.; Hollenberg, L. C. L.

2011-06-01

Fluorescent particles are routinely used to probe biological processes. The quantum properties of single spins within fluorescent particles have been explored in the field of nanoscale magnetometry, but not yet in biological environments. Here, we demonstrate optically detected magnetic resonance of individual fluorescent nanodiamond nitrogen-vacancy centres inside living human HeLa cells, and measure their location, orientation, spin levels and spin coherence times with nanoscale precision. Quantum coherence was measured through Rabi and spin-echo sequences over long (>10 h) periods, and orientation was tracked with effective 1° angular precision over acquisition times of 89 ms. The quantum spin levels served as fingerprints, allowing individual centres with identical fluorescence to be identified and tracked simultaneously. Furthermore, monitoring decoherence rates in response to changes in the local environment may provide new information about intracellular processes. The experiments reported here demonstrate the viability of controlled single spin probes for nanomagnetometry in biological systems, opening up a host of new possibilities for quantum-based imaging in the life sciences.

Improved localization accuracy in stochastic super-resolution fluorescence microscopy by K-factor image deshadowing

PubMed Central

Ilovitsh, Tali; Meiri, Amihai; Ebeling, Carl G.; Menon, Rajesh; Gerton, Jordan M.; Jorgensen, Erik M.; Zalevsky, Zeev

2013-01-01

Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution. PMID:24466491

Cy5.5 conjugated MnO nanoparticles for magnetic resonance/near-infrared fluorescence dual-modal imaging of brain gliomas.

PubMed

Chen, Ning; Shao, Chen; Li, Shuai; Wang, Zihao; Qu, Yanming; Gu, Wei; Yu, Chunjiang; Ye, Ling

2015-11-01

The fusion of molecular and anatomical modalities facilitates more reliable and accurate detection of tumors. Herein, we prepared the PEG-Cy5.5 conjugated MnO nanoparticles (MnO-PEG-Cy5.5 NPs) with magnetic resonance (MR) and near-infrared fluorescence (NIRF) imaging modalities. The applicability of MnO-PEG-Cy5.5 NPs as a dual-modal (MR/NIRF) imaging nanoprobe for the detection of brain gliomas was investigated. In vivo MR contrast enhancement of the MnO-PEG-Cy5.5 nanoprobe in the tumor region was demonstrated. Meanwhile, whole-body NIRF imaging of glioma bearing nude mouse exhibited distinct tumor localization upon injection of MnO-PEG-Cy5.5 NPs. Moreover, ex vivo CLSM imaging of the brain slice hosting glioma indicated the preferential accumulation of MnO-PEG-Cy5.5 NPs in the glioma region. Our results therefore demonstrated the potential of MnO-PEG-Cy5.5 NPs as a dual-modal (MR/NIRF) imaging nanoprobe in improving the diagnostic efficacy by simultaneously providing anatomical information from deep inside the body and more sensitive information at the cellular level. Copyright © 2015 Elsevier Inc. All rights reserved.

Color image analysis of contaminants and bacteria transport in porous media

NASA Astrophysics Data System (ADS)

Rashidi, Mehdi; Dehmeshki, Jamshid; Daemi, Mohammad F.; Cole, Larry; Dickenson, Eric

1997-10-01

Transport of contaminants and bacteria in aqueous heterogeneous saturated porous systems have been studied experimentally using a novel fluorescent microscopic imaging technique. The approach involves color visualization and quantification of bacterium and contaminant distributions within a transparent porous column. By introducing stained bacteria and an organic dye as a contaminant into the column and illuminating the porous regions with a planar sheet of laser beam, contaminant and bacterial transport processes through the porous medium can be observed and measured microscopically. A computer controlled color CCD camera is used to record the fluorescent images as a function of time. These images are recorded by a frame accurate high resolution VCR and are then analyzed using a color image analysis code written in our laboratories. The color images are digitized this way and simultaneous concentration and velocity distributions of both contaminant and bacterium are evaluated as a function of time and pore characteristics. The approach provides a unique dynamic probe to observe these transport processes microscopically. These results are extremely valuable in in-situ bioremediation problems since microscopic particle-contaminant- bacterium interactions are the key to understanding and optimization of these processes.

A device for real-time live-cell microscopy during dynamic dual-modal mechanostimulation

NASA Astrophysics Data System (ADS)

Lorusso, D.; Nikolov, H. N.; Chmiel, T.; Beach, R. J.; Sims, S. M.; Dixon, S. J.; Holdsworth, D. W.

2017-03-01

Mechanotransduction - the process by which cells sense and respond to mechanical stimuli - is essential for several physiological processes including skeletal homeostasis. Mammalian cells are thought to be sensitive to different modes of mechanical stimuli, including vibration and fluid shear. To better understand the mechanisms underlying the early stages of mechanotransduction, we describe the development of devices for mechanostimulation (by vibration and fluid shear) of live cells that can be integrated with real-time optical microscopy. The integrated system can deliver up to 3 Pa of fluid shear simultaneous with high-frequency sinusoidal vibrations up to 1 g. Stimuli can be applied simultaneously or independently to cells during real-time microscopic imaging. A custom microfluidic chamber was prepared from polydimethylsiloxane on a glass-bottom cell culture dish. Fluid flow was applied with a syringe pump to induce shear stress. This device is compatible with a custom-designed motion control vibration system. A voice coil actuates the system that is suspended on linear air bushings. Accelerations produced by the system were monitored with an on-board accelerometer. Displacement was validated optically using particle tracking digital high-speed imaging (1200 frames per second). During operation at nominally 45 Hz and 0.3 g, displacements were observed to be within 3.56% of the expected value. MC3T3-E1 osteoblast like cells were seeded into the microfluidic device and loaded with the calcium sensitive fluorescent probe fura-2, then mounted onto the dual-modal mechanostimulation platform. Cells were then imaged and monitored for fluorescence emission. In summary, we have developed a system to deliver physiologically relevant vibrations and fluid shear to live cells during real-time imaging and photometry. Monitoring the behavior of live cells loaded with appropriate fluorescent probes will enable characterization of the signals activated during the initial stages of mechanotransduction.

Assessing the sensitivity of diffusion MRI to detect neuronal activity directly.

PubMed

Bai, Ruiliang; Stewart, Craig V; Plenz, Dietmar; Basser, Peter J

2016-03-22

Functional MRI (fMRI) is widely used to study brain function in the neurosciences. Unfortunately, conventional fMRI only indirectly assesses neuronal activity via hemodynamic coupling. Diffusion fMRI was proposed as a more direct and accurate fMRI method to detect neuronal activity, yet confirmative findings have proven difficult to obtain. Given that the underlying relation between tissue water diffusion changes and neuronal activity remains unclear, the rationale for using diffusion MRI to monitor neuronal activity has yet to be clearly established. Here, we studied the correlation between water diffusion and neuronal activity in vitro by simultaneous calcium fluorescence imaging and diffusion MR acquisition. We used organotypic cortical cultures from rat brains as a biological model system, in which spontaneous neuronal activity robustly emerges free of hemodynamic and other artifacts. Simultaneous fluorescent calcium images of neuronal activity are then directly correlated with diffusion MR signals now free of confounds typically encountered in vivo. Although a simultaneous increase of diffusion-weighted MR signals was observed together with the prolonged depolarization of neurons induced by pharmacological manipulations (in which cell swelling was demonstrated to play an important role), no evidence was found that diffusion MR signals directly correlate with normal spontaneous neuronal activity. These results suggest that, whereas current diffusion MR methods could monitor pathological conditions such as hyperexcitability, e.g., those seen in epilepsy, they do not appear to be sensitive or specific enough to detect or follow normal neuronal activity.

Assessing the sensitivity of diffusion MRI to detect neuronal activity directly

PubMed Central

Bai, Ruiliang; Stewart, Craig V.; Plenz, Dietmar; Basser, Peter J.

2016-01-01

Functional MRI (fMRI) is widely used to study brain function in the neurosciences. Unfortunately, conventional fMRI only indirectly assesses neuronal activity via hemodynamic coupling. Diffusion fMRI was proposed as a more direct and accurate fMRI method to detect neuronal activity, yet confirmative findings have proven difficult to obtain. Given that the underlying relation between tissue water diffusion changes and neuronal activity remains unclear, the rationale for using diffusion MRI to monitor neuronal activity has yet to be clearly established. Here, we studied the correlation between water diffusion and neuronal activity in vitro by simultaneous calcium fluorescence imaging and diffusion MR acquisition. We used organotypic cortical cultures from rat brains as a biological model system, in which spontaneous neuronal activity robustly emerges free of hemodynamic and other artifacts. Simultaneous fluorescent calcium images of neuronal activity are then directly correlated with diffusion MR signals now free of confounds typically encountered in vivo. Although a simultaneous increase of diffusion-weighted MR signals was observed together with the prolonged depolarization of neurons induced by pharmacological manipulations (in which cell swelling was demonstrated to play an important role), no evidence was found that diffusion MR signals directly correlate with normal spontaneous neuronal activity. These results suggest that, whereas current diffusion MR methods could monitor pathological conditions such as hyperexcitability, e.g., those seen in epilepsy, they do not appear to be sensitive or specific enough to detect or follow normal neuronal activity. PMID:26941239

Serendipitous images of methanol in Comet Levy (1900 XX)

NASA Technical Reports Server (NTRS)

Hoban, Susan

1993-01-01

Reuter's (1992) model of the IR fluorescence of methanol is used to retrieve a methanol production rate of 3 +/- 1 x 10 exp 26/s and an abundance relative to water of about 0.1 percent. It is argued that calibration is of paramount importance and that a near-simultaneous spectrum is necessary for achieving a reliable estimate of the continuum underlying the emission feature.

Molecular Imaging of Tumor Angiogenesis and Therapeutic Effects with Dual Bioluminescence.

PubMed

Wang, Ran; Zhang, Kaiyue; Tao, Hongyan; Du, Wei; Wang, Di; Huang, Ziwei; Zhou, Manqian; Xu, Yang; Wang, Yuebing; Liu, Na; Wang, Hui; Li, Zongjin

2017-01-01

Angiogenesis is critical for the growth of tumor by supplying nutrients and oxygen that exacerbates the metastasis and progression of cancer. Noninvasive imaging of angiogenesis during the tumor therapeutic processes may provide novel opportunities for image-guided tumor management. Here, we want to develop a mouse animal model for assessing cancer progression and angiogenesis in the same individuals by molecular imaging. Breast cancer model was developed with mouse breast cancer cell line 4T1 carrying a reporter system encoding a triple fusion (TF) reporter gene consisting of renilla luciferase (Rluc), red fluorescent protein (RFP) and herpes simplex virus truncated thymidine kinase (HSV-ttk) in transgenic mice, which expressed firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (Vegfr2-luc). The mice were subsequently treated with ganciclovir (GCV) and the tumor angiogenesis was tracked by Fluc imaging and the growth status of tumor was monitored by imaging of Rluc simultaneously. Overall, this traceable breast cancer model can simultaneously image the tumor growth and angiogenesis in single individual, which may facilitate a better understanding the mechanisms of angiogenesis in the progression and regression of tumor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Simultaneous multiplane imaging of human ovarian cancer by volume holographic imaging

PubMed Central

Orsinger, Gabriel V.; Watson, Jennifer M.; Gordon, Michael; Nymeyer, Ariel C.; de Leon, Erich E.; Brownlee, Johnathan W.; Hatch, Kenneth D.; Chambers, Setsuko K.; Barton, Jennifer K.; Kostuk, Raymond K.; Romanowski, Marek

2014-01-01

Abstract. Ovarian cancer is the most deadly gynecologic cancer, a fact which is attributable to poor early detection and survival once the disease has reached advanced stages. Intraoperative laparoscopic volume holographic imaging has the potential to provide simultaneous visualization of surface and subsurface structures in ovarian tissues for improved assessment of developing ovarian cancer. In this ex vivo ovarian tissue study, we assembled a benchtop volume holographic imaging system (VHIS) to characterize the microarchitecture of 78 normal and 40 abnormal tissue specimens derived from ovarian, fallopian tube, uterine, and peritoneal tissues, collected from 26 patients aged 22 to 73 undergoing bilateral salpingo-oophorectomy, hysterectomy with bilateral salpingo-oophorectomy, or abdominal cytoreductive surgery. All tissues were successfully imaged with the VHIS in both reflectance- and fluorescence-modes revealing morphological features which can be used to distinguish between normal, benign abnormalities, and cancerous tissues. We present the development and successful application of VHIS for imaging human ovarian tissue. Comparison of VHIS images with corresponding histopathology allowed for qualitatively distinguishing microstructural features unique to the studied tissue type and disease state. These results motivate the development of a laparoscopic VHIS for evaluating the surface and subsurface morphological alterations in ovarian cancer pathogenesis. PMID:24676382

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy.

PubMed

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun; Chang, Po-Ling

2017-09-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target ( HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells.

"Smart" theranostic lanthanide nanoprobes with simultaneous up-conversion fluorescence and tunable T1-T2 magnetic resonance imaging contrast and near-infrared activated photodynamic therapy.

PubMed

Zhang, Yan; Das, Gautom Kumar; Vijayaragavan, Vimalan; Xu, Qing Chi; Padmanabhan, Parasuraman; Bhakoo, Kishore K; Selvan, Subramanian Tamil; Tan, Timothy Thatt Yang

2014-11-07

The current work reports a type of "smart" lanthanide-based theranostic nanoprobe, NaDyF4:Yb(3+)/NaGdF4:Yb(3+),Er(3+), which is able to circumvent the up-converting poisoning effect of Dy(3+) ions to give efficient near infrared (980 nm) triggered up-conversion fluorescence, and offers not only excellent dark T2-weighted MR contrast but also tunable bright and T1-weighted MR contrast properties. Due to the efficient up-converted energy transfer from the nanocrystals to chlorin e6 (Ce6) photosensitizers loaded onto the nanocrystals, cytotoxic singlet oxygen was generated and photodynamic therapy was demonstrated. Therefore, the current multifunctional nanocrystals could be potentially useful in various image-guided diagnoses where bright or dark MRI contrast could be selectively tuned to optimize image quality, but also as an efficient and more penetrative near-infrared activated photodynamic therapy agent.

Simultaneously Synchrotron X-ray Fluorescence and Ptychographic Imaging of Frozen Biological Single Cells

DOE PAGES

Chen, S.; Deng, J.; Nashed, Y. S. G.; ...

2016-07-25

Bionanoprobe (BNP), a hard x-ray fluorescence sample-scanning nanoprobe at the Advanced Photon Source of Argonne National Laboratory, has been used to quantitatively study elemental distributions in biological cells with sub-100 nm spatial resolution and high sensitivity. Cryogenic conditions enable biological samples to be studied in their frozen-hydrated state with both ultrastructure and elemental distributions more faithfully preserved compared to conventional chemical fixation or dehydration methods. Furthermore, radiation damage is reduced in two ways: the diffusion rate of free radicals is decreased at low temperatures; and the sample is embedded in vitrified ice, which reduces mass loss.

Spatially resolved density and ionization measurements of shocked foams using x-ray fluorescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacDonald, M. J.; Keiter, P. A.; Montgomery, D. S.

2016-09-22

We present experiments at the Trident laser facility demonstrating the use of x-ray fluorescence (XRF) to simultaneously measure density, ionization state populations, and electron temperature in shocked foams. An imaging x-ray spectrometer was used to obtain spatially-resolved measurements of Ti K-more » $$\\alpha$$ emission. Density profiles were measured from K-$$\\alpha$$ intensity. Ti ionization state distributions and electron temperatures were inferred by fitting K-$$\\alpha$$ spectra to spectra from CRETIN simulations. This study shows that XRF provides a powerful tool to complement other diagnostics to make equation of state measurements of shocked materials containing a suitable tracer element.« less

Construction and evaluation of a capillary array DNA sequencer based on a micromachined sheath-flow cuvette.

PubMed

Crabtree, H J; Bay, S J; Lewis, D F; Zhang, J; Coulson, L D; Fitzpatrick, G A; Delinger, S L; Harrison, D J; Dovichi, N J

2000-04-01

A capillary array electrophoresis DNA sequencer is reported based on a micromachined sheath-flow cuvette as the detection chamber. This cuvette is equipped with a set of micromachined features that hold the capillaries in precise registration to ensure uniform spacing between the capillaries, in order to generate uniform hydrodynamic flow in the cuvette. A laser beam excites all of the samples simultaneously, and a microscope objective images fluorescence onto a set of avalanche photodiodes, which operate in the analog mode. A high-gain transimpedance amplifier is used for each photodiode, providing high duty-cycle detection of fluorescence.

Imaging on a Shoestring: Cost-Effective Technologies for Probing Vadose Zone Transport Processes

NASA Astrophysics Data System (ADS)

Corkhill, C.; Bridge, J. W.; Barns, G.; Fraser, R.; Romero-Gonzalez, M.; Wilson, R.; Banwart, S.

2010-12-01

Key barriers to the widespread uptake of imaging technology for high spatial resolution monitoring of porous media systems are cost and accessibility. X-ray tomography, magnetic resonance imaging (MRI), gamma and neutron radiography require highly specialised equipment, controlled laboratory environments and/or access to large synchrotron facilities. Here we present results from visible light, fluorescence and autoradiographic imaging techniques developed at low cost and applied in standard analytical laboratories, adapted where necessary at minimal capital expense. UV-visible time lapse fluorescence imaging (UV-vis TLFI) in a transparent thin bed chamber enabled microspheres labelled with fluorescent dye and a conservative fluorophore solute (disodium fluorescein) to be measured simultaneously in saturated, partially-saturated and actively draining quartz sand to elucidate empirical values for colloid transport and deposition parameters distributed throughout the flow field, independently of theoretical approximations. Key results include the first experimental quantification of the effects of ionic strength and air-water interfacial area on colloid deposition above a capillary fringe, and the first direct observations of particle mobilisation and redeposition by moving saturation gradients during drainage. UV-vis imaging was also used to study biodegradation and reactive transport in a variety of saturated conditions, applying fluorescence as a probe for oxygen and nitrate concentration gradients, pH, solute transport parameters, reduction of uranium, and mapping of two-dimensional flow fields around a model dipole flow borehole system to validate numerical models. Costs are low: LED excitation sources (< US 50), flow chambers (US 200) and detectors (although a complete scientific-grade CCD set-up costs around US$ 8000, robust datasets can be obtained using a commercial digital SLR camera) mean that set-ups can be flexible to meet changing experimental requirements. The critical limitations of UV-vis fluorescence imaging are the need for reliable fluorescent probes suited to the experimental objective, and the reliance on thin-bed (2D) transparent porous media. Autoradiographic techniques address some of these limitations permit imaging of key biogeochemical processes in opaque media using radioactive probes, without the need for specialised radiation sources. We present initial calibration data for the use of autoradiography to monitor transport parameters for radionuclides (99-technetium), and a novel application of a radioactive salt tracer as a probe for pore water content, in model porous media systems.

Polymer-Based Dense Fluidic Networks for High Throughput Screening with Ultrasensitive Fluorescence Detection

PubMed Central

Okagbare, Paul I.; Soper, Steven A.

2011-01-01

Microfluidics represents a viable platform for performing High Throughput Screening (HTS) due to its ability to automate fluid handling and generate fluidic networks with high number densities over small footprints appropriate for the simultaneous optical interrogation of many screening assays. While most HTS campaigns depend on fluorescence, readers typically use point detection and serially address the assay results significantly lowering throughput or detection sensitivity due to a low duty cycle. To address this challenge, we present here the fabrication of a high density microfluidic network packed into the imaging area of a large field-of-view (FoV) ultrasensitive fluorescence detection system. The fluidic channels were 1, 5 or 10 μm (width), 1 μm (depth) with a pitch of 1–10 μm and each fluidic processor was individually addressable. The fluidic chip was produced from a molding tool using hot embossing and thermal fusion bonding to enclose the fluidic channels. A 40X microscope objective (numerical aperture = 0.75) created a FoV of 200 μm, providing the ability to interrogate ~25 channels using the current fluidic configuration. An ultrasensitive fluorescence detection system with a large FoV was used to transduce fluorescence signals simultaneously from each fluidic processor onto the active area of an electron multiplying charge-coupled device (EMCCD). The utility of these multichannel networks for HTS was demonstrated by carrying out the high throughput monitoring of the activity of an enzyme, APE1, used as a model screening assay. PMID:20872611

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds

NASA Astrophysics Data System (ADS)

Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H. G.; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

2016-06-01

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01908k

Fast online deconvolution of calcium imaging data

PubMed Central

Zhou, Pengcheng; Paninski, Liam

2017-01-01

Fluorescent calcium indicators are a popular means for observing the spiking activity of large neuronal populations, but extracting the activity of each neuron from raw fluorescence calcium imaging data is a nontrivial problem. We present a fast online active set method to solve this sparse non-negative deconvolution problem. Importantly, the algorithm 3progresses through each time series sequentially from beginning to end, thus enabling real-time online estimation of neural activity during the imaging session. Our algorithm is a generalization of the pool adjacent violators algorithm (PAVA) for isotonic regression and inherits its linear-time computational complexity. We gain remarkable increases in processing speed: more than one order of magnitude compared to currently employed state of the art convex solvers relying on interior point methods. Unlike these approaches, our method can exploit warm starts; therefore optimizing model hyperparameters only requires a handful of passes through the data. A minor modification can further improve the quality of activity inference by imposing a constraint on the minimum spike size. The algorithm enables real-time simultaneous deconvolution of O(105) traces of whole-brain larval zebrafish imaging data on a laptop. PMID:28291787

Tissue-like phantoms

DOEpatents

Frangioni, John V.; De Grand, Alec M.

2007-10-30

The invention is based, in part, on the discovery that by combining certain components one can generate a tissue-like phantom that mimics any desired tissue, is simple and inexpensive to prepare, and is stable over many weeks or months. In addition, new multi-modal imaging objects (e.g., beads) can be inserted into the phantoms to mimic tissue pathologies, such as cancer, or merely to serve as calibration standards. These objects can be imaged using one, two, or more (e.g., four) different imaging modalities (e.g., x-ray computed tomography (CT), positron emission tomography (PET), single photon emission computed tomography (SPECT), and near-infrared (NIR) fluorescence) simultaneously.

Ex-vivo imaging of excised tissue using vital dyes and confocal microscopy

PubMed Central

Johnson, Simon; Rabinovitch, Peter

2012-01-01

Vital dyes routinely used for staining cultured cells can also be used to stain and image live tissue slices ex-vivo. Staining tissue with vital dyes allows researchers to collect structural and functional data simultaneously and can be used for qualitative or quantitative fluorescent image collection. The protocols presented here are useful for structural and functional analysis of viable properties of cells in intact tissue slices, allowing for the collection of data in a structurally relevant environment. With these protocols, vital dyes can be applied as a research tool to disease processes and properties of tissue not amenable to cell culture based studies. PMID:22752953

Multiphoton imaging with a nanosecond supercontinuum source

NASA Astrophysics Data System (ADS)

Lefort, Claire; O'Connor, Rodney P.; Blanquet, Véronique; Baraige, Fabienne; Tombelaine, Vincent; Lévêque, Philippe; Couderc, Vincent; Leproux, Philippe

2016-03-01

Multiphoton microscopy is a well-established technique for biological imaging of several kinds of targets. It is classically based on multiphoton processes allowing two means of contrast simultaneously: two-photon fluorescence (TPF) and second harmonic generation (SHG). Today, the quasi exclusive laser technology used in that aim is femtosecond titanium sapphire (Ti: Sa) laser. We experimentally demonstrate that a nanosecond supercontinuum laser source (STM-250-VIS-IR-custom, Leukos, France; 1 ns, 600-2400 nm, 250 kHz, 1 W) allows to obtain the same kind of image quality in the case of both TPF and SHG, since it is properly filtered. The first set of images concerns the muscle of a mouse. It highlights the simultaneous detection of TPF and SHG. TPF is obtained thanks to the labelling of alpha-actinin with Alexa Fluor® 546 by immunochemistry. SHG is created from the non-centrosymmetric organization of myosin. As expected, discs of actin and myosin are superimposed alternatively. The resulting images are compared with those obtained from a standard femtosecond Ti: Sa source. The physical parameters of the supercontinuum are discussed. Finally, all the interest of using an ultra-broadband source is presented with images obtained in vivo on the brain of a mouse where tumor cells labeled with eGFP are grafted. Texas Red® conjugating Dextran is injected into the blood vessels network. Thus, two fluorophores having absorption wavelengths separated by 80 nm are imaged simultaneously with a single laser source.

Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ying; Li, Zhaohui; Liu, Misha

Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeledmore » single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.« less

Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation.

PubMed

Werley, Christopher A; Chien, Miao-Ping; Cohen, Adam E

2017-12-01

The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our 'Firefly' microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology ('Optopatch') in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes.

Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation

PubMed Central

Werley, Christopher A.; Chien, Miao-Ping; Cohen, Adam E.

2017-01-01

The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our ‘Firefly’ microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology (‘Optopatch’) in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes. PMID:29296505

Synchronous Bioimaging of Intracellular pH and Chloride Based on LSS Fluorescent Protein.

PubMed

Paredes, Jose M; Idilli, Aurora I; Mariotti, Letizia; Losi, Gabriele; Arslanbaeva, Lyaysan R; Sato, Sebastian Sulis; Artoni, Pietro; Szczurkowska, Joanna; Cancedda, Laura; Ratto, Gian Michele; Carmignoto, Giorgio; Arosio, Daniele

2016-06-17

Ion homeostasis regulates critical physiological processes in the living cell. Intracellular chloride concentration not only contributes in setting the membrane potential of quiescent cells but it also plays a role in modulating the dynamic voltage changes during network activity. Dynamic chloride imaging demands new tools, allowing faster acquisition rates and correct accounting of concomitant pH changes. Joining a long-Stokes-shift red-fluorescent protein to a GFP variant with high sensitivity to pH and chloride, we obtained LSSmClopHensor, a genetically encoded fluorescent biosensor optimized for the simultaneous chloride and pH imaging and requiring only two excitation wavelengths (458 and 488 nm). LSSmClopHensor allowed us to monitor the dynamic changes of intracellular pH and chloride concentration during seizure like discharges in neocortical brain slices. Only cells with tightly controlled resting potential revealed a narrow distribution of chloride concentration peaking at about 5 and 8 mM, in neocortical neurons and SK-N-SH cells, respectively. We thus showed that LSSmClopHensor represents a new versatile tool for studying the dynamics of chloride and proton concentration in living systems.

CH PLIF and PIV implementation using C-X (0,0) and intra-vibrational band filtered detection

NASA Astrophysics Data System (ADS)

Hammack, Stephen D.; Skiba, Aaron W.; Lee, Tonghun; Carter, Campbell D.

2018-02-01

This study demonstrates advancement in a low-pulse energy methylidyne (CH) planar laser-induced fluorescence (PLIF) method that facilitates its application alongside flows seeded for particle image velocimetry (PIV) or other particle scattering based methods, as well as in high scattering environments. The C-X (0,0) R-branch excitation and filtered detection are carefully selected such that the laser line frequency is heavily attenuated by an edge filter while allowing transmission of most of the (0,0) band fluorescence. There are strong OH A-X (0,0) lines in the vicinity, but they can be avoided or utilized through dye laser tuning. As a demonstration of efficacy, PIV is performed simultaneously with the PLIF imaging. Using the edge filter, particle scattering signal is reduced to sub-fluorescence levels, allowing for flame-front analysis. This achievement enables flame-front tracking at high repetition rates (due to the low-pulse energy required) in combination with a scattering method such as PIV or use in high scattering environments such as enclosed combustors or near burner surfaces.

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

PubMed

Kadam, Parnika; McAllister, Ryan; Urbach, Jeffrey S; Sandberg, Kathryn; Mueller, Susette C

2017-03-27

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

An in situ optical imaging system for measuring lipid uptake, vessel contraction, and lymph flow in small animal lymphatic vessels

NASA Astrophysics Data System (ADS)

Kassis, Timothy; Weiler, Michael J.; Dixon, J. Brandon

2012-03-01

All dietary lipids are transported to venous circulation through the lymphatic system, yet the underlying mechanisms that regulate this process remain unclear. Understanding how the lymphatics functionally respond to changes in lipid load is important in the diagnosis and treatment of lipid and lymphatic related diseases such as obesity, hypercholesterolemia, and lymphedema. Therefore, we sought to develop an in situ imaging system to quantify and correlate lymphatic function as it relates to lipid transport. A custom-built optical set-up provides us with the capability of dual-channel imaging of both high-speed bright-field video and fluorescence simultaneously. This is achieved by dividing the light path into two optical bands. Utilizing high-speed and back-illuminated CCD cameras and post-acquisition image processing algorithms, we have the potential quantify correlations between vessel contraction, lymph flow and lipid concentration of mesenteric lymphatic vessels in situ. Local flow velocity is measured through lymphocyte tracking, vessel contraction through measurements of the vessel walls and lipid uptake through fluorescence intensity tracking of a fluorescent long chain fatty acid analogue, Bodipy FL C16. This system will prove to be an invaluable tool for both scientists studying lymphatic function in health and disease, and those investigating strategies for targeting the lymphatic system with orally delivered drugs.

A facile synthesis of strong near infrared fluorescent layered double hydroxide nanovehicles with an anticancer drug for tumor optical imaging and therapy

NASA Astrophysics Data System (ADS)

Chen, Chunping; Yee, Lee Kim; Gong, Hua; Zhang, Yong; Xu, Rong

2013-05-01

In this work, a new multifunctional nanovehicle for tumor optical imaging and therapy was developed using Y2O3:Er3+,Yb3+ nanoparticles as near infrared fluorescent nanophosphors, and MgAl-layered double hydroxide (LDH) nanosheets as anticancer drug nanovehicles. Monodispersed Y2O3:Er3+,Yb3+ nanophosphors were readily synthesized by the urea assisted homogenous precipitation method. Hierarchically structured LDH nanosheets intercalated with an anticancer drug, fluorouracil (5FU), were deposited on the surface of Y2O3:Er3+,Yb3+@SiO2 by a simple precipitation method followed by hydrothermal treatment. The resultant Y2O3:Er3+,Yb3+@SiO2@LDH-5FU nanovehicles exhibit strong red upconversion fluorescence under the excitation of a 980 nm laser, which allows tracking of the nanovehicles after localization in cancer cells. A better anticancer efficiency was obtained over the nanovehicles than the free drug which can be attributed to their positively charged surfaces for favorable interaction with the negatively charged cell membranes. The multifunctional nanovehicles designed in this work are expected to be promising material candidates for simultaneous tumor optical imaging and therapy.In this work, a new multifunctional nanovehicle for tumor optical imaging and therapy was developed using Y2O3:Er3+,Yb3+ nanoparticles as near infrared fluorescent nanophosphors, and MgAl-layered double hydroxide (LDH) nanosheets as anticancer drug nanovehicles. Monodispersed Y2O3:Er3+,Yb3+ nanophosphors were readily synthesized by the urea assisted homogenous precipitation method. Hierarchically structured LDH nanosheets intercalated with an anticancer drug, fluorouracil (5FU), were deposited on the surface of Y2O3:Er3+,Yb3+@SiO2 by a simple precipitation method followed by hydrothermal treatment. The resultant Y2O3:Er3+,Yb3+@SiO2@LDH-5FU nanovehicles exhibit strong red upconversion fluorescence under the excitation of a 980 nm laser, which allows tracking of the nanovehicles after localization in cancer cells. A better anticancer efficiency was obtained over the nanovehicles than the free drug which can be attributed to their positively charged surfaces for favorable interaction with the negatively charged cell membranes. The multifunctional nanovehicles designed in this work are expected to be promising material candidates for simultaneous tumor optical imaging and therapy. Electronic supplementary information (ESI) available: TEM images of Y2O3:Er3+,Yb3+@SiO2 synthesized by using different amounts of TEOS, and confocal scanning laser microscopy images (Z stack) of MCF-7 cells incubated with Y2O3:Er3+,Yb3+@SiO2@LDH-5FU for 30 min and 24 h. See DOI: 10.1039/c3nr00781b

A theranostic nanoplatform: magneto-gold@fluorescence polymer nanoparticles for tumor targeting T1&T2-MRI/CT/NIR fluorescence imaging and induction of genuine autophagy mediated chemotherapy.

PubMed

Wang, Guannan; Qian, Kun; Mei, Xifan

2018-06-14

Multifunctional nanoparticles, bearing low toxicity and tumor-targeting properties, coupled with multifunctional diagnostic imaging and enhanced treatment efficacy, have drawn tremendous attention due to their enormous potential for medical applications. Herein, we report a new kind of biocompatible and tumor-targeting magneto-gold@fluorescent polymer nanoparticle (MGFs-LyP-1), which is based on ultra-small magneto-gold (Fe 3 O 4 -Au) nanoparticles and NIR emissive fluorescent polymers by a solvent-mediated method. This kind of nanoparticle could be taken up efficiently and simultaneously serve for in vivo tumor targeting T 1 &T 2 -MRI/CT/near infrared (NIR) fluorescence bioimaging. Furthermore, the nanoparticles exhibit small size, higher tumor targeting accumulation, excellent cytocompatibility for long-term tracking, and no disturbing cell proliferation and differentiation. Moreover, clear and convincing evidence proves that as-synthesized MGFs-LyP-1 could elicit genuine autophagy via inducing autophagosome formation, which offers a definite synergistic effect to enhance cancer therapy with doxorubicin (DOX) at a nontoxic concentration through enhancement of the autophagy flux. Meanwhile, the as-prepared nanoparticles could be rapidly cleared from mice without any obvious organ impairment. The results indeed reveal a promising prospect of an MGFs-LyP-1 contrast agent with low toxicity and high efficiency for promising application in biomedicine.

Construction of a femtosecond laser microsurgery system.

PubMed

Steinmeyer, Joseph D; Gilleland, Cody L; Pardo-Martin, Carlos; Angel, Matthew; Rohde, Christopher B; Scott, Mark A; Yanik, Mehmet Fatih

2010-03-01

Femtosecond laser microsurgery is a powerful method for studying cellular function, neural circuits, neuronal injury and neuronal regeneration because of its capability to selectively ablate sub-micron targets in vitro and in vivo with minimal damage to the surrounding tissue. Here, we present a step-by-step protocol for constructing a femtosecond laser microsurgery setup for use with a widely available compound fluorescence microscope. The protocol begins with the assembly and alignment of beam-conditioning optics at the output of a femtosecond laser. Then a dichroic mount is assembled and installed to direct the laser beam into the objective lens of a standard inverted microscope. Finally, the laser is focused on the image plane of the microscope to allow simultaneous surgery and fluorescence imaging. We illustrate the use of this setup by presenting axotomy in Caenorhabditis elegans as an example. This protocol can be completed in 2 d.

Hyperspectral image reconstruction for x-ray fluorescence tomography

DOE PAGES

Gürsoy, Doǧa; Biçer, Tekin; Lanzirotti, Antonio; ...

2015-01-01

A penalized maximum-likelihood estimation is proposed to perform hyperspectral (spatio-spectral) image reconstruction for X-ray fluorescence tomography. The approach minimizes a Poisson-based negative log-likelihood of the observed photon counts, and uses a penalty term that has the effect of encouraging local continuity of model parameter estimates in both spatial and spectral dimensions simultaneously. The performance of the reconstruction method is demonstrated with experimental data acquired from a seed of arabidopsis thaliana collected at the 13-ID-E microprobe beamline at the Advanced Photon Source. The resulting element distribution estimates with the proposed approach show significantly better reconstruction quality than the conventional analytical inversionmore » approaches, and allows for a high data compression factor which can reduce data acquisition times remarkably. In particular, this technique provides the capability to tomographically reconstruct full energy dispersive spectra without compromising reconstruction artifacts that impact the interpretation of results.« less

Novel Immunohistochemical Techniques Using Discrete Signal Amplification Systems for Human Cutaneous Peripheral Nerve Fiber Imaging

PubMed Central

Wang, Ningshan; Gibbons, Christopher H.; Freeman, Roy

2011-01-01

Confocal imaging uses immunohistochemical binding of specific antibodies to visualize tissues, but technical obstacles limit more widespread use of this technique in the imaging of peripheral nerve tissue. These obstacles include same-species antibody cross-reactivity and weak fluorescent signals of individual and co-localized antigens. The aims of this study were to develop new immunohistochemical techniques for imaging of peripheral nerve fibers. Three-millimeter punch skin biopsies of healthy individuals were fixed, frozen, and cut into 50-µm sections. Tissues were stained with a variety of antibody combinations with two signal amplification systems, streptavidin-biotin-fluorochrome (sABC) and tyramide-horseradish peroxidase-fluorochrome (TSA), used simultaneously to augment immunohistochemical signals. The combination of the TSA and sABC amplification systems provided the first successful co-localization of sympathetic adrenergic and sympathetic cholinergic nerve fibers in cutaneous human sweat glands and vasomotor and pilomotor systems. Primary antibodies from the same species were amplified individually without cross-reactivity or elevated background interference. The confocal fluorescent signal-to-noise ratio increased, and image clarity improved. These modifications to signal amplification systems have the potential for widespread use in the study of human neural tissues. PMID:21411809

Three-dimensional imaging of sulfides in silicate rocks at submicron resolution with multiphoton microscopy.

PubMed

Bénard, Antoine; Palle, Sabine; Doucet, Luc Serge; Ionov, Dmitri A

2011-12-01

We report the first application of multiphoton microscopy (MPM) to generate three-dimensional (3D) images of natural minerals (micron-sized sulfides) in thick (∼120 μm) rock sections. First, reflection mode (RM) using confocal laser scanning microscopy (CLSM), combined with differential interference contrast (DIC), was tested on polished sections. Second, two-photon fluorescence (TPF) and second harmonic signal (SHG) images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences.

Simultaneous laser-induced fluorescence and scattering detection of individual particles separated by capillary electrophoresis.

PubMed

Andreyev, Dmitry; Arriaga, Edgar A

2007-07-15

This technical note describes a detector capable of simultaneously monitoring scattering and fluorescence signals of individual particles separated by capillary electrophoresis. Due to its nonselective nature, scattering alone is not sufficient to identify analyte particles. However, when the analyte particles are fluorescent, the detector described here is able to identify simultaneously occurring scattering and fluorescent signals, even when contaminating particles (i.e., nonfluorescent) are present. Both fluorescent polystyrene particles and 10-nonyl acridine orange (NAO)-labeled mitochondria were used as models. Fluorescence versus scattering (FVS) plots made it possible to identify two types of particles and a contaminant in a mixture of polystyrene particles. We also analyzed NAO-labeled mitochondria before and after cryogenic storage; the mitochondria FVS plots changed with storage, which suggests that the detector reported here is suitable for monitoring subtle changes in mitochondrial morphology that would not be revealed by monitoring only fluorescence or scattering signals.

Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye.

PubMed

Sharma, Robin; Williams, David R; Palczewska, Grazyna; Palczewski, Krzysztof; Hunter, Jennifer J

2016-02-01

Although extrinsic fluorophores can be introduced to label specific cell types in the retina, endogenous fluorophores, such as NAD(P)H, FAD, collagen, and others, are present in all retinal layers. These molecules are a potential source of optical contrast and can enable noninvasive visualization of all cellular layers. We used a two-photon fluorescence adaptive optics scanning light ophthalmoscope (TPF-AOSLO) to explore the native autofluorescence of various cell classes spanning several layers in the unlabeled retina of a living primate eye. Three macaques were imaged on separate occasions using a custom TPF-AOSLO. Two-photon fluorescence was evoked by pulsed light at 730 and 920 nm excitation wavelengths, while fluorescence emission was collected in the visible range from several retinal layers and different locations. Backscattered light was recorded simultaneously in confocal modality and images were postprocessed to remove eye motion. All retinal layers yielded two-photon signals and the heterogeneous distribution of fluorophores provided optical contrast. Several structural features were observed, such as autofluorescence from vessel walls, Müller cell processes in the nerve fibers, mosaics of cells in the ganglion cell and other nuclear layers of the inner retina, as well as photoreceptor and RPE layers in the outer retina. This in vivo survey of two-photon autofluorescence throughout the primate retina demonstrates a wider variety of structural detail in the living eye than is available through conventional imaging methods, and broadens the use of two-photon imaging of normal and diseased eyes.

Multiparametric evaluation of hindlimb ischemia using time-series indocyanine green fluorescence imaging.

PubMed

Guang, Huizhi; Cai, Chuangjian; Zuo, Simin; Cai, Wenjuan; Zhang, Jiulou; Luo, Jianwen

2017-03-01

Peripheral arterial disease (PAD) can further cause lower limb ischemia. Quantitative evaluation of the vascular perfusion in the ischemic limb contributes to diagnosis of PAD and preclinical development of new drug. In vivo time-series indocyanine green (ICG) fluorescence imaging can noninvasively monitor blood flow and has a deep tissue penetration. The perfusion rate estimated from the time-series ICG images is not enough for the evaluation of hindlimb ischemia. The information relevant to the vascular density is also important, because angiogenesis is an essential mechanism for post-ischemic recovery. In this paper, a multiparametric evaluation method is proposed for simultaneous estimation of multiple vascular perfusion parameters, including not only the perfusion rate but also the vascular perfusion density and the time-varying ICG concentration in veins. The target method is based on a mathematical model of ICG pharmacokinetics in the mouse hindlimb. The regression analysis performed on the time-series ICG images obtained from a dynamic reflectance fluorescence imaging system. The results demonstrate that the estimated multiple parameters are effective to quantitatively evaluate the vascular perfusion and distinguish hypo-perfused tissues from well-perfused tissues in the mouse hindlimb. The proposed multiparametric evaluation method could be useful for PAD diagnosis. The estimated perfusion rate and vascular perfusion density maps (left) and the time-varying ICG concentration in veins of the ankle region (right) of the normal and ischemic hindlimbs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

NASA Astrophysics Data System (ADS)

Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

2013-03-01

Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

Photo-convertible fluorescent proteins as tools for fresh insights on subcellular interactions in plants.

PubMed

Griffiths, N; Jaipargas, E-A; Wozny, M R; Barton, K A; Mathur, N; Delfosse, K; Mathur, J

2016-08-01

Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development. These proteins have become increasingly useful for understanding the role of transient and sustained interactions between similar organelles. Tracking of single organelles after green-to-red conversion has provided novel insights on plastids and their stroma-filled extensions and on the formation of mega-mitochondria. Similarly colour recovery after photo-conversion has permitted the estimation of nuclear endo-reduplication events and is being developed further to image protein trafficking within the lumen of the endoplasmic reticulum. We have also applied photo-convertible proteins to create colour-differentiation between similar cell types to follow their development. Both the green and red fluorescent forms of these proteins are compatible with other commonly used single coloured FPs. This has allowed us to develop simultaneous visualization schemes for up to five types of organelles and investigate organelle interactivity. The advantages and caveats associated with the use of photo-convertible fluorescent proteins are discussed. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Rapid fabrication of carbon quantum dots as multifunctional nanovehicles for dual-modal targeted imaging and chemotherapy.

PubMed

Chiu, Sheng-Hui; Gedda, Gangaraju; Girma, Wubshet Mekonnen; Chen, Jem-Kun; Ling, Yong-Chien; Ghule, Anil V; Ou, Keng-Liang; Chang, Jia-Yaw

2016-12-01

Herein, we synthesized an S, N, and Gd tri-element doped magnetofluorescent carbon quantum dots (GdNS@CQDs) within 10min by using a one-pot microwave method. Our results showed that these magnetofluorescent GdNS@CQDs have excellent fluorescent and magnetic properties. Moreover, GdNS@CQDs exhibited high stability at physiological conditions and ionic strength. These magnetofluorescent GdNS@CQDs were conjugated with a folic acid, denoted as FA-GdNS@CQDs, for targeting dual modal fluorescence/magnetic resonance (MR) imaging. The in vitro and in vivo studies confirmed the high biocompatibility and low toxicity of FA-GdNS@CQDs. FA-GdNS@CQDs enhanced the MR response as compared to that for commercial Gd-DTPA. The targeting capabilities of FA-GdNS@CQDs were confirmed in HeLa and HepG2 cells using in vitro fluorescence and MR dual modality imaging. Additionally, an anticancer drug, doxorubicin, was incorporated into the FA-GdNS@CQDs forming FA-GdNS@CQDs-DOX, which enables targeted drug delivery. Importantly, the prepared FA-GdNS@CQDs-DOX showed a high quantity of doxorubicin loading capacity (about 80%) and pH-sensitive drug release. The uptake into cancer cells and the intracellular location of the FA-GdNS@CQDs were observed by confocal laser scanning microscopy. We also successfully demonstrated in vivo fluorescence bio imaging of the FA-GdNS@CQDs, using zebrafish as an animal model. In this manuscript, we reported a facial, rapid, and environmental friendly method to fabricate hetero atoms including gadolinium, nitrogen, and sulfur doped multi-functional magnetofluorescent carbon quantum dots (GdNS@CQDs) nanocomposite. These multifunctional GdNS@CQDs were conjugated with a folic acid for targeting dual modal fluorescence/magnetic resonance imaging. Additionally, an anticancer drug, doxorubicin, was incorporated into the nanocomposite forming FA-GdNS@CQDs-DOX, which enables targeted drug delivery. We have developed GdNS@CQDs with integrated functions for simultaneous in vitro cell imaging, targeting, and pH-sensitive controlled drug release in HeLa cells. Furthermore, we successfully demonstrated the use of this material for in vivo fluorescence imaging, using zebrafish as an animal model. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Simultaneous measurements of velocity, temperature, and pressure using rapid CW wavelength-modulation laser-induced fluorescence of OH

NASA Technical Reports Server (NTRS)

Chang, A. Y.; Battles, B. E.; Hanson, R. K.

1990-01-01

In high speed flows, laser induced fluorescence (LIF) on Doppler shifted transitions is an attractive technique for velocity measurement. LIF velocimetry was applied to combined single-point measurements of velocity, temperature, and pressure and 2-D imaging of velocity and pressure. Prior to recent research using NO, LIF velocimetry in combustion related flows relied largely on the use of seed molecules. Simultaneous, single-point LIF measurements is reported of velocity, temperature, and pressure using the naturally occurring combustion species OH. This experiment is an extension of earlier research in which a modified ring dye laser was used to make time resolved temperature measurements behind reflected shock waves by using OH absorption an in postflame gases by using OH LIF. A pair of fused-silica rhombs mounted on a single galvanonmeter in an intracavity-doubled Spectra-Physics 380 ring laser permit the UV output to be swept continuously over a few wave numbers at an effective frequency of 3kHz.

Two-Photon Probes for Lysosomes and Mitochondria: Simultaneous Detection of Lysosomes and Mitochondria in Live Tissues by Dual-Color Two-Photon Microscopy Imaging.

PubMed

Lim, Chang Su; Hong, Seung Taek; Ryu, Seong Shick; Kang, Dong Eun; Cho, Bong Rae

2015-10-01

Novel two-photon (TP) probes were developed for lysosomes (PLT-yellow) and mitochondria (BMT-blue and PMT-yellow). These probes emitted strong TP-excited fluorescence in cells at widely separated wavelength regions and displayed high organelle selectivity, good cell permeability, low cytotoxicity, and pH insensitivity. The BMT-blue and PLT-yellow probes could be utilized to detect lysosomes and mitochondria simultaneously in live tissues by using dual-color two-photon microscopy, with minimum interference from each other. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Studies on Structural, Optical, Thermal and Electrical Properties of Perylene-Doped p-terphenyl Luminophors.

PubMed

Desai, Netaji K; Mahajan, Prasad G; Bhopate, Dhanaji P; Dalavi, Dattatray K; Kamble, Avinash A; Gore, Anil H; Dongale, Tukaram D; Kolekar, Govind B; Patil, Shivajirao R

2018-01-01

A simple solid state reaction technique was employed for the preparation of polycrystalline luminophors of p-terphenyl containing different amounts of perylene followed by spectral characterization techniques viz. XRD, SEM, TGA-DSC, UV-Visible spectroscopy, thermo-electrical conductivity, fluorescence spectroscopy, fluorescence life time spectroscopy and temperature dependent fluorescence. X-ray diffraction profiles of the doped p-terphenyl reveal well-defined and sharp peaks indicate homogeneity and crystallinity. The SEM micrograph of pure p-terphenyl exhibit flakes like grains and then compact and finally gets separately with perylene amounts. The observed results indicate that closed packed crystal structures of doped p-terphenyl during crystal formation. The band gaps estimated from UV-visible spectroscopy decreased from 5.20 to 4.10 eV, while thermo-electrical conductivity increases with perylene content. The fluorescence spectra showed partial quenching of p-terphenyl fluorescence and simultaneously sensitization of perylene fluorescence at the excitation wavelength of p-terphenyl (290 nm) due to excitation energy transfer from p-terphenyl to perylene. The observed sensitization results are in harmony with intense blue color seen in fluorescence microscopy images and has high demand in scintillation process.

Coumarinocoumarin-Based Two-Photon Fluorescent Cysteine Biosensor for Targeting Lysosome.

PubMed

Chen, Chunyang; Zhou, Liuqing; Liu, Wei; Liu, Weisheng

2018-05-15

Coumarinocoumarin, one of the coumarin derivatives, is easy to synthesize and has rich modification sites. The large conjugate plane of coumarinocoumarin gives it a more excellent optical property than conventional coumarin, for example, the two-photon fluorescence property. So, the coumarinocoumarin-based probe (CCy) has been designed and synthesized, which is the first lysosomal targeting fluorescent biosensor for cysteine. This probe was prepared by a three-step procedure as a latent fluorescence probe to achieve high sensitivity and fluorescence turn-on response toward cysteine (Cys) over homocysteine (Hcy), glutathione (GSH), and other various natural amino acids under physiological conditions. Upon addition of Cys to the solution of CCy, remarkable enhancement on 520 nm of fluorescence spectra can be monitored. This probe was then successfully used for fluorescence imaging of Cys in mice organ tissues and HeLa cells, and quantitative determination has been achieved within a certain range, which proved the permeability of CCy. The concentration of Cys in animal serum was measured and the viability exceeded 80%, indicating that CCy can be a biocompatible and rapid probe for Cys in vivo. Simultaneously, its ability to detect Cys in lysosome has been validated by its lysosomal targeting.

OH Planar Laser Induced Fluorescence (PLIF) Measurements for the Study of High Pressure Flames: An Evaluation of a New Laser and a New Camera System

NASA Technical Reports Server (NTRS)

Tedder, Sarah; Hicks, Yolanda

2012-01-01

Planar laser induced fluorescence (PLIF) is used by the Combustion Branch at the NASA Glenn Research Center (NASA Glenn) to assess the characteristics of the flowfield produced by aircraft fuel injectors. To improve and expand the capabilities of the PLIF system new equipment was installed. The new capabilities of the modified PLIF system are assessed by collecting OH PLIF in a methane/air flame produced by a flat flame burner. Specifically, the modifications characterized are the addition of an injection seeder to a Nd:YAG laser pumping an optical parametric oscillator (OPO) and the use of a new camera with an interline CCD. OH fluorescence results using the injection seeded OPO laser are compared to results using a Nd:YAG pumped dye laser with ultraviolet extender (UVX). Best settings of the new camera for maximum detection of PLIF signal are reported for the controller gain and microchannel plate (MCP) bracket pulsing. Results are also reported from tests of the Dual Image Feature (DIF) mode of the new camera which allows image pairs to be acquired in rapid succession. This allows acquisition of a PLIF image and a background signal almost simultaneously. Saturation effects in the new camera were also investigated and are reported.

Two-photon luminescence lifetime imaging microscopy (LIM) to follow up cell metabolism and oxygen consumption during theranostic applications

NASA Astrophysics Data System (ADS)

Rück, A.; Breymayer, J.; Lilge, L.; Mandel, A.; Schäfer, P.; von Einem, B.; von Arnim, C.; Kalinina, S.

2018-02-01

A common property during tumor development is altered energy metabolism, which could lead to a switch from oxidative phosphorylation and glycolysis. The impact of this switch for theranostic applications could be significant. Interestingly altered metabolism could be correlated with a change in the fluorescence lifetimes of both NAD(P)H and FAD. However, as observed in a variety of investigations, the situation is complex and the result is influenced by parameters like oxidative stress, pH or viscosity. Besides metabolism, oxygen levels and consumption has to be taken into account in order to understand treatment responses. For this, correlated imaging of phosphorescence and fluorescence lifetime parameters has been investigated by us and used to observe metabolic markers simultaneously with oxygen concentrations. The technique is based on time correlated single photon counting to detect the fluorescence lifetime of NAD(P)H and FAD by FLIM and the phosphorescence lifetime of newly developed phosphors and photosensitizers by PLIM. For this, the photosensitizer TLD1433 from Theralase, which is based on a ruthenium (II) coordination complex, was used. TLD1433 which acts as a redox indicator was mainly found in cytoplasmatic organelles. The most important observation was that TLD1433 can be used as a phosphor to follow up local oxygen concentration and consumption during photodynamic therapy. Oxygen consumption was accompanied by a change in cell metabolism, observed by simultaneous FLIM/PLIM. The combination of autofluorescence-FLIM and phosphor-PLIM in luminescence lifetime microscopy provides new insights in light induced reactions.

Reflective afocal broadband adaptive optics scanning ophthalmoscope

PubMed Central

Dubra, Alfredo; Sulai, Yusufu

2011-01-01

A broadband adaptive optics scanning ophthalmoscope (BAOSO) consisting of four afocal telescopes, formed by pairs of off-axis spherical mirrors in a non-planar arrangement, is presented. The non-planar folding of the telescopes is used to simultaneously reduce pupil and image plane astigmatism. The former improves the adaptive optics performance by reducing the root-mean-square (RMS) of the wavefront and the beam wandering due to optical scanning. The latter provides diffraction limited performance over a 3 diopter (D) vergence range. This vergence range allows for the use of any broadband light source(s) in the 450-850 nm wavelength range to simultaneously image any combination of retinal layers. Imaging modalities that could benefit from such a large vergence range are optical coherence tomography (OCT), multi- and hyper-spectral imaging, single- and multi-photon fluorescence. The benefits of the non-planar telescopes in the BAOSO are illustrated by resolving the human foveal photoreceptor mosaic in reflectance using two different superluminescent diodes with 680 and 796 nm peak wavelengths, reaching the eye with a vergence of 0.76 D relative to each other. PMID:21698035

Reflective afocal broadband adaptive optics scanning ophthalmoscope.

PubMed

Dubra, Alfredo; Sulai, Yusufu

2011-06-01

A broadband adaptive optics scanning ophthalmoscope (BAOSO) consisting of four afocal telescopes, formed by pairs of off-axis spherical mirrors in a non-planar arrangement, is presented. The non-planar folding of the telescopes is used to simultaneously reduce pupil and image plane astigmatism. The former improves the adaptive optics performance by reducing the root-mean-square (RMS) of the wavefront and the beam wandering due to optical scanning. The latter provides diffraction limited performance over a 3 diopter (D) vergence range. This vergence range allows for the use of any broadband light source(s) in the 450-850 nm wavelength range to simultaneously image any combination of retinal layers. Imaging modalities that could benefit from such a large vergence range are optical coherence tomography (OCT), multi- and hyper-spectral imaging, single- and multi-photon fluorescence. The benefits of the non-planar telescopes in the BAOSO are illustrated by resolving the human foveal photoreceptor mosaic in reflectance using two different superluminescent diodes with 680 and 796 nm peak wavelengths, reaching the eye with a vergence of 0.76 D relative to each other.

Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging

PubMed Central

Entenberg, David; Wyckoff, Jeffrey; Gligorijevic, Bojana; Roussos, Evanthia T; Verkhusha, Vladislav V; Pollard, Jeffrey W; Condeelis, John

2014-01-01

Characterizing biological mechanisms dependent upon the interaction of many cell types in vivo requires both multiphoton microscope systems capable of expanding the number and types of fluorophores that can be imaged simultaneously while removing the wavelength and tunability restrictions of existing systems, and enhanced software for extracting critical cellular parameters from voluminous 4D data sets. We present a procedure for constructing a two-laser multiphoton microscope that extends the wavelength range of excitation light, expands the number of simultaneously usable fluorophores and markedly increases signal to noise via ‘over-clocking’ of detection. We also utilize a custom-written software plug-in that simplifies the quantitative tracking and analysis of 4D intravital image data. We begin by describing the optics, hardware, electronics and software required, and finally the use of the plug-in for analysis. We demonstrate the use of the setup and plug-in by presenting data collected via intravital imaging of a mouse model of breast cancer. The procedure may be completed in ~24 h. PMID:21959234

Infrared fluorescence microscopy of stained tissues: principles and technic.

PubMed

Puchtler, H; Meloan, S N; Paschal, L D

1980-01-01

Infrared photomicrography was used extensively from 1927 to the 1940's, but received little attention during the last decades. However, studies of infrared fluorescence of stained sections could not be found in the accessible literature. Ramsley (1968) published quantitative data on infrared fluorescence of approximately 250 dyes bound to textile fibers. The intensity of infrared fluorescence of many dyes varied widely with the substrate. It was therefore deemed of interest to determine whether or not similar differences in infrared fluorescence may occur when dyes are bound to histochemically distinct tissue structures. Myofibrils and collagens stained with triarylmethane dyes were chosen as test objects. Kodak infrared cut-off filter No. 301 and Wratten filter #16 were used as exciter filters to remove infrared and UV-blue and the light of a xenon lamp. Wratten filter #70 and #89B were employed as barrier filters. Infrared radiation was recorded with Kodak Ektachrome infrared film. To facilitate correlation of infrared fluorescence patterns with visible images, tissues were photographed also with conventional color film. Stained myofibrils, e.g. in myoepithelium, smooth and striated muscle emitted strong infrared fluorescence; collagen showed little or no fluorescence. Barrier filter Wratten #70 permitted simultaneous demonstration of infrared fluorescence and of non-fluorescent structures and thus facilitated histopathological studies. Preliminary findings indicate decrease or loss of infrared fluorescence of stained muscle fibers in various lesions, e.g. myocardial infarction, Duchenne-type muscular dystrophy.

Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems.

PubMed

Wang, E; Babbey, C M; Dunn, K W

2005-05-01

Fluorescence microscopy of the dynamics of living cells presents a special challenge to a microscope imaging system, simultaneously requiring both high spatial resolution and high temporal resolution, but with illumination levels low enough to prevent fluorophore damage and cytotoxicity. We have compared the high-speed Yokogawa CSU10 spinning disc confocal system with several conventional single-point scanning confocal (SPSC) microscopes, using the relationship between image signal-to-noise ratio and fluorophore photobleaching as an index of system efficiency. These studies demonstrate that the efficiency of the CSU10 consistently exceeds that of the SPSC systems. The high efficiency of the CSU10 means that quality images can be collected with much lower levels of illumination; the CSU10 was capable of achieving the maximum signal-to-noise of an SPSC system at illumination levels that incur only at 1/15th of the rate of the photobleaching of the SPSC system. Although some of the relative efficiency of the CSU10 system may be attributed to the use of a CCD rather than a photomultiplier detector system, our analyses indicate that high-speed imaging with the SPSC system is limited by fluorescence saturation at the high levels of illumination frequently needed to collect images at high frame rates. The high speed, high efficiency and freedom from fluorescence saturation combine to make the CSU10 effective for extended imaging of living cells at rates capable of capturing the three-dimensional motion of endosomes moving up to several micrometres per second.

Design, construction, and validation of a rotary multifunctional intravascular diagnostic catheter combining multispectral fluorescence lifetime imaging and intravascular ultrasound.

PubMed

Bec, Julien; Xie, Hongtao; Yankelevich, Diego R; Zhou, Feifei; Sun, Yang; Ghata, Narugopal; Aldredge, Ralph; Marcu, Laura

2012-10-01

We report the development and validation of an intravascular rotary catheter for bimodal interrogation of arterial pathologies. This is based on a point-spectroscopy scanning time-resolved fluorescence spectroscopy technique enabling reconstruction of fluorescence lifetime images (FLIm) and providing information on arterial intima composition and intravascular ultrasound (IVUS) providing information on arterial wall morphology. The catheter design allows for independent rotation of the ultrasonic and optical channels within an 8 Fr outer diameter catheter sheath and integrates a low volume flushing channel for blood removal in the optical pathways. In the current configuration, the two channels consist of (a) a standard 3 Fr IVUS catheter with single element transducer (40 MHz) and (b) a side-viewing fiber optic (400 μm core). Experiments conducted in tissue phantoms showed the ability of the catheter to operate in an intraluminal setting and to generate coregistered FLIm and IVUS in one pull-back scan. Current results demonstrate the feasibility of the catheter for simultaneous bimodal interrogation of arterial lumen and for generation of robust fluorescence lifetime data under IVUS guidance. These results facilitate further development of a FLIm-IVUS technique for intravascular diagnosis of atherosclerotic cardiovascular diseases including vulnerable plaques.

Fluorescence-Doped Particles for Simultaneous Temperature and Velocity Imaging

NASA Technical Reports Server (NTRS)

Danehy, Paul M.; Tiemsin, Pacita I.; Wohl, Chrostopher J.; Verkamp, Max; Lowe, T.; Maisto, P.; Byun, G.; Simpson, R.

2012-01-01

Polystyrene latex microspheres (PSLs) have been used for particle image velocimetry (PIV) and laser Doppler velocimetry (LDV) measurements for several decades. With advances in laser technologies, instrumentation, and data processing, the capability to collect more information about fluid flow beyond velocity is possible using new seed materials. To provide additional measurement capability, PSLs were synthesized with temperature-sensitive fluorescent dyes incorporated within the particle. These multifunctional PSLs would have the greatest impact if they could be used in large scale facilities with minimal modification to the facilities or the existing instrumentation. Consequently, several potential dyes were identified that were amenable to existing laser systems currently utilized in wind tunnels at NASA Langley Research Center as well as other wind and fluid (water) tunnels. PSLs incorporated with Rhodamine B, dichlorofluorescein (DCF, also known as fluorescein 548 or fluorescein 27) and other dyes were synthesized and characterized for morphology and spectral properties. The resulting particles were demonstrated to exhibit fluorescent emission, which would enable determination of both fluid velocity and temperature. They also would allow near-wall velocity measurements whereas laser scatter from surfaces currently prevents near-wall measurements using undoped seed materials. Preliminary results in a wind tunnel facility located at Virginia Polytechnic Institute and State University (Virginia Tech) have verified fluorescent signal detection and temperature sensitivity of fluorophore-doped PSLs.

Single-Cell in Situ RNA Analysis With Switchable Fluorescent Oligonucleotides.

PubMed

Xiao, Lu; Guo, Jia

2018-01-01

Comprehensive RNA analyses in individual cells in their native spatial contexts promise to transform our understanding of normal physiology and disease pathogenesis. Here we report a single-cell in situ RNA analysis approach using switchable fluorescent oligonucleotides (SFO). In this method, transcripts are first hybridized by pre-decoding oligonucleotides. These oligonucleotides subsequently recruit SFO to stain their corresponding RNA targets. After fluorescence imaging, all the SFO in the whole specimen are simultaneously removed by DNA strand displacement reactions. Through continuous cycles of target staining, fluorescence imaging, and SFO removal, a large number of different transcripts can be identified by unique fluorophore sequences and visualized at the optical resolution. To demonstrate the feasibility of this approach, we show that the hybridized SFO can be efficiently stripped by strand displacement reactions within 30 min. We also demonstrate that this SFO removal process maintains the integrity of the RNA targets and the pre-decoding oligonucleotides, and keeps them hybridized. Applying this approach, we show that transcripts can be restained in at least eight hybridization cycles with high analysis accuracy, which theoretically would enable the whole transcriptome to be quantified at the single molecule sensitivity in individual cells. This in situ RNA analysis technology will have wide applications in systems biology, molecular diagnosis, and targeted therapies.

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Jiang, Yiyue; Ozeki, Yasuyuki; Goda, Keisuke

2016-01-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

Photoelectrocyclization as an activation mechanism for organelle-specific live-cell imaging probes.

PubMed

Tran, Mai N; Chenoweth, David M

2015-05-26

Photoactivatable fluorophores are useful tools in live-cell imaging owing to their potential for precise spatial and temporal control. In this report, a new photoactivatable organelle-specific live-cell imaging probe based on a 6π electrocyclization/oxidation mechanism is described. It is shown that this new probe is water-soluble, non-cytotoxic, cell-permeable, and useful for mitochondrial imaging. The probe displays large Stokes shifts in both pre-activated and activated forms, allowing simultaneous use with common dyes and fluorescent proteins. Sequential single-cell activation experiments in dense cellular environments demonstrate high spatial precision and utility in single- or multi-cell labeling experiments. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Wide Field Spectroscopy of Diffusing and Interacting DNA Using Tunable Nanoscale Geometries

NASA Astrophysics Data System (ADS)

Scott, Shane; Leith, Jason; Brandao, Hugo; Sehayek, Simon; Hofkirchner, Alexander; Laurin, Jill; Berard, Daniel; Verge, Alexander; Wiseman, Paul; Leslie, Sabrina

2015-03-01

It remains an outstanding challenge to directly image interacting and diffusing biomolecules under physiological conditions. Many biochemical questions can be posed in the form: Does A interact with B? What are the energetics, kinetics, stoichiometry, and cooperativity of this interaction? To tackle this challenge, we use tunable nanoscale confinement to perform wide-field imaging of interacting DNA molecules in free solution, under an extended range of reagent concentrations and interaction rates. We present the integration of ``Convex Lens-induced Confinement (CLiC)'' microscopy with image correlation analysis, simultaneously suppressing background fluorescence and extending imaging times. The measured DNA-DNA interactions would be inaccessible to standard techniques but are important for developing a mechanistic understanding of life-preserving processes such as DNA transcription. NSERC.

Multimodality hard-x-ray imaging of a chromosome with nanoscale spatial resolution

DOE PAGES

Yan, Hanfei; Nazaretski, Evgeny; Lauer, Kenneth R.; ...

2016-02-05

Here, we developed a scanning hard x-ray microscope using a new class of x-ray nano-focusing optic called a multilayer Laue lens and imaged a chromosome with nanoscale spatial resolution. The combination of the hard x-ray's superior penetration power, high sensitivity to elemental composition, high spatial-resolution and quantitative analysis creates a unique tool with capabilities that other microscopy techniques cannot provide. Using this microscope, we simultaneously obtained absorption-, phase-, and fluorescence-contrast images of Pt-stained human chromosome samples. The high spatial-resolution of the microscope and its multi-modality imaging capabilities enabled us to observe the internal ultra-structures of a thick chromosome without sectioningmore » it.« less

Multimodality hard-x-ray imaging of a chromosome with nanoscale spatial resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Hanfei; Nazaretski, Evgeny; Lauer, Kenneth R.

Here, we developed a scanning hard x-ray microscope using a new class of x-ray nano-focusing optic called a multilayer Laue lens and imaged a chromosome with nanoscale spatial resolution. The combination of the hard x-ray's superior penetration power, high sensitivity to elemental composition, high spatial-resolution and quantitative analysis creates a unique tool with capabilities that other microscopy techniques cannot provide. Using this microscope, we simultaneously obtained absorption-, phase-, and fluorescence-contrast images of Pt-stained human chromosome samples. The high spatial-resolution of the microscope and its multi-modality imaging capabilities enabled us to observe the internal ultra-structures of a thick chromosome without sectioningmore » it.« less

Simultaneous neuron- and astrocyte-specific fluorescent marking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko

2015-03-27

Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignmentmore » of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein.« less

Evaluation of laser speckle contrast imaging as an intrinsic method to monitor blood brain barrier integrity

PubMed Central

Dufour, Suzie; Atchia, Yaaseen; Gad, Raanan; Ringuette, Dene; Sigal, Iliya; Levi, Ofer

2013-01-01

The integrity of the blood brain barrier (BBB) can contribute to the development of many brain disorders. We evaluate laser speckle contrast imaging (LSCI) as an intrinsic modality for monitoring BBB disruptions through simultaneous fluorescence and LSCI with vertical cavity surface emitting lasers (VCSELs). We demonstrated that drug-induced BBB opening was associated with a relative change of the arterial and venous blood velocities. Cross-sectional flow velocity ratio (veins/arteries) decreased significantly in rats treated with BBB-opening drugs, ≤0.81 of initial values. PMID:24156049

Three dimensional two-photon brain imaging in freely moving mice using a miniature fiber coupled microscope with active axial-scanning.

PubMed

Ozbay, Baris N; Futia, Gregory L; Ma, Ming; Bright, Victor M; Gopinath, Juliet T; Hughes, Ethan G; Restrepo, Diego; Gibson, Emily A

2018-05-25

We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. We show three-dimensional two-photon imaging of neuronal structure and record neuronal activity from GCaMP6s fluorescence from multiple focal planes in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling neurons in multiple depths to be imaged in a single plane. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 μm with an additional 180 μm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 μm, an axial resolution of 10 μm, and a field-of-view of 240 μm diameter. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.

Dynamic tissue analysis using time- and wavelength-resolved fluorescence spectroscopy for atherosclerosis diagnosis

PubMed Central

Sun, Yinghua; Sun, Yang; Stephens, Douglas; Xie, Hongtao; Phipps, Jennifer; Saroufeem, Ramez; Southard, Jeffrey; Elson, Daniel S.; Marcu, Laura

2011-01-01

Simultaneous time- and wavelength-resolved fluorescence spectroscopy (STWRFS) was developed and tested for the dynamic characterization of atherosclerotic tissue ex vivo and arterial vessels in vivo. Autofluorescence, induced by a 337 nm, 700 ps pulsed laser, was split to three wavelength sub-bands using dichroic filters, with each sub-band coupled into a different length of optical fiber for temporal separation. STWRFS allows for fast recording/analysis (few microseconds) of time-resolved fluorescence emission in these sub-bands and rapid scanning. Distinct compositions of excised human atherosclerotic aorta were clearly discriminated over scanning lengths of several centimeters based on fluorescence lifetime and the intensity ratio between 390 and 452 nm. Operation of STWRFS blood flow was further validated in pig femoral arteries in vivo using a single-fiber probe integrated with an ultrasound imaging catheter. Current results demonstrate the potential of STWRFS as a tool for real-time optical characterization of arterial tissue composition and for atherosclerosis research and diagnosis. PMID:21369214

Array biosensor for detection of toxins

NASA Technical Reports Server (NTRS)

Ligler, Frances S.; Taitt, Chris Rowe; Shriver-Lake, Lisa C.; Sapsford, Kim E.; Shubin, Yura; Golden, Joel P.

2003-01-01

The array biosensor is capable of detecting multiple targets rapidly and simultaneously on the surface of a single waveguide. Sandwich and competitive fluoroimmunoassays have been developed to detect high and low molecular weight toxins, respectively, in complex samples. Recognition molecules (usually antibodies) were first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate up to 12 different samples for the presence of multiple different analytes. Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots was detected using a CCD camera. Automated image analysis was used to determine a mean fluorescence value for each assay spot and to subtract the local background signal. The location of the spot and its mean fluorescence value were used to determine the toxin identity and concentration. Toxins were measured in clinical fluids, environmental samples and foods, with minimal sample preparation. Results are shown for rapid analyses of staphylococcal enterotoxin B, ricin, cholera toxin, botulinum toxoids, trinitrotoluene, and the mycotoxin fumonisin. Toxins were detected at levels as low as 0.5 ng mL(-1).

Frequency-domain photoacoustic and fluorescence microscopy: application on labeled and unlabeled cells

NASA Astrophysics Data System (ADS)

Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Pfeffer, Karoline; Wohlfarth, Sven; Hannesschläger, Günther; Klar, Thomas A.; Berer, Thomas

2018-02-01

In this paper, multimodal optical-resolution frequency-domain photoacoustic and fluorescence scanning microscopy is presented on labeled and unlabeled cells. In many molecules, excited electrons relax radiatively and non-radiatively, leading to fluorescence and photoacoustic signals, respectively. Both signals can then be detected simultaneously. There also exist molecules, e.g. hemoglobin, which do not exhibit fluorescence, but provide photoacoustic signals solely. Other molecules, especially fluorescent dyes, preferentially exhibit fluorescence. The fluorescence quantum yield of a molecule and with it the strength of photoacoustic and fluorescence signals depends on the local environment, e.g. on the pH. Therefore, the local distribution of the simultaneously recorded photoacoustic and fluorescence signals may be used in order to obtain information about the local chemistry.

Early tumor detection afforded by in vivo imaging of near-infrared II fluorescence.

PubMed

Tao, Zhimin; Dang, Xiangnan; Huang, Xing; Muzumdar, Mandar D; Xu, Eric S; Bardhan, Neelkanth Manoj; Song, Haiqin; Qi, Ruogu; Yu, Yingjie; Li, Ting; Wei, Wei; Wyckoff, Jeffrey; Birrer, Michael J; Belcher, Angela M; Ghoroghchian, P Peter

2017-07-01

Cell-intrinsic reporters such as luciferase (LUC) and red fluorescent protein (RFP) have been commonly utilized in preclinical studies to image tumor growth and to monitor therapeutic responses. While extrinsic reporters that emit near infrared I (NIR-I: 650-950 nm) or near-infrared II (NIR-II: 1000-1700 nm) optical signals have enabled minimization of tissue autofluorescence and light scattering, it has remained unclear as to whether their use has afforded more accurate tumor imaging in small animals. Here, we developed a novel optical imaging construct comprised of rare earth lanthanide nanoparticles coated with biodegradable diblock copolymers and doped with organic fluorophores, generating NIR-I and NIR-II emissive bands upon optical excitation. Simultaneous injection of multiple spectrally-unique nanoparticles into mice bearing tumor implants established via intraperitoneal dissemination of LUC + /RFP + OVCAR-8 ovarian cancer cells enabled direct comparisons of imaging with extrinsic vs. intrinsic reporters, NIR-II vs. NIR-I signals, as well as targeted vs. untargeted exogenous contrast agents in the same animal and over time. We discovered that in vivo optical imaging at NIR-II wavelengths facilitates more accurate detection of smaller and earlier tumor deposits, offering enhanced sensitivity, improved spatial contrast, and increased depths of tissue penetration as compared to imaging with visible or NIR-I fluorescent agents. Our work further highlights the hitherto underappreciated enhancements in tumor accumulation that may be achieved with intraperitoneal as opposed to intravenous administration of nanoparticles. Lastly, we found discrepancies in the fidelity of tumor uptake that could be obtained by utilizing small molecules for in vivo as opposed to in vitro targeting of nanoparticles to disseminated tumors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Image-guided intraocular injection using multimodality optical coherence tomography and fluorescence confocal scanning laser ophthalmoscopy in rodent ophthalmological models

NASA Astrophysics Data System (ADS)

Terrones, Benjamin D.; Benavides, Oscar R.; Leeburg, Kelsey C.; Mehanathan, Sankarathi B.; Levine, Edward M.; Tao, Yuankai K.

2018-02-01

Intraocular injections are routinely performed for delivery of anti-VEGF and anti-inflammatory therapies in humans. While these injections are also performed in mice to develop novel models of ophthalmic diseases and screen novel therapeutics, the injection location and volume are not well-controlled and reproducible. We overcome limitations of conventional injections methods by developing a multimodality, long working distance, non-contact optical coherence tomography (OCT) and fluorescence confocal scanning laser ophthalmoscopy (cSLO) system for retinal imaging before and after injections. Our OCT+cSLO system combines a custom-built spectraldomain OCT engine (875+/-85 nm) with 125 kHz line-rate with a modified commercial cSLO with a maximum frame-rate of 30 fps (512 x 512 pix.). The system was designed for an overlapping OCT+cSLO field-of-view of 1.1 mm with a 7.76 mm working distance to the pupil. cSLO excitation light sources and filters were optimized for simultaneous GFP and tdTomato imaging. Lateral resolution was 3.02 µm for OCT and 2.74 μm for cSLO. Intravitreal injections of 5%, 10%, and 20% intralipid with Alex Fluor 488 were manually injected intraocularly in C57BL/6 mice. Post-injection imaging showed structural changes associated with retinal puncture, including the injection track, a retinal elevation, and detachment of the posterior hyaloid. OCT enables quantitative analysis of injection location and volumes whereas complementary cSLO improves specificity for identifying fluorescently labeled injected compounds and transgenic cells. The long working distance of our non-contact OCT+cSLO system is uniquely-suited for concurrent imaging with intraocular injections and may be applied for imaging of ophthalmic surgical dynamics and real-time image-guided injections.

In-vivo imaging of retinal nerve fiber layer vasculature: imaging - histology comparison

PubMed Central

Scoles, Drew; Gray, Daniel C; Hunter, Jennifer J; Wolfe, Robert; Gee, Bernard P; Geng, Ying; Masella, Benjamin D; Libby, Richard T; Russell, Stephen; Williams, David R; Merigan, William H

2009-01-01

Background Although it has been suggested that alterations of nerve fiber layer vasculature may be involved in the etiology of eye diseases, including glaucoma, it has not been possible to examine this vasculature in-vivo. This report describes a novel imaging method, fluorescence adaptive optics (FAO) scanning laser ophthalmoscopy (SLO), that makes possible for the first time in-vivo imaging of this vasculature in the living macaque, comparing in-vivo and ex-vivo imaging of this vascular bed. Methods We injected sodium fluorescein intravenously in two macaque monkeys while imaging the retina with an FAO-SLO. An argon laser provided the 488 nm excitation source for fluorescence imaging. Reflectance images, obtained simultaneously with near infrared light, permitted precise surface registration of individual frames of the fluorescence imaging. In-vivo imaging was then compared to ex-vivo confocal microscopy of the same tissue. Results Superficial focus (innermost retina) at all depths within the NFL revealed a vasculature with extremely long capillaries, thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, typical of the radial peripapillary capillaries (RPCs). However, at a deeper focus beneath the NFL, (toward outer retina) the polygonal pattern typical of the ganglion cell layer (inner) and outer retinal vasculature was seen. These distinguishing patterns were also seen on histological examination of the same retinas. Furthermore, the thickness of the RPC beds and the caliber of individual RPCs determined by imaging closely matched that measured in histological sections. Conclusion This robust method demonstrates in-vivo, high-resolution, confocal imaging of the vasculature through the full thickness of the NFL in the living macaque, in precise agreement with histology. FAO provides a new tool to examine possible primary or secondary role of the nerve fiber layer vasculature in retinal vascular disorders and other eye diseases, such as glaucoma. PMID:19698151

Automatic neuron segmentation and neural network analysis method for phase contrast microscopy images.

PubMed

Pang, Jincheng; Özkucur, Nurdan; Ren, Michael; Kaplan, David L; Levin, Michael; Miller, Eric L

2015-11-01

Phase Contrast Microscopy (PCM) is an important tool for the long term study of living cells. Unlike fluorescence methods which suffer from photobleaching of fluorophore or dye molecules, PCM image contrast is generated by the natural variations in optical index of refraction. Unfortunately, the same physical principles which allow for these studies give rise to complex artifacts in the raw PCM imagery. Of particular interest in this paper are neuron images where these image imperfections manifest in very different ways for the two structures of specific interest: cell bodies (somas) and dendrites. To address these challenges, we introduce a novel parametric image model using the level set framework and an associated variational approach which simultaneously restores and segments this class of images. Using this technique as the basis for an automated image analysis pipeline, results for both the synthetic and real images validate and demonstrate the advantages of our approach.

Experimental demonstration of direct L-shell x-ray fluorescence imaging of gold nanoparticles using a benchtop x-ray source

PubMed Central

Manohar, Nivedh; Reynoso, Francisco J.; Cho, Sang Hyun

2013-01-01

Purpose: To develop a proof-of-principle L-shell x-ray fluorescence (XRF) imaging system that locates and quantifies sparse concentrations of gold nanoparticles (GNPs) using a benchtop polychromatic x-ray source and a silicon (Si)-PIN diode x-ray detector system. Methods: 12-mm-diameter water-filled cylindrical tubes with GNP concentrations of 20, 10, 5, 0.5, 0.05, 0.005, and 0 mg/cm3 served as calibration phantoms. An imaging phantom was created using the same cylindrical tube but filled with tissue-equivalent gel containing structures mimicking a GNP-loaded blood vessel and approximately 1 cm3 tumor. Phantoms were irradiated by a 3-mm-diameter pencil-beam of 62 kVp x-rays filtered by 1 mm aluminum. Fluorescence/scatter photons from phantoms were detected at 90° with respect to the beam direction using a Si-PIN detector placed behind a 2.5-mm-diameter lead collimator. The imaging phantom was translated horizontally and vertically in 0.3-mm steps to image a 6 mm × 15 mm region of interest (ROI). For each phantom, the net L-shell XRF signal from GNPs was extracted from background, and then corrected for detection efficiency and in-phantom attenuation using a fluorescence-to-scatter normalization algorithm. Results: XRF measurements with calibration phantoms provided a calibration curve showing a linear relationship between corrected XRF signal and GNP mass per imaged voxel. Using the calibration curve, the detection limit (at the 95% confidence level) of the current experimental setup was estimated to be a GNP mass of 0.35 μg per imaged voxel (1.73 × 10−2 cm3). A 2D XRF map of the ROI was also successfully generated, reasonably matching the known spatial distribution as well as showing the local variation of GNP concentrations. Conclusions:L-shell XRF imaging can be a highly sensitive tool that has the capability of simultaneously imaging the spatial distribution and determining the local concentration of GNPs presented on the order of parts-per-million level within subcentimeter-sized ex vivo samples and superficial tumors during preclinical animal studies. PMID:23927295

Developing single-laser sources for multimodal coherent anti-Stokes Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Pegoraro, Adrian Frank

Coherent anti-Stokes Raman scattering (CARS) microscopy has developed rapidly and is opening the door to new types of experiments. This work describes the development of new laser sources for CARS microscopy and their use for different applications. It is specifically focused on multimodal nonlinear optical microscopy—the simultaneous combination of different imaging techniques. This allows us to address a diverse range of applications, such as the study of biomaterials, fluid inclusions, atherosclerosis, hepatitis C infection in cells, and ice formation in cells. For these applications new laser sources are developed that allow for practical multimodal imaging. For example, it is shown that using a single Ti:sapphire oscillator with a photonic crystal fiber, it is possible to develop a versatile multimodal imaging system using optimally chirped laser pulses. This system can perform simultaneous two photon excited fluorescence, second harmonic generation, and CARS microscopy. The versatility of the system is further demonstrated by showing that it is possible to probe different Raman modes using CARS microscopy simply by changing a time delay between the excitation beams. Using optimally chirped pulses also enables further simplification of the laser system required by using a single fiber laser combined with nonlinear optical fibers to perform effective multimodal imaging. While these sources are useful for practical multimodal imaging, it is believed that for further improvements in CARS microscopy sensitivity, new excitation schemes are necessary. This has led to the design of a new, high power, extended cavity oscillator that should be capable of implementing new excitation schemes for CARS microscopy as well as other techniques. Our interest in multimodal imaging has led us to other areas of research as well. For example, a fiber-coupling scheme for signal collection in the forward direction is demonstrated that allows for fluorescence lifetime imaging without significant temporal distortion. Also highlighted is an imaging artifact that is unique to CARS microscopy that can alter image interpretation, especially when using multimodal imaging. By combining expertise in nonlinear optics, laser development, fiber optics, and microscopy, we have developed systems and techniques that will be of benefit for multimodal CARS microscopy.

Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain.

PubMed

Alves, Sandro; Bode, Julia; Bemelmans, Alexis-Pierre; von Kalle, Christof; Cartier, Nathalie; Tews, Björn

2016-06-20

Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. UM was applied to obtain high-resolution 3D analysis of AAV transduction in adult mouse brains, especially in the hippocampus, a region of interest for Alzheimer's disease therapy. We separately or simultaneously compared transduction efficacies for commonly used serotypes (AAV9 and AAVrh10) using fluorescent reporter expression. We provide a detailed comparative and quantitative analysis of the transduction profiles. UM allowed a rapid analysis of marker fluorescence expression in neurons with intact projections deep inside the brain, in defined anatomical structures. Major hippocampal neuronal transduction was observed with both vectors, with slightly better efficacy for AAV9 in UM. Glial response and synaptic marker expression did not change post transduction.We propose UM as a novel valuable complementary tool to efficiently and simultaneously unravel tropism of different viruses in a single non-dissected adult rodent brain.

Designing a multicolor long range nanoscopic ruler for the imaging of heterogeneous tumor cells

NASA Astrophysics Data System (ADS)

Chavva, Suhash Reddy; Viraka Nellore, Bhanu Priya; Pramanik, Avijit; Sinha, Sudarson Sekhar; Jones, Stacy; Ray, Paresh Chandra

2016-07-01

Tumor heterogeneity is one of the biggest challenges in cancer treatment and diagnosis. A multicolor optical ruler is essential to address the heterogeneous tumor cell complexity. Driven by this need, the current article reports the design of a multicolor long range nanoscopic ruler for screening tumor heterogeneity by accurately identifying epithelial cells and cancer stem cells (CSCs) simultaneously. A nanoscopic surface energy transfer (NSET) ruler has been developed using blue fluorescence polymer dots (PDs) and red fluorescence gold cluster dots (GCDs) as multicolor fluorescence donor and plasmonic gold nanoparticle (GNP) acts as an excellent acceptor. Reported experimental results demonstrated that the multicolor nanoscopic ruler's working window is above 35 nm distances, which is more than three times farther than that of Förster resonance energy transfer (FRET) distance limit. Theoretical modeling using Förster dipole-dipole coupling and dipole to nanoparticle surface energy transfer have been used to discuss the possible mechanism for multicolor nanoscopic ruler's long-range capability. Using RNA aptamers that are specific for the target cancer cells, experimental data demonstrate that the nanoscopic ruler can be used for screening epithelial and CSCs simultaneously from a whole blood sample with a detection capability of 10 cells per mL. Experimental data show that the nanoscopic ruler can distinguish targeted cells from non-targeted cells.

Simultaneous Gram and viability staining on activated sludge exposed to erythromycin: 3D CLSM time-lapse imaging of bacterial disintegration.

PubMed

Louvet, Jean-Noël; Attik, Ghania; Dumas, Dominique; Potier, Olivier; Pons, Marie-Noëlle

2011-11-01

The effect of erythromycin on activated sludge bacteria according to their Gram type was investigated with 3-dimensional Confocal Laser Scanning Microscopy (CLSM) time-lapse imaging. The fluorescent stains SYTOX Green and Texas Red-X conjugate of wheat germ agglutinin stained dying bacteria and Gram(+) bacteria respectively. Time-lapse imaging allowed an understanding of the staining mechanism and the measurement of the death rate. In presence of erythromycin (10mg/L), Gram(+) bacteria had a higher mortality rate than the Gram(-) bacteria. This result suggests that antibiotic in wastewater could change the activated sludge bacteria composition, according to their Gram type by selecting the bacteria which are the least sensitive to the antibiotics. However bacterial death was followed by bacterial disintegration leading to a decrease in the fluorescence. Results suggested that the viability indicators based on membrane integrity should be used with a correct sampling method, which can give the initial quantity of living bacteria. Copyright © 2011 Elsevier GmbH. All rights reserved.

Folate and Heptamethine Cyanine Modified Chitosan-Based Nanotheranostics for Tumor Targeted Near-Infrared Fluorescence Imaging and Photodynamic Therapy.

PubMed

Zhang, Yingying; Lv, Tingting; Zhang, Huijuan; Xie, Xiaodong; Li, Ziying; Chen, Haijun; Gao, Yu

2017-07-10

Folate (FA) and heptamethine cyanine (Cy7)-modified chitosan (CF7) was synthesized by click chemistry and its self-assembled nanoparticles (CF7Ns) were developed for tumor-specific imaging and photodynamic therapy. The characterization spectrum confirmed CF7 had a good FA and Cy7 conjugation efficacy. The diameter of CF7Ns measured by DLS was about 291.6 nm, and the morphology observed with AFM showed filamentous clusters of particles. The results of targeting ability of CF7Ns demonstrated enhanced targeting behaviors of CF7Ns compared with non-FA-modified nanoparticles C7Ns in FA receptor-positive HeLa cells. The cytotoxicity and cell apoptosis assay showed that CF7Ns under near-infrared light irradiation led to more apoptotic cell death in HeLa cells to improve the therapeutic efficacy. The mechanisms of the photodynamic effects of CF7Ns were demonstrated through measurement of intracellular reactive oxygen species and the apoptosis-related cytokines. These results suggested that CF7Ns are promising tumor targeting carriers for simultaneous fluorescence imaging and photodynamic therapy.

Fluorescence exclusion: A simple versatile technique to calculate cell volumes and local heights (Conference Presentation)

NASA Astrophysics Data System (ADS)

Thouvenin, Olivier; Fink, Mathias; Boccara, A. Claude

2017-02-01

Understanding volume regulation during mitosis is technically challenging. Indeed, a very sensitive non invasive imaging over time scales ranging from seconds to hours and over large fields is required. Therefore, Quantitative Phase Imaging (QPI) would be a perfect tool for such a project. However, because of asymmetric protein segregation during mitosis, an efficient separation of the refractive index and the height in the phase signal is required. Even though many strategies to make such a separation have been developed, they usually are difficult to implement, have poor sensitivity, or cannot be performed in living cells, or in a single shot. In this paper, we will discuss the use of a new technique called fluorescence exclusion to perform volume measurements. By coupling such technique with a simultaneous phase measurement, we were also able to recover the refractive index inside the cells. Fluorescence exclusion is a versatile and powerful technique that allows the volume measurement of many types of cells. A fluorescent dye, which cannot penetrate inside the cells, is mixed with the external medium in a confined environment. Therefore, the fluorescent signal depends on the inverse of the object's height. We could demonstrate both experimentally and theoretically that fluorescence exclusion can accurately measure cell volumes, even for cells much higher than the depth of focus of the objective. A local accurate height and RI measurement can also be obtained for smaller cells. We will also discuss the way to optimize the confinement of the observation chamber, either mechanically or optically.

Gold Nanobeacons for Tracking Gene Silencing in Zebrafish

PubMed Central

Cordeiro, Milton; Carvalho, Lara; Silva, Joana; Saúde, Leonor; Fernandes, Alexandra R.; Baptista, Pedro V.

2017-01-01

The use of gold nanoparticles for effective gene silencing has demonstrated its potential as a tool for gene expression experiments and for the treatment of several diseases. Here, we used a gold nanobeacon designed to specifically silence the enhanced green fluorescence protein (EGFP) mRNA in embryos of a fli-EGFP transgenic zebrafish line, while simultaneously allowing the tracking and localization of the silencing events via the beacon’s emission. Fluorescence imaging measurements demonstrated a decrease of the EGFP emission with a concomitant increase in the fluorescence of the Au-nanobeacon. Furthermore, microinjection of the Au-nanobeacon led to a negligible difference in mortality and malformations in comparison to the free oligonucleotide, indicating that this system is a biocompatible platform for the administration of gene silencing moieties. Together, these data illustrate the potential of Au-nanobeacons as tools for in vivo zebrafish gene modulation with low toxicity which may be used towards any gene of interest. PMID:28336844

Compact fixed wavelength femtosecond oscillators as an add-on for tunable Ti:sapphire lasers extend the range of applications towards multimodal imaging and optogenetics

NASA Astrophysics Data System (ADS)

Hakulinen, T.; Klein, J.

2016-03-01

Two-photon (2P) microscopy based on tunable Ti:sapphire lasers has become a widespread tool for 3D imaging with sub-cellular resolution in living tissues. In recent years multi-photon microscopy with simpler fixed-wavelength femtosecond oscillators using Yb-doped tungstenates as gain material has raised increasing interest in life-sciences, because these lasers offer one order of magnitude more average power than Ti:sapphire lasers in the wavelength range around 1040 nm: Two-photon (2P) excitation of mainly red or yellow fluorescent dyes and proteins (e.g. YFP, mFruit series) simultaneously has been proven with a single IR laser wavelength. A new approach is to extend the usability of existing tunable Titanium sapphire lasers by adding a fixed IR wavelength with an Yb femtosecond oscillator. By that means a multitude of applications for multimodal imaging and optogenetics can be supported. Furthermore fs Yb-lasers are available with a repetition rate of typically 10 MHz and an average power of typically 5 W resulting in pulse energy of typically 500 nJ, which is comparably high for fs-oscillators. This makes them an ideal tool for two-photon spinning disk laser scanning microscopy and holographic patterning for simultaneous photoactivation of large cell populations. With this work we demonstrate that economical, small-footprint Yb fixed-wavelength lasers can present an interesting add-on to tunable lasers that are commonly used in multiphoton microscopy. The Yb fs-lasers hereby offer higher power for imaging of red fluorescent dyes and proteins, are ideally enhancing existing Ti:sapphire lasers with more power in the IR, and are supporting pulse energy and power hungry applications such as spinning disk microscopy and holographic patterning.

Fabricating multifunctional microbubbles and nanobubbles for concurrent ultrasound and photoacoustic imaging

NASA Astrophysics Data System (ADS)

Qin, Ruogu; Xu, Jeff; Xu, Ronald; Kim, Chulhong; Wang, Lihong V.

2010-02-01

Background: Clinical ultrasound (US) uses ultrasonic scattering contrast to characterize subcutaneous anatomic structures. Photoacoustic (PA) imaging detects the functional properties of thick biological tissue with high optical contrast. In the case of image-guided cancer ablation therapy, simultaneous US and PA imaging can be useful for intraoperative assessment of tumor boundaries and ablation margins. In this regard, accurate co-registration between imaging modalities and high sensitivity to cancer cells are important. Methods: We synthesized poly-lactic-co-glycolic acid (PLGA) microbubbles (MBs) and nanobubbles (NBs) encapsulating India ink or indocyanine green (ICG). Multiple tumor simulators were fabricated by entrapping ink MBs or NBs at various concentrations in gelatin phantoms for simultaneous US and PA imaging. MBs and NBs were also conjugated with CC49 antibody to target TAG-72, a human glycoprotein complex expressed in many epithelial-derived cancers. Results: Accurate co-registration and intensity correlation were observed in US and PA images of MB and NB tumor simulators. MBs and NBs conjugating with CC49 effectively bound with over-expressed TAG-72 in LS174T colon cancer cell cultures. ICG was also encapsulated in MBs and NBs for the potential to integrate US, PA, and fluorescence imaging. Conclusions: Multifunctional MBs and NBs can be potentially used as a general contrast agent for multimodal intraoperative imaging of tumor boundaries and therapeutic margins.

Multiple functionalization of fluorescent nanoparticles for specific biolabeling and drug delivery of dopamine

NASA Astrophysics Data System (ADS)

Malvindi, Maria Ada; di Corato, Riccardo; Curcio, Annalisa; Melisi, Daniela; Rimoli, Maria Grazia; Tortiglione, Claudia; Tino, Angela; George, Chandramohan; Brunetti, Virgilio; Cingolani, Roberto; Pellegrino, Teresa; Ragusa, Andrea

2011-12-01

The development of fluorescent biolabels for specific targeting and controlled drug release is of paramount importance in biological applications due to their potential in the generation of novel tools for simultaneous diagnosis and treatment of diseases. Dopamine is a neurotransmitter involved in several neurological diseases, such as Parkinson's disease and attention deficit hyperactivity disorder (ADHD), and the controlled delivery of its agonists already proved to have beneficial effects both in vitro and in vivo. Here, we report the synthesis and multiple functionalization of highly fluorescent CdSe/CdS quantum rods for specific biolabeling and controlled drug release. After being transferred into aqueous media, the nanocrystals were made highly biocompatible through PEG conjugation and covered by a carbohydrate shell, which allowed specific GLUT-1 recognition. Controlled attachment of dopamine through an ester bond also allowed hydrolysis by esterases, yielding a smart nanotool for specific biolabeling and controlled drug release.The development of fluorescent biolabels for specific targeting and controlled drug release is of paramount importance in biological applications due to their potential in the generation of novel tools for simultaneous diagnosis and treatment of diseases. Dopamine is a neurotransmitter involved in several neurological diseases, such as Parkinson's disease and attention deficit hyperactivity disorder (ADHD), and the controlled delivery of its agonists already proved to have beneficial effects both in vitro and in vivo. Here, we report the synthesis and multiple functionalization of highly fluorescent CdSe/CdS quantum rods for specific biolabeling and controlled drug release. After being transferred into aqueous media, the nanocrystals were made highly biocompatible through PEG conjugation and covered by a carbohydrate shell, which allowed specific GLUT-1 recognition. Controlled attachment of dopamine through an ester bond also allowed hydrolysis by esterases, yielding a smart nanotool for specific biolabeling and controlled drug release. Electronic supplementary information (ESI) available: TEM images, absorption and emission spectra, ζ-potential and DLS graphics, gel electrophoresis images, cyclic voltammograms, western blot and RT-PCR data. See DOI: 10.1039/c1nr10797f

Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots.

PubMed

Kakizuka, Taishi; Ikezaki, Keigo; Kaneshiro, Junichi; Fujita, Hideaki; Watanabe, Tomonobu M; Ichimura, Taro

2016-07-01

Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery.

Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots

PubMed Central

Kakizuka, Taishi; Ikezaki, Keigo; Kaneshiro, Junichi; Fujita, Hideaki; Watanabe, Tomonobu M.; Ichimura, Taro

2016-01-01

Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery. PMID:27446684

Feedback mechanism for smart nozzles and nebulizers

DOEpatents

Montaser, Akbar [Potomac, MD; Jorabchi, Kaveh [Arlington, VA; Kahen, Kaveh [Kleinburg, CA

2009-01-27

Nozzles and nebulizers able to produce aerosol with optimum and reproducible quality based on feedback information obtained using laser imaging techniques. Two laser-based imaging techniques based on particle image velocimetry (PTV) and optical patternation map and contrast size and velocity distributions for indirect and direct pneumatic nebulizations in plasma spectrometry. Two pulses from thin laser sheet with known time difference illuminate droplets flow field. Charge coupled device (CCL)) captures scattering of laser light from droplets, providing two instantaneous particle images. Pointwise cross-correlation of corresponding images yields two-dimensional velocity map of aerosol velocity field. For droplet size distribution studies, solution is doped with fluorescent dye and both laser induced florescence (LIF) and Mie scattering images are captured simultaneously by two CCDs with the same field of view. Ratio of LIF/Mie images provides relative droplet size information, then scaled by point calibration method via phase Doppler particle analyzer.

Flame imaging using planar laser induced fluorescence of sulfur dioxide

NASA Astrophysics Data System (ADS)

Honza, Rene; Ding, Carl-Philipp; Dreizler, Andreas; Böhm, Benjamin

2017-09-01

Laser induced fluorescence of sulfur dioxide (SO2-PLIF) has been demonstrated as a useful tool for flame imaging. Advantage was taken from the strong temperature dependence of the SO2 fluorescence signal. SO2 fluorescence intensity increases by more than one order of magnitude if the temperature changes from ambient conditions to adiabatic flame temperatures of stoichiometric methane-air flames. This results in a steep gradient of SO2-PLIF intensities at the reaction zone and therefore can be used as a reliable flame marker. SO2 can be excited electronically using the fourth-harmonic of an Nd:YAG laser at 266 nm. This is an attractive alternative to OH-LIF, a well-recognized flame front marker, because no frequency-doubled dye lasers are needed. This simplifies the experimental setup and is advantageous for measurements at high repetition rates where dye bleaching can become an issue. To prove the performance of this approach, SO2-PLIF measurements were performed simultaneously with OH-PLIF on laminar premixed methane-air Bunsen flames for equivalence ratios between 0.9 and 1.25. These measurements were compared to 1D laminar flamelet simulations. The SO2 fluorescence signal was found to follow the temperature rise of the flame and is located closer to the steep temperature gradient than OH. Finally, the combined SO2- and OH-PLIF setup was applied to a spark ignition IC-engine to visualize the development of the early flame kernel.

Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye

PubMed Central

Sharma, Robin; Williams, David R.; Palczewska, Grazyna; Palczewski, Krzysztof; Hunter, Jennifer J.

2016-01-01

Purpose Although extrinsic fluorophores can be introduced to label specific cell types in the retina, endogenous fluorophores, such as NAD(P)H, FAD, collagen, and others, are present in all retinal layers. These molecules are a potential source of optical contrast and can enable noninvasive visualization of all cellular layers. We used a two-photon fluorescence adaptive optics scanning light ophthalmoscope (TPF-AOSLO) to explore the native autofluorescence of various cell classes spanning several layers in the unlabeled retina of a living primate eye. Methods Three macaques were imaged on separate occasions using a custom TPF-AOSLO. Two-photon fluorescence was evoked by pulsed light at 730 and 920 nm excitation wavelengths, while fluorescence emission was collected in the visible range from several retinal layers and different locations. Backscattered light was recorded simultaneously in confocal modality and images were postprocessed to remove eye motion. Results All retinal layers yielded two-photon signals and the heterogeneous distribution of fluorophores provided optical contrast. Several structural features were observed, such as autofluorescence from vessel walls, Müller cell processes in the nerve fibers, mosaics of cells in the ganglion cell and other nuclear layers of the inner retina, as well as photoreceptor and RPE layers in the outer retina. Conclusions This in vivo survey of two-photon autofluorescence throughout the primate retina demonstrates a wider variety of structural detail in the living eye than is available through conventional imaging methods, and broadens the use of two-photon imaging of normal and diseased eyes. PMID:26903224

Evidence of excited state localization and static disorder in LH2 investigated by 2D-polarization single-molecule imaging at room temperature.

PubMed

Tubasum, Sumera; Camacho, Rafael; Meyer, Matthias; Yadav, Dheerendra; Cogdell, Richard J; Pullerits, Tõnu; Scheblykin, Ivan G

2013-12-07

Two-dimensional polarization fluorescence imaging of single light harvesting complexes 2 (LH2) of Rps. acidophila was carried out to investigate the polarization properties of excitation and fluorescence emission simultaneously, at room temperature. In two separate experiments we excited LH2 with a spectrally narrow laser line matched to the absorption bands of the two chromophore rings, B800 and B850, thereby indirectly and directly triggering fluorescence of the B850 exciton state. A correlation analysis of the polarization modulation depths in excitation and emission for a large number of single complexes was performed. Our results show, in comparison to B800, that the B850 ring is a more isotropic absorber due to the excitonic nature of its excited states. At the same time, we observed a strong tendency for LH2 to emit with dipolar character, from which preferential localization of the emissive exciton, stable for minutes, is inferred. We argue that the observed effects can consistently be explained by static energetic disorder and/or deformation of the complex, with possible involvement of exciton self-trapping.

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

PubMed Central

Provost, Christopher R.; Sun, Luo

2010-01-01

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells. PMID:20485262

Monte Carlo-based fluorescence molecular tomography reconstruction method accelerated by a cluster of graphic processing units.

PubMed

Quan, Guotao; Gong, Hui; Deng, Yong; Fu, Jianwei; Luo, Qingming

2011-02-01

High-speed fluorescence molecular tomography (FMT) reconstruction for 3-D heterogeneous media is still one of the most challenging problems in diffusive optical fluorescence imaging. In this paper, we propose a fast FMT reconstruction method that is based on Monte Carlo (MC) simulation and accelerated by a cluster of graphics processing units (GPUs). Based on the Message Passing Interface standard, we modified the MC code for fast FMT reconstruction, and different Green's functions representing the flux distribution in media are calculated simultaneously by different GPUs in the cluster. A load-balancing method was also developed to increase the computational efficiency. By applying the Fréchet derivative, a Jacobian matrix is formed to reconstruct the distribution of the fluorochromes using the calculated Green's functions. Phantom experiments have shown that only 10 min are required to get reconstruction results with a cluster of 6 GPUs, rather than 6 h with a cluster of multiple dual opteron CPU nodes. Because of the advantages of high accuracy and suitability for 3-D heterogeneity media with refractive-index-unmatched boundaries from the MC simulation, the GPU cluster-accelerated method provides a reliable approach to high-speed reconstruction for FMT imaging.

Super-resolution atomic force photoactivated microscopy of biological samples (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lee, Seunghyun; Kim, Hyemin; Shin, Seungjun; Doh, Junsang; Kim, Chulhong

2017-03-01

Optical microscopy (OM) and photoacoustic microscopy (PAM) have previously been used to image the optical absorption of intercellular features of biological cells. However, the optical diffraction limit ( 200 nm) makes it difficult for these modalities to image nanoscale inner cell structures and the distribution of internal cell components. Although super-resolution fluorescence microscopy, such as stimulated emission depletion microscopy (STED) and stochastic optical reconstruction microscopy (STORM), has successfully performed nanoscale biological imaging, these modalities require the use of exogenous fluorescence agents, which are unfavorable for biological samples. Our newly developed atomic force photoactivated microscopy (AFPM) can provide optical absorption images with nanoscale lateral resolution without any exogenous contrast agents. AFPM combines conventional atomic force microscopy (AFM) and an optical excitation system, and simultaneously provides multiple contrasts, such as the topography and magnitude of optical absorption. AFPM can detect the intrinsic optical absorption of samples with 8 nm lateral resolution, easily overcoming the diffraction limit. Using the label-free AFPM system, we have successfully imaged the optical absorption properties of a single melanoma cell (B16F10) and a rosette leaf epidermal cell of Arabidopsis (ecotype Columbia (Col-0)) with nanoscale lateral resolution. The remarkable images show the melanosome distribution of a melanoma cell and the biological structures of a plant cell. AFPM provides superior imaging of optical absorption with a nanoscale lateral resolution, and it promises to become widely used in biological and chemical research.

Development of an endoscopic fluorescence image-guided OCT probe for oral cancer detection

NASA Astrophysics Data System (ADS)

McNichols, Roger J.; Gowda, Ashok; Bell, Brent A.; Johnigan, Richard M.; Calhoun, Karen H.; Motamedi, Massoud

2001-06-01

Oral squamous cell carcinoma is a disease which progresses through a number of well-defined morphological and biochemical changes. Optical coherence tomography (OCT) is a rapidly-evolving, non-invasive imaging modality which allows detailed probing of subsurface tissue structures with resolution on the order of microns. While this technique offers tremendous potential as a diagnostic tool for detection and characterization of oral cancer, OCT imaging is presently associated with a field of view on the order of millimeters, and acquisition time on the order of seconds. Thus, OCT's utility as a rapid cancer screening technique is presently limited. On the other hand, imaging of tissue autofluorescence provides a very rapid, high-throughput method for cancer screening. However, while autofluorescence measures may be sensitive to cancer, they are often non- specific and lead to a large number of false positives. In the present work, we have developed a fluorescence image guided optical coherence tomographic (FIG-OCT) probe in which tissue autofluorescence images are simultaneously used to guide OCT image acquisition of suspicious regions in real time. We have begun pre-clinical pilot studies with this instrument in a DMBA-induced model of oral cancer in the hamster cheek pouch. Initial results indicate that the FIG- OCT approach shows promise as a rapid and effective tool for screening of oral cancer.

Experimental Investigation of the Richtmyer-Meshkov Instability Through Simultaneous Measurements of Concentration and Velocity

NASA Astrophysics Data System (ADS)

Reese, Daniel; Ames, Alex; Noble, Chris; Oakley, Jason; Rothamer, Dave; Bonazza, Riccardo

2016-11-01

The present work investigates the evolution of the Richtmyer-Meshkov instability through simultaneous measurements of concentration and velocity. In the Wisconsin Shock Tube Laboratory at the University of Wisconsin, a broadband, shear-layer initial condition is created at the interface between helium and argon (Atwood number A = 0.7). The helium is seeded with acetone vapor for use in planar laser-induced fluorescence (PLIF), while each gas in the shear layer cross flow is seeded with particulate TiO2, which is used to track the flow and allow for the Mie scattering of light. Once impulsively accelerated by a M = 1.57 shock wave, the interface is imaged twice in close succession using a planar laser sheet containing both the second and fourth harmonic output (532 nm and 266 nm, respectively) of a dual-cavity Nd:YAG laser. Particle image pairs are captured on a dual-frame CCD camera, for use in particle image velocimetry (PIV), while PLIF images are corrected to show concentration. Velocity fields are obtained from particle images using the Insight 4G software package by TSI, and velocity field structure is investigated and compared against concentration images. Probability density functions (PDFs) and planar energy spectra (of both velocity fluctuations and concentration) are then calculated and results are discussed.

A bioanalytical platform for simultaneous detection and quantification of biological toxins.

PubMed

Weingart, Oliver G; Gao, Hui; Crevoisier, François; Heitger, Friedrich; Avondet, Marc-André; Sigrist, Hans

2012-01-01

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin's identity and concentration. The system's performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5-1 ng · mL(-1) in buffer or in raw milk.

A Bioanalytical Platform for Simultaneous Detection and Quantification of Biological Toxins

PubMed Central

Weingart, Oliver G.; Gao, Hui; Crevoisier, François; Heitger, Friedrich; Avondet, Marc-André; Sigrist, Hans

2012-01-01

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin’s identity and concentration. The system’s performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5–1 ng·mL−1 in buffer or in raw milk. PMID:22438766

Dual modality instrument for simultaneous optical coherence tomography imaging and fluorescence spectroscopy.

PubMed

Barton, Jennifer Kehlet; Guzman, Francisco; Tumlinson, Alexandre

2004-01-01

We develop a dual-modality device that combines the anatomical imaging capabilities of optical coherence tomography (OCT) with the functional capabilities of laser-induced fluorescence (LIF) spectroscopy. OCT provides cross-sectional images of tissue structure to a depth of up to 2 mm with approximately 10-microm resolution. LIF spectroscopy provides histochemical information in the form of emission spectra from a given tissue location. The OCT subsystem utilizes a superluminescent diode with a center wavelength of 1300 nm, whereas a helium cadmium laser provides the LIF excitation source at wavelengths of 325 and 442 nm. Preliminary data are obtained on eight postmortem aorta samples, each 10 mm in length. OCT images and LIF spectra give complementary information from normal and atherosclerotic portions of aorta wall. OCT images show structures such as intima, media, internal elastic lamina, and fibrotic regions. Emission spectra ratios of 520/490 (325-nm excitation) and 595/635 (442-nm excitation) could be used to identify normal and plaque regions with 97 and 91% correct classification rates, respectively. With miniaturization of the delivery probe and improvements in system speed, this dual-modality device could provide a valuable tool for identification and characterization of atherosclerotic plaques. (c) 2004 Society of Photo-Optical Instrumentation Engineers.

Development and application of the near-infrared and white-light thoracoscope system for minimally invasive lung cancer surgery

NASA Astrophysics Data System (ADS)

Mao, Yamin; Wang, Kun; He, Kunshan; Ye, Jinzuo; Yang, Fan; Zhou, Jian; Li, Hao; Chen, Xiuyuan; Wang, Jun; Chi, Chongwei; Tian, Jie

2017-06-01

In minimally invasive surgery, the white-light thoracoscope as a standard imaging tool is facing challenges of the low contrast between important anatomical or pathological regions and surrounding tissues. Recently, the near-infrared (NIR) fluorescence imaging shows superior advantages over the conventional white-light observation, which inspires researchers to develop imaging systems to improve overall outcomes of endoscopic imaging. We developed an NIR and white-light dual-channel thoracoscope system, which achieved high-fluorescent signal acquisition efficiency and the simultaneously optimal visualization of the NIR and color dual-channel signals. The system was designed to have fast and accurate image registration and high signal-to-background ratio by optimizing both software algorithms and optical hardware components for better performance in the NIR spectrum band. The system evaluation demonstrated that the minimally detectable concentration of indocyanine green (ICG) was 0.01 μM, and the spatial resolution was 35 μm. The in vivo feasibility of our system was verified by the preclinical experiments using six porcine models with the intravenous injection of ICG. Furthermore, the system was successfully applied for guiding the minimally invasive segmentectomy in three lung cancer patients, which revealed that our system held great promise for the clinical translation in lung cancer surgeries.

In vivo imaging of microscopic structures in the rat retina

PubMed Central

Geng, Ying; Greenberg, Kenneth P.; Wolfe, Robert; Gray, Daniel C.; Hunter, Jennifer J.; Dubra, Alfredo; Flannery, John G.; Williams, David R.; Porter, Jason

2010-01-01

Purpose The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. We have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo. Methods Green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy. Results Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics (AO). AO correction reduced the total root mean square wavefront error, on average, from 0.30 μm to 0.05 μm (1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was ∼1.84 μm, approximately 82% greater than the FWHM of the diffraction-limited LSF. Conclusions With perfect aberration compensation, the in vivo resolution in the rat eye could be ∼2× greater than that in the human eye due to its large numerical aperture (∼0.43). While the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using AO to investigate the rodent eye in vivo with high resolution. PMID:19578019

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy† †Electronic supplementary information (ESI) available: The LED device for the sample photobleaching, a schematic presentation of HILO microscopy, fluorescence spectra and hybridization curves of the molecular beacons, the linear correlation between the miRNA fluorescence intensity and the miRNA copy number, a validation of the miRNA adsorption and miRNA target gene expression via RT-qPCR, a validation of RT-qPCR using capillary electrophoresis, the reproducibility of RT-qPCR and Poisson distribution of the miRNA pipetting as well as a complete list of the oligonucleotides used in this study. See DOI: 10.1039/c7sc02701j Click here for additional data file.

PubMed Central

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun

2017-01-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target (HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells. PMID:28989695

Imaging hydrogen flames by two-photon, laser-induced fluorescence

NASA Technical Reports Server (NTRS)

Miles, R.; Lempert, W.; Kumar, V.; Diskin, G.

1991-01-01

A nonintrusive multicomponent imaging system is developed which can image hydrogen, hot oxygen, and air simultaneously. An Ar-F excimer laser is injection-locked to cover the Q1 two-photon transition in molecular hydrogen which allows the observation of both hot oxygen and cold hydrogen. Rayleigh scattering from the water molecules occurs at the same frequency as the illuminating laser allowing analysis of the air density. Images of ignited and nonignited hydrogen jets are recorded with a high-sensitivity gated video camera. The images permit the analysis of turbulent hydrogen-core jet, the combustion zone, and the surrounding air, and two-dimensional spatial correlations can be made to study the turbulent structure and couplings between different regions of the flow field. The method is of interest to the study of practical combustion systems which employ hydrogen-air diffusion flames.

One-Dimensional Spontaneous Raman Measurements Made in a Gas Turbine Combustor

NASA Technical Reports Server (NTRS)

DeGroot, Wilhelmus A.; Hicks, Yolanda R.; Locke, Randy J.; Anderson, Robert C.

2001-01-01

The NASA Glenn Research Center and the aerospace industry are designing and testing low-emission combustor concepts to build the next generation of cleaner, more fuel efficient aircraft powerplants. These combustors will operate at much higher inlet temperatures and at pressures that are up to 3 to 5 times greater than combustors in the current fleet. From a test and analysis viewpoint, there is an increasing need for measurements from these combustors that are nonintrusive, simultaneous, multipoint, and more quantitative. Glenn researchers have developed several unique test facilities (refs. 1 and 2) that allow, for the first time, optical interrogation of combustor flow fields, including subcomponent performance, at pressures ranging from 1 to 60 bar (1 to 60 atm). Experiments conducted at Glenn are the first application of a visible laser-pumped, one-dimensional, spontaneous Raman-scattering technique to analyze the flow in a high-pressure, advanced-concept fuel injector at pressures thus far reaching 12 bar (12 atm). This technique offers a complementary method to the existing two- and three-dimensional imaging methods used, such as planar laser-induced fluorescence. Raman measurements benefit from the fact that the signal from each species is a linear function of its density, and the relative densities of all major species can be acquired simultaneously with good precision. The Raman method has the added potential to calibrate multidimensional measurements by providing an independent measurement of species number-densities at known points within the planar laser-induced fluorescence images. The visible Raman method is similar to an ultraviolet-Raman technique first tried in the same test facility (ref. 3). However, the visible method did not suffer from the ultraviolet technique's fuel-born polycyclic aromatic hydrocarbon fluorescence interferences.

Live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix.

PubMed

Shih, Wenting; Yamada, Soichiro

2011-12-22

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network. By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Development and Evaluation of a Fluorescent Antibody-Drug Conjugate for Molecular Imaging and Targeted Therapy of Pancreatic Cancer

PubMed Central

Knutson, Steve; Raja, Erum; Bomgarden, Ryan; Nlend, Marie; Chen, Aoshuang; Kalyanasundaram, Ramaswamy; Desai, Surbhi

2016-01-01

Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming. Furthermore, antibody-drug conjugates (ADCs) used for tumor treatment generally remain distinct from conjugates used for diagnosis. Thus, there exists a need to develop simple dual-labeling methods for efficient therapeutic and diagnostic evaluation of antibody conjugates in pre-clinical model systems. Here, we present a rapid and simple method utilizing commercially available reagents for synthesizing a dual-labeled fluorescent ADC. Further, we demonstrate the fluorescent ADC’s utility for simultaneous targeted therapy and molecular imaging of cancer both in vitro and in vivo. Employing non-site-specific, amine-reactive chemistry, our novel biopharmaceutical theranostic is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to both paclitaxel and a near-infrared (NIR), polyethylene glycol modified (PEGylated) fluorophore (DyLight™ 680-4xPEG). Using in vitro systems, we demonstrate that this fluorescent ADC selectively binds a CEA-positive pancreatic cancer cell line (BxPC-3) in immunofluorescent staining and flow cytometry, exhibits efficient internalization kinetics, and is cytotoxic. Model studies using a xenograft of BxPC-3 cells in athymic mice also show the fluorescent ADC’s efficacy in detecting tumors in vivo and inhibiting tumor growth more effectively than equimolar amounts of unconjugated drug. Overall, our results demonstrate that non-selective, amine-targeting chemistry is an effective dual-labeling method for synthesizing and evaluating a bifunctional fluorescent antibody-drug conjugate, allowing concurrent detection, monitoring and treatment of cancer. PMID:27336622

Calibrating the imaging and therapy performance of magneto-fluorescent gold nanoshells for breast cancer

NASA Astrophysics Data System (ADS)

Dowell, Adam; Chen, Wenxue; Biswal, Nrusingh; Ayala-Orozco, Ciceron; Giuliano, Mario; Schiff, Rachel; Halas, Naomi J.; Joshi, Amit

2012-03-01

Gold nanoshells with NIR plasmon resonance can be modified to simultaneously enhance conjugated NIR fluorescence dyes and T2 contrast of embedded iron-oxide nanoparticles, and molecularly targeted to breast and other cancers. We calibrated the theranostic performance of magneto-fluorescent nanoshells, and contrasted the performance of molecularly targeted and untargeted nanoshells for breast cancer therapy, employing MCF-7L and their HER2 overexpressing derivative MCF-7/HER2-18 breast cancer cells as in vitro model systems. Silica core gold nanoshells with plasmon resonance on ~810 nm were doped with NIR dye ICG and ~10 nm iron-oxide nanoparticles in a ~20 nm epilayer of silica. A subset of nanoshells was conjugated to antibodies targeting HER2. Cell viability with varying laser power levels in presence and absence of bare and HER2-targeted nanoshells was assessed by calcein and propidium iodide staining. For MCF-7L cells, increasing power resulted in increased cell death (F=5.63, p=0.0018), and bare nanoshells caused more cell death than HER2-targeted nanoshells or laser treatment alone (F=30.13, p<0.001). For MCF-7/HER2-18 cells, death was greater with HER2-targeted nanoshells and was independent of laser power. This study demonstrates the capability of magneto-fluorescent nanocomplexes for imaging and therapy of breast cancer cells, and the advantages of targeting receptors unique to cancer cells.

Interface-Targeting Strategy Enables Two-Photon Fluorescent Lipid Droplet Probes for High-Fidelity Imaging of Turbid Tissues and Detecting Fatty Liver.

PubMed

Guo, Lifang; Tian, Minggang; Feng, Ruiqing; Zhang, Ge; Zhang, Ruoyao; Li, Xuechen; Liu, Zhiqiang; He, Xiuquan; Sun, Jing Zhi; Yu, Xiaoqiang

2018-04-04

Lipid droplets (LDs) with unique interfacial architecture not only play crucial roles in protecting a cell from lipotoxicity and lipoapoptosis but also closely relate with many diseases such as fatty liver and diabetes. Thus, as one of the important applied biomaterials, fluorescent probes with ultrahigh selectivity for in situ and high-fidelity imaging of LDs in living cells and tissues are critical to elucidate relevant physiological and pathological events as well as detect related diseases. However, available probes only utilizing LDs' waterless neutral cores but ignoring the unique phospholipid monolayer interfaces exhibit low selectivity. They cannot differentiate neutral cores of LDs from intracellular other lipophilic microenvironments, which results in extensively cloud-like background noise and severely limited their bioapplications. Herein, to design LD probes with ultrahigh selectivity, the exceptional interfacial architecture of LDs is considered adequately and thus an interface-targeting strategy is proposed for the first time. According to the novel strategy, we have developed two amphipathic fluorescent probes (N-Cy and N-Py) by introducing different cations into a lipophilic fluorophore (nitrobenzoxadiazole (NBD)). Consequently, their cationic moiety precisely locates the interfaces through electrostatic interaction and simultaneously NBD entirely embeds into the waterless core via hydrophobic interaction. Thus, high-fidelity and background-free fluorescence imaging of LDs are expectably realized in living cells in situ. Moreover, LDs in turbid tissues like skeletal muscle slices have been clearly imaged (up to 82 μm depth) by a two-photon microscope. Importantly, using N-Cy, we not only intuitively monitored the variations of LDs in number, size, and morphology but also clearly revealed their abnormity in hepatic tissues resulting from fatty liver. Therefore, these unique probes provide excellent imaging tools for elucidating LD-related physiological and pathological processes and the interface-targeting strategy possesses universal significance for designing probes with ultrahigh selectivity.

Full Field X-Ray Fluorescence Imaging Using Micro Pore Optics for Planetary Surface Exploration

NASA Technical Reports Server (NTRS)

Sarrazin, P.; Blake, D. F.; Gailhanou, M.; Walter, P.; Schyns, E.; Marchis, F.; Thompson, K.; Bristow, T.

2016-01-01

Many planetary surface processes leave evidence as small features in the sub-millimetre scale. Current planetary X-ray fluorescence spectrometers lack the spatial resolution to analyse such small features as they only provide global analyses of areas greater than 100 mm(exp 2). A micro-XRF spectrometer will be deployed on the NASA Mars 2020 rover to analyse spots as small as 120m. When using its line-scanning capacity combined to perpendicular scanning by the rover arm, elemental maps can be generated. We present a new instrument that provides full-field XRF imaging, alleviating the need for precise positioning and scanning mechanisms. The Mapping X-ray Fluorescence Spectrometer - "Map-X" - will allow elemental imaging with approximately 100µm spatial resolution and simultaneously provide elemental chemistry at the scale where many relict physical, chemical and biological features can be imaged in ancient rocks. The arm-mounted Map-X instrument is placed directly on the surface of an object and held in a fixed position during measurements. A 25x25 mm(exp 2) surface area is uniformly illuminated with X-rays or alpha-particles and gamma-rays. A novel Micro Pore Optic focusses a fraction of the emitted X-ray fluorescence onto a CCD operated at a few frames per second. On board processing allows measuring the energy and coordinates of each X-ray photon collected. Large sets of frames are reduced into 2d histograms used to compute higher level data products such as elemental maps and XRF spectra from selected regions of interest. XRF spectra are processed on the ground to further determine quantitative elemental compositions. The instrument development will be presented with an emphasis on the characterization and modelling of the X-ray focussing Micro Pore Optic. An outlook on possible alternative XRF imaging applications will be discussed.

Redox-Responsive Biomimetic Polymeric Micelle for Simultaneous Anticancer Drug Delivery and Aggregation-Induced Emission Active Imaging.

PubMed

Hu, Jun; Zhuang, Weihua; Ma, Boxuan; Su, Xin; Yu, Tao; Li, Gaocan; Hu, Yanfei; Wang, Yunbing

2018-05-10

Intelligent polymeric micelles have been developed as potential nanoplatforms for efficient drug delivery and diagnosis. Herein, we successfully prepared redox-sensitive polymeric micelles combined aggregation-induced emission (AIE) imaging as an outstanding anticancer drug carrier system for simultaneous chemotherapy and bioimaging. The amphiphilic copolymer TPE-SS-PLAsp- b-PMPC could self-assemble into spherical micelles, and these biomimetic micelles exhibited great biocompatibility and remarkable ability in antiprotein adsorption, showing great potential for biomedical application. Anticancer drug doxorubicin (DOX) could be encapsulated during the self-assembly process, and these drug-loaded micelles showed intelligent drug release and improved antitumor efficacy due to the quick disassembly in response to high levels of glutathione (GSH) in the environment. Moreover, the intracellular DOX release could be traced through the fluorescent imaging of these AIE micelles. As expected, the in vivo antitumor study exhibited that these DOX-carried micelles showed better antitumor efficacy and less adverse effects than that of free DOX. These results strongly indicated that this smart biomimetic micelle system would be a prominent candidate for chemotherapy and bioimaging.