A pragmatic guide to multiphoton microscope design

PubMed Central

Young, Michael D.; Field, Jeffrey J.; Sheetz, Kraig E.; Bartels, Randy A.; Squier, Jeff

2016-01-01

Multiphoton microscopy has emerged as a ubiquitous tool for studying microscopic structure and function across a broad range of disciplines. As such, the intent of this paper is to present a comprehensive resource for the construction and performance evaluation of a multiphoton microscope that will be understandable to the broad range of scientific fields that presently exploit, or wish to begin exploiting, this powerful technology. With this in mind, we have developed a guide to aid in the design of a multiphoton microscope. We discuss source selection, optical management of dispersion, image-relay systems with scan optics, objective-lens selection, single-element light-collection theory, photon-counting detection, image rendering, and finally, an illustrated guide for building an example microscope. PMID:27182429

Improvement of depth resolution on photoacoustic imaging using multiphoton absorption

NASA Astrophysics Data System (ADS)

Yamaoka, Yoshihisa; Fujiwara, Katsuji; Takamatsu, Tetsuro

2007-07-01

Commercial imaging systems, such as computed tomography and magnetic resonance imaging, are frequently used powerful tools for observing structures deep within the human body. However, they cannot precisely visualized several-tens micrometer-sized structures for lack of spatial resolution. In this presentation, we propose photoacoustic imaging using multiphoton absorption technique to generate ultrasonic waves as a means of improving depth resolution. Since the multiphoton absorption occurs at only the focus point and the employed infrared pulses deeply penetrate living tissues, it enables us to extract characteristic features of structures embedded in the living tissue. When nanosecond pulses from a 1064-nm Nd:YAG laser were focused on Rhodamine B/chloroform solution (absorption peak: 540 nm), the peak intensity of the generated photoacoustic signal was proportional to the square of the input pulse energy. This result shows that the photoacoustic signals can be induced by the two-photon absorption of infrared nanosecond pulse laser and also can be detected by a commercial low-frequency MHz transducer. Furthermore, in order to evaluate the depth resolution of multiphoton-photoacoustic imaging, we investigated the dependence of photoacoustic signal on depth position using a 1-mm-thick phantom in a water bath. We found that the depth resolution of two-photon photoacoustic imaging (1064 nm) is greater than that of one-photon photoacoustic imaging (532 nm). We conclude that evolving multiphoton-photoacoustic imaging technology renders feasible the investigation of biomedical phenomena at the deep layer in living tissue.

Advanced multiphoton methods for in vitro and in vivo functional imaging of mouse retinal neurons (Conference Presentation)

NASA Astrophysics Data System (ADS)

Cohen, Noam; Schejter, Adi; Farah, Nairouz; Shoham, Shy

2016-03-01

Studying the responses of retinal ganglion cell (RGC) populations has major significance in vision research. Multiphoton imaging of optogenetic probes has recently become the leading approach for visualizing neural populations and has specific advantages for imaging retinal activity during visual stimulation, because it leads to reduced direct photoreceptor excitation. However, multiphoton retinal activity imaging is not straightforward: point-by-point scanning leads to repeated neural excitation while optical access through the rodent eye in vivo has proven highly challenging. Here, we present two enabling optical designs for multiphoton imaging of responses to visual stimuli in mouse retinas expressing calcium indicators. First, we present an imaging solution based on Scanning Line Temporal Focusing (SLITE) for rapidly imaging neuronal activity in vitro. In this design, we scan a temporally focused line rather than a point, increasing the scan speed and reducing the impact of repeated excitation, while maintaining high optical sectioning. Second, we present the first in vivo demonstration of two-photon imaging of RGC activity in the mouse retina. To obtain these cellular resolution recordings we integrated an illumination path into a correction-free imaging system designed using an optical model of the mouse eye. This system can image at multiple depths using an electronically tunable lens integrated into its optical path. The new optical designs presented here overcome a number of outstanding obstacles, allowing the study of rapid calcium- and potentially even voltage-indicator signals both in vitro and in vivo, thereby bringing us a step closer toward distributed monitoring of action potentials.

Design and implementation of fiber-based multiphoton endoscopy with microelectromechanical systems scanning

PubMed Central

Tang, Shuo; Jung, Woonggyu; McCormick, Daniel; Xie, Tuqiang; Su, Jiangping; Ahn, Yeh-Chan; Tromberg, Bruce J.; Chen, Zhongping

2010-01-01

A multiphoton endoscopy system has been developed using a two-axis microelectromechanical systems (MEMS) mirror and double-cladding photonic crystal fiber (DCPCF). The MEMS mirror has a 2-mm-diam, 20-deg optical scanning angle, and 1.26-kHz and 780-Hz resonance frequencies on the x and y axes. The maximum number of resolvable focal spots of the MEMS scanner is 720×720 on the x and y axes, which indicates that the MEMS scanner can potentially support high-resolution multiphoton imaging. The DCPCF is compared with standard single-mode fiber and hollow-core photonic bandgap fiber on the basis of dispersion, attenuation, and coupling efficiency properties. The DCPCF has high collection efficiency, and its dispersion can be compensated by grating pairs. Three configurations of probe design are investigated, and their imaging quality and field of view are compared. A two-lens configuration with a collimation and a focusing lens provides the optimum imaging performance and packaging flexibility. The endoscope is applied to image fluorescent microspheres and bovine knee joint cartilage. PMID:19566298

Miniature fiber-optic multiphoton microscopy system using frequency-doubled femtosecond Er-doped fiber laser

PubMed Central

Huang, Lin; Mills, Arthur K.; Zhao, Yuan; Jones, David J.; Tang, Shuo

2016-01-01

We report on a miniature fiber-optic multiphoton microscopy (MPM) system based on a frequency-doubled femtosecond Er-doped fiber laser. The femtosecond pulses from the laser source are delivered to the miniature fiber-optic probe at 1.58 µm wavelength, where a standard single mode fiber is used for delivery without the need of free-space dispersion compensation components. The beam is frequency-doubled inside the probe by a periodically poled MgO:LiNbO3 crystal. Frequency-doubled pulses at 786 nm with a maximum power of 80 mW and a pulsewidth of 150 fs are obtained and applied to excite intrinsic signals from tissues. A MEMS scanner, a miniature objective, and a multimode collection fiber are further used to make the probe compact. The miniature fiber-optic MPM system is highly portable and robust. Ex vivo multiphoton imaging of mammalian skins demonstrates the capability of the system in imaging biological tissues. The results show that the miniature fiber-optic MPM system using frequency-doubled femtosecond fiber laser can potentially bring the MPM imaging for clinical applications. PMID:27231633

Visualizing Viral Infection In Vivo by Multi-Photon Intravital Microscopy.

PubMed

Sewald, Xaver

2018-06-20

Viral pathogens have adapted to the host organism to exploit the cellular machinery for virus replication and to modulate the host cells for efficient systemic dissemination and immune evasion. Much of our knowledge of the effects that virus infections have on cells originates from in vitro imaging studies using experimental culture systems consisting of cell lines and primary cells. Recently, intravital microscopy using multi-photon excitation of fluorophores has been applied to observe virus dissemination and pathogenesis in real-time under physiological conditions in living organisms. Critical steps during viral infection and pathogenesis could be studied by direct visualization of fluorescent virus particles, virus-infected cells, and the immune response to viral infection. In this review, I summarize the latest research on in vivo studies of viral infections using multi-photon intravital microscopy (MP-IVM). Initially, the underlying principle of multi-photon microscopy is introduced and experimental challenges during microsurgical animal preparation and fluorescent labeling strategies for intravital imaging are discussed. I will further highlight recent studies that combine MP-IVM with optogenetic tools and transcriptional analysis as a powerful approach to extend the significance of in vivo imaging studies of viral pathogens.

Collaborative Initiative in Biomedical Imaging to Study Complex Diseases

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

Lin, Weili; Fiddy, Michael A.

2012-03-31

The work reported addressed these topics: Fluorescence imaging; Optical coherence tomography; X-ray interferometer/phase imaging system; Quantitative imaging from scattered fields, Terahertz imaging and spectroscopy; and Multiphoton and Raman microscopy.

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

PubMed Central

Poland, Simon P.; Krstajić, Nikola; Monypenny, James; Coelho, Simao; Tyndall, David; Walker, Richard J.; Devauges, Viviane; Richardson, Justin; Dutton, Neale; Barber, Paul; Li, David Day-Uei; Suhling, Klaus; Ng, Tony; Henderson, Robert K.; Ameer-Beg, Simon M.

2015-01-01

We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction. PMID:25780724

In vivo histology: optical biopsies with chemical contrast using clinical multiphoton/coherent anti-Stokes Raman scattering tomography

NASA Astrophysics Data System (ADS)

Weinigel, M.; Breunig, H. G.; Kellner-Höfer, M.; Bückle, R.; Darvin, M. E.; Klemp, M.; Lademann, J.; König, K.

2014-05-01

The majority of existing coherent anti-Stokes Raman scattering (CARS) imaging systems are still huge and complicated laboratory systems and neither compact nor user-friendly nor mobile medically certified CARS systems. We have developed a new flexible multiphoton/CARS tomograph for imaging in a clinical environment. The system offers exceptional 360° flexibility with a very stable setup and enables label free ‘in vivo histology’ with chemical contrast within seconds. It can be completely operated by briefly trained non-laser experts. The imaging capability and flexibility of the novel in vivo tomograph are shown on optical biopsies with subcellular resolution and chemical contrast of patients suffering from psoriasis and squamous cell carcinoma.

Ophthalmic imaging using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Teng, Shu-Wen; Peng, Ju-Li; Lin, Huei-Hsing; Wu, Hai-Yin; Lo, Wen; Sun, Yen; Lin, Wei-Chou; Lin, Sung-Jan; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Dong, Chen-Yuan

2005-04-01

This purpose of this study is to demonstrate the feasibility of using multiphoton microscopy in ophthalmologic imaging. Without the introduction of extrinsic fluorescence molecules, multiphoton induced autofluorescence and second harmonic generation signals can be used to obtain useful structural information of normal and diseased corneas. Our work can potentially lead to the in vivo application of multiphoton microscopy in investigating corneal physiology and pathologies.

Visible-to-visible four-photon ultrahigh resolution microscopic imaging with 730-nm diode laser excited nanocrystals.

PubMed

Wang, Baoju; Zhan, Qiuqiang; Zhao, Yuxiang; Wu, Ruitao; Liu, Jing; He, Sailing

2016-01-25

Further development of multiphoton microscopic imaging is confronted with a number of limitations, including high-cost, high complexity and relatively low spatial resolution due to the long excitation wavelength. To overcome these problems, for the first time, we propose visible-to-visible four-photon ultrahigh resolution microscopic imaging by using a common cost-effective 730-nm laser diode to excite the prepared Nd(3+)-sensitized upconversion nanoparticles (Nd(3+)-UCNPs). An ordinary multiphoton scanning microscope system was built using a visible CW diode laser and the lateral imaging resolution as high as 161-nm was achieved via the four-photon upconversion process. The demonstrated large saturation excitation power for Nd(3+)-UCNPs would be more practical and facilitate the four-photon imaging in the application. A sample with fine structure was imaged to demonstrate the advantages of visible-to-visible four-photon ultrahigh resolution microscopic imaging with 730-nm diode laser excited nanocrystals. Combining the uniqueness of UCNPs, the proposed visible-to-visible four-photon imaging would be highly promising and attractive in the field of multiphoton imaging.

Microstructure imaging of human rectal mucosa using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Liu, N. R.; Chen, G.; Chen, J. X.; Yan, J.; Zhuo, S. M.; Zheng, L. Q.; Jiang, X. S.

2011-01-01

Multiphoton microscopy (MPM) has high resolution and sensitivity. In this study, MPM was used to image microstructure of human rectal mucosa. The morphology and distribution of the main components in mucosa layer, absorptive cells and goblet cells in the epithelium, abundant intestinal glands in the lamina propria and smooth muscle fibers in the muscularis mucosa were clearly monitored. The variations of these components were tightly relevant to the pathology in gastrointestine system, especially early rectal cancer. The obtained images will be helpful for the diagnosis of early colorectal cancer.

Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks

NASA Astrophysics Data System (ADS)

Dana, Hod; Marom, Anat; Paluch, Shir; Dvorkin, Roman; Brosh, Inbar; Shoham, Shy

2014-06-01

Planar neural networks and interfaces serve as versatile in vitro models of central nervous system physiology, but adaptations of related methods to three dimensions (3D) have met with limited success. Here, we demonstrate for the first time volumetric functional imaging in a bioengineered neural tissue growing in a transparent hydrogel with cortical cellular and synaptic densities, by introducing complementary new developments in nonlinear microscopy and neural tissue engineering. Our system uses a novel hybrid multiphoton microscope design combining a 3D scanning-line temporal-focusing subsystem and a conventional laser-scanning multiphoton microscope to provide functional and structural volumetric imaging capabilities: dense microscopic 3D sampling at tens of volumes per second of structures with mm-scale dimensions containing a network of over 1,000 developing cells with complex spontaneous activity patterns. These developments open new opportunities for large-scale neuronal interfacing and for applications of 3D engineered networks ranging from basic neuroscience to the screening of neuroactive substances.

Deep Tissue Fluorescent Imaging in Scattering Specimens Using Confocal Microscopy

PubMed Central

Clendenon, Sherry G.; Young, Pamela A.; Ferkowicz, Michael; Phillips, Carrie; Dunn, Kenneth W.

2015-01-01

In scattering specimens, multiphoton excitation and nondescanned detection improve imaging depth by a factor of 2 or more over confocal microscopy; however, imaging depth is still limited by scattering. We applied the concept of clearing to deep tissue imaging of highly scattering specimens. Clearing is a remarkably effective approach to improving image quality at depth using either confocal or multiphoton microscopy. Tissue clearing appears to eliminate the need for multiphoton excitation for deep tissue imaging. PMID:21729357

Video-rate resonant scanning multiphoton microscopy

PubMed Central

Kirkpatrick, Nathaniel D.; Chung, Euiheon; Cook, Daniel C.; Han, Xiaoxing; Gruionu, Gabriel; Liao, Shan; Munn, Lance L.; Padera, Timothy P.; Fukumura, Dai; Jain, Rakesh K.

2013-01-01

The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates—only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment. PMID:24353926

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

Imaging of cardiovascular structures using near-infrared femtosecond multiphoton laser scanning microscopy.

PubMed

Schenke-Layland, Katja; Riemann, Iris; Stock, Ulrich A; König, Karsten

2005-01-01

Multiphoton imaging represents a novel and very promising medical diagnostic technology for the high-resolution analysis of living biological tissues. We performed multiphoton imaging to analyzed structural features of extracellular matrix (ECM) components, e.g., collagen and elastin, of vital pulmonary and aortic heart valves. High-resolution autofluorescence images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and optical sectioning, without the requirement of embedding, fixation, or staining. Collagenous structures were selectively imaged by detection of second harmonic generation (SHG). Additionally, routine histology and electron microscopy were integrated to verify the observed results. In comparison with pulmonary tissues, aortic heart valve specimens show very similar matrix formations. The quality of the resulting three-dimensional (3-D) images enabled the differentiation between collagenous and elastic fibers. These experimental results indicate that multiphoton imaging with near-infrared (NIR) femtosecond laser pulses may prove to be a useful tool for the nondestructive monitoring and characterization of cardiovascular structures. Copyright 2005 Society of Photo-Optical Instrumentation Engineers.

Clinical optical coherence tomography combined with multiphoton tomography for evaluation of several skin disorders

NASA Astrophysics Data System (ADS)

König, Karsten; Speicher, Marco; Bückle, Rainer; Reckfort, Julia; McKenzie, Gordon; Welzel, Julia; Koehler, Martin J.; Elsner, Peter; Kaatz, Martin

2010-02-01

The first clinical trial of optical coherence tomography (OCT) combined with multiphoton tomography (MPT) and dermoscopy is reported. State-of-the-art (i) OCT systems for dermatology (e.g. multibeam swept source OCT), (ii) the femtosecond laser multiphoton tomograph DermaInspectTM, and (iii) digital dermoscopes were applied to 47 patients with a diversity of skin diseases and disorders such as skin cancer, psoriasis, hemangioma, connective tissue diseases, pigmented lesions, and autoimmune bullous skin diseases. Dermoscopy, also called 'epiluminescent microscopy', provides two-dimensional color images of the skin surface. OCT imaging is based on the detection of optical reflections within the tissue measured interferometrically whereas nonlinear excitation of endogenous fluorophores and the second harmonic generation are the bases of MPT images. OCT cross sectional "wide field" image provides a typical field of view of 5 x 2 mm2 and offers fast information on the depth and the volume of the investigated lesion. In comparison, multiphoton tomography presents 0.36 x 0.36 mm2 horizontal or diagonal sections of the region of interest within seconds with submicron resolution and down to a tissue depth of 200 μm. The combination of OCT and MPT provides a synergistic optical imaging modality for early detection of skin cancer and other skin diseases.

Maximizing fluorescence collection efficiency in multiphoton microscopy

PubMed Central

Zinter, Joseph P.; Levene, Michael J.

2011-01-01

Understanding fluorescence propagation through a multiphoton microscope is of critical importance in designing high performance systems capable of deep tissue imaging. Optical models of a scattering tissue sample and the Olympus 20X 0.95NA microscope objective were used to simulate fluorescence propagation as a function of imaging depth for physiologically relevant scattering parameters. The spatio-angular distribution of fluorescence at the objective back aperture derived from these simulations was used to design a simple, maximally efficient post-objective fluorescence collection system. Monte Carlo simulations corroborated by data from experimental tissue phantoms demonstrate collection efficiency improvements of 50% – 90% over conventional, non-optimized fluorescence collection geometries at large imaging depths. Imaging performance was verified by imaging layer V neurons in mouse cortex to a depth of 850 μm. PMID:21934897

Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

PubMed Central

Carriles, Ramón; Schafer, Dawn N.; Sheetz, Kraig E.; Field, Jeffrey J.; Cisek, Richard; Barzda, Virginijus; Sylvester, Anne W.; Squier, Jeffrey A.

2009-01-01

We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences. PMID:19725639

Multiphoton imaging microscopy at deeper layers with adaptive optics control of spherical aberration.

PubMed

Bueno, Juan M; Skorsetz, Martin; Palacios, Raquel; Gualda, Emilio J; Artal, Pablo

2014-01-01

Despite the inherent confocality and optical sectioning capabilities of multiphoton microscopy, three-dimensional (3-D) imaging of thick samples is limited by the specimen-induced aberrations. The combination of immersion objectives and sensorless adaptive optics (AO) techniques has been suggested to overcome this difficulty. However, a complex plane-by-plane correction of aberrations is required, and its performance depends on a set of image-based merit functions. We propose here an alternative approach to increase penetration depth in 3-D multiphoton microscopy imaging. It is based on the manipulation of the spherical aberration (SA) of the incident beam with an AO device while performing fast tomographic multiphoton imaging. When inducing SA, the image quality at best focus is reduced; however, better quality images are obtained from deeper planes within the sample. This is a compromise that enables registration of improved 3-D multiphoton images using nonimmersion objectives. Examples on ocular tissues and nonbiological samples providing different types of nonlinear signal are presented. The implementation of this technique in a future clinical instrument might provide a better visualization of corneal structures in living eyes.

Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation

NASA Astrophysics Data System (ADS)

Lien, Chi-Hsiang; Abrigo, Gerald; Chen, Pei-Hsuan; Chien, Fan-Ching

2017-02-01

Wavelength tunable temporal focusing multiphoton excitation microscopy (TFMPEM) is conducted to visualize optical sectioning images of multiple fluorophore-labeled specimens through the optimal two-photon excitation (TPE) of each type of fluorophore. The tunable range of excitation wavelength was determined by the groove density of the grating, the diffraction angle, the focal length of lenses, and the shifting distance of the first lens in the beam expander. Based on a consideration of the trade-off between the tunable-wavelength range and axial resolution of temporal focusing multiphoton excitation imaging, the presented system demonstrated a tunable-wavelength range from 770 to 920 nm using a diffraction grating with groove density of 830 lines/mm. TPE fluorescence imaging examination of a fluorescent thin film indicated that the width of the axial confined excitation was 3.0±0.7 μm and the shifting distance of the temporal focal plane was less than 0.95 μm within the presented wavelength tunable range. Fast different wavelength excitation and three-dimensionally rendered imaging of Hela cell mitochondria and cytoskeletons and mouse muscle fibers were demonstrated. Significantly, the proposed system can improve the quality of two-color TFMPEM images through different excitation wavelengths to obtain higher-quality fluorescent signals in multiple-fluorophore measurements.

Intra-operative label-free multimodal multiphoton imaging of breast cancer margins and microenvironment (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sun, Yi; You, Sixian; Tu, Haohua; Spillman, Darold R.; Marjanovic, Marina; Chaney, Eric J.; Liu, George Z.; Ray, Partha S.; Higham, Anna; Boppart, Stephen A.

2017-02-01

Label-free multi-photon imaging has been a powerful tool for studying tissue microstructures and biochemical distributions, particularly for investigating tumors and their microenvironments. However, it remains challenging for traditional bench-top multi-photon microscope systems to conduct ex vivo tumor tissue imaging in the operating room due to their bulky setups and laser sources. In this study, we designed, built, and clinically demonstrated a portable multi-modal nonlinear label-free microscope system that combined four modalities, including two- and three- photon fluorescence for studying the distributions of FAD and NADH, and second and third harmonic generation, respectively, for collagen fiber structures and the distribution of micro-vesicles found in tumors and the microenvironment. Optical realignments and switching between modalities were motorized for more rapid and efficient imaging and for a light-tight enclosure, reducing ambient light noise to only 5% within the brightly lit operating room. Using up to 20 mW of laser power after a 20x objective, this system can acquire multi-modal sets of images over 600 μm × 600 μm at an acquisition rate of 60 seconds using galvo-mirror scanning. This portable microscope system was demonstrated in the operating room for imaging fresh, resected, unstained breast tissue specimens, and for assessing tumor margins and the tumor microenvironment. This real-time label-free nonlinear imaging system has the potential to uniquely characterize breast cancer margins and the microenvironment of tumors to intraoperatively identify structural, functional, and molecular changes that could indicate the aggressiveness of the tumor.

Clinical optical coherence tomography combined with multiphoton tomography of patients with skin diseases.

PubMed

König, Karsten; Speicher, Marco; Bückle, Rainer; Reckfort, Julia; McKenzie, Gordon; Welzel, Julia; Koehler, Martin J; Elsner, Peter; Kaatz, Martin

2009-07-01

We report on the first clinical study based on optical coherence tomography (OCT) in combination with multiphoton tomography (MPT) and dermoscopy. 47 patients with a variety of skin diseases and disorders such as skin cancer, psoriasis, hemangioma, connective tissue diseases, pigmented lesions, and autoimmune bullous skin diseases have been investigated with (i) state-of-the-art OCT systems for dermatology including multibeam swept source OCT, (ii) the femtosecond laser multiphoton tomograph, and (iii) dermoscopes. Dermoscopy provides two-dimensional color images of the skin surface. OCT images reflect modifications of the intratissue refractive index whereas MPT is based on nonlinear excitation of endogenous fluorophores and second harmonic generation. A stack of cross-sectional OCT "wide field" images with a typical field of view of 5 x 2 mm(2) gave fast information on the depth and the volume of the lesion. Multiphoton tomography provided 0.36 x 0.36 mm(2) horizontal/diagonal optical sections within seconds of a particular region of interest with superior submicron resolution down to a tissue depth of 200 mum. The combination of OCT and MPT provides a unique powerful optical imaging modality for early detection of skin cancer and other skin diseases as well as for the evaluation of the efficiency of treatments.

Multiphoton microscopy and image guided light activated therapy using nanomaterials (Conference Presentation)

NASA Astrophysics Data System (ADS)

Prasad, Paras N.

2017-02-01

This talk will focus on design and applications of nanomaterials exhibiting strong multiphoton upconversion for multiphoton microscopy as well as for image-guided and light activated therapy .1-3 Such processes can occur by truly nonlinear optical interactions proceeding through virtual intermediate states or by stepwise coupled linear excitations through real intermediate states. Multiphoton processes in biocompatible multifunctional nanoparticles allow for 3D deep tissue imaging. In addition, they can produce in-situ photon conversion of deep tissue penetrating near IR light into a needed shorter wavelength light for photo-activated therapy at a targeted site, thus overcoming the limited penetration of UV or visible light into biological media. We are using near IR emitters such as silicon quantum dots which also exhibit strong multiphoton excitation for multiphoton microscopy. Another approach involves nonlinear nanocrystals such as ZnO which can produce four wave mixing, sum frequency generation as well as second harmonic generation to convert a deep tissue penetrating Near IR light at the targeted biological site to a desired shorter wavelength light suitable for bio imaging or activation of a therapy. We have utilized this approach to activate a photosensitizer for photodynamic therapy. Yet another type of upconversion materials is rare-earth ion doped optical nanotransformers which transform a Near IR (NIR) light from an external source by sequential single photon absorption, in situ and on demand, to a needed wavelength. Applications of these nanotransformers in multiphoton photoacoustic imaging will also be presented. An exciting direction pursued by us using these multiphoton nanoparticles, is functional imaging of brain. Simultaneously, they can effect optogenetics for regioselective stimulation of neurons for providing an effective intervention/augmentation strategy to enhance the cognitive state and lead to a foundation for futuristic vision of super human capabilities. Challenges and opportunities will be discussed.

Imaging rat esophagus using combination of reflectance confocal and multiphoton microscopy

NASA Astrophysics Data System (ADS)

Zhuo, S. M.; Chen, J. X.; Jiang, X. S.; Lu, K. C.; Xie, S. S.

2008-08-01

We combine reflectance confocal microscopy (RCM) with multiphoton microscopy (MPM) to image rat esophagus. The two imaging modalities allow detection of layered-resolved complementary information from esophagus. In the keratinizing layer, the keratinocytes boundaries can be characterized by RCM, while the keratinocytes cytoplasm (keratin) can be further imaged by multiphoton autofluorescence signal. In the epithelium, the epithelial cellular boundaries and nucleus can be detected by RCM, and MPM can be used for imaging epithelial cell cytoplasm and monitoring metabolic state of epithelium. In the stroma, multiphoton autofluorescence signal is used to image elastin and second harmonic generation signal is utilized to detect collagen, while RCM is used to determine the optical property of stroma. Overall, these results suggest that the combination of RCM and MPM has potential to provide more important and comprehensive information for early diagnosis of esophageal cancer.

Single-photon counting multicolor multiphoton fluorescence microscope.

PubMed

Buehler, Christof; Kim, Ki H; Greuter, Urs; Schlumpf, Nick; So, Peter T C

2005-01-01

We present a multicolor multiphoton fluorescence microscope with single-photon counting sensitivity. The system integrates a standard multiphoton fluorescence microscope, an optical grating spectrograph operating in the UV-Vis wavelength region, and a 16-anode photomultiplier tube (PMT). The major technical innovation is in the development of a multichannel photon counting card (mC-PhCC) for direct signal collection from multi-anode PMTs. The electronic design of the mC-PhCC employs a high-throughput, fully-parallel, single-photon counting scheme along with a high-speed electrical or fiber-optical link interface to the data acquisition computer. There is no electronic crosstalk among the detection channels of the mC-PhCC. The collected signal remains linear up to an incident photon rate of 10(8) counts per second. The high-speed data interface offers ample bandwidth for real-time readout: 2 MByte lambda-stacks composed of 16 spectral channels, 256 x 256 pixel image with 12-bit dynamic range can be transferred at 30 frames per second. The modular design of the mC-PhCC can be readily extended to accommodate PMTs of more anodes. Data acquisition from a 64-anode PMT has been verified. As a demonstration of system performance, spectrally resolved images of fluorescent latex spheres and ex-vivo human skin are reported. The multicolor multiphoton microscope is suitable for highly sensitive, real-time, spectrally-resolved three-dimensional imaging in biomedical applications.

Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast.

PubMed

Kao, Ya-Ting; Zhu, Xinxin; Xu, Fang; Min, Wei

2012-08-01

Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes.

Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast

PubMed Central

Kao, Ya-Ting; Zhu, Xinxin; Xu, Fang; Min, Wei

2012-01-01

Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes. PMID:22876358

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy.

PubMed

Ficht, Xenia; Thelen, Flavian; Stolp, Bettina; Stein, Jens V

2018-05-07

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

Fast volumetric imaging with patterned illumination via digital micro-mirror device-based temporal focusing multiphoton microscopy.

PubMed

Chang, Chia-Yuan; Hu, Yvonne Yuling; Lin, Chun-Yu; Lin, Cheng-Han; Chang, Hsin-Yu; Tsai, Sheng-Feng; Lin, Tzu-Wei; Chen, Shean-Jen

2016-05-01

Temporal focusing multiphoton microscopy (TFMPM) has the advantage of area excitation in an axial confinement of only a few microns; hence, it can offer fast three-dimensional (3D) multiphoton imaging. Herein, fast volumetric imaging via a developed digital micromirror device (DMD)-based TFMPM has been realized through the synchronization of an electron multiplying charge-coupled device (EMCCD) with a dynamic piezoelectric stage for axial scanning. The volumetric imaging rate can achieve 30 volumes per second according to the EMCCD frame rate of more than 400 frames per second, which allows for the 3D Brownian motion of one-micron fluorescent beads to be spatially observed. Furthermore, it is demonstrated that the dynamic HiLo structural multiphoton microscope can reject background noise by way of the fast volumetric imaging with high-speed DMD patterned illumination.

Hybrid label-free multiphoton and optoacoustic microscopy (MPOM)

NASA Astrophysics Data System (ADS)

Soliman, Dominik; Tserevelakis, George J.; Omar, Murad; Ntziachristos, Vasilis

2015-07-01

Many biological applications require a simultaneous observation of different anatomical features. However, unless potentially harmful staining of the specimens is employed, individual microscopy techniques do generally not provide multi-contrast capabilities. We present a hybrid microscope integrating optoacoustic microscopy and multiphoton microscopy, including second-harmonic generation, into a single device. This combined multiphoton and optoacoustic microscope (MPOM) offers visualization of a broad range of structures by employing different contrast mechanisms and at the same time enables pure label-free imaging of biological systems. We investigate the relative performance of the two microscopy modalities and demonstrate their multi-contrast abilities through the label-free imaging of a zebrafish larva ex vivo, simultaneously visualizing muscles and pigments. This hybrid microscopy application bears great potential for developmental biology studies, enabling more comprehensive information to be obtained from biological specimens without the necessity of staining.

Polymer dots enable deep in vivo multiphoton fluorescence imaging of cerebrovascular architecture

NASA Astrophysics Data System (ADS)

Hassan, Ahmed M.; Wu, Xu; Jarrett, Jeremy W.; Xu, Shihan; Miller, David R.; Yu, Jiangbo; Perillo, Evan P.; Liu, Yen-Liang; Chiu, Daniel T.; Yeh, Hsin-Chih; Dunn, Andrew K.

2018-02-01

Deep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep multiphoton imaging has never before been demonstrated. Here we characterize the multiphoton properties of three pdot variants (CNPPV, PFBT, and PFPV) and demonstrate deep imaging of cortical microvasculature in C57 mice. Specifically, we measure the two- versus three-photon power dependence of these pdots and observe a clear three-photon excitation signature at wavelengths longer than 1300 nm, and a transition from two-photon to three-photon excitation within a 1060 - 1300 nm excitation range. Furthermore, we show that pdots enable in vivo two-photon imaging of cerebrovascular architecture in mice up to 850 μm beneath the pial surface using 800 nm excitation. In contrast with traditional multiphoton probes, we also demonstrate that the broad multiphoton absorption spectrum of pdots permits imaging at longer wavelengths (λex = 1,060 and 1225 nm). These wavelengths approach an ideal biological imaging wavelength near 1,300 nm and confer compatibility with a high-power ytterbium-fiber laser and a high pulse energy optical parametric amplifier, resulting in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths of 900 μm and 1300 μm. Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, which will undoubtedly unlock the ability to interrogate deep structures in vivo.

Multiphoton microscopy for the in-situ investigation of cellular processes and integrity in cryopreservation.

PubMed

Doerr, Daniel; Stark, Martin; Ehrhart, Friederike; Zimmermann, Heiko; Stracke, Frank

2009-08-01

In this study we demonstrate a new noninvasive imaging method to monitor freezing processes in biological samples and to investigate life in the frozen state. It combines a laser scanning microscope with a computer-controlled cryostage. Nearinfrared (NIR) femtosecond laser pulses evoke the fluorescence of endogenous fluorophores and fluorescent labels due to multiphoton absorption.The inherent optical nonlinearity of multiphoton absorption allows 3D fluorescence imaging for optical tomography of frozen biological material in-situ. As an example for functional imaging we use fluorescence lifetime imaging (FLIM) to create images with chemical and physical contrast.

Multiphoton microscopy system with a compact fiber-based femtosecond-pulse laser and handheld probe

PubMed Central

Liu, Gangjun; Kieu, Khanh; Wise, Frank W.; Chen, Zhongping

2012-01-01

We report on the development of a compact multiphoton microscopy (MPM) system that integrates a compact and robust fiber laser with a miniature probe. The all normal dispersion fiber femtosecond laser has a central wavelength of 1.06 μm, pulse width of 125 fs and average power of more than 1 W. A double cladding photonic crystal fiber was used to deliver the excitation beam and to collect the two-photon signal. The hand-held probe included galvanometer-based mirror scanners, relay lenses and a focusing lens. The packaged probe had a diameter of 16 mm. Second harmonic generation (SHG) images and two-photon excited fluorescence (TPEF) images of biological tissues were demonstrated using the system. MPM images of different biological tissues acquired by the compact system which integrates an FBFP laser, an DCPCF and a miniature handheld probe. PMID:20635426

Remote focusing for programmable multi-layer differential multiphoton microscopy

PubMed Central

Hoover, Erich E.; Young, Michael D.; Chandler, Eric V.; Luo, Anding; Field, Jeffrey J.; Sheetz, Kraig E.; Sylvester, Anne W.; Squier, Jeff A.

2010-01-01

We present the application of remote focusing to multiphoton laser scanning microscopy and utilize this technology to demonstrate simultaneous, programmable multi-layer imaging. Remote focusing is used to independently control the axial location of multiple focal planes that can be simultaneously imaged with single element detection. This facilitates volumetric multiphoton imaging in scattering specimens and can be practically scaled to a large number of focal planes. Further, it is demonstrated that the remote focusing control can be synchronized with the lateral scan directions, enabling imaging in orthogonal scan planes. PMID:21326641

Hybrid reflecting objectives for functional multiphoton microscopy in turbid media

PubMed Central

Vučinić, Dejan; Bartol, Thomas M.; Sejnowski, Terrence J.

2010-01-01

Most multiphoton imaging of biological specimens is performed using microscope objectives optimized for high image quality under wide-field illumination. We present a class of objectives designed de novo without regard for these traditional constraints, driven exclusively by the needs of fast multiphoton imaging in turbid media: the delivery of femtosecond pulses without dispersion and the efficient collection of fluorescence. We model the performance of one such design optimized for a typical brain-imaging setup and show that it can greatly outperform objectives commonly used for this task. PMID:16880851

In vivo multiphoton imaging of bile duct ligation

NASA Astrophysics Data System (ADS)

Liu, Yuan; Li, Feng-Chieh; Chen, Hsiao-Chin; Chang, Po-shou; Yang, Shu-Mei; Lee, Hsuan-Shu; Dong, Chen-Yuan

2008-02-01

Bile is the exocrine secretion of liver and synthesized by hepatocytes. It is drained into duodenum for the function of digestion or drained into gallbladder for of storage. Bile duct obstruction is a blockage in the tubes that carry bile to the gallbladder and small intestine. However, Bile duct ligation results in the changes of bile acids in serum, liver, urine, and feces1, 2. In this work, we demonstrate a novel technique to image this pathological condition by using a newly developed in vivo imaging system, which includes multiphoton microscopy and intravital hepatic imaging chamber. The images we acquired demonstrate the uptake, processing of 6-CFDA in hepatocytes and excretion of CF in the bile canaliculi. In addition to imaging, we can also measure kinetics of the green fluorescence intensity.

Distinguishing human normal or cancerous esophagus tissue ex vivo using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Liu, N. R.; Chen, G. N.; Wu, S. S.; Chen, R.

2014-02-01

Application of multiphoton microscopy (MPM) to clinical cancer research has greatly developed over the last few years. In this paper, we mainly focus on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) for investigating esophageal cancer. We chiefly discuss the SHG/TPEF image and spectral characteristics of normal and cancerous esophagus submucosa with the combined multi-channel imaging mode and Lambda mode of a multiphoton microscope (LSM 510 META). Great differences can be detected, such as collagen content and morphology, glandular-shaped cancer cells, TPEF/SHG intensity ratio, and so on, which demonstrate that the multiphoton imaging technique has the potential ability for minimally-invasive early cancer diagnosis.

In vivo multiphoton microscopy beyond 1 mm in the brain

NASA Astrophysics Data System (ADS)

Miller, David R.; Medina, Flor A.; Hassan, Ahmed; Perillo, Evan P.; Hagan, Kristen; Kazmi, S. M. Shams; Zemelman, Boris V.; Dunn, Andrew K.

2017-02-01

We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. We demonstrate an imaging depth of 1,200 μm in vasculature and 1,160 μm in neurons. We also demonstrate deep-tissue imaging using Indocyanine Green (ICG), which is FDA approved and a promising route to translate multiphoton microscopy to human applications.

Multiphoton in vivo imaging with a femtosecond semiconductor disk laser

PubMed Central

Voigt, Fabian F.; Emaury, Florian; Bethge, Philipp; Waldburger, Dominik; Link, Sandro M.; Carta, Stefano; van der Bourg, Alexander; Helmchen, Fritjof; Keller, Ursula

2017-01-01

We use an ultrafast diode-pumped semiconductor disk laser (SDL) to demonstrate several applications in multiphoton microscopy. The ultrafast SDL is based on an optically pumped Vertical External Cavity Surface Emitting Laser (VECSEL) passively mode-locked with a semiconductor saturable absorber mirror (SESAM) and generates 170-fs pulses at a center wavelength of 1027 nm with a repetition rate of 1.63 GHz. We demonstrate the suitability of this laser for structural and functional multiphoton in vivo imaging in both Drosophila larvae and mice for a variety of fluorophores (including mKate2, tdTomato, Texas Red, OGB-1, and R-CaMP1.07) and for endogenous second-harmonic generation in muscle cell sarcomeres. We can demonstrate equivalent signal levels compared to a standard 80-MHz Ti:Sapphire laser when we increase the average power by a factor of 4.5 as predicted by theory. In addition, we compare the bleaching properties of both laser systems in fixed Drosophila larvae and find similar bleaching kinetics despite the large difference in pulse repetition rates. Our results highlight the great potential of ultrafast diode-pumped SDLs for creating a cost-efficient and compact alternative light source compared to standard Ti:Sapphire lasers for multiphoton imaging. PMID:28717563

Multiphoton imaging with high peak power VECSELs

NASA Astrophysics Data System (ADS)

Mirkhanov, Shamil; Quarterman, Adrian H.; Swift, Samuel; Praveen, Bavishna B.; Smyth, Conor J. C.; Wilcox, Keith G.

2016-03-01

Multiphoton imaging (MMPI) has become one of thee key non-invasive light microscopy techniques. This technique allows deep tissue imaging with high resolution and less photo-damage than conventional confocal microscopy. MPI is type of laser-scanning microscopy that employs localized nonlinear excitation, so that fluorescence is excited only with is scanned focal volume. For many years, Ti: sapphire femtosecond lasers have been the leading light sources for MPI applications. However, recent developments in laser sources and new types of fluorophores indicate that longer wavelength excitation could be a good alternative for these applications. Mode-locked VECSEELs have the potential to be low cost, compact light sources for MPI systems, with the additional advantage of broad wavelength coverage through use of different semiconductor material systems. Here, we use a femtosecond fibber laser to investigate the effect average power and repetition rate has on MPI image quality, to allow us to optimize our mode-locked VVECSELs for MPI.

Characteristics of subgingival calculus detection by multiphoton fluorescence microscopy

NASA Astrophysics Data System (ADS)

Tung, Oi-Hong; Lee, Shyh-Yuan; Lai, Yu-Lin; Chen, How-Foo

2011-06-01

Subgingival calculus has been recognized as a major cause of periodontitis, which is one of the main chronic infectious diseases of oral cavities and a principal cause of tooth loss in humans. Bacteria deposited in subgingival calculus or plaque cause gingival inflammation, function deterioration, and then periodontitis. However, subgingival calculus within the periodontal pocket is a complicated and potentially delicate structure to be detected with current dental armamentaria, namely dental x-rays and dental probes. Consequently, complete removal of subgingival calculus remains a challenge to periodontal therapies. In this study, the detection of subgingival calculus employing a multiphoton autofluorescence imaging method was characterized in comparison with a one-photon confocal fluorescence imaging technique. Feasibility of such a system was studied based on fluorescence response of gingiva, healthy teeth, and calculus with and without gingiva covered. The multiphoton fluorescence technology perceived the tissue-covered subgingival calculus that cannot be observed by the one-photon confocal fluorescence method.

Detection of the multiphoton signals in stained tissue using nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Zeng, Yaping; Xu, Jian; Kang, Deyong; Lin, Jiangbo; Chen, Jianxin

2016-10-01

Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging, has become a powerful, important tool for tissue imaging at the molecular level. Recently, MPM is also used to image hematoxylin and eosin (H and E)-stained sections in cancer diagnostics. However, several studies have showed that the MPM images of tissue stained with H and E are significantly different from unstained tissue sections. Our aim was to detect of the multiphoton signals in stained tissue by using MPM. In this paper, MPM was used to image histological sections of esophageal invasive carcinoma tissues stained with H, E, H and E and fresh tissue. To detect of the multiphoton signals in stained tissue, the emission spectroscopic of tissue stained with H, E, H and E were obtained. For comparison, the fresh tissues were also investigated. Our results showed that the tissue stained with H, E, H and E could be detected by their TPEF signals. While the tissue stained with H and fresh tissue could be detected by their TPEF and SHG signals. In this work, we detect of the multiphoton signals in stained tissue. These findings will be useful for choosing suitable staining method so to improve the quality of MPM imaging in the future.

Label-free imaging of rat spinal cords based on multiphoton microscopy

NASA Astrophysics Data System (ADS)

Liao, Chenxi; Wang, Zhenyu; Zhou, Linquan; Zhu, Xiaoqin; Liu, Wenge; Chen, Jianxin

2016-10-01

As an integral part of the central nervous system, the spinal cord is a communication cable between the body and the brain. It mainly contains neurons, glial cells, nerve fibers and fiber tracts. The recent development of the optical imaging technique allows high-resolution imaging of biological tissues with the great potential for non-invasively looking inside the body. In this work, we evaluate the imaging capacity of multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) for the cells and extracellular matrix in the spinal cord at molecular level. Rat spinal cord tissues were sectioned and imaged by MPM to demonstrate that MPM is able to show the microstructure including white matter, gray matter, ventral horns, dorsal horns, and axons based on the distinct intrinsic sources in each region of spinal cord. In the high-resolution and high-contrast MPM images, the cell profile can be clearly identified as dark shadows caused by nuclei and encircled by cytoplasm. The nerve fibers in white matter region emitted both SHG and TPEF signals. The multiphoton microscopic imaging technique proves to be a fast and effective tool for label-free imaging spinal cord tissues, based on endogenous signals in biological tissue. It has the potential to extend this optical technique to clinical study, where the rapid and damage-free imaging is needed.

Nanoparticle-assisted-multiphoton microscopy for in vivo brain imaging of mice

NASA Astrophysics Data System (ADS)

Qian, Jun

2015-03-01

Neuro/brain study has attracted much attention during past few years, and many optical methods have been utilized in order to obtain accurate and complete neural information inside the brain. Relying on simultaneous absorption of two or more near-infrared photons by a fluorophore, multiphoton microscopy can achieve deep tissue penetration and efficient light detection noninvasively, which makes it very suitable for thick-tissue and in vivo bioimaging. Nanoparticles possess many unique optical and chemical properties, such as anti-photobleaching, large multiphoton absorption cross-section, and high stability in biological environment, which facilitates their applications in long-term multiphoton microscopy as contrast agents. In this paper, we will introduce several typical nanoparticles (e.g. organic dye doped polymer nanoparticles and gold nanorods) with high multiphoton fluorescence efficiency. We further applied them in two- and three-photon in vivo functional brain imaging of mice, such as brain-microglia imaging, 3D architecture reconstruction of brain blood vessel, and blood velocity measurement.

In vivo microscopy of the mouse brain using multiphoton laser scanning techniques

NASA Astrophysics Data System (ADS)

Yoder, Elizabeth J.

2002-06-01

The use of multiphoton microscopy for imaging mouse brain in vivo offers several advantages and poses several challenges. This tutorial begins by briefly comparing multiphoton microscopy with other imaging modalities used to visualize the brain and its activity. Next, an overview of the techniques for introducing fluorescence into whole animals to generate contrast for in vivo microscopy using two-photon excitation is presented. Two different schemes of surgically preparing mice for brain imaging with multiphoton microscopy are reviewed. Then, several issues and problems with in vivo microscopy - including motion artifact, respiratory and cardiac rhythms, maintenance of animal health, anesthesia, and the use of fiducial markers - are discussed. Finally, examples of how these techniques have been applied to visualize the cerebral vasculature and its response to hypercapnic stimulation are provided.

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue.

PubMed

Yoshitake, Tadayuki; Giacomelli, Michael G; Cahill, Lucas C; Schmolze, Daniel B; Vardeh, Hilde; Faulkner-Jones, Beverly E; Connolly, James L; Fujimoto, James G

2016-12-01

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

PubMed Central

Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

2016-01-01

Abstract. Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue. PMID:28032121

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

NASA Astrophysics Data System (ADS)

Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

2016-12-01

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

In vivo multiphoton imaging of a diverse array of fluorophores to investigate deep neurovascular structure

PubMed Central

Miller, David R.; Hassan, Ahmed M.; Jarrett, Jeremy W.; Medina, Flor A.; Perillo, Evan P.; Hagan, Kristen; Shams Kazmi, S. M.; Clark, Taylor A.; Sullender, Colin T.; Jones, Theresa A.; Zemelman, Boris V.; Dunn, Andrew K.

2017-01-01

We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. By combining the high repetition rate (511 kHz) and high pulse energy (400 nJ) of our amplifier laser system, we demonstrate imaging of vasculature labeled with Texas Red and Indocyanine Green, and neurons expressing tdTomato and yellow fluorescent protein. We measure the blood flow speed of a single capillary at a depth of 1.2 mm, and image vasculature to a depth of 1.53 mm with fine axial steps (5 μm) and reasonable acquisition times. The high image quality enabled analysis of vascular morphology at depths to 1.45 mm. PMID:28717582

Clinical application of multiphoton tomography in combination with high-frequency ultrasound for evaluation of skin diseases.

PubMed

König, Karsten; Speicher, Marco; Köhler, Martin J; Scharenberg, Rüdiger; Kaatz, Martin

2010-12-01

The first-ever application of high-frequency ultrasound combined with multiphoton tomography (MPT) and dermoscopy in a clinical trial is reported. 47 patients with different dermatoses such as benign and malign skin cancers, connective tissue diseases, inflammatory skin diseases, and autoimmune bullous skin diseases have been investigated with (i) state-of-the-art and highly sophisticated ultrasound systems for dermatology, (ii) the femtosecond laser multiphoton tomograph and (iii) dermoscopes. Dermoscopy provides two-dimensional color images of the skin surface with a magnification up to 70 x. Depending on the ultrasonic frequencies from 7.5 MHz to 100 MHz, the signal depth varies from about 1 mm to 80 mm. Vertical ultrasound wide-field images provide fast information on depth and volume of the lesion. The 100 MHz ultrasound allows imaging with resolutions down to 16 μm (axial) and 32 μm (lateral). Multiphoton tomography provides 0.36 x 0.36 x 0.001 mm³ horizontal optical sections of a particular region of interest with submicron resolution down to 200 μm tissue depth. The autofluorescence of mitochondrial coenzymes, keratin, melanin, and elastin as well as the network of collagen structures can be imaged. The combination of ultrasound and MPT opens novel synergistic possibilities in diagnostics of skin diseases with a special focus on the early detection of skin cancer as well as the evaluation of treatments. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cornea surgery with nanojoule femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Koenig, Karsten; Wang, Bagui; Riemann, Iris; Kobow, Jens

2005-04-01

We report on a novel optical method for (i) flap-generation in LASIK procedures as well as (ii) for flap-free intrastromal refractive surgery based on nanojoule femtosecond laser pulses. The near infrared 200 fs pulses for multiphoton ablation have been provided by ultracompact turn-key MHz laser resonators. LASIK flaps and intracorneal cavities have been realized with high precision within living New Zealand rabbits using the system FemtoCutO (JenLab GmbH, Jena, Germany) at 800 nm laser wavelength. Using low-energy sub-2 nJ laser pulses, collateral damage due to photodisruptive and self-focusing effects was avoided. The laser ablation system consists of fast galvoscanners, focusing optics of high numerical aperture as well as a sensitive imaging system and provides also the possibility of 3D multiphoton imaging of fluorescent cellular organelles and SHG signals from collagen. Multiphoton tomography of the cornea was used to determine the exact intratissue beam position and to visualize intraocular post-laser effects. The wound healing process has been investigated up to 90 days after instrastromal laser ablation by histological analysis. Regeneration of damaged collagen structures and the migration of inflammation cells have been detected.

Monitoring wound healing by multiphoton tomography/endoscopy

NASA Astrophysics Data System (ADS)

König, Karsten; Weinigel, Martin; Bückle, Rainer; Kaatz, Martin; Hipler, Christina; Zens, Katharina; Schneider, Stefan W.; Huck, Volker

2015-02-01

Certified clinical multiphoton tomographs are employed to perform rapid label-free high-resolution in vivo histology. Novel tomographs include a flexible 360° scan head attached to a mechano-optical arm for autofluorescence and SHG imaging as well as rigid two-photon GRIN microendoscope. Mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen can be imaged with submicron resolution in human skin. The system was employed to study the healing of chronic wounds (venous leg ulcer) and acute wounds (curettage of actinic or seborrheic keratosis) on a subcellular level. Furthermore, a flexible sterile foil as interface between wound and focusing optic was tested.

Characterization of a reflective objective with multiphoton microscopy

NASA Astrophysics Data System (ADS)

Kabir, Mohammad M.; Choubal, Aakash M.; Sivaguru, Mayandi; Toussaint, Kimani C.

2018-02-01

Reflective objectives (ROs) can reduce chromatic aberration across a wide wavelength range in multiphoton microscopy (MPM). However, a systematic characterization of the performance of ROs has not been carried out. In this paper, we analyze the performance of a 0.5 numerical-aperture (NA) RO and compare it with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments extending 1 octave in visible and NIR wavelengths, the SO introduces defocusing errors of 25% for TPF images of sub-diffraction fluorescent beads and 10% for SHG images of collagen fibers. For both imaging systems, the RO provides a corresponding error of 4%. This work highlights the potential usefulness of ROs for multimodal MPM applications.

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.

Multiphoton imaging: a view to understanding sulfur mustard lesions

NASA Astrophysics Data System (ADS)

Werrlein, Robert J. S.; Madren-Whalley, Janna S.

2003-07-01

It is well known that topical exposure to sulfur mustard (SM) produces persistent, incapacitating blisters of the skin. However, the primary lesions effecting epidermal-dermal separation and disabling of mechanisms for cutaneous repair remain uncertain. Immunofluorescent staining plus multiphoton imaging of human epidermal tissues and keratinocytes exposed to SM (400 μM x 5 min)have revealed that SM disrupts adhesion-complex molecules which are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Images of keratin-14 showed early, progressive, postexposure collapse of the K5/K14 cytoskeleton that resulted in ventral displacement of the nuclei beneath its collapsing filaments. This effectively corrupted the dynamic filament assemblies that link basal-cell nuclei to the extracellular matrix via α6β4-integrin and laminin-5. At 1 h postexposure, there was disruption in the surface organization of α6β4 integrins, associated displacement of laminin-5 anchoring sites and a concomitant loss of functional asymmetry. Accordingly, our multiphoton images are providing compelling evidence that SM induces prevesicating lesions that disrupt the receptor-ligand organization and cytoskeletal systems required for maintaining dermal-epidermal attachment, signal transduction, and polarized mobility.

COMPACT NON-CONTACT TOTAL EMISSION DETECTION FOR IN-VIVO MULTI-PHOTON EXCITATION MICROSCOPY

PubMed Central

Glancy, Brian; Karamzadeh, Nader S.; Gandjbakhche, Amir H.; Redford, Glen; Kilborn, Karl; Knutson, Jay R.; Balaban, Robert S.

2014-01-01

Summary We describe a compact, non-contact design for a Total Emission Detection (c-TED) system for intra-vital multi-photon imaging. To conform to a standard upright two-photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non-contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c-TED device compared to heavily optimized objective-based emission collection. The best light collection enhancement was seen from murine fat (5×-2× gains as a function of depth), while murine skeletal muscle and rat kidney showed gains of over two and just under two-fold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a two-fold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers (enabled by greater light collection efficiency) yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo-damage, as well as the applicability of this device to other multi-photon imaging methods is discussed. PMID:24251437

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

NASA Astrophysics Data System (ADS)

Wang, Taejun; Jang, Won Hyuk; Lee, Seunghun; Yoon, Calvin J.; Lee, Jun Ho; Kim, Bumju; Hwang, Sekyu; Hong, Chun-Pyo; Yoon, Yeoreum; Lee, Gilgu; Le, Viet-Hoan; Bok, Seoyeon; Ahn, G.-One; Lee, Jaewook; Gho, Yong Song; Chung, Euiheon; Kim, Sungjee; Jang, Myoung Ho; Myung, Seung-Jae; Kim, Myoung Joon; So, Peter T. C.; Kim, Ki Hean

2016-06-01

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

Multiphoton fluorescence lifetime imaging of chemotherapy distribution in solid tumors

NASA Astrophysics Data System (ADS)

Carlson, Marjorie; Watson, Adrienne L.; Anderson, Leah; Largaespada, David A.; Provenzano, Paolo P.

2017-11-01

Doxorubicin is a commonly used chemotherapeutic employed to treat multiple human cancers, including numerous sarcomas and carcinomas. Furthermore, doxorubicin possesses strong fluorescent properties that make it an ideal reagent for modeling drug delivery by examining its distribution in cells and tissues. However, while doxorubicin fluorescence and lifetime have been imaged in live tissue, its behavior in archival samples that frequently result from drug and treatment studies in human and animal patients, and murine models of human cancer, has to date been largely unexplored. Here, we demonstrate imaging of doxorubicin intensity and lifetimes in archival formalin-fixed paraffin-embedded sections from mouse models of human cancer with multiphoton excitation and multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton excitation imaging reveals robust doxorubicin emission in tissue sections and captures spatial heterogeneity in cells and tissues. However, quantifying the amount of doxorubicin signal in distinct cell compartments, particularly the nucleus, often remains challenging due to strong signals in multiple compartments. The addition of FLIM analysis to display the spatial distribution of excited state lifetimes clearly distinguishes between signals in distinct compartments such as the cell nuclei versus cytoplasm and allows for quantification of doxorubicin signal in each compartment. Furthermore, we observed a shift in lifetime values in the nuclei of transformed cells versus nontransformed cells, suggesting a possible diagnostic role for doxorubicin lifetime imaging to distinguish normal versus transformed cells. Thus, data here demonstrate that multiphoton FLIM is a highly sensitive platform for imaging doxorubicin distribution in normal and diseased archival tissues.

Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores.

PubMed

Chen, Zhixing; Wei, Lu; Zhu, Xinxin; Min, Wei

2012-08-13

It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.

Ex-vivo multiphoton analysis of rabbit corneal wound healing following photorefractive keratectomy

NASA Astrophysics Data System (ADS)

Wang, Tsung-Jen; Lo, Wen; Dong, Chen-Yuan; Hu, Fung-Rong

2008-02-01

The aim of this study is to assess the application of multiphoton autofluorescence and second harmonic generation (SHG) microscopy for investigating corneal wound healing after high myopic (-10.0D) photorefractive keratectomy (PRK) procedures on the rabbit eyes. The effect of PRK on the morphology and distribution of keratocytes were investigated using multiphoton excited autofluorescence imaging, while the effect of PRK on the arrangement of collagen fibers was monitored by second-harmonic generation imaging. Without histological processing, multiphoton microscopy is able to characterize corneal damage and wound healing from PRK. Our results show that this technique has potential application in the clinical evaluation of corneal damage due to refractive surgery, and may be used to study the unwanted side effects of these procedures.

Multiphoton microscopy can visualize zonal damage and decreased cellular metabolic activity in hepatic ischemia-reperfusion injury in rats

NASA Astrophysics Data System (ADS)

Thorling, Camilla A.; Liu, Xin; Burczynski, Frank J.; Fletcher, Linda M.; Gobe, Glenda C.; Roberts, Michael S.

2011-11-01

Ischemia-reperfusion (I/R) injury is a common occurrence in liver surgery. In orthotopic transplantation, the donor liver is exposed to periods of ischemia and when oxygenated blood is reintroduced to the liver, oxidative stress may develop and lead to graft failure. The aim of this project was to investigate whether noninvasive multiphoton and fluorescence lifetime imaging microscopy, without external markers, were useful in detecting early liver damage caused by I/R injury. Localized hepatic ischemia was induced in rats for 1 h followed by 4 h reperfusion. Multiphoton and fluorescence lifetime imaging microscopy was conducted prior to ischemia and up to 4 h of reperfusion and compared to morphological and biochemical assessment of liver damage. Liver function was significantly impaired at 2 and 4 h of reperfusion. Multiphoton microscopy detected liver damage at 1 h of reperfusion, manifested by vacuolated cells and heterogeneous spread of damage over the liver. The damage was mainly localized in the midzonal region of the liver acinus. In addition, fluorescence lifetime imaging showed a decrease in cellular metabolic activity. Multiphoton and fluorescence lifetime imaging microscopy detected evidence of early I/R injury both structurally and functionally. This provides a simple noninvasive technique useful for following progressive liver injury without external markers.

Multiphoton microscopy in every lab: the promise of ultrafast semiconductor disk lasers

NASA Astrophysics Data System (ADS)

Emaury, Florian; Voigt, Fabian F.; Bethge, Philipp; Waldburger, Dominik; Link, Sandro M.; Carta, Stefano; van der Bourg, Alexander; Helmchen, Fritjof; Keller, Ursula

2017-07-01

We use an ultrafast diode-pumped semiconductor disk laser (SDL) to demonstrate several applications in multiphoton microscopy. The ultrafast SDL is based on an optically pumped Vertical External Cavity Surface Emitting Laser (VECSEL) passively mode-locked with a semiconductor saturable absorber mirror (SESAM) and generates 170-fs pulses at a center wavelength of 1027 nm with a repetition rate of 1.63 GHz. We demonstrate the suitability of this laser for structural and functional multiphoton in vivo imaging in both Drosophila larvae and mice for a variety of fluorophores (including mKate2, tdTomato, Texas Red, OGB-1, and R-CaMP1.07) and for endogenous second-harmonic generation in muscle cell sarcomeres. We can demonstrate equivalent signal levels compared to a standard 80-MHz Ti:Sapphire laser when we increase the average power by a factor of 4.5 as predicted by theory. In addition, we compare the bleaching properties of both laser systems in fixed Drosophila larvae and find similar bleaching kinetics despite the large difference in pulse repetition rates. Our results highlight the great potential of ultrafast diode-pumped SDLs for creating a cost-efficient and compact alternative light source compared to standard Ti:Sapphire lasers for multiphoton imaging.

Multiphoton imaging of low grade, high grade intraepithelial neoplasia and intramucosal invasive cancer of esophagus

NASA Astrophysics Data System (ADS)

Xu, Jian; Jiang, Liwei; Kang, Deyong; Wu, Xuejing; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Lin, Jiangbo; Chen, Jianxin

2017-04-01

Esophageal squamous cell carcinoma (ESCC) is devastating because of its aggressive lymphatic spread and clinical course. It is believed to occur through low-grade intraepithelial neoplasia (LGIN), high-grade intraepithelial neoplasia (HGIN), and intramucosal invasive cancer (IMC) before transforming to submucosal cancer. In particular, these early lesions (LGIN, HGIN and IMC), which involve no lymph node nor distant metastasis, can be cured by endoscopic treatment. Therefore, early identification of these lesions is important so as to offer a curative endoscopic resection, thus slowing down the development of ESCC. In this work, spectral information and morphological features of the normal esophageal mucosa are first studied. Then, the morphological changes of LGIN, HGIN and IMC are described. Lastly, quantitative parameters are also extracted by calculating the nuclear-to-cytoplasmic ratio of epithelial cells and the pixel density of collagen in the lamina propria. These results show that multiphoton microscopy (MPM) has the ability to identify normal esophageal mucosa, LGIN, HGIN and IMC. With the development of multiphoton endoscope systems for in vivo imaging, combined with a laser ablation system, MPM has the potential to provide immediate pathologic diagnosis and curative treatment of ESCC before the transformation to submucosal cancer in the future.

Imaging-guided two-photon excitation-emission-matrix measurements of human skin tissues

NASA Astrophysics Data System (ADS)

Yu, Yingqiu; Lee, Anthony M. D.; Wang, Hequn; Tang, Shuo; Zhao, Jianhua; Lui, Harvey; Zeng, Haishan

2012-07-01

There are increased interests on using multiphoton imaging and spectroscopy for skin tissue characterization and diagnosis. However, most studies have been done with just a few excitation wavelengths. Our objective is to perform a systematic study of the two-photon fluorescence (TPF) properties of skin fluorophores, normal skin, and diseased skin tissues. A nonlinear excitation-emission-matrix (EEM) spectroscopy system with multiphoton imaging guidance was constructed. A tunable femtosecond laser was used to vary excitation wavelengths from 730 to 920 nm for EEM data acquisition. EEM measurements were performed on excised fresh normal skin tissues, seborrheic keratosis tissue samples, and skin fluorophores including: NADH, FAD, keratin, melanin, collagen, and elastin. We found that in the stratum corneum and upper epidermis of normal skin, the cells have large sizes and the TPF originates from keratin. In the lower epidermis, cells are smaller and TPF is dominated by NADH contributions. In the dermis, TPF is dominated by elastin components. The depth resolved EEM measurements also demonstrated that keratin structure has intruded into the middle sublayers of the epidermal part of the seborrheic keratosis lesion. These results suggest that the imaging guided TPF EEM spectroscopy provides useful information for the development of multiphoton clinical devices for skin disease diagnosis.

Multifocal multiphoton microscopy with adaptive optical correction

NASA Astrophysics Data System (ADS)

Coelho, Simao; Poland, Simon; Krstajic, Nikola; Li, David; Monypenny, James; Walker, Richard; Tyndall, David; Ng, Tony; Henderson, Robert; Ameer-Beg, Simon

2013-02-01

Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled MegaFLI. Beam parallelization performed via a 3D Gerchberg-Saxton (GS) algorithm using a Spatial Light Modulator (SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction. Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.

Coherent beam control through inhomogeneous media in multi-photon microscopy

NASA Astrophysics Data System (ADS)

Paudel, Hari Prasad

Multi-photon fluorescence microscopy has become a primary tool for high-resolution deep tissue imaging because of its sensitivity to ballistic excitation photons in comparison to scattered excitation photons. The imaging depth of multi-photon microscopes in tissue imaging is limited primarily by background fluorescence that is generated by scattered light due to the random fluctuations in refractive index inside the media, and by reduced intensity in the ballistic focal volume due to aberrations within the tissue and at its interface. We built two multi-photon adaptive optics (AO) correction systems, one for combating scattering and aberration problems, and another for compensating interface aberrations. For scattering correction a MEMS segmented deformable mirror (SDM) was inserted at a plane conjugate to the objective back-pupil plane. The SDM can pre-compensate for light scattering by coherent combination of the scattered light to make an apparent focus even at a depths where negligible ballistic light remains (i.e. ballistic limit). This problem was approached by investigating the spatial and temporal focusing characteristics of a broad-band light source through strongly scattering media. A new model was developed for coherent focus enhancement through or inside the strongly media based on the initial speckle contrast. A layer of fluorescent beads under a mouse skull was imaged using an iterative coherent beam control method in the prototype two-photon microscope to demonstrate the technique. We also adapted an AO correction system to an existing in three-photon microscope in a collaborator lab at Cornell University. In the second AO correction approach a continuous deformable mirror (CDM) is placed at a plane conjugate to the plane of an interface aberration. We demonstrated that this "Conjugate AO" technique yields a large field-of-view (FOV) advantage in comparison to Pupil AO. Further, we showed that the extended FOV in conjugate AO is maintained over a relatively large axial misalignment of the conjugate planes of the CDM and the aberrating interface. This dissertation advances the field of microscopy by providing new models and techniques for imaging deeply within strongly scattering tissue, and by describing new adaptive optics approaches to extending imaging FOV due to sample aberrations.

Wavefront sensorless adaptive optics temporal focusing-based multiphoton microscopy

PubMed Central

Chang, Chia-Yuan; Cheng, Li-Chung; Su, Hung-Wei; Hu, Yvonne Yuling; Cho, Keng-Chi; Yen, Wei-Chung; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen

2014-01-01

Temporal profile distortions reduce excitation efficiency and image quality in temporal focusing-based multiphoton microscopy. In order to compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated into the microscope. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions. With the AOS correction, not only is the axial excitation symmetrically refocused, but the axial resolution with full two-photon excited fluorescence (TPEF) intensity is also maintained. Hence, the contrast of the TPEF image of a R6G-doped PMMA thin film is enhanced along with a 3.7-fold increase in intensity. Furthermore, the TPEF image quality of 1μm fluorescent beads sealed in agarose gel at different depths is improved. PMID:24940539

A multiphoton laser scanning microscope setup for transcranial in vivo brain imaging on mice

NASA Astrophysics Data System (ADS)

Nase, Gabriele; Helm, P. Johannes; Reppen, Trond; Ottersen, Ole Petter

2005-12-01

We describe a multiphoton laser scanning microscope setup for transcranial in vivo brain imaging in mice. The modular system is based on a modified industrial standard Confocal Scanning Laser Microscope (CSLM) and is assembled mainly from commercially available components. A special multifunctional stage, which is optimized for both laser scanning microscopic observation and preparative animal surgery, has been developed and built. The detection unit includes a highly efficient photomultiplier tube installed in a Peltier-cooled thermal box shielding the detector from changes in room temperature and from distortions caused by external electromagnetic fields. The images are recorded using a 12-bit analog-to-digital converter. Depending on the characteristics of the staining, individual nerve cells can be imaged down to at least 100μm below the intact cranium and down to at least 200μm below the opened cranium.

Comparison of Cornea Module and DermaInspect for noninvasive imaging of ocular surface pathologies

NASA Astrophysics Data System (ADS)

Steven, Philipp; Müller, Maya; Koop, Norbert; Rose, Christian; Hüttmann, Gereon

2009-11-01

Minimally invasive imaging of ocular surface pathologies aims at securing clinical diagnosis without actual tissue probing. For this matter, confocal microscopy (Cornea Module) is in daily use in ophthalmic practice. Multiphoton microscopy is a new optical technique that enables high-resolution imaging and functional analysis of living tissues based on tissue autofluorescence. This study was set up to compare the potential of a multiphoton microscope (DermaInspect) to the Cornea Module. Ocular surface pathologies such as pterygia, papillomae, and nevi were investigated in vivo using the Cornea Module and imaged immediately after excision by DermaInspect. Two excitation wavelengths, fluorescence lifetime imaging and second-harmonic generation (SHG), were used to discriminate different tissue structures. Images were compared with the histopathological assessment of the samples. At wavelengths of 730 nm, multiphoton microscopy exclusively revealed cellular structures. Collagen fibrils were specifically demonstrated by second-harmonic generation. Measurements of fluorescent lifetimes enabled the highly specific detection of goblet cells, erythrocytes, and nevus-cell clusters. At the settings used, DermaInspect reaches higher resolutions than the Cornea Module and obtains additional structural information. The parallel detection of multiphoton excited autofluorescence and confocal imaging could expand the possibilities of minimally invasive investigation of the ocular surface toward functional analysis at higher resolutions.

Towards in vivo breast skin characterization using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Batista, Ana; Uchugonova, Aisada; Breunig, Hans Georg; König, Karsten

2017-02-01

Breast cancer, the most common type of cancer in women worldwide, as well as its treatment (e.g. radiation therapy) can affect the human skin. Multiphoton imaging could provide new insights into these skin alterations non-invasively and with high-resolution. As a preparation for a later investigation involving patients, areas of the breast and forearm skin of healthy volunteers were imaged using the clinically certified multiphoton imaging tomograph MPTflex based on endogenous skin autofluorescence and second-harmonic signals. Depth-resolved image stacks were acquired in consecutive weeks to explore the influence of hormonal variations on the skin properties. Both breasts were considered and up to three different areas were imaged per session. Acquisition parameters were optimized to minimize artifacts caused by breathing-motion. As a first result, skin properties, such as the epidermal thickness, appear to be influenced by hormonal variations.

Quantitative structural markers of colorectal dysplasia in a cross sectional study of ex vivo murine tissue using label-free multiphoton microscopy

NASA Astrophysics Data System (ADS)

Prieto, Sandra P.; Greening, Gage J.; Lai, Keith K.; Muldoon, Timothy J.

2016-03-01

Two-photon excitation of label-free tissue is of increasing interest, as advances have been made in endoscopic clinical application of multiphoton microscopy, such as second harmonic generation (SHG) scanning endoscopy used to monitor cervical collagen in mice1. We used C57BL mice as a model to investigate the progression of gastrointestinal structures, specifically glandular area and circularity. We used multiphoton microscopy to image ex-vivo label-free murine colon, focusing on the collagen structure changes over time, in mice ranging from 10 to 20 weeks of age. Series of images were acquired within the colonic and intestinal tissue at depth intervals of 20 microns from muscularis to the epithelium, up to a maximum depth of 180 microns. The imaging system comprised a two-photon laser tuned to 800nm wavelength excitation, and the SHG emission was filtered with a 400/40 bandpass filter before reaching the photomultiplier tube. Images were acquired at 15 frames per second, for 200 to 300 cumulative frames, with a field of view of 261um by 261um, and 40mW at sample. Image series were compared to histopathology H&E slides taken from adjacent locations. Quantitative metrics for determining differences between murine glandular structures were applied, specifically glandular area and circularity.

Characterizing lamina propria of human gastric mucosa by multiphoton microscopy

NASA Astrophysics Data System (ADS)

Liu, Y. C.; Yang, H. Q.; Chen, G.; Zhuo, S. M.; Chen, J. X.; Yan, J.

2011-01-01

Lamina propria (LP) of gastric mucosa plays an important role in progression of gastric cancer because of the site at where inflammatory reactions occur. Multiphoton imaging has been recently employed for microscopic examination of intact tissue. In this paper, using multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), high resolution multiphoton microscopic images of lamina propria (LP) are obtained in normal human gastric mucosa at excitation wavelength λex = 800 nm. The main source of tissue TPEF originated from the cells of gastric glands, and loose connective tissue, collagen, produced SHG signals. Our results demonstrated that MPM can be effective for characterizing the microstructure of LP in human gastric mucosa. The findings will be helpful for diagnosing and staging early gastric cancer in the clinics.

Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning of thick tissues

NASA Astrophysics Data System (ADS)

Cheng, Li-Chung; Chang, Chia-Yuan; Yen, Wei-Chung; Chen, Shean-Jen

2012-10-01

Conventional multiphoton microscopy employs beam scanning; however, in this study a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. The microscope integrates a 10 kHz repetition rate ultrafast amplifier featuring strong instantaneous peak power (maximum 400 μJ/pulse at 90 fs pulse width) with a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled device camera. This configuration can produce multiphoton excited images with an excitation area larger than 200 × 100 μm2 at a frame rate greater than 100 Hz. Brownian motions of fluorescent microbeads as small as 0.5 μm have been instantaneously observed with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Moreover, we combine the widefield multiphoton microscopy with structure illuminated technique named HiLo to reject the background scattering noise to get better quality for bioimaging.

Raman Microscopy: A Noninvasive Method to Visualize the Localizations of Biomolecules in the Cornea.

PubMed

Kaji, Yuichi; Akiyama, Toshihiro; Segawa, Hiroki; Oshika, Tetsuro; Kano, Hideaki

2017-11-01

In vivo and in situ visualization of biomolecules without pretreatment will be important for diagnosis and treatment of ocular disorders in the future. Recently, multiphoton microscopy, based on the nonlinear interactions between molecules and photons, has been applied to reveal the localizations of various molecules in tissues. We aimed to use multimodal multiphoton microscopy to visualize the localizations of specific biomolecules in rat corneas. Multiphoton images of the corneas were obtained from nonlinear signals of coherent anti-Stokes Raman scattering, third-order sum frequency generation, and second-harmonic generation. The localizations of the adhesion complex-containing basement membrane and Bowman layer were clearly visible in the third-order sum frequency generation images. The fine structure of type I collagen was observed in the corneal stroma in the second-harmonic generation images. The localizations of lipids, proteins, and nucleic acids (DNA/RNA) was obtained in the coherent anti-Stokes Raman scattering images. Imaging technologies have progressed significantly and been applied in medical fields. Optical coherence tomography and confocal microscopy are widely used but do not provide information on the molecular structure of the cornea. By contrast, multiphoton microscopy provides information on the molecular structure of living tissues. Using this technique, we successfully visualized the localizations of various biomolecules including lipids, proteins, and nucleic acids in the cornea. We speculate that multiphoton microscopy will provide essential information on the physiological and pathological conditions of the cornea, as well as molecular localizations in tissues without pretreatment.

Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning.

PubMed

Cheng, Li-Chung; Chang, Chia-Yuan; Lin, Chun-Yu; Cho, Keng-Chi; Yen, Wei-Chung; Chang, Nan-Shan; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen

2012-04-09

In this study, a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. Key features of this microscope are the integrations of a 10 kHz repetition rate ultrafast amplifier featuring high instantaneous peak power (maximum 400 μJ/pulse at a 90 fs pulse width) and a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled camera into a spatiotemporal focusing microscope. This configuration can produce multiphoton images with an excitation area larger than 200 × 100 μm² at a frame rate greater than 100 Hz (current maximum of 200 Hz). Brownian motions of fluorescent microbeads as small as 0.5 μm were observed in real-time with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Furthermore, second harmonic images of chicken tendons demonstrate that the developed widefield multiphoton microscope can provide high resolution z-sectioning for bioimaging.

In vivo multiphoton imaging of human skin: assessment of topical corticosteroid-induced epidermis atrophy and depigmentation

NASA Astrophysics Data System (ADS)

Ait El Madani, Hassan; Tancrède-Bohin, Emmanuelle; Bensussan, Armand; Colonna, Anne; Dupuy, Alain; Bagot, Martine; Pena, Ana-Maria

2012-02-01

Multiphoton microscopy has emerged in the past decade as a promising tool for noninvasive skin imaging. Our aim was to evaluate the potential of multiphoton microscopy to detect topical corticosteroids side effects within the epidermis and to provide new insights into their dynamics. Healthy volunteers were topically treated with clobetasol propionate on a small region of their forearms under overnight occlusion for three weeks. The treated region of each patient was investigated at D0, D7, D15, D22 (end of the treatment), and D60. Our study shows that multiphoton microscopy allows for the detection of corticoid-induced epidermis modifications: thinning of stratum corneum compactum and epidermis, decrease of keratinocytes size, and changes in their morphology from D7 to D22. We also show that multiphoton microscopy enables in vivo three-dimensional (3-D) quantitative assessment of melanin content. We observe that melanin density decreases during treatment and almost completely disappears at D22. Moreover, these alterations are reversible as they are no longer present at D60. Our study demonstrates that multiphoton microscopy is a convenient and powerful tool for noninvasive 3-D dynamical studies of skin integrity and pigmentation.

A novel multiphoton microscopy images segmentation method based on superpixel and watershed.

PubMed

Wu, Weilin; Lin, Jinyong; Wang, Shu; Li, Yan; Liu, Mingyu; Liu, Gaoqiang; Cai, Jianyong; Chen, Guannan; Chen, Rong

2017-04-01

Multiphoton microscopy (MPM) imaging technique based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) shows fantastic performance for biological imaging. The automatic segmentation of cellular architectural properties for biomedical diagnosis based on MPM images is still a challenging issue. A novel multiphoton microscopy images segmentation method based on superpixels and watershed (MSW) is presented here to provide good segmentation results for MPM images. The proposed method uses SLIC superpixels instead of pixels to analyze MPM images for the first time. The superpixels segmentation based on a new distance metric combined with spatial, CIE Lab color space and phase congruency features, divides the images into patches which keep the details of the cell boundaries. Then the superpixels are used to reconstruct new images by defining an average value of superpixels as image pixels intensity level. Finally, the marker-controlled watershed is utilized to segment the cell boundaries from the reconstructed images. Experimental results show that cellular boundaries can be extracted from MPM images by MSW with higher accuracy and robustness. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

PubMed Central

Gaylo, Alison; Overstreet, Michael G.; Fowell, Deborah J.

2016-01-01

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections. PMID:27078264

Multiphoton tomography of the human eye

NASA Astrophysics Data System (ADS)

König, Karsten; Batista, Ana; Hager, Tobias; Seitz, Berthold

2017-02-01

Multiphoton tomography (MPT) is a novel label-free clinical imaging method for non-invasive tissue imaging with high spatial (300 nm) and temporal (100 ps) resolutions. In vivo optical histology can be realized due to the nonlinear excitation of endogenous fluorophores and second-harmonic generation (SHG) of collagen. Furthermore, optical metabolic imaging (OMI) is performed by two-photon autofluorescence lifetime imaging (FLIM). So far, applications of the multiphoton tomographs DermaInspect and MPTflex were limited to dermatology. Novel applications include intraoperative brain tumor imaging as well as cornea imaging. In this work we describe two-photon imaging of ex vivo human corneas unsuitable for transplantation. Furthermore, the cross-linking (CXL) process of corneal collagen based on UVA exposure and 0.1 % riboflavin was studied. The pharmacokinetics of the photosensitizer could be detected with high spatial resolution. Interestingly, an increase in the stromal autofluorescence intensity and modifications of the autofluorescence lifetimes were observed in the human corneal samples within a few days following CXL.

Rapid in vivo vertical tissue sectioning by multiphoton tomography

NASA Astrophysics Data System (ADS)

Batista, Ana; Breunig, Hans Georg; König, Karsten

2018-02-01

A conventional tool in the pathological field is histology which involves the analysis of thin sections of tissue in which specific cellular structures are stained with different dyes. The process to obtain these stained tissue sections is time consuming and invasive as it requires tissue removal, fixation, sectioning, and staining. Moreover, imaging of live tissue is not possible. We demonstrate that multiphoton tomography can provide within seconds, non-invasive, label-free, vertical images of live tissue which are in quality similar to conventional light micrographs of histologic stained specimen. In contrast to conventional setups based on laser scanning which image horizontally sections, the vertical in vivo images are directly recorded by combined line scanning and timed adjustments of the height of the focusing optics. In addition, multiphoton tomography provides autofluorescence lifetimes which can be used to determine the metabolic states of cells.

Transverse correlations in multiphoton entanglement

NASA Astrophysics Data System (ADS)

Wen, Jianming; Rubin, Morton H.; Shih, Yanhua

2007-10-01

We have analyzed the transverse correlation in multiphoton entanglement. The generalization of quantum ghost imaging is extended to the N -photon state. The Klyshko’s two-photon advanced-wave picture is generalized to the N -photon case.

Non-invasive assessment of the liver using imaging

NASA Astrophysics Data System (ADS)

Thorling Thompson, Camilla; Wang, Haolu; Liu, Xin; Liang, Xiaowen; Crawford, Darrell H.; Roberts, Michael S.

2016-12-01

Chronic liver disease causes 2,000 deaths in Australia per year and early diagnosis is crucial to avoid progression to cirrhosis and end stage liver disease. There is no ideal method to evaluate liver function. Blood tests and liver biopsies provide spot examinations and are unable to track changes in function quickly. Therefore better techniques are needed. Non-invasive imaging has the potential to extract increased information over a large sampling area, continuously tracking dynamic changes in liver function. This project aimed to study the ability of three imaging techniques, multiphoton and fluorescence lifetime imaging microscopy, infrared thermography and photoacoustic imaging, in measuring liver function. Collagen deposition was obvious in multiphoton and fluorescence lifetime imaging in fibrosis and cirrhosis and comparable to conventional histology. Infrared thermography revealed a significantly increased liver temperature in hepatocellular carcinoma. In addition, multiphoton and fluorescence lifetime imaging and photoacoustic imaging could both track uptake and excretion of indocyanine green in rat liver. These results prove that non-invasive imaging can extract crucial information about the liver continuously over time and has the potential to be translated into clinic in the assessment of liver disease.

Epifluorescence light collection for multiphoton microscopic endoscopy

NASA Astrophysics Data System (ADS)

Brown, Christopher M.; Rivera, David R.; Xu, Chris; Webb, Watt W.

2011-03-01

Multiphoton microscopic endoscopy (MPM-E) is a promising medical in vivo diagnostic imaging technique because it captures intrinsic fluorescence and second harmonic generation signals to reveal anatomical and histological information about disease states in tissue. However, maximizing light collection from multiphoton endoscopes remains a challenge: weak nonlinear emissions from endogenous structures, miniature optics, large imaging depths, and light scattering in tissue all hamper light collection. The quantity of light that may be collected using a dual-clad fiber system from scattering phantoms that mimic the properties of the in vivo environment is measured. In this experiment, 800nm excitation light from a Ti:Sapphire laser is dispersion compensated and focused through a SM800 optical fiber and lens system into the tissue phantom. Emission light from the phantom passes through the lens system, reflects off the dichroic and is then collected by a second optical fiber actuated by a micromanipulator. The lateral position of the collection fiber varies, measuring the distribution of emitted light 2000μm on either side of the focal point reimaged to the object plane. This spatial collection measurement is performed at depths up to 200μm from the phantom surface. The tissue phantoms are composed of a 15.8 μM fluorescein solution mixed with microspheres, approximating the scattering properties of human bladder and dermis tissue. Results show that commercially available dual-clad optical fibers collect more than 47% of the total emission returning to the object plane from both phantoms. Based on these results, initial MPM-E devices will image the surface of epithelial tissues.

Video-rate hyperspectral two-photon fluorescence microscopy for in vivo imaging

NASA Astrophysics Data System (ADS)

Deng, Fengyuan; Ding, Changqin; Martin, Jerald C.; Scarborough, Nicole M.; Song, Zhengtian; Eakins, Gregory S.; Simpson, Garth J.

2018-02-01

Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beamscanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam's position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.

High-resolution multiphoton microscopy with a low-power continuous wave laser pump.

PubMed

Chen, Xiang-Dong; Li, Shen; Du, Bo; Dong, Yang; Wang, Ze-Hao; Guo, Guang-Can; Sun, Fang-Wen

2018-02-15

Multiphoton microscopy (MPM) has been widely used for three-dimensional biological imaging. Here, based on the photon-induced charge state conversion process, we demonstrated a low-power high-resolution MPM with a nitrogen vacancy (NV) center in diamond. Continuous wave green and orange lasers were used to pump and detect the two-photon charge state conversion, respectively. The power of the laser for multiphoton excitation was 40 μW. Both the axial and lateral resolutions were improved approximately 1.5 times compared with confocal microscopy. The results can be used to improve the resolution of the NV center-based quantum sensing and biological imaging.

Multiphoton fluorescence imaging of NADH to quantify metabolic changes in epileptic tissue in vitro

NASA Astrophysics Data System (ADS)

Chia, Thomas H.; Zinter, Joseph; Spencer, Dennis D.; Williamson, Anne; Levene, Michael J.

2007-02-01

A powerful advantage of multiphoton microscopy is its ability to image endogenous fluorophores such as the ubiquitous coenzyme NADH in discrete cellular populations. NADH is integral in both oxidative and non-oxidative cellular metabolism. NADH loses fluorescence upon oxidation to NAD +; thus changes in NADH fluorescence can be used to monitor metabolism. Recent studies have suggested that hypo metabolic astrocytes play an important role in cases of temporal lobe epilepsy (TLE). Current theories suggest this may be due to defective and/or a reduced number of mitochondria or dysfunction of the neuronal-astrocytic metabolic coupling. Measuring NADH fluorescence changes following chemical stimulation enables the quantification of the cellular distribution of metabolic anomalies in epileptic brain tissue compared to healthy tissue. We present what we believe to be the first multiphoton microscopy images of NADH from the human brain. We also present images of NADH fluorescence from the hippocampus of the kainate-treated rat TLE model. In some experiments, human and rat astrocytes were selectively labeled with the fluorescent dye sulforhodamine 101 (SR101). Our results demonstrate that multiphoton microscopy is a powerful tool for assaying the metabolic pathologies associated with temporal lobe epilepsy in humans and in rodent models.

In vivo 3D measurement of moxifloxacin and gatifloxacin distributions in the mouse cornea using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Lee, Seunghun; Lee, Jun Ho; Park, Jin Hyoung; Yoon, Yeoreum; Chung, Wan Kyun; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

2016-05-01

Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. Intrinsic multiphoton fluorescence properties of moxifloxacin and gatifloxacin were characterized, and their distributions in mouse cornea in vivo were measured by 3D MPM imaging. Both moxifloxacin and gatifloxacin had similar multiphoton spectra, while moxifloxacin had stronger fluorescence than gatifloxacin. MPM imaging of mouse cornea in vivo showed (1) moxifloxacin had good penetration through the superficial corneal epithelium, while gatifloxacin had relatively poor penetration, (2) both ophthalmic solutions had high intracellular distribution. In vivo MPM results were consistent with previous studies. This study demonstrates the feasibility of MPM as a method for in vivo direct measurement of moxifloxacin and gatifloxacin in the cornea.

In vivo 3D measurement of moxifloxacin and gatifloxacin distributions in the mouse cornea using multiphoton microscopy

PubMed Central

Lee, Seunghun; Lee, Jun Ho; Park, Jin Hyoung; Yoon, Yeoreum; Chung, Wan Kyun; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

2016-01-01

Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. Intrinsic multiphoton fluorescence properties of moxifloxacin and gatifloxacin were characterized, and their distributions in mouse cornea in vivo were measured by 3D MPM imaging. Both moxifloxacin and gatifloxacin had similar multiphoton spectra, while moxifloxacin had stronger fluorescence than gatifloxacin. MPM imaging of mouse cornea in vivo showed (1) moxifloxacin had good penetration through the superficial corneal epithelium, while gatifloxacin had relatively poor penetration, (2) both ophthalmic solutions had high intracellular distribution. In vivo MPM results were consistent with previous studies. This study demonstrates the feasibility of MPM as a method for in vivo direct measurement of moxifloxacin and gatifloxacin in the cornea. PMID:27138688

The nature of multiphoton fluorescence from red blood cells

NASA Astrophysics Data System (ADS)

Saytashev, Ilyas; Murphy, Michael; Osseiran, Sam; Spence, Dana M.; Evans, Conor L.; Dantus, Marcos

2016-03-01

We report on the nature of multiphoton excited fluorescence observed from human erythrocytes (red blood cells RBC's) and their "ghosts" following 800nm sub-15 fs excitation. The detected optical signal is assigned as two-photon excited fluorescence from hemoglobin. Our findings are supported by wavelength-resolved fluorescence lifetime decay measurements using time-correlated single photon counting system from RBC's, their ghosts as well as in vitro samples of various fluorophores including riboflavin, NADH, NAD(P)H, hemoglobin. We find that low-energy and short-duration pulses allow two-photon imaging of RBC's, but longer more intense pulses lead to their destruction.

Optical Spectroscopy and Multiphoton Imaging for the Diagnosis and Characterization of Hyperplasias in the Mouse Mammary

DTIC Science & Technology

2006-09-01

was inhibited with 3 - bromopyruvate , which inhibits glyceraldehyde- 3 -phosphate dehydrogenase and 3 -phosphoglycerate kinase in a competitive manner (8...consistent with FAD fluorescence (12). Multiphoton FLIM of NADH showed that 3 - bromopyruvate caused an increase in the fluorescence lifetime of protein...images from 4 dishes), cells treated with 3 - bromopyruvate (n=6 images from 2 dishes), which inhibits glycolysis, and cells treated with CoCl2 (n=6

All-near-infrared multiphoton microscopy interrogates intact tissues at deeper imaging depths than conventional single- and two-photon near-infrared excitation microscopes

PubMed Central

Sarder, Pinaki; Yazdanfar, Siavash; Akers, Walter J.; Tang, Rui; Sudlow, Gail P.; Egbulefu, Christopher

2013-01-01

Abstract. The era of molecular medicine has ushered in the development of microscopic methods that can report molecular processes in thick tissues with high spatial resolution. A commonality in deep-tissue microscopy is the use of near-infrared (NIR) lasers with single- or multiphoton excitations. However, the relationship between different NIR excitation microscopic techniques and the imaging depths in tissue has not been established. We compared such depth limits for three NIR excitation techniques: NIR single-photon confocal microscopy (NIR SPCM), NIR multiphoton excitation with visible detection (NIR/VIS MPM), and all-NIR multiphoton excitation with NIR detection (NIR/NIR MPM). Homologous cyanine dyes provided the fluorescence. Intact kidneys were harvested after administration of kidney-clearing cyanine dyes in mice. NIR SPCM and NIR/VIS MPM achieved similar maximum imaging depth of ∼100  μm. The NIR/NIR MPM enabled greater than fivefold imaging depth (>500  μm) using the harvested kidneys. Although the NIR/NIR MPM used 1550-nm excitation where water absorption is relatively high, cell viability and histology studies demonstrate that the laser did not induce photothermal damage at the low laser powers used for the kidney imaging. This study provides guidance on the imaging depth capabilities of NIR excitation-based microscopic techniques and reveals the potential to multiplex information using these platforms. PMID:24150231

Direct trabecular meshwork imaging in porcine eyes through multiphoton gonioscopy

PubMed Central

Masihzadeh, Omid; Ammar, David A.; Kahook, Malik Y.; Gibson, Emily A.; Lei, Tim C.

2013-01-01

Abstract. The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido-corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans. PMID:23515864

Direct trabecular meshwork imaging in porcine eyes through multiphoton gonioscopy.

PubMed

Masihzadeh, Omid; Ammar, David A; Kahook, Malik Y; Gibson, Emily A; Lei, Tim C

2013-03-01

The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido-corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans.

Direct trabecular meshwork imaging in porcine eyes through multiphoton gonioscopy

NASA Astrophysics Data System (ADS)

Masihzadeh, Omid; Ammar, David A.; Kahook, Malik Y.; Gibson, Emily A.; Lei, Tim C.

2013-03-01

The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido-corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans.

Influence of Vacuum Cooling on Escherichia coli O157:H7 Infiltration in Fresh Leafy Greens via a Multiphoton-Imaging Approach

PubMed Central

Vonasek, Erica

2015-01-01

Microbial pathogen infiltration in fresh leafy greens is a significant food safety risk factor. In various postharvest operations, vacuum cooling is a critical process for maintaining the quality of fresh produce. The overall goal of this study was to evaluate the risk of vacuum cooling-induced infiltration of Escherichia coli O157:H7 into lettuce using multiphoton microscopy. Multiphoton imaging was chosen as the method to locate E. coli O157:H7 within an intact lettuce leaf due to its high spatial resolution, low background fluorescence, and near-infrared (NIR) excitation source compared to those of conventional confocal microscopy. The variables vacuum cooling, surface moisture, and leaf side were evaluated in a three-way factorial study with E. coli O157:H7 on lettuce. A total of 188 image stacks were collected. The images were analyzed for E. coli O157:H7 association with stomata and E. coli O157:H7 infiltration. The quantitative imaging data were statistically analyzed using analysis of variance (ANOVA). The results indicate that the low-moisture condition led to an increased risk of microbial association with stomata (P < 0.05). Additionally, the interaction between vacuum cooling levels and moisture levels led to an increased risk of infiltration (P < 0.05). This study also demonstrates the potential of multiphoton imaging for improving sensitivity and resolution of imaging-based measurements of microbial interactions with intact leaf structures, including infiltration. PMID:26475109

First in vivo animal studies on intraocular nanosurgery and multiphoton tomography with low-energy 80-MHz near-infrared femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Konig, Karsten; Wang, Bagui; Krauss, Oliver; Riemann, Iris; Schubert, Harald; Kirste, Sigrun; Fischer, Peter

2004-07-01

We report on a method for refractive laser surgery based on low-energy femtosecond laser pulses provided by ultracompact turn-key non-amplified laser systems. An additional excimer laser is not required for ablation of the stroma. The novel method has the potential to be used for (i) optical flap creation as well as stroma ablation and (ii) for non-invasive flap-free intrastromal ablation. In addition, 3D multiphoton imaging of the cornea can be performed. In particular, we used sub-nanojoule near infrared 80 MHz femtosecond laser pulses for multiphoton imaging of corneal structures with ultrahigh resolution (< 1μm) as well as for highly precise intraocular refractive surgery. Imaging based on two-photon excited cellular autofluorescence and SHG formation in collagen structures was performed at GW/cm2 intensities, whereas destructive optical breakdown for nanoprocessing occurred at TW/cm2 light intensities. These high intensities were realized with sub-nJ pulses within a subfemtoliter intrastromal volume by diffraction-limited focussing with high NA objectives and beam scanning 50 to 140 μm below the epithelial surface. Multiphoton tomography of the cornea was used to determine the target of interest and to visualize intraocular post-laser effects. Histological examination with light- and electron microscopes of laser-exposed porcine and rabbit eyes reveal a minimum intratissue cut size below 1 μm without destructive effects to surrounding collagen structures. LASIK flaps and intracorneal cavities could be realized with high precision using 200 fs, 80 MHz, sub-nanojoule pulses at 800 nm. First studies on 80 MHz femtosecond laser surgery on living rabbits have been performed.

Multiphoton gonioscopy to image the trabecular meshwork of porcine eyes

NASA Astrophysics Data System (ADS)

Masihzadeh, Omid; Ammar, David A.; Kahook, Malik Y.; Gibson, Emily A.; Lei, Tim C.

2013-03-01

The aqueous outflow system (AOS), including the trabecular meshwork (TM), the collector channels (CC) and the Schlemm's canal (SC), regulates intraocular pressure (IOP) through the drainage of the aqueous humor (AH). Abnormal IOP elevation leads to increased pressure stress to retinal ganglion cells, resulting in cell loss that can ultimately lead to complete loss of eyesight. Therefore, development of imaging tools to detect abnormal structural and functional changes of the AOS is important in early diagnosis and prevention of glaucoma. Multiphoton microscopy (MPM), including twophoton autofluorescence (TPAF) and second harmonic generation (SHG), is a label-free microscopic technique that allows molecular specific imaging of biological tissues like the TM. Since the TM and other AOS structures are located behind the highly scattering scleral tissue, transscleral imaging of the TM does not provide enough optical resolution. In this work, a gonioscopic lens is used to allow direct optical access of the TM through the cornea for MPM imaging. Compared to transscleral imaging, the acquired MPM images show improved resolution as individual collagen fiber bundles of the TM can be observed. MPM gonioscopy may have the potential to be developed as a future clinical imaging tool for glaucoma diagnostics.

Optimizing ultrafast wide field-of-view illumination for high-throughput multi-photon imaging and screening of mutant fluorescent proteins

NASA Astrophysics Data System (ADS)

Stoltzfus, Caleb; Mikhailov, Alexandr; Rebane, Aleksander

2017-02-01

Fluorescence induced by 1wo-photon absorption (2PA) and three-photon absorption (3PA) is becoming an increasingly important tool for deep-tissue microscopy, especially in conjunction with genetically-encoded functional probes such as fluorescent proteins (FPs). Unfortunately, the efficacy of the multi-photon excitation of FPs is notoriously low, and because relations between a biological fluorophore's nonlinear-optical properties and its molecular structure are inherently complex, there are no practical avenues available that would allow boosting the performance of current FPs. Here we describe a novel method, where we apply directed evolution to optimize the 2PA properties of EGFP. Key to the success of this approach consists in high-throughput screening of mutants that would allow selection of variants with promising 2PA and 3PA properties in a broad near-IR excitation range of wavelength. For this purpose, we construct and test a wide field-of-view (FOV), femtosecond imaging system that we then use to quantify the multi-photon excited fluorescence in the 550- 1600 nm range of tens of thousands of E. coli colonies expressing randomly mutated FPs in a standard 10 cm diameter Petri dish configuration. We present a quantitative analysis of different factors that are currently limiting the maximum throughput of the femtosecond multi-photon screening techniques and also report on quantitative measurement of absolute 2PA and 3PA cross sections spectra.

Continuum generation in ultra high numerical aperture fiber with application to multiphoton microscopy

NASA Astrophysics Data System (ADS)

Sayler, Nicholas

Nonlinear microscopy benefits from broadband laser sources, enabling efficient excitation of an array of fluorophores, for example. This work demonstrates broadening of a narrow band input pulse (6 nm to 40 nm) centered at 1040 nm with excellent shot-to-shot stability. In a preliminary demonstration, multiphoton imaging with pulses from the fiber is performed. In particular second harmonic imaging of corn starch is performed.

Macroscopic multiphoton biomedical imaging using semiconductor saturable Bragg reflector mode-locked lasers

NASA Astrophysics Data System (ADS)

Girkin, John M.; Burns, David; Dawson, Martin D.

1999-06-01

We report on the development of practical and user friendly lasers for multiphoton imaging of biological material. The laser developed for the work is a laser diode pumped Cr:LiSAF source modelocked using a saturable Bragg reflector as the passive modelocking element. For this system we routinely obtain 100 fs pulses at a repetition rate 200 MHz with an average output power of 20 mW. The laser has a single operator control and is particularly suitable for use by non-laser specialists. We have used the source developed to image a range of biologically significant samples. The initial work has centered on the imaging of intact human dental tissue. The first two-photon images of dental tissue are reported showing the development of early dental disease from depths up to 500 micrometers into the tooth. These results demonstrate the detection of carious lesions before the more conventional techniques currently used by dental practitioners. Work on other living intact biological tissue is also reported, in particular plants containing a genetically bred fluorescent marker to enable the examination of complete and intact living plant tissue.

Photonic crystal fiber-generated coherent supercontinuum for fast stain-free histopathology and intraoperative multiphoton imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Tu, Haohua; You, Sixian; Sun, Yi; Spillman, Darold R.; Ray, Partha S.; Liu, George; Boppart, Stephen A.

2017-03-01

In contrast to a broadband Ti:sapphire laser that mode locks a continuum of emission and enables broadband biophotonic applications, supercontinuum generation moves the spectral broadening outside the laser cavity into a nonlinear medium, and may thus improve environmental stability and more readily enable clinical translation. Using a photonic crystal fiber for passive spectral broadening, this technique becomes widely accessible from a narrowband fixed-wavelength mode-locked laser. Currently, fiber supercontinuum sources have benefited single-photon biological imaging modalities, including light-sheet or confocal microscopy, diffuse optical tomography, and retinal optical coherence tomography. However, they have not fully benefited multiphoton biological imaging modalities with proven capability for high-resolution label-free molecular imaging. The reason can be attributed to the amplitude/phase noise of fiber supercontinuum, which is amplified from the intrinsic noise of the input laser and responsible for spectral decoherence. This instability deteriorates the performance of multiphoton imaging modalities more than that of single-photon imaging modalities. Building upon a framework of coherent fiber supercontinuum generation, we have avoided this instability or decoherence, and balanced the often conflicting needs to generate strong signal, prevent sample photodamage, minimize background noise, accelerate imaging speed, improve imaging depth, accommodate different modalities, and provide user-friendly operation. Our prototypical platforms have enabled fast stain-free histopathology of fresh tissue in both laboratory and intraoperative settings to discover a wide variety of imaging-based cancer biomarkers, which may reduce the cost and waiting stress associated with disease/cancer diagnosis. A clear path toward intraoperative multiphoton imaging can be envisioned to help pathologists and surgeons improve cancer surgery.

A least squares approach to estimating the probability distribution of unobserved data in multiphoton microscopy

NASA Astrophysics Data System (ADS)

Salama, Paul

2008-02-01

Multi-photon microscopy has provided biologists with unprecedented opportunities for high resolution imaging deep into tissues. Unfortunately deep tissue multi-photon microscopy images are in general noisy since they are acquired at low photon counts. To aid in the analysis and segmentation of such images it is sometimes necessary to initially enhance the acquired images. One way to enhance an image is to find the maximum a posteriori (MAP) estimate of each pixel comprising an image, which is achieved by finding a constrained least squares estimate of the unknown distribution. In arriving at the distribution it is assumed that the noise is Poisson distributed, the true but unknown pixel values assume a probability mass function over a finite set of non-negative values, and since the observed data also assumes finite values because of low photon counts, the sum of the probabilities of the observed pixel values (obtained from the histogram of the acquired pixel values) is less than one. Experimental results demonstrate that it is possible to closely estimate the unknown probability mass function with these assumptions.

In vivo, two-color multiphoton microscopy using a femtosecond diamond Raman laser

NASA Astrophysics Data System (ADS)

Jarrett, Jeremy W.; Perillo, Evan P.; Hassan, Ahmed; Miller, David R.; Dunn, Andrew K.

2018-02-01

Multiphoton microscopy is an essential tool for detailed study of neurovascular structure and function. Wavelength mixing of synchronized laser sources—two-color multiphoton microscopy—increases the spectral window of excitable fluorophores without the need for wavelength tuning. However, implementation of two-color microscopy requires a dual output laser source, which is typically costly and complicated. We have developed a relatively simple and low-cost diamond Raman laser pumped with a ytterbium fiber amplifier. The dual output system generates excitation light at both 1060 nm (pump wavelength) and 1250 nm (first Stokes emission of diamond laser) which, when temporally and spatially overlapped, yield an effective two-color excitation wavelength of 1160 nm. This source provides an almost complete coverage of fluorophores excitable within the range of 1000-1300 nm. When compared with 1060 nm excitation, twocolor excitation at 1160 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (e.g. tdKatushka2). We demonstrate multicolor imaging of tdKatushka2 and Hoechst 33342 via simultaneous two-color twophoton, and two-color three-photon microscopy in engineered 3-D multicellular spheroids. Additionally, we show that this laser system is capable of in vivo imaging in mouse cortex to nearly 1 mm in depth with two-color excitation. This system can also be used to excite genetically encoded calcium indicators (e.g. RCaMP and GCaMP), which will be paramount in studying neuronal activity.

Multiphoton imaging for assessing renal disposition in acute kidney injury

NASA Astrophysics Data System (ADS)

Liu, Xin; Liang, Xiaowen; Wang, Haolu; Roberts, Darren M.; Roberts, Michael S.

2016-11-01

Estimation of renal function and drug renal disposition in acute kidney injury (AKI), is important for appropriate dosing of drugs and adjustment of therapeutic strategies, but is challenging due to fluctuations in kidney function. Multiphoton microscopy has been shown to be a useful tool in studying drug disposition in liver and can reflect dynamic changes of liver function. We extend this imaging technique to investigate glomerular filtration rate (GFR) and tubular transporter functional change in various animal models of AKI, which mimic a broad range of causes of AKI such as hypoxia (renal ischemia- reperfusion), therapeutic drugs (e.g. cisplatin), rhabdomyolysis (e.g. glycerol-induced) and sepsis (e.g. LPSinduced). The MPM images revealed acute injury of tubular cells as indicated by reduced autofluorescence and cellular vacuolation in AKI groups compared to control group. In control animal, systemically injected FITC-labelled inulin was rapidly cleared from glomerulus, while the clearance of FITC-inulin was significantly delayed in most of animals in AKI group, which may reflect the reduced GFR in AKI. Following intravenous injection, rhodamine 123, a fluorescent substrate of p-glycoprotein (one of tubular transporter), was excreted into urine in proximal tubule via p-glycoprotein; in response to AKI, rhodamine 123 was retained in tubular cells as revealed by slower decay of fluorescence intensity, indicating P-gp transporter dysfunction in AKI. Thus, real-time changes in GFR and transporter function can be imaged in rodent kidney with AKI using multiphoton excitation of exogenously injected fluorescent markers.

Automatic 3D segmentation of multiphoton images: a key step for the quantification of human skin.

PubMed

Decencière, Etienne; Tancrède-Bohin, Emmanuelle; Dokládal, Petr; Koudoro, Serge; Pena, Ana-Maria; Baldeweck, Thérèse

2013-05-01

Multiphoton microscopy has emerged in the past decade as a useful noninvasive imaging technique for in vivo human skin characterization. However, it has not been used until now in evaluation clinical trials, mainly because of the lack of specific image processing tools that would allow the investigator to extract pertinent quantitative three-dimensional (3D) information from the different skin components. We propose a 3D automatic segmentation method of multiphoton images which is a key step for epidermis and dermis quantification. This method, based on the morphological watershed and graph cuts algorithms, takes into account the real shape of the skin surface and of the dermal-epidermal junction, and allows separating in 3D the epidermis and the superficial dermis. The automatic segmentation method and the associated quantitative measurements have been developed and validated on a clinical database designed for aging characterization. The segmentation achieves its goals for epidermis-dermis separation and allows quantitative measurements inside the different skin compartments with sufficient relevance. This study shows that multiphoton microscopy associated with specific image processing tools provides access to new quantitative measurements on the various skin components. The proposed 3D automatic segmentation method will contribute to build a powerful tool for characterizing human skin condition. To our knowledge, this is the first 3D approach to the segmentation and quantification of these original images. © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Demodulation signal processing in multiphoton imaging

NASA Astrophysics Data System (ADS)

Fisher, Walter G.; Wachter, Eric A.; Piston, David W.

2002-06-01

Multiphoton laser scanning microscopy offers numerous advantages, but sensitivity can be seriously affected by contamination from ambient room light. Typically, this forces experiments to be performed in an absolutely dark room. Since mode-locked lasers are used to generate detectable signals, signal-processing can be used to avoid such problems by taking advantage of the pulsed characteristics of such lasers. Demodulation of the fluorescence signal generated at the mode-locked frequency can result in significant reduction of interference from ambient noise sources. Such demodulation can be readily adapted to existing microscopes by inserting appropriate processor circuitry between the detector and data collection system, yielding a more robust microscope.

Fluorescence lifetime imaging with near-infrared dyes

NASA Astrophysics Data System (ADS)

Becker, Wolfgang; Shcheslavskiy, Vladislav

2013-02-01

Near-infrared (NIR) dyes are used as fluorescence markers in small-animal imaging and in diffuse optical tomography of the human brain. In these applications it is important to know whether the dyes bind to proteins or other tissue constituents, and whether their fluorescence lifetimes depend on the targets they are bound to. Unfortunately, neither the lasers nor the detectors of commonly used confocal and multiphoton laser scanning microscopes allow for excitation and detection of NIR fluorescence. We therefore upgraded existing confocal TCSPC FLIM systems with NIR lasers and NIR sensitive detectors. In multiphoton systems we used the Ti:Sa laser as a one-photon excitation source in combination with an NIR-sensitive detector in the confocal beam path. We tested a number of NIR dyes in biological tissue. Some of them showed clear lifetime changes depending on the tissue structures they are bound to. We therefore believe that NIR FLIM can deliver supplementary information on the tissue constitution and on local biochemical parameters.

Intravital multiphoton imaging of mouse tibialis anterior muscle

PubMed Central

Lau, Jasmine; Goh, Chi Ching; Devi, Sapna; Keeble, Jo; See, Peter; Ginhoux, Florent; Ng, Lai Guan

2016-01-01

ABSTRACT Intravital imaging by multiphoton microscopy is a powerful tool to gain invaluable insight into tissue biology and function. Here, we provide a step-by-step tissue preparation protocol for imaging the mouse tibialis anterior skeletal muscle. Additionally, we include steps for jugular vein catheterization that allow for well-controlled intravenous reagent delivery. Preparation of the tibialis anterior muscle is minimally invasive, reducing the chances of inducing damage and inflammation prior to imaging. The tibialis anterior muscle is useful for imaging leukocyte interaction with vascular endothelium, and to understand muscle contraction biology. Importantly, this model can be easily adapted to study neuromuscular diseases and myopathies. PMID:28243520

Clinical multiphoton FLIM tomography

NASA Astrophysics Data System (ADS)

König, Karsten

2012-03-01

This paper gives an overview on current clinical high resolution multiphoton fluorescence lifetime imaging in volunteers and patients. Fluorescence lifetime imaging (FLIM) in Life Sciences was introduced in Jena/Germany in 1988/89 based on a ZEISS confocal picosecond dye laser scanning microscope equipped with a single photon counting unit. The porphyrin distribution in living cells and living tumor-bearing mice was studied with high spatial, temporal, and spectral resolution. Ten years later, time-gated cameras were employed to detect dental caries in volunteers based on one-photon excitation of autofluorescent bacteria with long fluorescence lifetimes. Nowadays, one-photon FLIM based on picosecond VIS laser diodes are used to study ocular diseases in humans. Already one decade ago, first clinical twophoton FLIM images in humans were taken with the certified clinical multiphoton femtosecond laser tomograph DermaInspectTM. Multiphoton tomographs with FLIM modules are now operating in hospitals at Brisbane, Tokyo, Berlin, Paris, London, Modena and other European cities. Multiple FLIM detectors allow spectral FLIM with a temporal resolution down to 20 ps (MCP) / 250 ps (PMT) and a spectral resolution of 10 nm. Major FLIM applications include the detection of intradermal sunscreen and tattoo nanoparticles, the detection of different melanin types, the early diagnosis of dermatitis and malignant melanoma, as well as the measurement of therapeutic effects in pateints suffering from dermatitis. So far, more than 1,000 patients and volunteers have been investigated with the clinical multiphoton FLIM tomographs DermaInspectTM and MPTflexTM.

In vivo stepwise multi-photon activation fluorescence imaging of melanin in human skin

NASA Astrophysics Data System (ADS)

Lai, Zhenhua; Gu, Zetong; Abbas, Saleh; Lowe, Jared; Sierra, Heidy; Rajadhyaksha, Milind; DiMarzio, Charles

2014-03-01

The stepwise multi-photon activated fluorescence (SMPAF) of melanin is a low cost and reliable method of detecting melanin because the activation and excitation can be a continuous-wave (CW) mode near infrared (NIR) laser. Our previous work has demonstrated the melanin SMPAF images in sepia melanin, mouse hair, and mouse skin. In this study, we show the feasibility of using SMPAF to detect melanin in vivo. in vivo melanin SMPAF images of normal skin and benign nevus are demonstrated. SMPAF images add specificity for melanin detection than MPFM images and CRM images. Melanin SMPAF is a promising technology to enable early detection of melanoma for dermatologists.

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.

Multi-photon vertical cross-sectional imaging with a dynamically-balanced thin-film PZT z-axis microactuator.

PubMed

Choi, Jongsoo; Duan, Xiyu; Li, Haijun; Wang, Thomas D; Oldham, Kenn R

2017-10-01

Use of a thin-film piezoelectric microactuator for axial scanning during multi-photon vertical cross-sectional imaging is described. The actuator uses thin-film lead-zirconate-titanate (PZT) to generate upward displacement of a central mirror platform, micro-machined from a silicon-on-insulator (SOI) wafer to dimensions compatible with endoscopic imaging instruments. Device modeling in this paper focuses on existence of frequencies near device resonance producing vertical motion with minimal off-axis tilt even in the presence of multiple vibration modes and non-uniformity in fabrication outcomes. Operation near rear resonance permits large stroke lengths at low voltages relative to other vertical microactuators. Highly uniform vertical motion of the mirror platform is a key requirement for vertical cross-sectional imaging in the remote scan architecture being used for multi-photon instrument prototyping. The stage is installed in a benchtop testbed in combination with an electrostatic mirror that performs in-plane scanning. Vertical sectional images are acquired from 15 μm diameter beads and excised mouse colon tissue.

Multimodal microscopy and the stepwise multi-photon activation fluorescence of melanin

NASA Astrophysics Data System (ADS)

Lai, Zhenhua

The author's work is divided into three aspects: multimodal microscopy, stepwise multi-photon activation fluorescence (SMPAF) of melanin, and customized-profile lenses (CPL) for on-axis laser scanners, which will be introduced respectively. A multimodal microscope provides the ability to image samples with multiple modalities on the same stage, which incorporates the benefits of all modalities. The multimodal microscopes developed in this dissertation are the Keck 3D fusion multimodal microscope 2.0 (3DFM 2.0), upgraded from the old 3DFM with improved performance and flexibility, and the multimodal microscope for targeting small particles (the "Target" system). The control systems developed for both microscopes are low-cost and easy-to-build, with all components off-the-shelf. The control system have not only significantly decreased the complexity and size of the microscope, but also increased the pixel resolution and flexibility. The SMPAF of melanin, activated by a continuous-wave (CW) mode near-infrared (NIR) laser, has potential applications for a low-cost and reliable method of detecting melanin. The photophysics of melanin SMPAF has been studied by theoretical analysis of the excitation process and investigation of the spectra, activation threshold, and photon number absorption of melanin SMPAF. SMPAF images of melanin in mouse hair and skin, mouse melanoma, and human black and white hairs are compared with images taken by conventional multi-photon fluorescence microscopy (MPFM) and confocal reflectance microscopy (CRM). SMPAF images significantly increase specificity and demonstrate the potential to increase sensitivity for melanin detection compared to MPFM images and CRM images. Employing melanin SMPAF imaging to detect melanin inside human skin in vivo has been demonstrated, which proves the effectiveness of melanin detection using SMPAF for medical purposes. Selective melanin ablation with micrometer resolution has been presented using the Target system. Compared to the traditional selective photothermolysis, this method demonstrates higher precision, higher specificity and deeper penetration. Therefore, the SMPAF guided selective ablation of melanin is a promising tool of removing melanin for both medical and cosmetic purposes. Three CPLs have been designed for low-cost linear-motion scanners, low-cost fast spinning scanners and high-precision fast spinning scanners. Each design has been tailored to the industrial manufacturing ability and market demands.

Combined multiphoton imaging and automated functional enucleation of porcine oocytes using femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Kuetemeyer, Kai; Lucas-Hahn, Andrea; Petersen, Bjoern; Lemme, Erika; Hassel, Petra; Niemann, Heiner; Heisterkamp, Alexander

2010-07-01

Since the birth of ``Dolly'' as the first mammal cloned from a differentiated cell, somatic cell cloning has been successful in several mammalian species, albeit at low success rates. The highly invasive mechanical enucleation step of a cloning protocol requires sophisticated, expensive equipment and considerable micromanipulation skill. We present a novel noninvasive method for combined oocyte imaging and automated functional enucleation using femtosecond (fs) laser pulses. After three-dimensional imaging of Hoechst-labeled porcine oocytes by multiphoton microscopy, our self-developed software automatically identified the metaphase plate. Subsequent irradiation of the metaphase chromosomes with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation (functional enucleation). We show that fs laser-based functional enucleation of porcine oocytes completely inhibited the parthenogenetic development without affecting the oocyte morphology. In contrast, nonirradiated oocytes were able to develop parthenogenetically to the blastocyst stage without significant differences to controls. Our results indicate that fs laser systems have great potential for oocyte imaging and functional enucleation and may improve the efficiency of somatic cell cloning.

USE OF MULTIPHOTON LASER SCANNING MICROSCOPY TO IMAGE BENZO[A]PYRENE AND METABOLITES IN FISH EGGS

EPA Science Inventory

Multiphoton laser scanning microscopy (MPLSM) is a promising tool to study the tissue distribution of environmental chemical contaminants during fish early life stages. One such chemical for which this is possible is benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon that a...

Multiphoton Imaging of Rabbit Cornea Treated with Mitomycin C after Photorefractive Keratectomy

NASA Astrophysics Data System (ADS)

Hsueh, Chiu-Mei; Lo, Wen; Wang, Tsung-Jen; Hu, Fung-Rong; Dong, Chen-Yuan

2007-07-01

In this work we use multiphoton microscopy to observe the post surgery structure variation of rabbit cornea after photorefractive keratectomy (PRK). In addition, we added mitomycin C (MMC) to the post surgery rabbit cornea in order to investigate the effect of MMC treatment on the postoperative regeneration.

Multiphoton spectroscopy of human skin in vivo

NASA Astrophysics Data System (ADS)

Breunig, Hans G.; Weinigel, Martin; König, Karsten

2012-03-01

In vivo multiphoton-intensity images and emission spectra of human skin are reported. Optical sections from different depths of the epidermis and dermis have been measured with near-infrared laser-pulse excitation. While the intensity images reveal information on the morphology, the spectra show emission characteristics of main endogenous skin fluorophores like keratin, NAD(P)H, melanin, elastin and collagen as well as of second harmonic generation induced by the excitation-light interaction with the dermal collagen network.

Ex vivo imaging and quantification of liver fibrosis using second-harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Sun, Tzu-Lin; Liu, Yuan; Sung, Ming-Chin; Chen, Hsiao-Ching; Yang, Chun-Hui; Hovhannisyan, Vladimir; Lin, Wei-Chou; Jeng, Yung-Ming; Chen, Wei-Liang; Chiou, Ling-Ling; Huang, Guan-Tarn; Kim, Ki-Hean; So, Peter T. C.; Chen, Yang-Fang; Lee, Hsuan-Shu; Dong, Chen-Yuan

2010-05-01

Conventionally, liver fibrosis is diagnosed using histopathological techniques. The traditional method is time-consuming in that the specimen preparation procedure requires sample fixation, slicing, and labeling. Our goal is to apply multiphoton microscopy to efficiently image and quantitatively analyze liver fibrosis specimens bypassing steps required in histological preparation. In this work, the combined imaging modality of multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) was used for the qualitative imaging of liver fibrosis of different METAVIR grades under label-free, ex vivo conditions. We found that while MAF is effective in identifying cellular architecture in the liver specimens, it is the spectrally distinct SHG signal that allows the characterization of the extent of fibrosis. We found that qualitative SHG imaging can be used for the effective identification of the associated features of liver fibrosis specimens graded METAVIR 0 to 4. In addition, we attempted to associate quantitative SHG signal to the different METAVIR grades and found that an objective determination of the extent of disease progression can be made. Our approach demonstrates the potential of using multiphoton imaging in rapid classification of ex vivo liver fibrosis in the clinical setting and investigation of liver fibrosis-associated physiopathology in animal models in vivo.

Ex vivo imaging and quantification of liver fibrosis using second-harmonic generation microscopy.

PubMed

Sun, Tzu-Lin; Liu, Yuan; Sung, Ming-Chin; Chen, Hsiao-Ching; Yang, Chun-Hui; Hovhannisyan, Vladimir; Lin, Wei-Chou; Jeng, Yung-Ming; Chen, Wei-Liang; Chiou, Ling-Ling; Huang, Guan-Tarn; Kim, Ki-Hean; So, Peter T C; Chen, Yang-Fang; Lee, Hsuan-Shu; Dong, Chen-Yuan

2010-01-01

Conventionally, liver fibrosis is diagnosed using histopathological techniques. The traditional method is time-consuming in that the specimen preparation procedure requires sample fixation, slicing, and labeling. Our goal is to apply multiphoton microscopy to efficiently image and quantitatively analyze liver fibrosis specimens bypassing steps required in histological preparation. In this work, the combined imaging modality of multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) was used for the qualitative imaging of liver fibrosis of different METAVIR grades under label-free, ex vivo conditions. We found that while MAF is effective in identifying cellular architecture in the liver specimens, it is the spectrally distinct SHG signal that allows the characterization of the extent of fibrosis. We found that qualitative SHG imaging can be used for the effective identification of the associated features of liver fibrosis specimens graded METAVIR 0 to 4. In addition, we attempted to associate quantitative SHG signal to the different METAVIR grades and found that an objective determination of the extent of disease progression can be made. Our approach demonstrates the potential of using multiphoton imaging in rapid classification of ex vivo liver fibrosis in the clinical setting and investigation of liver fibrosis-associated physiopathology in animal models in vivo.

Applications of rigid and flexible GRIN-endoscopes

NASA Astrophysics Data System (ADS)

Schenkl, Selma; Ehlers, Alexander; Riemann, Iris; Messerschmidt, Bernhard; Bückle, Rainer; König, Karsten

2007-02-01

Multiphoton autofluorescence imaging became an important technique for minimal invasive examination of cells in biological tissue. Rigid and flexible endoscopes based on gradient index lenses (GRIN-lenses) extend this minimalinvasive technique to deep lying cell layers, inner body and specimens, difficult to access. In the rigid endoscope, a GRIN-lens overcomes the limited depth range, given by the working distance of the microscope objective. The focus of the conventional laser scanning tomography is reproduced tens of millimeters in the specimen under study by the GRIN-lens (diameter 1.8 and 3 μm). We will present images of fluorescent beads, proteins cells and skin tissue, as well as first in vivo measurements on human skin. The autofluorescence signal stems from the endogenous fluorophore elastin and SHG from collagen. The flexible endoscope dispenses completely the need of a microscope next to the specimen of interest. The excitation laser pulses is delivered via a well-characterized photonic crystal fiber and subsequently focused by a newly designed GRIN-lens system. The fluorescence, also transferred by a fiber is detected by a PMT detector. We will show the excellent imaging qualities of a newly developed GRIN-lens system with high-resolution images of proteins, cells and plant tissue and give an out-look on multiphoton endoscopy.

Clinical coherent anti-Stokes Raman scattering and multiphoton tomography of human skin with a femtosecond laser and photonic crystal fiber

NASA Astrophysics Data System (ADS)

Breunig, Hans Georg; Weinigel, Martin; Bückle, Rainer; Kellner-Höfer, Marcel; Lademann, Jürgen; Darvin, Maxim E.; Sterry, Wolfram; König, Karsten

2013-02-01

We report on in vivo coherent anti-Stokes Raman scattering spectroscopy (CARS), two-photon fluorescence and second-harmonic-generation imaging on human skin with a novel multimodal clinical CARS/multiphoton tomograph. CARS imaging is realized by a combination of femtosecond pulses with broadband continuum pulses generated by a photonic crystal fiber. The images reveal the microscopic distribution of (i) non-fluorescent lipids, (ii) endogenous fluorophores and (iii) the collagen network inside the human skin in vivo with subcellular resolution. Examples of healthy as well as cancer-affected skin are presented.

USE OF MULTIPHOTON LASER SCANNING MICROSCOPY TO IMAGE BENZO[A]PYRENE AND METABOLITES IN FISH EARLY LIFE STAGES

EPA Science Inventory

Multiphoton laser scanning micrsocopy holds promise as a tool to study the tissue distribution of environmental chemical contaminants during fish early life stage development. One such chemical for which this is possible is benzo[a]pyrene (BaP), a polyaromatic hydrocarbon that a...

Comparison of higher-order multiphoton signal generation and collection at the 1700-nm window based on transmittance measurement of objective lenses.

PubMed

Wen, Wenhui; Wang, Yuxin; Liu, Hongji; Wang, Kai; Qiu, Ping; Wang, Ke

2018-01-01

One benefit of excitation at the 1700-nm window is the more accessible modalities of multiphoton signal generation. It is demonstrated here that the transmittance performance of the objective lens is of vital importance for efficient higher-order multiphoton signal generation and collection excited at the 1700-nm window. Two commonly used objective lenses for multiphoton microscopy (MPM) are characterized and compared, one with regular coating and the other with customized coating for high transmittance at the 1700-nm window. Our results show that, fourth harmonic generation imaging of mouse tail tendon and 5-photon fluorescence of carbon quantum dots using the regular objective lens shows an order of magnitude signal higher than those using the customized objective lens. Besides, the regular objective lens also enables a 3-photon fluorescence imaging depth of >1600 μm in mouse brain in vivo. Our results will provide guidelines for objective lens selection for MPM at the 1700-nm window. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photodissociation dynamics of nitromethane and methyl nitrite by infrared multiphoton dissociation imaging with quasiclassical trajectory calculations: signatures of the roaming pathway.

PubMed

Dey, Arghya; Fernando, Ravin; Abeysekera, Chamara; Homayoon, Zahra; Bowman, Joel M; Suits, Arthur G

2014-02-07

We combine the techniques of infrared multiphoton dissociation (IRMPD) with state selective ion imaging to probe roaming dynamics in the unimolecular dissociation of nitromethane and methyl nitrite. Recent theoretical calculations suggest a "roaming-mediated isomerization" pathway of nitromethane to methyl nitrite prior to decomposition. State-resolved imaging of the NO product coupled with infrared multiphoton dissociation was carried out to examine this unimolecular decomposition near threshold. The IRMPD images for the NO product from nitromethane are consistent with the earlier IRMPD studies that first suggested the importance of an isomerization pathway. A significant Λ-doublet propensity is seen in nitromethane IRMPD but not methyl nitrite. The experimental observations are augmented by quasiclassical trajectory calculations for nitromethane and methyl nitrite near threshold for each dissociation pathway. The observation of distinct methoxy vibrational excitation for trajectories from nitromethane and methyl nitrite dissociation at the same total energy show that the nitromethane dissociation bears a nonstatistical signature of the roaming isomerization pathway, and this is possibly responsible for the nitromethane Λ-doublet propensity as well.

Multiphoton Intravital Calcium Imaging.

PubMed

Cheetham, Claire E J

2018-06-26

Multiphoton intravital calcium imaging is a powerful technique that enables high-resolution longitudinal monitoring of cellular and subcellular activity hundreds of microns deep in the living organism. This unit addresses the application of 2-photon microscopy to imaging of genetically encoded calcium indicators (GECIs) in the mouse brain. The protocols in this unit enable real-time intravital imaging of intracellular calcium concentration simultaneously in hundreds of neurons, or at the resolution of single synapses, as mice respond to sensory stimuli or perform behavioral tasks. Protocols are presented for implantation of a cranial imaging window to provide optical access to the brain and for 2-photon image acquisition. Protocols for implantation of both open skull and thinned skull windows for single or multi-session imaging are described. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

Imaging the morphological change of tissue structure during the early phase of esophageal tumor progression using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Xu, Jian; Kang, Deyong; Xu, Meifang; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Jianxin

2012-12-01

Esophageal cancer is a common malignancy with a very poor prognosis. Successful strategies for primary prevention and early detection are critically needed to control this disease. Multiphoton microscopy (MPM) is becoming a novel optical tool of choice for imaging tissue architecture and cellular morphology by two-photon excited fluorescence. In this study, we used MPM to image microstructure of human normal esophagus, carcinoma in situ (CIS), and early invasive carcinoma in order to establish the morphological features to differentiate these tissues. The diagnostic features such as the appearance of cancerous cells, the significant loss of stroma, the absence of the basement membrane were extracted to distinguish between normal and cancerous esophagus tissue. These results correlated well with the paired histological findings. With the advancement of clinically miniaturized MPM and the multi-photon probe, combining MPM with standard endoscopy will therefore allow us to make a real-time in vivo diagnosis of early esophageal cancer at the cellular level.

Differential Multiphoton Laser Scanning Microscopy

PubMed Central

Field, Jeffrey J.; Sheetz, Kraig E.; Chandler, Eric V.; Hoover, Erich E.; Young, Michael D.; Ding, Shi-you; Sylvester, Anne W.; Kleinfeld, David; Squier, Jeff A.

2016-01-01

Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot. PMID:27390511

A review of biomedical multiphoton microscopy and its laser sources

NASA Astrophysics Data System (ADS)

Lefort, Claire

2017-10-01

Multiphoton microscopy (MPM) has been the subject of major development efforts for about 25 years for imaging biological specimens at micron scale and presented as an elegant alternative to classical fluorescence methods such as confocal microscopy. In this topical review, the main interests and technical requirements of MPM are addressed with a focus on the crucial role of excitation source for optimization of multiphoton processes. Then, an overview of the different sources successfully demonstrated in literature for MPM is presented, and their physical parameters are inventoried. A classification of these sources in function with their ability to optimize multiphoton processes is proposed, following a protocol found in literature. Starting from these considerations, a suggestion of a possible identikit of the ideal laser source for MPM concludes this topical review. Dedicated to Martin.

Non-descanned multifocal multiphoton microscopy with a multianode photomultiplier tube

PubMed Central

Cha, Jae Won; Yew, Elijah Y. S.; Kim, Daekeun; Subramanian, Jaichandar; Nedivi, Elly; So, Peter T. C.

2015-01-01

Multifocal multiphoton microscopy (MMM) improves imaging speed over a point scanning approach by parallelizing the excitation process. Early versions of MMM relied on imaging detectors to record emission signals from multiple foci simultaneously. For many turbid biological specimens, the scattering of emission photons results in blurred images and degrades the signal-to-noise ratio (SNR). We have recently demonstrated that a multianode photomultiplier tube (MAPMT) placed in a descanned configuration can effectively collect scattered emission photons from each focus into their corresponding anodes significantly improving image SNR for highly scattering specimens. Unfortunately, a descanned MMM has a longer detection path resulting in substantial emission photon loss. Optical design constraints in a descanned geometry further results in significant optical aberrations especially for large field-of-view (FOV), high NA objectives. Here, we introduce a non-descanned MMM based on MAPMT that substantially overcomes most of these drawbacks. We show that we improve signal efficiency up to fourfold with limited image SNR degradation due to scattered emission photons. The excitation foci can also be spaced wider to cover the full FOV of the objective with minimal aberrations. The performance of this system is demonstrated by imaging interneuron morphological structures deep in the brains of living mice. PMID:25874160

Multiphoton microscopic imaging of human normal and cancerous oesophagus tissue.

PubMed

Chen, W S; Wang, Y; Liu, N R; Zhang, J X; Chen, R

2014-01-01

In this paper, microstructures of human oesophageal submucosa are evaluated using multiphoton microscopy, based on two-photon excited fluorescence and second harmonic generation. The content and distribution of collagen, elastic fibers and cancer cells in normal and cancerous submucosa layer have been distinctly obtained and briefly discussed. The variation of these components is very relevant to the pathology in oesophagus, especially in early oesophageal cancer. Our results further indicate that the multiphoton microscopy technique has the potential application in vivo in clinical diagnosis and monitoring of early oesophageal cancer. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Label-free imaging of cortical structures with multiphoton microscopy

NASA Astrophysics Data System (ADS)

Wang, Shu; Chen, Xiuqiang; Wu, Weilin; Chen, Zhida; Lin, Ruolan; Lin, Peihua; Wang, Xingfu; Fu, Yu Vincent; Chen, Jianxin

2017-02-01

Cortical structures in the central nervous system exhibit an ordered laminar organization. Defined cell layers are significant to our understanding of brain structure and function. In this work, multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), which was applied for qualitatively visualizing the structure of cerebral and cerebellar cortex from the fresh, unfixed, and unstained specimen. MPM is able to effectively identify neurons and neurites in cerebral cortex, as well as glial cells, Purkinje cells, and granule cells in cerebellar cortex at subcellular resolution. In addition, the use of automated image processing algorithms can quantify the circularity of neurons and the density distribution of neurites based on the intrinsic nonlinear optical contrast, further providing quantitative characteristics for automatically analyzing the laminar structure of cortical structures. These results suggest that with the development of the feasibility of two-photon fiberscopes and microendoscope probes, the combined MPM and image analysis holds potential to provide supplementary information to augment the diagnostic accuracy of neuropathology and in vivo identification of various neurological illnesses in clinic.

A microwave-assisted solution combustion synthesis to produce europium-doped calcium phosphate nanowhiskers for bioimaging applications.

PubMed

Wagner, Darcy E; Eisenmann, Kathryn M; Nestor-Kalinoski, Andrea L; Bhaduri, Sarit B

2013-09-01

Biocompatible nanoparticles possessing fluorescent properties offer attractive possibilities for multifunctional bioimaging and/or drug and gene delivery applications. Many of the limitations with current imaging systems center on the properties of the optical probes in relation to equipment technical capabilities. Here we introduce a novel high aspect ratio and highly crystalline europium-doped calcium phosphate nanowhisker produced using a simple microwave-assisted solution combustion synthesis method for use as a multifunctional bioimaging probe. X-ray diffraction confirmed the material phase as europium-doped hydroxyapatite. Fluorescence emission and excitation spectra and their corresponding peaks were identified using spectrofluorimetry and validated with fluorescence, confocal and multiphoton microscopy. The nanowhiskers were found to exhibit red and far red wavelength fluorescence under ultraviolet excitation with an optimal peak emission of 696 nm achieved with a 350 nm excitation. Relatively narrow emission bands were observed, which may permit their use in multicolor imaging applications. Confocal and multiphoton microscopy confirmed that the nanoparticles provide sufficient intensity to be utilized in imaging applications. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Structure of multiphoton quantum optics. I. Canonical formalism and homodyne squeezed states

NASA Astrophysics Data System (ADS)

dell'Anno, Fabio; de Siena, Silvio; Illuminati, Fabrizio

2004-03-01

We introduce a formalism of nonlinear canonical transformations for general systems of multiphoton quantum optics. For single-mode systems the transformations depend on a tunable free parameter, the homodyne local-oscillator angle; for n -mode systems they depend on n heterodyne mixing angles. The canonical formalism realizes nontrivial mixing of pairs of conjugate quadratures of the electromagnetic field in terms of homodyne variables for single-mode systems, and in terms of heterodyne variables for multimode systems. In the first instance the transformations yield nonquadratic model Hamiltonians of degenerate multiphoton processes and define a class of non-Gaussian, nonclassical multiphoton states that exhibit properties of coherence and squeezing. We show that such homodyne multiphoton squeezed states are generated by unitary operators with a nonlinear time evolution that realizes the homodyne mixing of a pair of conjugate quadratures. Tuning of the local-oscillator angle allows us to vary at will the statistical properties of such states. We discuss the relevance of the formalism for the study of degenerate (up-)down-conversion processes. In a companion paper [

Structure of multiphoton quantum optics. I. Canonical formalism and homodyne squeezed states

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

Dell'Anno, Fabio; De Siena, Silvio; Illuminati, Fabrizio

2004-03-01

We introduce a formalism of nonlinear canonical transformations for general systems of multiphoton quantum optics. For single-mode systems the transformations depend on a tunable free parameter, the homodyne local-oscillator angle; for n-mode systems they depend on n heterodyne mixing angles. The canonical formalism realizes nontrivial mixing of pairs of conjugate quadratures of the electromagnetic field in terms of homodyne variables for single-mode systems, and in terms of heterodyne variables for multimode systems. In the first instance the transformations yield nonquadratic model Hamiltonians of degenerate multiphoton processes and define a class of non-Gaussian, nonclassical multiphoton states that exhibit properties of coherencemore » and squeezing. We show that such homodyne multiphoton squeezed states are generated by unitary operators with a nonlinear time evolution that realizes the homodyne mixing of a pair of conjugate quadratures. Tuning of the local-oscillator angle allows us to vary at will the statistical properties of such states. We discuss the relevance of the formalism for the study of degenerate (up-)down-conversion processes. In a companion paper [F. Dell'Anno, S. De Siena, and F. Illuminati, 69, 033813 (2004)], we provide the extension of the nonlinear canonical formalism to multimode systems, we introduce the associated heterodyne multiphoton squeezed states, and we discuss their possible experimental realization.« less

Pushing the Limit of Infrared Multiphoton Dissociation to Megadalton-Size DNA Ions.

PubMed

Doussineau, Tristan; Antoine, Rodolphe; Santacreu, Marion; Dugourd, Philippe

2012-08-16

We report the use of infrared multiphoton dissociation (IRMPD) for the determination of relative activation energies for unimolecular dissociation of megadalton DNA ions. Single ions with masses in the megadalton range were stored in an electrostatic ion trap for a few tens of milliseconds and the image current generated by the roundtrips of ions in the trap was recorded. While being trapped, single ions were irradiated by a CO2 laser and fragmented, owing to multiphoton IR activation. The analysis of the single-ion image current during the heating period allows us to measure changes in the charge of the trapped ion. We estimated the activation energy associated with the dissociation of megadalton-size DNA ions in the frame of an Arrhenius-like model by analyzing a large set of individual ions in order to construct a frequency histogram of the dissociation rates for a collection of ions.

Adaptive multiphoton endomicroscopy through a dynamically deformed multicore optical fiber using proximal detection.

PubMed

Warren, Sean C; Kim, Youngchan; Stone, James M; Mitchell, Claire; Knight, Jonathan C; Neil, Mark A A; Paterson, Carl; French, Paul M W; Dunsby, Chris

2016-09-19

This paper demonstrates multiphoton excited fluorescence imaging through a polarisation maintaining multicore fiber (PM-MCF) while the fiber is dynamically deformed using all-proximal detection. Single-shot proximal measurement of the relative optical path lengths of all the cores of the PM-MCF in double pass is achieved using a Mach-Zehnder interferometer read out by a scientific CMOS camera operating at 416 Hz. A non-linear least squares fitting procedure is then employed to determine the deformation-induced lateral shift of the excitation spot at the distal tip of the PM-MCF. An experimental validation of this approach is presented that compares the proximally measured deformation-induced lateral shift in focal spot position to an independent distally measured ground truth. The proximal measurement of deformation-induced shift in focal spot position is applied to correct for deformation-induced shifts in focal spot position during raster-scanning multiphoton excited fluorescence imaging.

Label-free identification of intestinal metaplasia in the stomach using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Wu, G.; Wei, J.; Zheng, Z.; Ye, J.; Zeng, S.

2014-06-01

The early diagnosis of intestinal metaplasia (IM) in the stomach together with effective therapeutic interventions is crucial to reducing the mortality-rates of the patients associated with gastric cancer. However, it is challenging during conventional white-light endoscopy, and histological analysis remains the ‘gold standard’ for the final diagnosis. Here, we describe a label-free imaging method, multiphoton microscopy (MPM), for the identification of IM in the stomach. It was found that multiphoton imaging provides cellular and subcellular details to the identification of IM from normal gastric tissues. In particular, there is significant difference in the population density of goblet cells between normal and IM gastric tissues, providing substantial potential to become a quantitative intrinsic marker for in vivo clinical diagnosis of early gastric lesions. To our knowledge, this is the first demonstration of the potential of MPM for the identification of IM.

Mitochondrial Permeability Transition in Pathogenesis of Hemorrhagic Injury: Targeted Therapy with Minocycline

DTIC Science & Technology

2012-03-01

minocy- cline treatment (Figures 1-4). Minocycline also improved mitochondrial function as assessed by intravital multiphoton imaging of the...will make direct measurements by intravital multiphoton microscopy to determine whether onset of the mitochondrial permeability transition and...oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization were assessed by intravital microscopy. After H/R with vehicle or

Cold Multiphoton Matrix Assisted Laser Desorption/Ionization (MALDI)

NASA Astrophysics Data System (ADS)

Harris, Peter; Cooke, William; Tracy, Eugene

2008-05-01

We present evidence of a cold multiphoton MALDI process occurring at a Room Temperature Ionic Liquid (RTIL)/metal interface. Our RTIL, 1-Butyl-3-methylimidazolium hexafluorophosphate, remains a stable liquid at room temperatures, even at pressures lower than 10-9 torr. We focus the 2^nd harmonic of a pulsed (2ns pulse length) Nd:YAG laser onto a gold grid coated with RTIL to generate a cold (narrow velocity spread) ion source with temporal resolution comparable to current MALDI ion sources. Unlike conventional MALDI, we believe multiphoton MALDI does not rely on collisional ionization within the ejection plume, and thus produces large signals at laser intensities just above threshold. Removing the collisional ionization process allow us to eject material from smaller regions of a sample, enhancing the suitability of multiphoton MALDI as an ion imaging technique.

Design of a fiber-optic multiphoton microscopy handheld probe

PubMed Central

Zhao, Yuan; Sheng, Mingyu; Huang, Lin; Tang, Shuo

2016-01-01

We have developed a fiber-optic multiphoton microscopy (MPM) system with handheld probe using femtosecond fiber laser. Here we present the detailed optical design and analysis of the handheld probe. The optical systems using Lightpath 352140 and 352150 as objective lens were analyzed. A custom objective module that includes Lightpath 355392 and two customized corrective lenses was designed. Their performances were compared by wavefront error, field curvature, astigmatism, F-θ error, and tolerance in Zemax simulation. Tolerance analysis predicted the focal spot size to be 1.13, 1.19 and 0.83 µm, respectively. Lightpath 352140 and 352150 were implemented in experiment and the measured lateral resolution was 1.22 and 1.3 µm, respectively, which matched with the prediction. MPM imaging by the handheld probe were conducted on leaf, fish scale and rat tail tendon. The MPM resolution can potentially be improved by the custom objective module. PMID:27699109

Design of a fiber-optic multiphoton microscopy handheld probe.

PubMed

Zhao, Yuan; Sheng, Mingyu; Huang, Lin; Tang, Shuo

2016-09-01

We have developed a fiber-optic multiphoton microscopy (MPM) system with handheld probe using femtosecond fiber laser. Here we present the detailed optical design and analysis of the handheld probe. The optical systems using Lightpath 352140 and 352150 as objective lens were analyzed. A custom objective module that includes Lightpath 355392 and two customized corrective lenses was designed. Their performances were compared by wavefront error, field curvature, astigmatism, F-θ error, and tolerance in Zemax simulation. Tolerance analysis predicted the focal spot size to be 1.13, 1.19 and 0.83 µm, respectively. Lightpath 352140 and 352150 were implemented in experiment and the measured lateral resolution was 1.22 and 1.3 µm, respectively, which matched with the prediction. MPM imaging by the handheld probe were conducted on leaf, fish scale and rat tail tendon. The MPM resolution can potentially be improved by the custom objective module.

High-resolution, 2- and 3-dimensional imaging of uncut, unembedded tissue biopsy samples.

PubMed

Torres, Richard; Vesuna, Sam; Levene, Michael J

2014-03-01

Despite continuing advances in tissue processing automation, traditional embedding, cutting, and staining methods limit our ability for rapid, comprehensive visual examination. These limitations are particularly relevant to biopsies for which immediate therapeutic decisions are most necessary, faster feedback to the patient is desired, and preservation of tissue for ancillary studies is most important. The recent development of improved tissue clearing techniques has made it possible to consider use of multiphoton microscopy (MPM) tools in clinical settings, which could address difficulties of established methods. To demonstrate the potential of MPM of cleared tissue for the evaluation of unembedded and uncut pathology samples. Human prostate, liver, breast, and kidney specimens were fixed and dehydrated by using traditional histologic techniques, with or without incorporation of nucleic acid fluorescent stains into dehydration steps. A benzyl alcohol/benzyl benzoate clearing protocol was substituted for xylene. Multiphoton microscopy was performed on a home-built system. Excellent morphologic detail was achievable with MPM at depths greater than 500 μm. Pseudocoloring produced images analogous to hematoxylin-eosin-stained images. Concurrent second-harmonic generation detection allowed mapping of collagen. Subsequent traditional section staining with hematoxylin-eosin did not reveal any detrimental morphologic effects. Sample immunostains on renal tissue showed preservation of normal reactivity. Complete reconstructions of 1-mm cubic samples elucidated 3-dimensional architectural organization. Multiphoton microscopy on cleared, unembedded, uncut biopsy specimens shows potential as a practical clinical tool with significant advantages over traditional histology while maintaining compatibility with gold standard techniques. Further investigation to address remaining implementation barriers is warranted.

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.

3D Printer Generated Tissue iMolds for Cleared Tissue Using Single- and Multi-Photon Microscopy for Deep Tissue Evaluation.

PubMed

Miller, Sean J; Rothstein, Jeffrey D

2017-01-01

Pathological analyses and methodology has recently undergone a dramatic revolution. With the creation of tissue clearing methods such as CLARITY and CUBIC, groups can now achieve complete transparency in tissue samples in nano-porous hydrogels. Cleared tissue is then imagined in a semi-aqueous medium that matches the refractive index of the objective being used. However, one major challenge is the ability to control tissue movement during imaging and to relocate precise locations post sequential clearing and re-staining. Using 3D printers, we designed tissue molds that fit precisely around the specimen being imaged. First, images are taken of the specimen, followed by importing and design of a structural mold, then printed with affordable plastics by a 3D printer. With our novel design, we have innovated tissue molds called innovative molds (iMolds) that can be generated in any laboratory and are customized for any organ, tissue, or bone matter being imaged. Furthermore, the inexpensive and reusable tissue molds are made compatible for any microscope such as single and multi-photon confocal with varying stage dimensions. Excitingly, iMolds can also be generated to hold multiple organs in one mold, making reconstruction and imaging much easier. Taken together, with iMolds it is now possible to image cleared tissue in clearing medium while limiting movement and being able to relocate precise anatomical and cellular locations on sequential imaging events in any basic laboratory. This system provides great potential for screening widespread effects of therapeutics and disease across entire organ systems.

Velocity-Map Imaging for Emittance Characterization of Multiphoton Electron Emission from a Gold Surface

NASA Astrophysics Data System (ADS)

Ye, Hong; Trippel, Sebastian; Di Fraia, Michele; Fallahi, Arya; Mücke, Oliver D.; Kärtner, Franz X.; Küpper, Jochen

2018-04-01

A velocity-map-imaging spectrometer is demonstrated to characterize the normalized emittance (root-mean-square, rms) of photoemitted electron bunches. Both the two-dimensional spatial distribution and the projected velocity distribution images of photoemitted electrons are recorded by the detection system and analyzed to obtain the normalized emittance (rms). With the presented distribution function of the electron photoemission angles, a mathematical method is implemented to reconstruct the three-dimensional velocity distribution. As a first example, multiphoton emission from a planar Au surface is studied via irradiation at a glancing angle by intense 45-fs laser pulses at a central wavelength of 800 nm. The reconstructed energy distribution agrees very well with the Berglund-Spicer theory of photoemission. The normalized emittance (rms) of the intrinsic electron bunch is characterized to be 128 and 14 nm rad in the X and Y directions, respectively. The demonstrated imaging spectrometer has the ability to characterize the normalized emittance (rms) in a few minutes with a fine energy resolution of 0.2 meV in the image center and will, thereby, foster the further development of x-ray free-electron-laser injectors and ultrafast electron diffraction, and it opens up opportunities for studying correlated electron emission from surfaces and vacuum nanoelectronic devices.

Top-down mass spectrometry imaging of intact proteins by laser ablation ESI FT-ICR MS.

PubMed

Kiss, András; Smith, Donald F; Reschke, Brent R; Powell, Matthew J; Heeren, Ron M A

2014-05-01

Laser ablation ESI (LAESI) is a recent development in MS imaging. It has been shown that lipids and small metabolites can be imaged in various samples such as plant material, tissue sections or bacterial colonies without any sample pretreatment. Further, LAESI has been shown to produce multiply charged protein ions from liquids or solid surfaces. This presents a means to address one of the biggest challenges in MS imaging; the identification of proteins directly from biological tissue surfaces. Such identification is hindered by the lack of multiply charged proteins in common MALDI ion sources and the difficulty of performing tandem MS on such large, singly charged ions. We present here top-down identification of intact proteins from tissue with a LAESI ion source combined with a hybrid ion-trap FT-ICR mass spectrometer. The performance of the system was first tested with a standard protein with electron capture dissociation and infrared multiphoton dissociation fragmentation to prove the viability of LAESI FT-ICR for top-down proteomics. Finally, the imaging of a tissue section was performed, where a number of intact proteins were measured and the hemoglobin α chain was identified directly from tissue using CID and infrared multiphoton dissociation fragmentation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Full optical model of micro-endoscope with optical coherence microscopy, multiphoton microscopy and visible capabilities

NASA Astrophysics Data System (ADS)

Vega, David; Kiekens, Kelli C.; Syson, Nikolas C.; Romano, Gabriella; Baker, Tressa; Barton, Jennifer K.

2018-02-01

While Optical Coherence Microscopy (OCM), Multiphoton Microscopy (MPM), and narrowband imaging are powerful imaging techniques that can be used to detect cancer, each imaging technique has limitations when used by itself. Combining them into an endoscope to work in synergy can help achieve high sensitivity and specificity for diagnosis at the point of care. Such complex endoscopes have an elevated risk of failure, and performing proper modelling ensures functionality and minimizes risk. We present full 2D and 3D models of a multimodality optical micro-endoscope to provide real-time detection of carcinomas, called a salpingoscope. The models evaluate the endoscope illumination and light collection capabilities of various modalities. The design features two optical paths with different numerical apertures (NA) through a single lens system with a scanning optical fiber. The dual path is achieved using dichroic coatings embedded in a triplet. A high NA optical path is designed to perform OCM and MPM while a low NA optical path is designed for the visible spectrum to navigate the endoscope to areas of interest and narrowband imaging. Different tests such as the reflectance profile of homogeneous epithelial tissue were performed to adjust the models properly. Light collection models for the different modalities were created and tested for efficiency. While it is challenging to evaluate the efficiency of multimodality endoscopes, the models ensure that the system is design for the expected light collection levels to provide detectable signal to work for the intended imaging.

Multiphoton autofluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada

2017-06-01

The multiphoton fluorescence lifetime imaging tomograph MPTflex with its flexible 360-deg scan head, articulated arm, and tunable femtosecond laser source was employed to study induced pluripotent stem cell (iPS) cultures. Autofluorescence (AF) lifetime imaging was performed with 250-ps temporal resolution and submicron spatial resolution using time-correlated single-photon counting. The two-photon excited AF was based on the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide/flavoproteins. iPS cells generated from mouse embryonic fibroblasts (MEFs) and cocultured with growth-arrested MEFs as feeder cells have been studied. Significant differences on AF lifetime signatures were identified between iPS and feeder cells as well as between their differentiating counterparts.

Ultrafast, large-field multiphoton microscopy based on an acousto-optic deflector and a spatial light modulator.

PubMed

Shao, Yonghong; Qin, Wan; Liu, Honghai; Qu, Junle; Peng, Xiang; Niu, Hanben; Gao, Bruce Z

2012-07-01

We present an ultrafast, large-field multiphoton excitation fluorescence microscope with high lateral and axial resolutions based on a two-dimensional (2-D) acousto-optical deflector (AOD) scanner and spatial light modulator (SLM). When a phase-only SLM is used to shape the near-infrared light from a mode-locked titanium:sapphire laser into a multifocus array including the 0-order beam, a 136 μm × 136 μm field of view is achieved with a 60× objective using a 2-D AOD scanner without any mechanical scan element. The two-photon fluorescence image of a neuronal network that was obtained using this system demonstrates that our microscopy permits observation of dynamic biological events in a large field with high-temporal and -spatial resolution.

Molecule-specific darkfield and multiphoton imaging using gold nanocages

NASA Astrophysics Data System (ADS)

Powless, Amy J.; Jenkins, Samir V.; McKay, Mary Lee; Chen, Jingyi; Muldoon, Timothy J.

2015-03-01

Due to their robust optical properties, biological inertness, and readily adjustable surface chemistry, gold nanostructures have been demonstrated as contrast agents in a variety of biomedical imaging applications. One application is dynamic imaging of live cells using bioconjugated gold nanoparticles to monitor molecule trafficking mechanisms within cells; for instance, the regulatory pathway of epidermal growth factor receptor (EGFR) undergoing endocytosis. In this paper, we have demonstrated a method to track endocytosis of EGFR in MDA-MB-468 breast adenocarcinoma cells using bioconjugated gold nanocages (AuNCs) and multiphoton microscopy. Dynamic imaging was performed using a time series capture of 4 images every minute for one hour. Specific binding and internalization of the bioconjugated AuNCs was observed while the two control groups showed non-specific binding at fewer surface sites, leading to fewer bound AuNCs and no internalization.

Application of Multiphoton Microscopy in Dermatological Studies: a Mini-Review

PubMed Central

Yew, Elijah; Rowlands, Christopher

2014-01-01

This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance. PMID:25075226

Analysis of the chicken retina with an adaptive optics multiphoton microscope.

PubMed

Bueno, Juan M; Giakoumaki, Anastasia; Gualda, Emilio J; Schaeffel, Frank; Artal, Pablo

2011-06-01

The structure and organization of the chicken retina has been investigated with an adaptive optics multiphoton imaging microscope in a backward configuration. Non-stained flat-mounted retinal tissues were imaged at different depths, from the retinal nerve fiber layer to the outer segment, by detecting the intrinsic nonlinear fluorescent signal. From the stacks of images corresponding to the different retinal layers, volume renderings of the entire retina were reconstructed. The density of photoreceptors and ganglion cells layer were directly estimated from the images as a function of the retinal eccentricity. The maximum anatomical resolving power at different retinal eccentricities was also calculated. This technique could be used for a better characterization of retinal alterations during myopia development, and may be useful for visualization of retinal pathologies and intoxication during pharmacological studies.

Reassignment of scattered emission photons in multifocal multiphoton microscopy.

PubMed

Cha, Jae Won; Singh, Vijay Raj; Kim, Ki Hean; Subramanian, Jaichandar; Peng, Qiwen; Yu, Hanry; Nedivi, Elly; So, Peter T C

2014-06-05

Multifocal multiphoton microscopy (MMM) achieves fast imaging by simultaneously scanning multiple foci across different regions of specimen. The use of imaging detectors in MMM, such as CCD or CMOS, results in degradation of image signal-to-noise-ratio (SNR) due to the scattering of emitted photons. SNR can be partly recovered using multianode photomultiplier tubes (MAPMT). In this design, however, emission photons scattered to neighbor anodes are encoded by the foci scan location resulting in ghost images. The crosstalk between different anodes is currently measured a priori, which is cumbersome as it depends specimen properties. Here, we present the photon reassignment method for MMM, established based on the maximum likelihood (ML) estimation, for quantification of crosstalk between the anodes of MAPMT without a priori measurement. The method provides the reassignment of the photons generated by the ghost images to the original spatial location thus increases the SNR of the final reconstructed image.

Multimodal, multiphoton microscopy and image correlation analysis for characterizing corneal thermal damage

NASA Astrophysics Data System (ADS)

Lo, Wen; Chang, Yu-Lin; Liu, Jia-Shiu; Hseuh, Chiu-Mei; Hovhannisyan, Vladimir; Chen, Shean-Jen; Tan, Hsin-Yuan; Dong, Chen-Yuan

2009-09-01

We used the combination of multiphoton autofluorescence (MAF), forward second-harmonic generation (FWSHG), and backward second-harmonic generation (BWSHG) imaging for the qualitative and quantitative characterization of thermal damage of ex vivo bovine cornea. We attempt to characterize the structural alterations by qualitative MAF, FWSHG, and BWSHG imaging in the temperature range of 37 to 90°C. In addition to measuring the absolute changes in the three types of signals at the stromal surface, we also performed image correlation analysis between FWSHG and BWSHG and demonstrate that with increasing thermal damage, image correlation between FWSHG and BWSHG significantly increases. Our results show that while MAF and BWSHG intensities may be used as preliminary indicators of the extent of corneal thermal damage, the most sensitive measures are provided by the decay in FWSHG intensity and the convergence of FWSHG and BWSHG images.

Nonlinear excitation fluorescence microscopy: source considerations for biological applications

NASA Astrophysics Data System (ADS)

Wokosin, David L.

2008-02-01

Ultra-short-pulse solid-state laser sources have improved contrast within fluorescence imaging and also opened new windows of investigation in biological imaging applications. Additionally, the pulsed illumination enables harmonic scattering microscopy which yields intrinsic structure, symmetry and contrast from viable embryos, cells and tissues. Numerous human diseases are being investigated by the combination of (more) intact dynamic tissue imaging of cellular function with gene-targeted specificity and electrophysiology context. The major limitation to more widespread use of multi-photon microscopy has been the complete system cost and added complexity above and beyond commercial camera and confocal systems. The current status of all-solid-state ultrafast lasers as excitation sources will be reviewed since these lasers offer tremendous potential for affordable, reliable, "turnkey" multiphoton imaging systems. This effort highlights the single box laser systems currently commercially available, with defined suggestions for the ranges for individual laser parameters as derived from a biological and fluorophore limited perspective. The standard two-photon dose is defined by 800nm, 10mW, 200fs, and 80Mhz - at the sample plane for tissue culture cells, i.e. after the full scanning microscope system. Selected application-derived excitation wavelengths are well represented by 700nm, 780nm, ~830nm, ~960nm, 1050nm, and 1250nm. Many of the one-box lasers have fixed or very limited excitation wavelengths available, so the lasers will be lumped near 780nm, 800nm, 900nm, 1050nm, and 1250nm. The following laser parameter ranges are discussed: average power from 200mW to 2W, pulse duration from 70fs to 700fs, pulse repetition rate from 20MHz to 200MHz, with the laser output linearly polarized with an extinction ratio at least 100:1.

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.

Multiphoton imaging of myogenic differentiation in gelatin-based hydrogels as tissue engineering scaffolds.

PubMed

Kim, Min Jeong; Shin, Yong Cheol; Lee, Jong Ho; Jun, Seung Won; Kim, Chang-Seok; Lee, Yunki; Park, Jong-Chul; Lee, Soo-Hong; Park, Ki Dong; Han, Dong-Wook

2016-01-01

Hydrogels can serve as three-dimensional (3D) scaffolds for cell culture and be readily injected into the body. Recent advances in the image technology for 3D scaffolds like hydrogels have attracted considerable attention to overcome the drawbacks of ordinary imaging technologies such as optical and fluorescence microscopy. Multiphoton microscopy (MPM) is an effective method based on the excitation of two-photons. In the present study, C2C12 myoblasts differentiated in 3D gelatin hydroxyphenylpropionic acid (GHPA) hydrogels were imaged by using a custom-built multiphoton excitation fluorescence microscopy to compare the difference in the imaging capacity between conventional microscopy and MPM. The physicochemical properties of GHPA hydrogels were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. In addition, the cell viability and proliferation of C2C12 myoblasts cultured in the GHPA hydrogels were analyzed by using Live/Dead Cell and CCK-8 assays, respectively. It was found that C2C12 cells were well grown and normally proliferated in the hydrogels. Furthermore, the hydrogels were shown to be suitable to facilitate the myogenic differentiation of C2C12 cells incubated in differentiation media, which had been corroborated by MPM. It was very hard to get clear images from a fluorescence microscope. Our findings suggest that the gelatin-based hydrogels can be beneficially utilized as 3D scaffolds for skeletal muscle engineering and that MPM can be effectively applied to imaging technology for tissue regeneration.

First multiphoton tomography of brain in man

NASA Astrophysics Data System (ADS)

König, Karsten; Kantelhardt, Sven R.; Kalasauskas, Darius; Kim, Ella; Giese, Alf

2016-03-01

We report on the first two-photon in vivo brain tissue imaging study in man. High resolution in vivo histology by multiphoton tomography (MPT) including two-photon FLIM was performed in the operation theatre during neurosurgery to evaluate the feasibility to detect label-free tumor borders with subcellular resolution. This feasibility study demonstrates, that MPT has the potential to identify tumor borders on a cellular level in nearly real-time.

Multiphoton dynamics of qutrits in the ultrastrong coupling regime with a quantized photonic field

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

Avetissian, H. K., E-mail: avetissian@ysu.am; Avetissian, A. K.; Mkrtchian, G. F.

2015-12-15

Multiphoton resonant excitation of a three-state quantum system (a qutrit) with a single-mode photonic field is considered in the ultrastrong coupling regime, when the qutrit–photonic field coupling rate is comparable to appreciable fractions of the photon frequency. For ultrastrong couplings, the obtained solutions of the Schrödinger equation that reveal multiphoton Rabi oscillations in qutrits with the interference effects leading to the collapse and revival of atomic excitation probabilities at the direct multiphoton resonant transitions.

Detection and mapping of trace explosives on surfaces under ambient conditions using multiphoton electron extraction spectroscopy (MEES).

PubMed

Tang, Shisong; Vinerot, Nataly; Fisher, Danny; Bulatov, Valery; Yavetz-Chen, Yehuda; Schechter, Israel

2016-08-01

Multiphoton electron extraction spectroscopy (MEES) is an analytical method in which UV laser pulses are utilized for extracting electrons from solid surfaces in multiphoton processes under ambient conditions. Counting the emitted electrons as a function of laser wavelength results in detailed spectral features, which can be used for material identification. The method has been applied to detection of trace explosives on a variety of surfaces. Detection was possible on dusty swabs spiked with explosives and also in the standard dry-transfer contamination procedure. Plastic explosives could also be detected. The analytical limits of detection (LODs) are in the sub pmole range, which indicates that MEES is one of the most sensitive detection methods for solid surface under ambient conditions. Scanning the surface with the laser allows for its imaging, such that explosives (as well as other materials) can be located. The imaging mode is also useful in forensic applications, such as detection of explosives in human fingerprints. Copyright © 2016 Elsevier B.V. All rights reserved.

Dynamical measurements of motion behavior of free fluorescent sphere using the wide field temporal focusing microscopy with astigmatism method (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lien, Chi-Hsiang; Lin, Chun-Yu; Chen, Shean-Jen; Chien, Fan-Ching

2017-02-01

A three-dimensional (3D) single fluorescent particle tracking strategy based on temporal focusing multiphoton excitation microscopy (TFMPEM) combined with astigmatism imaging is proposed for delivering nanoscale-level axial information that reveals 3D trajectories of single fluorospheres in the axially-resolved multiphoton excitation volume without z-axis scanning. It provides the dynamical ability by measuring the diffusion coefficient of fluorospheres in glycerol solutions with a position standard deviation of 14 nm and 21 nm in the lateral and axial direction and a frame rate of 100 Hz. Moreover, the optical trapping force based on the TFMPEM is minimized to avoid the interference in the tracing measurements compared to that in the spatial focusing MPE approaches. Therefore, we presented a three dimensional single particle tracking strategy to overcome the limitation of the time resolution of the multiphoton imaging using fast frame rate of TFMPEM, and provide three dimensional locations of multiple particles using an astigmatism method.

Multiphoton Rydberg and valence dynamics of CH3Br probed by mass spectrometry and slice imaging.

PubMed

Hafliðason, Arnar; Glodic, Pavle; Koumarianou, Greta; Samartzis, Peter C; Kvaran, Ágúst

2018-06-18

The multiphoton dynamics of CH3Br were probed by Mass Resolved MultiPhoton Ionization (MR-MPI), Slice Imaging and Photoelectron Imaging in the two-photon excitation region of 66 000 to 80 000 cm-1. Slice images of the CH3+ and Br+ photoproducts of ten two-photon resonant transitions to np and nd Rydberg states of the parent molecule were recorded. CH3+ ions dominate the mass spectra. Kinetic energy release spectra (KERs) were derived from slice and photoelectron images and anisotropy parameters were extracted from the angular distributions of the ions to identify the processes and the dynamics involved. At all wavelengths we observe three-photon excitations, via the two-photon resonant transitions to molecular Rydberg states, forming metastable, superexcited (CH3Br#) states which dissociate to form CH3 Rydberg states (CH3**) along with Br/Br*. A correlation between the parent Rydberg states excited and CH3** formed is evident. For the three highest excitation energies used, the CH3Br# metastable states also generate high kinetic energy fragments of CH3(X) and Br/Br*. In addition for two out of these three wavelengths we also measure one-photon photolysis of CH3Br in the A band forming CH3(X) in various vibrational modes and bromine atoms in the ground (Br) and spin-orbit excited (Br*) states.

Multiphoton imaging the disruptive nature of sulfur mustard lesions

NASA Astrophysics Data System (ADS)

Werrlein, Robert J.; Braue, Catherine R.; Dillman, James F.

2005-03-01

Sulfur mustard [bis-2-chloroethyl sulfide] is a vesicating agent first used as a weapon of war in WWI. It causes debilitating blisters at the epidermal-dermal junction and involves molecules that are also disrupted by junctional epidermolysis bullosa (JEB) and other blistering skin diseases. Despite its recurring use in global conflicts, there is still no completely effective treatment. We have shown by imaging human keratinocytes in cell culture and in intact epidermal tissues that the basal cells of skin contain well-organized molecules (keratins K5/K14, α6β4 integrin, laminin 5 and α3β1 integrin) that are early targets of sulfur mustard. Disruption and collapse of these molecules is coincident with nuclear displacement, loss of functional asymmetry, and loss of polarized mobility. The progression of this pathology precedes basal cell detachment by 8-24 h, a time equivalent to the "clinical latent phase" that defines the extant period between agent exposure and vesication. Our images indicate that disruption of adhesion-complex molecules also impairs cytoskeletal proteins and the integration of structures required for signal transduction and tissue repair. We have recently developed an optical system to test this hypothesis, i.e., to determine whether and how the early disruption of target molecules alters signal transduction. This environmentally controlled on-line system provides a nexus for real-time correlation of imaged lesions with DNA microarray analysis, and for using multiphoton microscopy to facilitate development of more effective treatment strategies.

Label-free structural characterization of mitomycin C-modulated wound healing after photorefractive keratectomy by the use of multiphoton microscopy

NASA Astrophysics Data System (ADS)

Lo, Wen; Wang, Tsung-Jen; Chen, Wei-Liang; Hsueh, Chiu-Mei; Chen, Shean-Jen; Chen, Yang-Fang; Chou, Hsiu-Chu; Lin, Pi-Jung; Hu, Fung-Rong; Dong, Chen-Yuan

2010-05-01

We applied multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) microscopy to monitor corneal wound healing after photorefractive keratectomy (PRK). Our results show that keratocyte activation can be observed by an increase in its MAF, while SHG imaging of corneal stroma can show the depletion of Bowman's layer after PRK and the reticular collagen deposition in the wound healing stage. Furthermore, quantification of the keratocyte activation and collagen deposition in conjunction with immunohistochemistry and histological images demonstrate the effectiveness of mitomycin C (MMC) in suppressing myofibroblast proliferation and collagen regeneration in the post-PRK wound healing process.

Analysis of the chicken retina with an adaptive optics multiphoton microscope

PubMed Central

Bueno, Juan M.; Giakoumaki, Anastasia; Gualda, Emilio J.; Schaeffel, Frank; Artal, Pablo

2011-01-01

The structure and organization of the chicken retina has been investigated with an adaptive optics multiphoton imaging microscope in a backward configuration. Non-stained flat-mounted retinal tissues were imaged at different depths, from the retinal nerve fiber layer to the outer segment, by detecting the intrinsic nonlinear fluorescent signal. From the stacks of images corresponding to the different retinal layers, volume renderings of the entire retina were reconstructed. The density of photoreceptors and ganglion cells layer were directly estimated from the images as a function of the retinal eccentricity. The maximum anatomical resolving power at different retinal eccentricities was also calculated. This technique could be used for a better characterization of retinal alterations during myopia development, and may be useful for visualization of retinal pathologies and intoxication during pharmacological studies. PMID:21698025

In vivo multiphoton kinetic imaging of the toxic effect of carbon tetrachloride on hepatobiliary metabolism.

PubMed

Lin, Chih-Ju; Lee, Sheng-Lin; Lee, Hsuan-Shu; Dong, Chen-Yuan

2018-06-01

We used intravital multiphoton microscopy to study the recovery of hepatobiliary metabolism following carbon tetrachloride (CCl4) induced hepatotoxicity in mice. The acquired images were processed by a first order kinetic model to generate rate constant resolved images of the mouse liver. We found that with progression of hepatotoxicity, the spatial gradient of hepatic function disappeared. A CCl4-induced damage mechanism involves the compromise of membrane functions, resulting in accumulation of processed 6-carboxyfluorescein molecules. At day 14 following induction, a restoration of the mouse hepatobiliary function was found. Our approach allows the study of the response of hepatic functions to chemical agents in real time and is useful for studying pharmacokinetics of drug molecules through optical microscopic imaging. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Multi-photon EIT

NASA Astrophysics Data System (ADS)

Laarits, Toomas; O'Gorman, Bryan; Crescimanno, Michael

2008-03-01

We describe and solve a quantum optics models for multiphoton interrogation of an electromagnetically induced transparency (EIT) resonance. Multiphoton EIT, like its well studied Lambda-system EIT progenitor, is a generalization of the N-resonance process recently studied for atomic time keeping. The solution of these models allows a preliminary determination of this processes utility as the basis of a frequency standard.

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

Avetissian, H. K.; Avchyan, B. R.; Mkrtchian, G. F.

The multiphoton resonant excitation of three-level atoms by the two laser fields of different frequencies is investigated. The time evolution of the system and analytical solutions expressing Rabi oscillations of the probability amplitudes at the two-color multiphoton resonant excitation are found using a nonperturbative resonant approach. The specific examples for experimental implementation of two-color multiphoton resonant excitation of hydrogen atoms are considered.

Photoelectric artefact from optogenetics and imaging on microelectrodes and bioelectronics: New Challenges and Opportunities

PubMed Central

Kozai, Takashi D.Y.; Vazquez, Alberto L.

2015-01-01

Bioelectronics, electronic technologies that interface with biological systems, are experiencing rapid growth in terms of technology development and applications, especially in neuroscience and neuroprosthetic research. The parallel growth with optogenetics and in vivo multi-photon microscopy has also begun to generate great enthusiasm for simultaneous applications with bioelectronic technologies. However, emerging research showing artefact contaminated data highlight the need for understanding the fundamental physical principles that critically impact experimental results and complicate their interpretation. This review covers four major topics: 1) material dependent properties of the photoelectric effect (conductor, semiconductor, organic, photoelectric work function (band gap)); 2) optic dependent properties of the photoelectric effect (single photon, multiphoton, entangled biphoton, intensity, wavelength, coherence); 3) strategies and limitations for avoiding/minimizing photoelectric effects; and 4) advantages of and applications for light-based bioelectronics (photo-bioelectronics). PMID:26167283

QED theory of multiphoton transitions in atoms and ions

NASA Astrophysics Data System (ADS)

Zalialiutdinov, Timur A.; Solovyev, Dmitry A.; Labzowsky, Leonti N.; Plunien, Günter

2018-03-01

This review surveys the quantum theory of electromagnetic radiation for atomic systems. In particular, a review of current theoretical studies of multiphoton processes in one and two-electron atoms and highly charged ions is provided. Grounded on the quantum electrodynamics description the multiphoton transitions in presence of cascades, spin-statistic behaviour of equivalent photons and influence of external electric fields on multiphoton in atoms and anti-atoms are discussed. Finally, the nonresonant corrections which define the validity of the concept of the excited state energy levels are introduced.

Time-resolved photoion imaging spectroscopy: Determining energy distribution in multiphoton absorption experiments

NASA Astrophysics Data System (ADS)

Qian, D. B.; Shi, F. D.; Chen, L.; Martin, S.; Bernard, J.; Yang, J.; Zhang, S. F.; Chen, Z. Q.; Zhu, X. L.; Ma, X.

2018-04-01

We propose an approach to determine the excitation energy distribution due to multiphoton absorption in the case of excited systems following decays to produce different ion species. This approach is based on the measurement of the time-resolved photoion position spectrum by using velocity map imaging spectrometry and an unfocused laser beam with a low fluence and homogeneous profile. Such a measurement allows us to identify the species and the origin of each ion detected and to depict the energy distribution using a pure Poisson's equation involving only one variable which is proportional to the absolute photon absorption cross section. A cascade decay model is used to build direct connections between the energy distribution and the probability to detect each ionic species. Comparison between experiments and simulations permits the energy distribution and accordingly the absolute photon absorption cross section to be determined. This approach is illustrated using C60 as an example. It may therefore be extended to a wide variety of molecules and clusters having decay mechanisms similar to those of fullerene molecules.

Resolution enhancement of 2-photon microscopy using high-refractive index microspheres

NASA Astrophysics Data System (ADS)

Tehrani, Kayvan Forouhesh; Darafsheh, Arash; Phang, Sendy; Mortensen, Luke J.

2018-02-01

Intravital microscopy using multiphoton processes is the standard tool for deep tissue imaging inside of biological specimens. Usually, near-infrared and infrared light is used to excite the sample, which enables imaging several mean free path inside a scattering tissues. Using longer wavelengths, however, increases the width of the effective multiphoton Point Spread Function (PSF). Many features inside of cells and tissues are smaller than the diffraction limit, and therefore not possible to distinguish using a large PSF. Microscopy using high refractive index microspheres has shown promise to increase the numerical aperture of an imaging system and enhance the resolution. It has been shown that microspheres can image features λ/7 using single photon process fluorescence. In this work, we investigate resolution enhancement for Second Harmonic Generation (SHG) and 2-photon fluorescence microscopy. We used Barium Titanate glass microspheres with diameters ˜20-30 μm and refractive index ˜1.9-2.1. We show microsphere-assisted SHG imaging in bone collagen fibers. Since bone is a very dense tissue constructed of bundles of collagen fibers, it is nontrivial to image individual fibers. We placed microspheres on a dense area of the mouse cranial bone, and achieved imaging of individual fibers. We found that microsphere assisted SHG imaging resolves features of the bone fibers that are not readily visible in conventional SHG imaging. We extended this work to 2-photon microscopy of mitochondria in mouse soleus muscle, and with the help of microsphere resolving power, we were able to trace individual mitochondrion from their ensemble.

Label-free in vivo imaging of Drosophila melanogaster by multiphoton microscopy

NASA Astrophysics Data System (ADS)

Lin, Chiao-Ying; Hovhannisyan, Vladimir; Wu, June-Tai; Lin, Sung-Jan; Lin, Chii-Wann; Chen, Jyh-Horng; Dong, Chen-Yuan

2008-02-01

The fruit fly Drosophila melanogaster is one of the most valuable organisms in genetic and developmental biology studies. Drosophila is a small organism with a short life cycle, and is inexpensive and easy to maintain. The entire genome of Drosophila has recently been sequenced (cite the reference). These advantages make fruit fly an attractive model organism for biomedical researches. Unlike humans, Drosophila can be subjected to genetic manipulation with relative ease. Originally, Drosophila was mostly used in classical genetics studies. In the model era of molecular biology, the fruit fly has become a model organ for developmental biology researches. In the past, numerous molecularly modified mutants with well defined genetic defects affecting different aspects of the developmental processes have been identified and studied. However, traditionally, the developmental defects of the mutant flies are mostly examined in isolated fixed tissues which preclude the observation of the dynamic interaction of the different cell types and the extracellular matrix. Therefore, the ability to image different organelles of the fruit fly without extrinsic labeling is invaluable for Drosophila biology. In this work, we successfully acquire in vivo images of both developing muscles and axons of motor neurons in the three larval stages by using the minimially invasive imaging modality of multiphoton (SHG) microscopy. We found that while SHG imaging is useful in revealing the muscular architecture of the developing larva, it is the autofluorescence signal that allows label-free imaging of various organelles to be achieved. Our results demonstrate that multiphoton imaging is a powerful technique for investigation the development of Drosophila.

Extracellular oxygen concentration mapping with a confocal multiphoton laser scanning microscope and TCSPC card

NASA Astrophysics Data System (ADS)

Hosny, Neveen A.; Lee, David A.; Knight, Martin M.

2010-02-01

Extracellular oxygen concentrations influence cell metabolism and tissue function. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods show limited spatial resolution and/or require custom made systems. This study describes a new optimised approach for quantitative extracellular oxygen detection, providing an off-the-shelf system with high spatial resolution and an improved lifetime determination over previous techniques, while avoiding systematic photon pile-up. Fluorescence lifetime detection of an oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]2+, was measured using a Becker&Hickl time-correlated single photon counting (TCSPC) card with excitation provided by a multi-photon laser. This technique was able to identify a subpopulation of isolated chondrocyte cells, seeded in three-dimensional agarose gel, displaying a significant spatial oxygen gradient. Thus this technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

Application of a reflective microscope objective for multiphoton microscopy.

PubMed

Kabir, Mohammad M; Choubal, Aakash M; Toussaint, Kimani C

2018-04-20

Reflective objectives (ROs) mitigate chromatic aberration across a broad wavelength range. Yet, a systematic performance characterisation of ROs has not been done. In this paper, we compare the performance of a 0.5 numerical-aperture (NA) reflective objective (RO) with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments spanning ∼1 octave in the visible and NIR wavelengths, the SO leads to defocusing errors of 25-40% for TPF images of subdiffraction fluorescent beads and 10-12% for SHG images of collagen fibres. The corresponding error for the RO is ∼4% for both imaging modalities. This work emphasises the potential utility of ROs for multimodal multiphoton microscopy applications. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Conjugate adaptive optics with remote focusing in multiphoton microscopy

NASA Astrophysics Data System (ADS)

Tao, Xiaodong; Lam, Tuwin; Zhu, Bingzhao; Li, Qinggele; Reinig, Marc R.; Kubby, Joel

2018-02-01

The small correction volume for conventional wavefront shaping methods limits their application in biological imaging through scattering media. In this paper, we take advantage of conjugate adaptive optics (CAO) and remote focusing (CAORF) to achieve three-dimensional (3D) scanning through a scattering layer with a single correction. Our results show that the proposed system can provide 10 times wider axial field of view compared with a conventional conjugate AO system when 16,384 segments are used on a spatial light modulator. We demonstrate two-photon imaging with CAORF through mouse skull. The fluorescent microspheres embedded under the scattering layers can be clearly observed after applying the correction.

Visualizing radiofrequency-skin interaction using multiphoton microscopy in vivo.

PubMed

Tsai, Tsung-Hua; Lin, Sung-Jan; Lee, Woan-Ruoh; Wang, Chun-Chin; Hsu, Chih-Ting; Chu, Thomas; Dong, Chen-Yuan

2012-02-01

Redundant skin laxity is a major feature of aging. Recently, radiofrequency has been introduced for nonablative tissue tightening by volumetric heating of the deep dermis. Despite the wide range of application based on this therapy, the effect of this technique on tissue and the subsequent tissue remodeling have not been investigated in detail. Our objective is to evaluate the potential of non-linear optics, including multiphoton autofluorescence and second harmonic generation (SHG) microscopy, as a non-invasive imaging modality for the real-time study of radiofrequency-tissue interaction. Electro-optical synergy device (ELOS) was used as the radiofrequency source in this study. The back skin of nude mouse was irradiated with radiofrequency at different passes. We evaluated the effect on skin immediately and 1 month after treatment with multiphoton microscopy. Corresponding histology was performed for comparison. We found that SHG is negatively correlated to radiofrequency passes, which means that collagen structural disruption happens immediately after thermal damage. After 1 month of collagen remodeling, SHG signals increased above baseline, indicating that collagen regeneration has occurred. Our findings may explain mechanism of nonablative skin tightening and were supported by histological examinations. Our work showed that monitoring the dermal heating status of RF and following up the detailed process of tissue reaction can be imaged and quantified with multiphoton microscopy non-invasively in vivo. Copyright © 2011. Published by Elsevier Ireland Ltd.

Development and characterization of non-resonant multiphoton photoacoustic spectroscopy (NMPPAS) for brain tumor margining

NASA Astrophysics Data System (ADS)

Dahal, Sudhir

During tumor removal surgery, due to the problems associated with obtaining high-resolution, real-time chemical images of where exactly the tumor ends and healthy tissue begins (tumor margining), it is often necessary to remove a much larger volume of tissue than the tumor itself. In the case of brain tumor surgery, however, it is extremely unsafe to remove excess tissue. Therefore, without an accurate image of the tumor margins, some of the tumor's finger-like projections are inevitably left behind in the surrounding parenchyma to grow again. For this reason, the development of techniques capable of providing high-resolution real-time images of tumor margins up to centimeters below the surface of a tissue is ideal for the diagnosis and treatment of tumors, as well as surgical guidance during brain tumor excision. A novel spectroscopic technique, non-resonant multiphoton photoacoustic spectroscopy (NMPPAS), is being developed with the capabilities of obtaining high-resolution subsurface chemical-based images of underlying tumors. This novel technique combines the strengths of multiphoton tissue spectroscopy and photoacoustic spectroscopy into a diagnostic methodology that will, ultimately, provide unparalleled chemical information and images to provide the state of sub-surface tissues. The NMPPAS technique employs near-infrared light (in the diagnostic window) to excite ultraviolet and/or visible light absorbing species deep below the tissue's surface. Once a multiphoton absorption event occurs, non-radiative relaxation processes generates a localized thermal expansion and subsequent acoustic wave that can be detected using a piezoelectric transducer. Since NMPPAS employs an acoustic detection modality, much deeper diagnoses can be performed than that is possible using current state of the art high-resolution chemical imaging techniques such as multiphoton fluorescence spectroscopy. NMPPAS was employed to differentiate between excised brain tumors (astrocytoma III) and healthy tissue with over 99% accuracy. NMPPAS spectral features showed evident differences between tumor and healthy tissues, and ratiometric analysis ensured that only a few wavelengths could be used for excitation instead of using numerous wavelength excitations to create spectra. This process would significantly reduce the analysis time while maintaining the same degree of accuracy. Tissue phantoms were fabricated in order to characterize the properties of NMPPAS. Scattering particles were doped into the phantoms to simulate their light scattering properties to real tissues. This allowed for better control over shape, size, reproducibility and doping in the sample while maintaining the light-tissue interaction properties of real tissue. To make NMPPAS viable for clinical applications, the technique was characterized to determine the spatial (lateral and longitudinal) resolution, depth of penetration and its ability to image in three-dimension through layers of tissue. Both resolutions were determined to be near-cellular level resolution (50-70 microm), obtained initially with the aid of the technique of multiphoton fluorescence, and later verified using NMPPAS imaging. Additionally, the maximum depth of penetration and detection was determined to be about 1.4cm, making the technique extremely suitable to margin tumors from underlying tissues in the brain. The capability of NMPPAS to detect and image layers that lie beneath other structures and blood vessels was also investigated. Three-dimensional images were obtained for the first time using NMPPAS. The images were obtained from different depths and structures were imaged through other layers of existing structures in the sample. This verified that NMPPAS was capable of detecting and imaging structures that lie embedded within the tissues. NMPPAS images of embedded structures were also obtained with the presence of hemoglobin, which is potentially the largest source of background in blood-perfused tissues, thus showing that the technique is capable of detecting and differentiating in blood-perfused samples.

Phase Sensitive Demodulation in Multiphoton Microscopy

NASA Astrophysics Data System (ADS)

Fisher, Walt G.; Piston, David W.; Wachter, Eric A.

2002-06-01

Multiphoton laser scanning microscopy offers advantages in depth of penetration into intact samples over other optical sectioning techniques. To achieve these advantages it is necessary to detect the emitted light without spatial filtering. In this nondescanned (nonconfocal) approach, ambient room light can easily contaminate the signal, forcing experiments to be performed in absolute darkness. For multiphoton microscope systems employing mode-locked lasers, signal processing can be used to reduce such problems by taking advantage of the pulsed characteristics of such lasers. Specifically, by recovering fluorescence generated at the mode-locked frequency, interference from stray light and other ambient noise sources can be significantly reduced. This technology can be adapted to existing microscopes by inserting demodulation circuitry between the detector and data collection system. The improvement in signal-to-noise ratio afforded by this approach yields a more robust microscope system and opens the possibility of moving multiphoton microscopy from the research lab to more demanding settings, such as the clinic.

Module for multiphoton high-resolution hyperspectral imaging and spectroscopy

NASA Astrophysics Data System (ADS)

Zeytunyan, Aram; Baldacchini, Tommaso; Zadoyan, Ruben

2018-02-01

We developed a module for dual-output, dual-wavelength lasers that facilitates multiphoton imaging and spectroscopy experiments and enables hyperspectral imaging with spectral resolution up to 5 cm-1. High spectral resolution is achieved by employing spectral focusing. Specifically, two sets of grating pairs are used to control the chirps in each laser beam. In contrast with the approach that uses fixed-length glass rods, grating pairs allow matching the spectral resolution and the linewidths of the Raman lines of interest. To demonstrate the performance of the module, we report the results of spectral focusing CARS and SRS microscopy experiments for various test samples and Raman shifts. The developed module can be used for a variety of multimodal imaging and spectroscopy applications, such as single- and multi-color two-photon fluorescence, second harmonic generation, third harmonic generation, pump-probe, transient absorption, and others.

Automated classification of multiphoton microscopy images of ovarian tissue using deep learning.

PubMed

Huttunen, Mikko J; Hassan, Abdurahman; McCloskey, Curtis W; Fasih, Sijyl; Upham, Jeremy; Vanderhyden, Barbara C; Boyd, Robert W; Murugkar, Sangeeta

2018-06-01

Histopathological image analysis of stained tissue slides is routinely used in tumor detection and classification. However, diagnosis requires a highly trained pathologist and can thus be time-consuming, labor-intensive, and potentially risk bias. Here, we demonstrate a potential complementary approach for diagnosis. We show that multiphoton microscopy images from unstained, reproductive tissues can be robustly classified using deep learning techniques. We fine-train four pretrained convolutional neural networks using over 200 murine tissue images based on combined second-harmonic generation and two-photon excitation fluorescence contrast, to classify the tissues either as healthy or associated with high-grade serous carcinoma with over 95% sensitivity and 97% specificity. Our approach shows promise for applications involving automated disease diagnosis. It could also be readily applied to other tissues, diseases, and related classification problems. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing.

PubMed

Chu, Jing; Shi, Panpan; Deng, Xiaoyuan; Jin, Ying; Liu, Hao; Chen, Maosheng; Han, Xue; Liu, Hanping

2018-03-25

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP-labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full-thickness cutaneous wound site in streptozotocin-induced diabetic mice. Wounds treated with MSC-ADM demonstrated an increased percentage of wound closure. The treatment of MSC-ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col-I) fibers synthesis via second harmonic generation imaging. The synthesis of Col-I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP-labeled MSCs during wound healing was simultaneously traced via two-photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo-multiplier tube. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Imaging sulfur mustard lesions in human epidermal tissues and keratinocytes by confocal and multiphoton microscopy

NASA Astrophysics Data System (ADS)

Werrlein, Robert; Madren-Whalley, Janna S.

2002-06-01

Topical exposure to sulfur mustard (HD), a known theat agent, produces persistent and debilitating cutaneous blisters. The blisters occur at the dermal-epidermal junction following a dose-dependent latent period of 8-24 h, however, the primary lesions causing vesication remain uncertain. Immunofluorescent images reveal that a 5-min exposure to 400 (mu) M HD disrupts molecules that are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Using keratinocyte cultures and fluorochomes conjugated to two different keratin-14 (K14) antibodies (clones CKB1 and LL002), results have shown a statistically significant (p<0.1) 1-h decrease of 29.2% in expression of the CKB1 epitope, a nearly complete loss of CKB1 expression within 2 h, and progressive cytoskeletal (K14) collapse without loss in expression of the LL002 epitope. With human epidermal tissues, multi-photon images of (alpha) 6 integrin and laminin 5 showed disruptive changes in the cell-surface organization and integrity of these adhesion molecules. At 1 H postexposure, analyses showed a statistically significant (p<0.1) decrease of 27.3% in (alpha) 6 integrin emissions, and a 32% decrease in laminin 5 volume. Multi-photon imaging indicates that molecules essential for epidermal-dermal attachment are early targets in the alkylating events leading to HD-induced vesication.

Simulation of multi-photon emission isotopes using time-resolved SimSET multiple photon history generator

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

Chiang, Chih-Chieh; Lin, Hsin-Hon; Lin, Chang-Shiun

Abstract-Multiple-photon emitters, such as In-111 or Se-75, have enormous potential in the field of nuclear medicine imaging. For example, Se-75 can be used to investigate the bile acid malabsorption and measure the bile acid pool loss. The simulation system for emission tomography (SimSET) is a well-known Monte Carlo simulation (MCS) code in nuclear medicine for its high computational efficiency. However, current SimSET cannot simulate these isotopes due to the lack of modeling of complex decay scheme and the time-dependent decay process. To extend the versatility of SimSET for simulation of those multi-photon emission isotopes, a time-resolved multiple photon history generatormore » based on SimSET codes is developed in present study. For developing the time-resolved SimSET (trSimSET) with radionuclide decay process, the new MCS model introduce new features, including decay time information and photon time-of-flight information, into this new code. The half-life of energy states were tabulated from the Evaluated Nuclear Structure Data File (ENSDF) database. The MCS results indicate that the overall percent difference is less than 8.5% for all simulation trials as compared to GATE. To sum up, we demonstrated that time-resolved SimSET multiple photon history generator can have comparable accuracy with GATE and keeping better computational efficiency. The new MCS code is very useful to study the multi-photon imaging of novel isotopes that needs the simulation of lifetime and the time-of-fight measurements. (authors)« less

From morphology to biochemical state – intravital multiphoton fluorescence lifetime imaging of inflamed human skin

PubMed Central

Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

2016-01-01

The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy. PMID:27004454

From morphology to biochemical state - intravital multiphoton fluorescence lifetime imaging of inflamed human skin

NASA Astrophysics Data System (ADS)

Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

2016-03-01

The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy.

Oleic acid-enhanced transdermal delivery pathways of fluorescent nanoparticles

NASA Astrophysics Data System (ADS)

Lo, Wen; Ghazaryan, Ara; Tso, Chien-Hsin; Hu, Po-Sheng; Chen, Wei-Liang; Kuo, Tsung-Rong; Lin, Sung-Jan; Chen, Shean-Jen; Chen, Chia-Chun; Dong, Chen-Yuan

2012-05-01

Transdermal delivery of nanocarriers provides an alternative pathway to transport therapeutic agents, alleviating pain, improving compliance of patients, and increasing overall effectiveness of delivery. In this work, enhancement of transdermal delivery of fluorescent nanoparticles and sulforhodamine B with assistance of oleic acid was visualized utilizing multiphoton microscopy (MPM) and analyzed quantitatively using multi-photon excitation-induced fluorescent signals. Results of MPM imaging and MPM intensity-based spatial depth-dependent analysis showed that oleic acid is effective in facilitating transdermal delivery of nanoparticles.

Multiphoton tomography of intratissue tattoo nanoparticles

NASA Astrophysics Data System (ADS)

König, Karsten

2012-02-01

Most of today's intratissue tattoo pigments are unknown nanoparticles. So far, there was no real control of their use due to the absence of regulations. Some of the tattoo pigments contain carcinogenic amines e.g. azo pigment Red 22. Nowadays, the European Union starts to control the administration of tattoo pigments. There is an interest to obtain information on the intratissue distribution, their interaction with living cells and the extracellular matrix, and the mechanisms behind laser tattoo removal. Multiphoton tomographs are novel biosafety and imaging tools that can provide such information non-invasively and without further labeling. When using the spectral FLIM module, spatially-resolved emission spectra, excitation spectra, and fluorescence lifetimes can pr provided. Multiphoton tomographs are used by all major cosmetic comapanies to test the biosafety of sunscreen nanoparticles.

Usefulness of Intravital Multiphoton Microscopy in Visualizing Study of Mouse Cochlea and Volume Changes in the Scala Media

PubMed Central

Ju, Hyun Mi; Lee, Sun Hee; Kong, Tae Hoon; Kwon, Seung-Hae; Choi, Jin Sil; Seo, Young Joon

2017-01-01

Conventional microscopy has limitations in viewing the cochlear microstructures due to three-dimensional spiral structure and the overlying bone. But these issues can be overcome by imaging the cochlea in vitro with intravital multiphoton microscopy (MPM). By using near-infrared lasers for multiphoton excitation, intravital MPM can detect endogenous fluorescence and second harmonic generation of tissues. In this study, we used intravital MPM to visualize various cochlear microstructures without any staining and non-invasively analyze the volume changes of the scala media (SM) without removing the overlying cochlear bone. The intravital MPM images revealed various tissue types, ranging from thin membranes to dense bone, as well as the spiral ganglion beneath the cochlear bone. The two-dimensional, cross-sectional, and serial z-stack intravital MPM images also revealed the spatial dilation of the SM in the temporal bone of pendrin-deficient mice. These findings suggest that intravital MPM might serve as a new method for obtaining microanatomical information regarding the cochlea, similar to standard histopathological analyses in the animal study for the cochlea. Given the capability of intravital MPM for detecting an increase in the volume of the SM in pendrin-deficient mice, it might be a promising new tool for assessing the pathophysiology of hearing loss in the future. PMID:28824523

Usefulness of Intravital Multiphoton Microscopy in Visualizing Study of Mouse Cochlea and Volume Changes in the Scala Media.

PubMed

Ju, Hyun Mi; Lee, Sun Hee; Kong, Tae Hoon; Kwon, Seung-Hae; Choi, Jin Sil; Seo, Young Joon

2017-01-01

Conventional microscopy has limitations in viewing the cochlear microstructures due to three-dimensional spiral structure and the overlying bone. But these issues can be overcome by imaging the cochlea in vitro with intravital multiphoton microscopy (MPM). By using near-infrared lasers for multiphoton excitation, intravital MPM can detect endogenous fluorescence and second harmonic generation of tissues. In this study, we used intravital MPM to visualize various cochlear microstructures without any staining and non-invasively analyze the volume changes of the scala media (SM) without removing the overlying cochlear bone. The intravital MPM images revealed various tissue types, ranging from thin membranes to dense bone, as well as the spiral ganglion beneath the cochlear bone. The two-dimensional, cross-sectional, and serial z-stack intravital MPM images also revealed the spatial dilation of the SM in the temporal bone of pendrin-deficient mice. These findings suggest that intravital MPM might serve as a new method for obtaining microanatomical information regarding the cochlea, similar to standard histopathological analyses in the animal study for the cochlea. Given the capability of intravital MPM for detecting an increase in the volume of the SM in pendrin-deficient mice, it might be a promising new tool for assessing the pathophysiology of hearing loss in the future.

Multiphoton microscopic imaging of fibrotic focus in invasive ductal carcinoma of the breast

NASA Astrophysics Data System (ADS)

Chen, Sijia; Nie, Yuting; Lian, Yuane; Wu, Yan; Fu, Fangmeng; Wang, Chuan; Zhuo, Shuangmu; Chen, Jianxin

2014-11-01

During the proliferation of breast cancer, the desmoplastic can evoke a fibrosis response by invading healthy tissue. Fibrotic focus (FF) in invasive ductal carcinoma (IDC) of the breast had been reported to be associated with significantly poorer survival rate than IDC without FF. As an important prognosis indicator, it's difficult to obtain the exact fibrotic information from traditional detection method such as mammography. Multiphoton imaging based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) has been recently employed for microscopic examination of unstained tissue. In this study, multiphoton microscopy (MPM) was used to image the fibrotic focus in invasive ductal carcinoma tissue. The morphology and distribution of collagen in fibrotic focus can be demonstrated by the SHG signal. Variation of collagen between IDC with and without FF will be examined and further characterized, which may be greatly related to the metastasis of breast cancer. Our result suggested that the MPM can be efficient in identifying and locating the fibrotic focus in IDC. Combining with the pathology analysis and other detecting methods, MPM owns potential in becoming an advanced histological tool for detecting the fibrotic focus in IDC and collecting prognosis information, which may guide the subsequent surgery option and therapy procedure for patients.

In vivo Delivery of Fluoresceinated Dextrans to the Murine Growth Plate: Imaging of Three Vascular Routes by Multiphoton Microscopy

PubMed Central

Farnum, Cornelia; Lenox, Michelle; Zipfel, Warren; Horton, William; Williams, Rebecca

2008-01-01

Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and via a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature, and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4-5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da), or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa) vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few per cent of vascular concentration) 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes. PMID:16342207

Insight into plant cell wall chemistry and structure by combination of multiphoton microscopy with Raman imaging.

PubMed

Heiner, Zsuzsanna; Zeise, Ingrid; Elbaum, Rivka; Kneipp, Janina

2018-04-01

Spontaneous Raman scattering microspectroscopy, second harmonic generation (SHG) and 2-photon excited fluorescence (2PF) were used in combination to characterize the morphology together with the chemical composition of the cell wall in native plant tissues. As the data obtained with unstained sections of Sorghum bicolor root and leaf tissues illustrate, nonresonant as well as pre-resonant Raman microscopy in combination with hyperspectral analysis reveals details about the distribution and composition of the major cell wall constituents. Multivariate analysis of the Raman data allows separation of different tissue regions, specifically the endodermis, xylem and lumen. The orientation of cellulose microfibrils is obtained from polarization-resolved SHG signals. Furthermore, 2-photon autofluorescence images can be used to image lignification. The combined compositional, morphological and orientational information in the proposed coupling of SHG, Raman imaging and 2PF presents an extension of existing vibrational microspectroscopic imaging and multiphoton microscopic approaches not only for plant tissues. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Intraoperative assisting diagnosis of esophageal submucosal cancer using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Zeng, Yaping; Xu, Jian; Zhou, Qun; Kang, Deyong; Zhuo, Shuangmu; Zhu, Xiaoqin; Lin, Jiangbo; Chen, Jianxin

2018-07-01

Multiphoton microscopy (MPM) based on two-photon excited fluorescence and second harmonic generation can achieve high-resolution images of biological tissues at the cellular and subcellular levels. In this work, we used MPM imaging of intraoperative hematoxylin and eosin (H&E)-stained frozen sections (FSs) of esophagus to explore whether MPM can provide complementary information to the auxiliary diagnosis of esophageal submucosal cancer during the intraoperative period. It was found that MPM has the ability not only to clearly reveal biological tissue microstructure and its morphological changes, but can reveal information not distinguishable in H&E-stained images. The complementary information of nonlinear spectral analysis, orientation and morphology changes in the collagen showed that MPM has important accessory diagnostic value for the differential diagnosis of submucosal carcinoma of the esophagus during the intraoperative period.

Multiphoton microscopic imaging of histological sections without hematoxylin and eosin staining differentiates carcinoma in situ lesion from normal oesophagus

NASA Astrophysics Data System (ADS)

Chen, Jianxin; Xu, Jian; Kang, Deyong; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Jiang, Xingshan

2013-10-01

Multiphoton microscopy (MPM) has become a powerful, important tool for tissues imaging at the molecular level. In this paper, this technique was extended to histological investigations, differentiating carcinoma in situ (CIS) lesion from normal oesophagus by imaging histological sections without hematoxylin and eosin (H&E) staining. The results show that the histology procedures of dehydration, paraffin embedding, and de-paraffinizing highlighted two photon excited fluorescence of cytoplasm and nucleolus of epithelial cell and collagen in stroma. MPM has the ability to identify the characteristics of CIS lesion including changes of squamous cells and full epithelium, identification of basement membrane, especially prominent nucleolus. The studies described here show that MPM has the potential for future retrospective studies of tumor staging by employing on histological section specimens without H&E staining.

Autofluorescence multiphoton microscopy for visualization of tissue morphology and cellular dynamics in murine and human airways.

PubMed

Kretschmer, Sarah; Pieper, Mario; Hüttmann, Gereon; Bölke, Torsten; Wollenberg, Barbara; Marsh, Leigh M; Garn, Holger; König, Peter

2016-08-01

The basic understanding of inflammatory airway diseases greatly benefits from imaging the cellular dynamics of immune cells. Current imaging approaches focus on labeling specific cells to follow their dynamics but fail to visualize the surrounding tissue. To overcome this problem, we evaluated autofluorescence multiphoton microscopy for following the motion and interaction of cells in the airways in the context of tissue morphology. Freshly isolated murine tracheae from healthy mice and mice with experimental allergic airway inflammation were examined by autofluorescence multiphoton microscopy. In addition, fluorescently labeled ovalbumin and fluorophore-labeled antibodies were applied to visualize antigen uptake and to identify specific cell populations, respectively. The trachea in living mice was imaged to verify that the ex vivo preparation reflects the in vivo situation. Autofluorescence multiphoton microscopy was also tested to examine human tissue from patients in short-term tissue culture. Using autofluorescence, the epithelium, underlying cells, and fibers of the connective tissue, as well as blood vessels, were identified in isolated tracheae. Similar structures were visualized in living mice and in the human airway tissue. In explanted murine airways, mobile cells were localized within the tissue and we could follow their migration, interactions between individual cells, and their phagocytic activity. During allergic airway inflammation, increased number of eosinophil and neutrophil granulocytes were detected that moved within the connective tissue and immediately below the epithelium without damaging the epithelial cells or connective tissues. Contacts between granulocytes were transient lasting 3 min on average. Unexpectedly, prolonged interactions between granulocytes and antigen-uptaking cells were observed lasting for an average of 13 min. Our results indicate that autofluorescence-based imaging can detect previously unknown immune cell interactions in the airways. The method also holds the potential to be used during diagnostic procedures in humans if integrated into a bronchoscope.

Characterization of human carotid atherosclerotic tissues imaged by combining multiple multiphoton microscopy techniques

NASA Astrophysics Data System (ADS)

Baria, E.; Cicchi, R.; Nesi, G.; Massi, D.; Pavone, F. S.

2017-07-01

We combined Second Harmonic Generation, Two-Photon Fluorescence and Fluorescence Lifetime Imaging Microscopy for studying human carotid ex vivo tissue sections affected by atherosclerosis, resulting in the discrimination of different arterial regions within the plaques.

Computational code in atomic and nuclear quantum optics: Advanced computing multiphoton resonance parameters for atoms in a strong laser field

NASA Astrophysics Data System (ADS)

Glushkov, A. V.; Gurskaya, M. Yu; Ignatenko, A. V.; Smirnov, A. V.; Serga, I. N.; Svinarenko, A. A.; Ternovsky, E. V.

2017-10-01

The consistent relativistic energy approach to the finite Fermi-systems (atoms and nuclei) in a strong realistic laser field is presented and applied to computing the multiphoton resonances parameters in some atoms and nuclei. The approach is based on the Gell-Mann and Low S-matrix formalism, multiphoton resonance lines moments technique and advanced Ivanov-Ivanova algorithm of calculating the Green’s function of the Dirac equation. The data for multiphoton resonance width and shift for the Cs atom and the 57Fe nucleus in dependence upon the laser intensity are listed.

Three-Dimensional Photoactivated Localization Microscopy with Genetically Expressed Probes

PubMed Central

Temprine, Kelsey; York, Andrew G.; Shroff, Hari

2017-01-01

Photoactivated localization microscopy (PALM) and related single-molecule imaging techniques enable biological image acquisition at ~20 nm lateral and ~50–100 nm axial resolution. Although such techniques were originally demonstrated on single imaging planes close to the coverslip surface, recent technical developments now enable the 3D imaging of whole fixed cells. We describe methods for converting a 2D PALM into a system capable of acquiring such 3D images, with a particular emphasis on instrumentation that is compatible with choosing relatively dim, genetically expressed photoactivatable fluorescent proteins (PA-FPs) as PALM probes. After reviewing the basics of 2D PALM, we detail astigmatic and multiphoton imaging approaches well suited to working with PA-FPs. We also discuss the use of open-source localization software appropriate for 3D PALM. PMID:25391803

Real-time digital signal processing in multiphoton and time-resolved microscopy

NASA Astrophysics Data System (ADS)

Wilson, Jesse W.; Warren, Warren S.; Fischer, Martin C.

2016-03-01

The use of multiphoton interactions in biological tissue for imaging contrast requires highly sensitive optical measurements. These often involve signal processing and filtering steps between the photodetector and the data acquisition device, such as photon counting and lock-in amplification. These steps can be implemented as real-time digital signal processing (DSP) elements on field-programmable gate array (FPGA) devices, an approach that affords much greater flexibility than commercial photon counting or lock-in devices. We will present progress toward developing two new FPGA-based DSP devices for multiphoton and time-resolved microscopy applications. The first is a high-speed multiharmonic lock-in amplifier for transient absorption microscopy, which is being developed for real-time analysis of the intensity-dependence of melanin, with applications in vivo and ex vivo (noninvasive histopathology of melanoma and pigmented lesions). The second device is a kHz lock-in amplifier running on a low cost (50-200) development platform. It is our hope that these FPGA-based DSP devices will enable new, high-speed, low-cost applications in multiphoton and time-resolved microscopy.

Multi-photon excited luminescence of magnetic FePt core-shell nanoparticles.

PubMed

Seemann, K M; Kuhn, B

2014-07-01

We present magnetic FePt nanoparticles with a hydrophilic, inert, and biocompatible silico-tungsten oxide shell. The particles can be functionalized, optically detected, and optically manipulated. To show the functionalization the fluorescent dye NOPS was bound to the FePt core-shell nanoparticles with propyl-triethoxy-silane linkers and fluorescence of the labeled particles were observed in ethanol (EtOH). In aqueous dispersion the NOPS fluorescence is quenched making them invisible using 1-photon excitation. However, we observe bright luminescence of labeled and even unlabeled magnetic core-shell nanoparticles with multi-photon excitation. Luminescence can be detected in the near ultraviolet and the full visible spectral range by near infrared multi-photon excitation. For optical manipulation, we were able to drag clusters of particles, and maybe also single particles, by a focused laser beam that acts as optical tweezers by inducing an electric dipole in the insulated metal nanoparticles. In a first application, we show that the luminescence of the core-shell nanoparticles is bright enough for in vivo multi-photon imaging in the mouse neocortex down to cortical layer 5.

In vivo multiphoton and fluorescence lifetime imaging microscopy of the healthy and cholestatic liver

NASA Astrophysics Data System (ADS)

Kuznetsova, Daria S.; Dudenkova, Varvara V.; Rodimova, Svetlana A.; Bobrov, Nikolai V.; Zagainov, Vladimir E.; Zagaynova, Elena V.

2018-02-01

A cholestatic liver disease presents one of the most common liver diseases and can potentially progress to cirrhosis or even cholangiocarcinoma. Conventional techniques are insufficient to precisely describe the complex internal structure, heterogeneous cell populations and the dynamics of biological processes of the liver. Currently, the methods of multiphoton and fluorescence lifetime imaging microscopy are actively introducing to biomedical research. Those methods are extremely informative and non-destructive that allows studying of a large number of processes occurring inside cells and tissues, analyzing molecular cellular composition, as well as evaluating the state of connective tissue fibers due to their ability to generate a second optical harmonic. Multiphoton and FLIM microscopy do not need additional staining of samples or the incorporation of any markers to study metabolism, lipid composition, microstructure analysis, evaluation of fibrous structures. These parameters have pronounced changes in hepatocytes of liver with common pathological diseases. Thereby in this study we investigated metabolic changes in the healthy and cholestatic liver based on the fluorescence of the metabolic co-factors NAD(P)H and FAD by multiphoton microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH was presented. The data can be used to develop new criteria for the identification of hepatic pathology at the level of hepatocyte changes directed to personalized medicine in the future.

5D-intravital tomography as a novel tool for non-invasive in-vivo analysis of human skin

NASA Astrophysics Data System (ADS)

König, Karsten; Weinigel, Martin; Breunig, Hans G.; Gregory, Axel; Fischer, Peter; Kellner-Höfer, Marcel; Bückle, Rainer; Schwarz, Martin; Riemann, Iris; Stracke, Frank; Huck, Volker; Gorzelanny, Christian; Schneider, Stefan W.

2010-02-01

Some years ago, CE-marked clinical multiphoton systems for 3D imaging of human skin with subcellular resolution have been launched. These tomographs provide optical biopsies with submicron resolution based on two-photon excited autofluorescence (NAD(P)H, flavoproteins, keratin, elastin, melanin, porphyrins) and second harmonic generation by collagen. The 3D tomograph was now transferred into a 5D imaging system by the additional detection of the emission spectrum and the fluorescence lifetime based on spatially and spectrally resolved time-resolved single photon counting. The novel 5D intravital tomograph (5D-IVT) was employed for the early detection of atopic dermatitis and the analysis of treatment effects.

Temporal focusing-based multiphoton excitation microscopy via digital micromirror device.

PubMed

Yih, Jenq-Nan; Hu, Yvonne Yuling; Sie, Yong Da; Cheng, Li-Chung; Lien, Chi-Hsiang; Chen, Shean-Jen

2014-06-01

This Letter presents an enhanced temporal focusing-based multiphoton excitation (MPE) microscope in which the conventional diffraction grating is replaced by a digital micromirror device (DMD). Experimental results from imaging a thin fluorescence film show that the 4.0 μm axial resolution of the microscope is comparable with that of a setup incorporating a 600  lines/mm grating; hence, the optical sectioning ability of the proposed setup is demonstrated. Similar to a grating, the DMD diffracts illuminating light frequencies for temporal focusing; additionally, it generates arbitrary patterns. Since the DMD is placed on the image-conjugate plane of the objective lens' focal plane, the MPE pattern can be projected on the focal plane precisely.

Imaging photoelectron circular dichroism of chiral molecules by femtosecond multiphoton coincidence detection.

PubMed

Lehmann, C Stefan; Ram, N Bhargava; Powis, Ivan; Janssen, Maurice H M

2013-12-21

Here, we provide a detailed account of novel experiments employing electron-ion coincidence imaging to discriminate chiral molecules. The full three-dimensional angular scattering distribution of electrons is measured after photoexcitation with either left or right circular polarized light. The experiment is performed using a simplified photoelectron-photoion coincidence imaging setup employing only a single particle imaging detector. Results are reported applying this technique to enantiomers of the chiral molecule camphor after three-photon ionization by circularly polarized femtosecond laser pulses at 400 nm and 380 nm. The electron-ion coincidence imaging provides the photoelectron spectrum of mass-selected ions that are observed in the time-of-flight mass spectra. The coincident photoelectron spectra of the parent camphor ion and the various fragment ions are the same, so it can be concluded that fragmentation of camphor happens after ionization. We discuss the forward-backward asymmetry in the photoelectron angular distribution which is expressed in Legendre polynomials with moments up to order six. Furthermore, we present a method, similar to one-photon electron circular dichroism, to quantify the strength of the chiral electron asymmetry in a single parameter. The circular dichroism in the photoelectron angular distribution of camphor is measured to be 8% at 400 nm. The electron circular dichroism using femtosecond multiphoton excitation is of opposite sign and about 60% larger than the electron dichroism observed before in near-threshold one-photon ionization with synchrotron excitation. We interpret our multiphoton ionization as being resonant at the two-photon level with the 3s and 3p Rydberg states of camphor. Theoretical calculations are presented that model the photoelectron angular distribution from a prealigned camphor molecule using density functional theory and continuum multiple scattering X alpha photoelectron scattering calculations. Qualitative agreement is observed between the experimental results and the theoretical calculations of the Legendre moments representing the angular distribution for the two enantiomers. The electron-ion coincidence technique using multiphoton ionization opens new directions in table-top analytical mass-spectrometric applications of mixtures of chiral molecules.

Imaging photoelectron circular dichroism of chiral molecules by femtosecond multiphoton coincidence detection

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

Lehmann, C. Stefan; Ram, N. Bhargava; Janssen, Maurice H. M., E-mail: m.h.m.janssen@vu.nl

2013-12-21

Here, we provide a detailed account of novel experiments employing electron-ion coincidence imaging to discriminate chiral molecules. The full three-dimensional angular scattering distribution of electrons is measured after photoexcitation with either left or right circular polarized light. The experiment is performed using a simplified photoelectron-photoion coincidence imaging setup employing only a single particle imaging detector. Results are reported applying this technique to enantiomers of the chiral molecule camphor after three-photon ionization by circularly polarized femtosecond laser pulses at 400 nm and 380 nm. The electron-ion coincidence imaging provides the photoelectron spectrum of mass-selected ions that are observed in the time-of-flightmore » mass spectra. The coincident photoelectron spectra of the parent camphor ion and the various fragment ions are the same, so it can be concluded that fragmentation of camphor happens after ionization. We discuss the forward-backward asymmetry in the photoelectron angular distribution which is expressed in Legendre polynomials with moments up to order six. Furthermore, we present a method, similar to one-photon electron circular dichroism, to quantify the strength of the chiral electron asymmetry in a single parameter. The circular dichroism in the photoelectron angular distribution of camphor is measured to be 8% at 400 nm. The electron circular dichroism using femtosecond multiphoton excitation is of opposite sign and about 60% larger than the electron dichroism observed before in near-threshold one-photon ionization with synchrotron excitation. We interpret our multiphoton ionization as being resonant at the two-photon level with the 3s and 3p Rydberg states of camphor. Theoretical calculations are presented that model the photoelectron angular distribution from a prealigned camphor molecule using density functional theory and continuum multiple scattering X alpha photoelectron scattering calculations. Qualitative agreement is observed between the experimental results and the theoretical calculations of the Legendre moments representing the angular distribution for the two enantiomers. The electron-ion coincidence technique using multiphoton ionization opens new directions in table-top analytical mass-spectrometric applications of mixtures of chiral molecules.« less

Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy

PubMed Central

Cha, Jae Won; Ballesta, Jerome; So, Peter T.C.

2010-01-01

The imaging depth of two-photon excitation fluorescence microscopy is partly limited by the inhomogeneity of the refractive index in biological specimens. This inhomogeneity results in a distortion of the wavefront of the excitation light. This wavefront distortion results in image resolution degradation and lower signal level. Using an adaptive optics system consisting of a Shack-Hartmann wavefront sensor and a deformable mirror, wavefront distortion can be measured and corrected. With adaptive optics compensation, we demonstrate that the resolution and signal level can be better preserved at greater imaging depth in a variety of ex-vivo tissue specimens including mouse tongue muscle, heart muscle, and brain. However, for these highly scattering tissues, we find signal degradation due to scattering to be a more dominant factor than aberration. PMID:20799824

Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy.

PubMed

Cha, Jae Won; Ballesta, Jerome; So, Peter T C

2010-01-01

The imaging depth of two-photon excitation fluorescence microscopy is partly limited by the inhomogeneity of the refractive index in biological specimens. This inhomogeneity results in a distortion of the wavefront of the excitation light. This wavefront distortion results in image resolution degradation and lower signal level. Using an adaptive optics system consisting of a Shack-Hartmann wavefront sensor and a deformable mirror, wavefront distortion can be measured and corrected. With adaptive optics compensation, we demonstrate that the resolution and signal level can be better preserved at greater imaging depth in a variety of ex-vivo tissue specimens including mouse tongue muscle, heart muscle, and brain. However, for these highly scattering tissues, we find signal degradation due to scattering to be a more dominant factor than aberration.

In vivo imaging of spinal cord in contusion injury model mice by multi-photon microscopy

NASA Astrophysics Data System (ADS)

Oshima, Y.; Horiuchi, H.; Ogata, T.; Hikita, A.; Miura, H.; Imamura, T.

2014-03-01

Fluorescent imaging technique is a promising method and has been developed for in vivo applications in cellular biology. In particular, nonlinear optical imaging technique, multi-photon microscopy has make it possible to analyze deep portion of tissues in living animals such as axons of spinal code. Traumatic spinal cord injuries (SCIs) are usually caused by contusion damages. Therefore, observation of spinal cord tissue after the contusion injury is necessary for understanding cellular dynamics in response to traumatic SCI and development of the treatment for traumatic SCI. Our goal is elucidation of mechanism for degeneration of axons after contusion injuries by establishing SCI model and chronic observation of injured axons in the living animals. Firstly we generated and observed acute SCI model by contusion injury. By using a multi-photon microscope, axons in dorsal cord were visualized approximately 140 micron in depth from the surface. Immediately after injury, minimal morphological change of spinal cord was observed. At 3 days after injury, spinal cord was swelling and the axons seem to be fragmented. At 7 days after injury, increased degradation of axons could be observed, although the image was blurred due to accumulation of the connective tissue. In the present study, we successfully observed axon degeneration after the contusion SCI in a living animal in vivo. Our final goal is to understand molecular mechanisms and cellular dynamics in response to traumatic SCIs in acute and chronic stage.

All-optical bidirectional neural interfacing using hybrid multiphoton holographic optogenetic stimulation.

PubMed

Paluch-Siegler, Shir; Mayblum, Tom; Dana, Hod; Brosh, Inbar; Gefen, Inna; Shoham, Shy

2015-07-01

Our understanding of neural information processing could potentially be advanced by combining flexible three-dimensional (3-D) neuroimaging and stimulation. Recent developments in optogenetics suggest that neurophotonic approaches are in principle highly suited for noncontact stimulation of network activity patterns. In particular, two-photon holographic optical neural stimulation (2P-HONS) has emerged as a leading approach for multisite 3-D excitation, and combining it with temporal focusing (TF) further enables axially confined yet spatially extended light patterns. Here, we study key steps toward bidirectional cell-targeted 3-D interfacing by introducing and testing a hybrid new 2P-TF-HONS stimulation path for accurate parallel optogenetic excitation into a recently developed hybrid multiphoton 3-D imaging system. The system is shown to allow targeted all-optical probing of in vitro cortical networks expressing channelrhodopsin-2 using a regeneratively amplified femtosecond laser source tuned to 905 nm. These developments further advance a prospective new tool for studying and achieving distributed control over 3-D neuronal circuits both in vitro and in vivo.

Multiphoton microscopy of transdermal quantum dot delivery using two photon polymerization-fabricated polymer microneedles

PubMed Central

Gittard, Shaun D; Miller, Philip R; Boehm, Ryan D; Ovsianikov, Aleksandr; Chichkov, Boris N; Heiser, Jeremy; Gordon, John; Monteiro-Riviere, Nancy A; Narayan, Roger J

2010-01-01

Due to their ability to serve as fluorophores and drug delivery vehicles, quantum dots are a powerful tool for theranostics-based clinical applications. In this study, microneedle devices for transdermal drug delivery were fabricated by means of two-photon polymerization of an acrylate-based polymer. We examined proliferation of cells on this polymer using neonatal human epidermal keratinocytes and human dermal fibroblasts. The microneedle device was used to inject quantum dots into porcine skin; imaging of the quantum dots was performed using multiphoton microscopy. PMID:21413181

Gaussian beam in two-photon fluorescence imaging of rat brain microvessel

NASA Astrophysics Data System (ADS)

Shi, Lingyan; Rodríguez-Contreras, Adrián; Alfano, Robert R.

2014-12-01

The critical optical properties of a Gaussian laser beam in two-photon or multiphoton fluorescence imaging, including the beam spot size, depth of focus, and intensity profile, are investigated for spatially locating nanoscale solutes in and surrounding the microvessels of rat brain.

Multi-photon microscopy with a low-cost and highly efficient Cr:LiCAF laser

PubMed Central

Sakadić, Sava; Demirbas, Umit; Mempel, Thorsten R.; Moore, Anna; Ruvinskaya, Svetlana; Boas, David A.; Sennaroglu, Alphan; Kartner, Franz X.; Fujimoto, James G.

2009-01-01

Multi-photon microscopy (MPM) is a powerful tool for biomedical imaging, enabling molecular contrast and integrated structural and functional imaging on the cellular and subcellular level. However, the cost and complexity of femtosecond laser sources that are required in MPM are significant hurdles to widespread adoption of this important imaging modality. In this work, we describe femtosecond diode pumped Cr:LiCAF laser technology as a low cost alternative to femtosecond Ti:Sapphire lasers for MPM. Using single mode pump diodes which cost only $150 each, a diode pumped Cr:LiCAF laser generates ~70-fs duration, 1.8-nJ pulses at ~800 nm wavelengths, with a repetition rate of 100 MHz and average output power of 180 mW. Representative examples of MPM imaging in neuroscience, immunology, endocrinology and cancer research using Cr:LiCAF laser technology are presented. These studies demonstrate the potential of this laser source for use in a broad range of MPM applications. PMID:19065223

Quantitative multiphoton imaging

NASA Astrophysics Data System (ADS)

König, Karsten; Weinigel, Martin; Breunig, Hans Georg; Uchugonova, Aisada

2014-02-01

Certified clinical multiphoton tomographs for label-free multidimensional high-resolution in vivo imaging have been introduced to the market several years ago. Novel tomographs include a flexible 360° scan head attached to a mechanooptical arm for autofluorescence and SHG imaging as well as a CARS module. Non-fluorescent lipids and water, mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen can be imaged in vivo with submicron resolution in human skin. Sensitive and rapid detectors allow single photon counting and the construction of 3D maps where the number of detected photons per voxel is depicted. Intratissue concentration profiles from endogenous as well exogenous substances can be generated when the number of detected photons can be correlated with the number of molecules with respect to binding and scattering behavior. Furthermore, the skin ageing index SAAID based on the ratio elastin/collagen as well as the epidermis depth based on the onset of SHG generation can be determined.

Pump-Probe Imaging Differentiates Melanoma from Melanocytic Nevi

PubMed Central

Matthews, Thomas E.; Piletic, Ivan R.; Selim, M. Angelica; Simpson, Mary Jane; Warren, Warren S.

2012-01-01

Melanoma diagnosis is clinically challenging; the accuracy of visual inspection by dermatologists is highly variable and heavily weighted toward false positives. Even the current gold standard of biopsy results in varying diagnoses among pathologists. We have developed a multiphoton technique (based on pump-probe spectroscopy) that directly determines the microscopic distribution of eumelanin and pheomelanin in pigmented lesions of human skin. Our initial results showed a marked difference in the chemical variety of melanin between nonmalignant nevi and melanoma, as well as a number of substantial architectural differences. We examined slices from 42 pigmented lesions and found that melanomas had an increased eumelanin content compared to nonmalignant nevi. When used as a diagnostic criterion, the ratio of eumelanin to pheomelanin captured all investigated melanomas but excluded three-quarters of dysplastic nevi and all benign dermal nevi. Evaluating architectural and cytological features revealed by multiphoton imaging, including the maturation of melanocytes, presence of pigmented melanocytes in the dermis, number and location of melanocytic nests, and confluency of pigmented cells in the epidermis, further increased specificity, allowing rejection of more than half of the remaining false-positive results. We then adapted this multiphoton imaging technique to hematoxylin and eosin (H&E)–stained slides. By adding melanin chemical contrast to H&E-stained slides, pathologists will gain complementary information to increase the ease and accuracy of melanoma diagnosis. PMID:21346168

Multiphoton microscopy based cryo-imaging of inflated frozen human lung sections at -60°C in healthy and COPD lungs

NASA Astrophysics Data System (ADS)

Abraham, Thomas; Kayra, Damian; Zhang, Angela; Suzuki, Masaru; McDonough, John; Elliott, W. M.; Cooper, Joel D.; Hogg, James C.

2013-02-01

Lung is a complex gas exchanger with interfacial area (where the gas exchange takes place) is about the size of a tennis court. Respiratory function is linked to the biomechanical stability of the gas exchange or alveolar regions which directly depends on the spatial distributions of the extracellular matrix fibers such fibrillar collagens and elastin fibers. It is very important to visualize and quantify these fibers at their native and inflated conditions to have correct morphometric information on differences between control and diseased states. This can be only achieved in the ex vivo states by imaging directly frozen lung specimens inflated to total lung capacity. Multiphoton microscopy, which uses ultra-short infrared laser pulses as the excitation source, produces multiphoton excitation fluorescence (MPEF) signals from endogenously fluorescent proteins (e.g. elastin) and induces specific second harmonic generation (SHG) signals from non-centrosymmetric proteins such as fibrillar collagens in fresh human lung tissues [J. Struct. Biol. (2010)171,189-196]. Here we report for the first time 3D image data obtained directly from thick frozen inflated lung specimens (~0.7- 1.0 millimeter thick) visualized at -60°C without prior fixation or staining in healthy and diseased states. Lung specimens donated for transplantation and released for research when no appropriate recipient was identified served as controls, and diseased lung specimens donated for research by patients receiving lung transplantation for very severe COPD (n=4) were prepared as previously described [N. Engl. J. Med. (2011) 201, 1567]. Lung slices evenly spaced between apex and base were examined using multiphoton microscopy while maintained at -60°C using a temperature controlled cold stage with a temperature resolution of 0.1°C. Infrared femto-second laser pulses tuned to 880nm, dry microscopic objectives, and non-de-scanned detectors/spectrophotometer located in the reflection geometry were used for generating the 3D images/spectral information. We found that this novel imaging approach can provide spatially resolved 3D images with spectral specificities from frozen inflated lungs that are sensitive enough to identity the micro-structural details of fibrillar collagens and elastin fibers in alveolar walls in both healthy and diseased tissues.

Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord

PubMed Central

Uckermann, Ortrud; Galli, Roberta; Beiermeister, Rudolf; Sitoci-Ficici, Kerim-Hakan; Later, Robert; Leipnitz, Elke; Chavakis, Triantafyllos; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

2015-01-01

Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes. PMID:26355949

Visualization of dermal alteration in skin lesions with discoid lupus erythematosus by multiphoton microscopy

NASA Astrophysics Data System (ADS)

Lin, L. H.; Yu, H. B.; Zhu, X. Q.; Zhuo, S. M.; Wang, Y. Y.; Yang, Y. H.; Chen, J. X.

2013-04-01

Discoid lupus erythematosus (DLE) is a chronic dermatological disease which lacks valid methods for early diagnosis and therapeutic monitoring. Considering the collagen and elastin disorder due to mucin deposition of DLE, multiphoton microscopy (MPM) imaging techniques were employed to obtain high-resolution collagen and elastin images from the dermis. The content and distribution of collagen and elastin were quantified to characterize the dermal pathological status of skin lesions with DLE in comparison with normal skin. Our results showed a significant difference between skin lesions with DLE and normal skin in terms of the morphological structure of collagen and elastin in the dermis, demonstrating the possibility of MPM for noninvasively tracking the pathological process of DLE even in its early stages and evaluating the therapeutic efficacy at the molecular level.

Clinical studies of pigmented lesions in human skin by using a multiphoton tomograph

NASA Astrophysics Data System (ADS)

Balu, Mihaela; Kelly, Kristen M.; Zachary, Christopher B.; Harris, Ronald M.; Krasieva, Tatiana B.; König, Karsten; Tromberg, Bruce J.

2013-02-01

In vivo imaging of pigmented lesions in human skin was performed with a clinical multiphoton microscopy (MPM)-based tomograph (MPTflex, JenLab, Germany). Two-photon excited fluorescence was used for visualizing endogenous fluorophores such as NADH/FAD, keratin, melanin in the epidermal cells and elastin fibers in the dermis. Collagen fibers were imaged by second harmonic generation. Our study involved in vivo imaging of benign melanocytic nevi, atypical nevi and melanoma. The goal of this preliminary study was to identify in vivo the characteristic features and their frequency in pigmented lesions at different stages (benign, atypical and malignant) and to evaluate the ability of in vivo MPM to distinguish atypical nevi from melanoma. Comparison with histopathology was performed for the biopsied lesions. Benign melanocytic nevi were characterized by the presence of nevus cell nests at the epidermal-dermal junction. In atypical nevi, features such as lentiginous hyperplasia, acanthosis and architectural disorder were imaged. Cytological atypia was present in all the melanoma lesions imaged, showing the strongest correlation with malignancy. The MPM images demonstrated very good correlation with corresponding histological images, suggesting that MPM could be a promising tool for in vivo non-invasive pigmented lesion diagnosis, particularly distinguishing atypical nevi from melanoma.

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

PubMed Central

Andresen, Volker; Sporbert, Anje

2014-01-01

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 µm tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells – on the level of a few protein molecules in germinal centers. PMID:24748007

Metabolic Mapping of Breast Cancer with Multiphoton Spectral and Lifetime Imaging

DTIC Science & Technology

2008-03-01

Biomedical Optics May/June 2008 Vol. 133031220-1 the most prevalent cancer among women . 1 Therefore, tech- nologies to detect, classify, study, and...and molecular function using optical imaging: applications to breast cancer,” Breast Cancer Res. Treat. 31, 41–46 2001. 3. M. Sidani , J. Wyckoff

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

The Hernandez lab is seeking a postdoctoral fellow to join the research program, which is focused on interrogating the molecular underpinnings of metastatic colonization. The lab utilizes multi-photon intravital microscopy to mechanistically interrogate and visualize the dynamics of metastatic outgrowth, including the roles of supporting stromal and immune cells. The lab has begun pioneering first-ever human tissue models by repurposing perfusion systems to sustain metastasis-bearing tissue (liver and peritoneum) ex vivo. We envision these models will allow us to 1) evaluate putative metastasis governing genes in human tissue, 2) personalize investigation of the metastatic cascade by leveraging multi-photon imaging with an individual patient’s tumor cells, which will be dissociated, labelled, and subsequently injected into the perfusate to seed that patient’s metastatic target tissue, and 3) utilized tumor-bearing tissue as a platform for drug discovery and evaluation of novel drug-delivery combinations. We believe our human tissue models have the potential to transcend multiple disciplines in translational medicine and permit investigations and manipulations not previously possible.

Tunable multiphoton Rabi oscillations in an electronic spin system

NASA Astrophysics Data System (ADS)

Bertaina, S.; Groll, N.; Chen, L.; Chiorescu, I.

2011-10-01

We report on multiphoton Rabi oscillations and controlled tuning of a multilevel system at room temperature (S=5/2 for Mn2+:MgO) in and out of a quasiharmonic level configuration. The anisotropy is much smaller than the Zeeman splittings, e.g., the six-level scheme shows only a small deviation from an equidistant diagram. This allows us to tune the spin dynamics by compensating for the cubic anisotropy with either a precise static-field orientation or a microwave field intensity. Using the rotating-frame approximation, the experiments are explained very well by both an analytical model and a generalized numerical model. The calculated multiphoton Rabi frequencies are in excellent agreement with the experimental data.

Adaptive optics plug-and-play setup for high-resolution microscopes with multi-actuator adaptive lens

NASA Astrophysics Data System (ADS)

Quintavalla, M.; Pozzi, P.; Verhaegen, Michelle; Bijlsma, Hielke; Verstraete, Hans; Bonora, S.

2018-02-01

Adaptive Optics (AO) has revealed as a very promising technique for high-resolution microscopy, where the presence of optical aberrations can easily compromise the image quality. Typical AO systems however, are almost impossible to implement on commercial microscopes. We propose a simple approach by using a Multi-actuator Adaptive Lens (MAL) that can be inserted right after the objective and works in conjunction with an image optimization software allowing for a wavefront sensorless correction. We presented the results obtained on several commercial microscopes among which a confocal microscope, a fluorescence microscope, a light sheet microscope and a multiphoton microscope.

A series of fluorene-based two-photon absorbing molecules: synthesis, linear and nonlinear characterization, and bioimaging

PubMed Central

Andrade, Carolina D.; Yanez, Ciceron O.; Rodriguez, Luis; Belfield, Kevin D.

2010-01-01

The synthesis, structural, and photophysical characterization of a series of new fluorescent donor–acceptor and acceptor-acceptor molecules, based on the fluorenyl ring system, with two-photon absorbing properties is described. These new compounds exhibited large Stokes shifts, high fluorescent quantum yields, and, significantly, high two-photon absorption cross sections, making them well suited for two-photon fluorescence microscopy (2PFM) imaging. Confocal and two-photon fluorescence microscopy imaging of COS-7 and HCT 116 cells incubated with probe I showed endosomal selectivity, demonstrating the potential of this class of fluorescent probes in multiphoton fluorescence microscopy. PMID:20481596

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.

Wavefront optimized nonlinear microscopy of ex vivo human retinas

NASA Astrophysics Data System (ADS)

Gualda, Emilio J.; Bueno, Juan M.; Artal, Pablo

2010-03-01

A multiphoton microscope incorporating a Hartmann-Shack (HS) wavefront sensor to control the ultrafast laser beam's wavefront aberrations has been developed. This instrument allowed us to investigate the impact of the laser beam aberrations on two-photon autofluorescence imaging of human retinal tissues. We demonstrated that nonlinear microscopy images are improved when laser beam aberrations are minimized by realigning the laser system cavity while wavefront controlling. Nonlinear signals from several human retinal anatomical features have been detected for the first time, without the need of fixation or staining procedures. Beyond the improved image quality, this approach reduces the required excitation power levels, minimizing the side effects of phototoxicity within the imaged sample. In particular, this may be important to study the physiology and function of the healthy and diseased retina.

Compact diode laser source for multiphoton biological imaging

PubMed Central

Niederriter, Robert D.; Ozbay, Baris N.; Futia, Gregory L.; Gibson, Emily A.; Gopinath, Juliet T.

2016-01-01

We demonstrate a compact, pulsed diode laser source suitable for multiphoton microscopy of biological samples. The center wavelength is 976 nm, near the peak of the two-photon cross section of common fluorescent markers such as genetically encoded green and yellow fluorescent proteins. The laser repetition rate is electrically tunable between 66.67 kHz and 10 MHz, with 2.3 ps pulse duration and peak powers >1 kW. The laser components are fiber-coupled and scalable to a compact package. We demonstrate >600 μm depth penetration in brain tissue, limited by laser power. PMID:28101420

Measurement of absorption spectrum of deuterium oxide (D{sub 2}O) and its application to signal enhancement in multiphoton microscopy at the 1700-nm window

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

Wang, Yuxin; Wen, Wenhui; Wang, Kai

2016-01-11

1700-nm window has been demonstrated to be a promising excitation window for deep-tissue multiphoton microscopy (MPM). Long working-distance water immersion objective lenses are typically used for deep-tissue imaging. However, absorption due to immersion water at 1700 nm is still high and leads to dramatic decrease in signals. In this paper, we demonstrate measurement of absorption spectrum of deuterium oxide (D{sub 2}O) from 1200 nm to 2600 nm, covering the three low water-absorption windows potentially applicable for deep-tissue imaging (1300 nm, 1700 nm, and 2200 nm). We apply this measured result to signal enhancement in MPM at the 1700-nm window. Compared with water immersion, D{sub 2}O immersionmore » enhances signal levels in second-harmonic generation imaging, 3-photon fluorescence imaging, and third-harmonic generation imaging by 8.1, 24.8, and 24.7 times with 1662-nm excitation, in good agreement with theoretical calculation based on our absorption measurement. This suggests D{sub 2}O a promising immersion medium for deep-tissue imaging.« less

Nanodissection of human chromosomes and ultraprecise eye surgery with nanojoule near-infrared femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Koenig, Karsten; Riemann, Iris; Krauss, Oliver; Fritzsche, Wolfgang

2002-04-01

Nanojoule and sub-nanojoule 80 MHz femtosecond laser pulses at 750-850 nm of a compact titanium:sapphire laser have been used for highly precise nanoprocessing of DNA as well as of intracellular and intratissue compartments. In particular, a mean power between 15 mW and 100 mW, 170 fs pulse width, submicron distance of illumination spots and microsecond beam dwell times on spots have been used for multiphoton- mediated nanoprocessing of human chromosomes, brain and ocular intrastromal tissue. By focusing the laser beam with high numerical aperture focusing optics of the laser scan system femt-O-cut and of modified multiphoton scanning microscopes to diffraction-limited spots and TW/cm2 light intensities, precise submicron holes and cuts have been processed by single spot exposure and line scans. A minimum FWHM cut size below 70 nm during the partial dissection of the human chromosome 3 was achieved. Complete chromosome dissection could be performed with FWHM cut sizes below 200 nm. Intracellular chromosome dissection was possible. Intratissue processing in depths of 50 - 100micrometers and deeper with a precision of about 1micrometers including cuts through a nuclei of a single intratissue cell without destructive photo-disruption effects to surrounding tissue layers have been demonstrated in brain and eye tissues. The femt-O-cut system includes a diagnostic system for optical tomography with submicron resolution based on multiphoton- excited autofluorescence imaging (MAI) and second harmonic generation. This system was used to localize the intracellular and intratissue targets and to control the effects of nanoprocessing. These studies show, that in contrast to conventional approaches of material processing with amplified femtosecond laser systems and (mu) J pulse energies, nanoprocessing of materials including biotissues can be performed with nJ and sub-nJ high repetition femtosecond laser pulses of turn-key compact lasers without collateral damage. Potential applications include highly precise cell and embryo surgery, gene diagnostics and gene therapy, intrastromal refractive surgery, cancer therapy and brain surgery.

Laser spectroscopic study of the Rydberg state structure of atomic lithium

NASA Astrophysics Data System (ADS)

Ballard, M. Kent

1998-07-01

Pulsed laser induced fluorescence spectroscopy was performed on both isotopic species of atomic lithium. Nonresonant multiphoton excitation spectra were recorded. The laser induced fluorescence of the lithium vapor was measured following excitation with a tunable, pulsed, nanosecond laser. Both two- and three-photon allowed transitions were observed resulting in four different transition series originating from the 22S and 22P levels, the latter likely originating from photodissociation products of the lithium dimer, Li2. Forty-seven identifiable transitions were assigned for 6Li. Evidence for a parity forbidden multiphoton transition is also present. For 7Li, fifty-three identifiable transitions were assigned including an additional series of parity forbidden multiphoton transitions. Laser polarization and power dependencies were measured and found to be consistent with the multiphoton transition probabilities. Due to the intense laser fields needed to produce the nonresonant multiphoton excitations, the lithium vapor was subjected to the laser induced ac Stark effect. The Autler-Townes doublets observed for the nF gets 2P transition series were found to exhibit normal asymmetry. The observed asymmetrical Autler-Townes profiles are explained in terms of the two-level and the three-level atomic systems which are based on different excitation schemes. A new computerized data acquisition system was developed as well as associated computer programs needed to analyze spectra.

Structure of multiphoton quantum optics. II. Bipartite systems, physical processes, and heterodyne squeezed states

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

Dell'Anno, Fabio; De Siena, Silvio; Illuminati, Fabrizio

2004-03-01

Extending the scheme developed for a single mode of the electromagnetic field in the preceding paper [F. Dell'Anno, S. De Siena, and F. Illuminati, Phys. Rev. A 69, 033812 (2004)], we introduce two-mode nonlinear canonical transformations depending on two heterodyne mixing angles. They are defined in terms of Hermitian nonlinear functions that realize heterodyne superpositions of conjugate quadratures of bipartite systems. The canonical transformations diagonalize a class of Hamiltonians describing nondegenerate and degenerate multiphoton processes. We determine the coherent states associated with the canonical transformations, which generalize the nondegenerate two-photon squeezed states. Such heterodyne multiphoton squeezed states are defined asmore » the simultaneous eigenstates of the transformed, coupled annihilation operators. They are generated by nonlinear unitary evolutions acting on two-mode squeezed states. They are non-Gaussian, highly nonclassical, entangled states. For a quadratic nonlinearity the heterodyne multiphoton squeezed states define two-mode cubic phase states. The statistical properties of these states can be widely adjusted by tuning the heterodyne mixing angles, the phases of the nonlinear couplings, as well as the strength of the nonlinearity. For quadratic nonlinearity, we study the higher-order contributions to the susceptibility in nonlinear media and we suggest possible experimental realizations of multiphoton conversion processes generating the cubic-phase heterodyne squeezed states.« less

Structure of multiphoton quantum optics. II. Bipartite systems, physical processes, and heterodyne squeezed states

NASA Astrophysics Data System (ADS)

dell'Anno, Fabio; de Siena, Silvio; Illuminati, Fabrizio

2004-03-01

Extending the scheme developed for a single mode of the electromagnetic field in the preceding paper [

Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging

NASA Astrophysics Data System (ADS)

Wei, Lu; Zhu, Xinxin; Chen, Zhixing; Min, Wei

2014-02-01

Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.

Pancreatic cancer cell detection by targeted lipid microbubbles and multiphoton imaging

NASA Astrophysics Data System (ADS)

Cromey, Benjamin; McDaniel, Ashley; Matsunaga, Terry; Vagner, Josef; Kieu, Khanh Quoc; Banerjee, Bhaskar

2018-04-01

Surgical resection of pancreatic cancer represents the only chance of cure and long-term survival in this common disease. Unfortunately, determination of a cancer-free margin at surgery is based on one or two tiny frozen section biopsies, which is far from ideal. Not surprisingly, cancer is usually left behind and is responsible for metastatic disease. We demonstrate a method of receptor-targeted imaging using peptide ligands, lipid microbubbles, and multiphoton microscopy that could lead to a fast and accurate way of examining the entire cut surface during surgery. Using a plectin-targeted microbubble, we performed a blinded in-vitro study to demonstrate avid binding of targeted microbubbles to pancreatic cancer cells but not noncancerous cell lines. Further work should lead to a much-needed point-of-care diagnostic test for determining clean margins in oncologic surgery.

Evaluation of Barrett esophagus by multiphoton microscopy.

PubMed

Chen, Jianxin; Wong, Serena; Nathanson, Michael H; Jain, Dhanpat

2014-02-01

Multiphoton microscopy (MPM) based on 2-photon excitation fluorescence and second-harmonic generation allows simultaneous visualization of cellular details and extracellular matrix components of fresh, unfixed, and unstained tissue. Portable multiphoton microscopes, which could be placed in endoscopy suites, and multiphoton endomicroscopes are in development, but their clinical utility is unknown. To examine fresh, unfixed endoscopic biopsies obtained from the distal esophagus and gastroesophageal junction to (1) define the MPM characteristics of normal esophageal squamous mucosa and gastric columnar mucosa, and (2) evaluate whether diagnosis of intestinal metaplasia/Barrett esophagus (BE) could be made reliably with MPM. The study examined 35 untreated, fresh biopsy specimens from 25 patients who underwent routine upper endoscopy. A Zeiss LSM 710 Duo microscope (Carl Zeiss, Thornwood, New York) coupled to a Spectra-Physics (Mountain View, California) Tsunami Ti:sapphire laser was used to obtain a MPM image within 4 hours of fresh specimen collection. After obtaining MPM images, the biopsy specimens were placed in 10% buffered formalin and submitted for routine histopathologic examination. Then, the MPM images were compared with the findings in the hematoxylin-eosin-stained, formalin-fixed, paraffin-embedded sections. The MPM characteristics of the squamous, gastric-type columnar and intestinal-type columnar epithelium were analyzed. In biopsies with discrepancy between MPM imaging and hematoxylin-eosin-stained sections, the entire tissue block was serially sectioned and reevaluated. A diagnosis of BE was made when endoscopic and histologic criteria were satisfied. Based on effective 2-photon excitation fluorescence of cellular reduced pyridine nucleotides and flavin adenine dinucleotide and lack of 2-photon excitation fluorescence of mucin and cellular nuclei, MPM could readily identify and distinguish among squamous epithelial cells, goblet cells, gastric foveolar-type mucous cells, and parietal cells in the area of gastroesophageal junction. Based on the cell types identified, the mucosa was defined as squamous, columnar gastric type (cardia/fundic-type), and metaplastic columnar intestinal-type/BE. Various types of mucosa seen in the study of 35 biopsies included normal squamous mucosa only (n = 14; 40%), gastric cardia-type mucosa only (n = 2; 6%), gastric fundic mucosa (n = 6; 17%), and both squamous and gastric mucosa (n = 13; 37%). Intestinal metaplasia was identified by the presence of goblet cells in 10 of 25 cases (40%) leading to a diagnosis of BE on MPM imaging and only in 7 cases (28%) by histopathology. In 3 of 35 biopsies (9%), clear-cut goblet cells were seen by MPM imaging but not by histopathology, even after the entire tissue block was sectioned. Based on effective 2-photon excitation fluorescence of elastin and second-harmonic generation of collagen, connective tissue in the lamina propria and the basement membrane was also visualized with MPM. Multiphoton microscopy has the ability to accurately distinguish squamous epithelium and different cellular elements of the columnar mucosa obtained from biopsies around the gastroesophageal junction, including goblet cells that are important for the diagnosis of BE. Thus, use of MPM in the endoscopy suite might provide immediate microscopic images during endoscopy, improving screening and surveillance of patients with BE.

Noninvasive Assessment of Collagen Gel Microstructure and Mechanics Using Multiphoton Microscopy

PubMed Central

Raub, Christopher B.; Suresh, Vinod; Krasieva, Tatiana; Lyubovitsky, Julia; Mih, Justin D.; Putnam, Andrew J.; Tromberg, Bruce J.; George, Steven C.

2007-01-01

Multiphoton microscopy of collagen hydrogels produces second harmonic generation (SHG) and two-photon fluorescence (TPF) images, which can be used to noninvasively study gel microstructure at depth (∼1 mm). The microstructure is also a primary determinate of the mechanical properties of the gel; thus, we hypothesized that bulk optical properties (i.e., SHG and TPF) could be used to predict bulk mechanical properties of collagen hydrogels. We utilized polymerization temperature (4–37°C) and glutaraldehyde to manipulate collagen hydrogel fiber diameter, space-filling properties, and cross-link density. Multiphoton microscopy and scanning electron microscopy reveal that as polymerization temperature decreases (37–4°C) fiber diameter and pore size increase, whereas hydrogel storage modulus (G′, from 23 ± 3 Pa to 0.28 ± 0.16 Pa, respectively, mean ± SE) and mean SHG decrease (minimal change in TPF). In contrast, glutaraldehyde significantly increases the mean TPF signal (without impacting the SHG signal) and the storage modulus (16 ± 3.5 Pa before to 138 ± 40 Pa after cross-linking, mean ± SD). We conclude that SHG and TPF can characterize differential microscopic features of the collagen hydrogel that are strongly correlated with bulk mechanical properties. Thus, optical imaging may be a useful noninvasive tool to assess tissue mechanics. PMID:17172303

In vivo detection of basal cell carcinoma: comparison of a reflectance confocal microscope and a multiphoton tomograph

NASA Astrophysics Data System (ADS)

Ulrich, Martina; Klemp, Marisa; Darvin, Maxim E.; König, Karsten; Lademann, Jürgen; Meinke, Martina C.

2013-06-01

The standard diagnostic procedure for basal cell carcinoma (BCC) is invasive tissue biopsy with time-consuming histological examination. To reduce the number of biopsies, noninvasive optical methods have been developed providing high-resolution skin examination. We present direct comparison of a reflectance confocal microscope (RLSM) and a multiphoton tomograph (MPT) for BCC diagnosis. Both systems are applied to nine patients prior to surgery, and the results are analyzed, including histological results. Both systems prove suitable for detecting typical characteristics of BCC in various stages. The RLSM allows large horizontal overview images to be obtained, enabling the investigator to find the regions of interest quickly, e.g., BCC nests. Elongated cells and palisading structures are easily recognized using both methods. Due to the higher resolution, changes in nucleus diameter or cytoplasm could be visualized with the MPT. Therefore, the nucleus diameter, nucleus/cytoplasm ratio, and cell density are estimated for normal and BCC cells using the MPT. The nucleus of elongated BCC cells is significantly longer than other measured normal skin cells, whereas the cell density and nucleus/cytoplasm ratio of BCC cannot be significantly distinguished from granular cells.

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.

Super-resolution fluorescence microscopy by stepwise optical saturation

PubMed Central

Zhang, Yide; Nallathamby, Prakash D.; Vigil, Genevieve D.; Khan, Aamir A.; Mason, Devon E.; Boerckel, Joel D.; Roeder, Ryan K.; Howard, Scott S.

2018-01-01

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a M-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples. PMID:29675306

Cutaneous porphyrins exhibit anti-stokes fluorescence that is detectable in sebum (Conference Presentation)

NASA Astrophysics Data System (ADS)

Tian, Giselle; Zeng, Haishan; Zhao, Jianhua; Wu, Zhenguo; Al Jasser, Mohammed; Lui, Harvey; Mclean, David I.

2016-02-01

Porphyrins produced by Propionibacterium acnes represent the principal fluorophore associated with acne, and appear as orange-red luminescence under the Wood's lamp. Assessment of acne based on Wood's lamp (UV) or visible light illumination is limited by photon penetration depth and has limited sensitivity for earlier stage lesions. Inducing fluorescence with near infrared (NIR) excitation may provide an alternative way to assess porphyrin-related skin disorders. We discovered that under 785 nm CW laser excitation PpIX powder exhibits fluorescence emission in the shorter wavelength range of 600-715 nm with an intensity that is linearly dependent on the excitation power. We attribute this shorter wavelength emission to anti-Stokes fluorescence. Similar anti-Stokes fluorescence was also detected focally in all skin-derived samples containing porphyrins. Regular (Stokes) fluorescence was present under UV and visible light excitation on ex vivo nasal skin and sebum from uninflamed acne, but not on nose surface smears or sebum from inflamed acne. Co-registered CW laser-excited anti-Stokes fluorescence and fs laser-excited multi-photon fluorescence images of PpIX powder showed similar features. In the skin samples because of the anti-Stokes effect, the NIR-induced fluorescence was presumably specific for porphyrins since there appeared to be no anti-Stokes emission signals from other typical skin fluorophores such as lipids, keratins and collagen. Anti-Stokes fluorescence under NIR CW excitation is more sensitive and specific for porphyrin detection than UV- or visible light-excited regular fluorescence and fs laser-excited multi-photon fluorescence. This approach also has higher image contrast compared to NIR fs laser-based multi-photon fluorescence imaging. The anti-Stokes fluorescence of porphyrins within sebum could potentially be applied to detecting and targeting acne lesions for treatment via fluorescence image guidance.

Controlling the transmitted information of a multi-photon interacting with a single-Cooper pair box

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

Kadry, Heba, E-mail: hkadry1@yahoo.com; Abdel-Aty, Abdel-Haleem, E-mail: hkadry1@yahoo.com; Zakaria, Nordin, E-mail: hkadry1@yahoo.com

2014-10-24

We study a model of a multi-photon interaction of a single Cooper pair box with a cavity field. The exchange of the information using this system is studied. We quantify the fidelity of the transmitted information. The effect of the system parameters (detuning parameter, field photons, state density and mean photon number) in the fidelity of the transmitted information is investigated. We found that the fidelity of the transmitted information can be controlled using the system parameters.

The Multiphoton Interaction of Lambda Model Atom and Two-Mode Fields

NASA Technical Reports Server (NTRS)

Liu, Tang-Kun

1996-01-01

The system of two-mode fields interacting with atom by means of multiphotons is addressed, and the non-classical statistic quality of two-mode fields with interaction is discussed. Through mathematical calculation, some new rules of non-classical effects of two-mode fields which evolue with time, are established.

Identification of normal and cancerous human colorectal muscularis propria by multiphoton microscopy in different sections

NASA Astrophysics Data System (ADS)

Zhou, Yi; Chen, Zhifen; Kang, Deyong; li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Guan, Guoxian; Chen, Jianxin

2016-01-01

Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a potential diagnostic tool is attractive. MPM can effectively provide information about morphological and biochemical changes in biological tissues at the molecular level. In this paper, we attempt to identify normal and cancerous human colorectal muscularis propria by multiphoton microscopy in different sections (both in transverse and longitudinal sections). The results show that MPM can display different microstructure changes in the transverse and longitudinal sections of colorectal muscularis propria. MPM also can quantitatively describe the alteration of collagen content between normal and cancerous muscle layers. These are important pathological findings that MPM images can bring more detailed complementary information about tissue architecture and cell morphology through observing the transverse and longitudinal sections of colorectal muscularis propria. This work demonstrates that MPM can be better for identifying the microstructural characteristics of normal and cancerous human colorectal muscularis propria in different sections.

Tuning the photon statistics of a strongly coupled nanophotonic system

NASA Astrophysics Data System (ADS)

Dory, Constantin; Fischer, Kevin A.; Müller, Kai; Lagoudakis, Konstantinos G.; Sarmiento, Tomas; Rundquist, Armand; Zhang, Jingyuan L.; Kelaita, Yousif; Sapra, Neil V.; Vučković, Jelena

2017-02-01

We investigate the dynamics of single- and multiphoton emission from detuned strongly coupled systems based on the quantum-dot-photonic-crystal resonator platform. Transmitting light through such systems can generate a range of nonclassical states of light with tunable photon counting statistics due to the nonlinear ladder of hybridized light-matter states. By controlling the detuning between emitter and resonator, the transmission can be tuned to strongly enhance either single- or two-photon emission processes. Despite the strongly dissipative nature of these systems, we find that by utilizing a self-homodyne interference technique combined with frequency filtering we are able to find a strong two-photon component of the emission in the multiphoton regime. In order to explain our correlation measurements, we propose rate equation models that capture the dominant processes of emission in both the single- and multiphoton regimes. These models are then supported by quantum-optical simulations that fully capture the frequency filtering of emission from our solid-state system.

Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device.

PubMed

Park, Jong Kang; Rowlands, Christopher J; So, Peter T C

2017-01-01

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice.

Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device

PubMed Central

Park, Jong Kang; Rowlands, Christopher J.; So, Peter T. C.

2017-01-01

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice. PMID:29387484

Three-Photon Luminescence of Gold Nanorods and Its Applications for High Contrast Tissue and Deep In Vivo Brain Imaging

PubMed Central

Wang, Shaowei; Xi, Wang; Cai, Fuhong; Zhao, Xinyuan; Xu, Zhengping; Qian, Jun; He, Sailing

2015-01-01

Gold nanoparticles can be used as contrast agents for bio-imaging applications. Here we studied multi-photon luminescence (MPL) of gold nanorods (GNRs), under the excitation of femtosecond (fs) lasers. GNRs functionalized with polyethylene glycol (PEG) molecules have high chemical and optical stability, and can be used as multi-photon luminescent nanoprobes for deep in vivo imaging of live animals. We have found that the depth of in vivo imaging is dependent upon the transmission and focal capability of the excitation light interacting with the GNRs. Our study focused on the comparison of MPL from GNRs with two different aspect ratios, as well as their ex vivo and in vivo imaging effects under 760 nm and 1000 nm excitation, respectively. Both of these wavelengths were located at an optically transparent window of biological tissue (700-1000 nm). PEGylated GNRs, which were intravenously injected into mice via the tail vein and accumulated in major organs and tumor tissue, showed high image contrast due to distinct three-photon luminescence (3PL) signals upon irradiation of a 1000 nm fs laser. Concerning in vivo mouse brain imaging, the 3PL imaging depth of GNRs under 1000 nm fs excitation could reach 600 μm, which was approximately 170 μm deeper than the two-photon luminescence (2PL) imaging depth of GNRs with a fs excitation of 760 nm. PMID:25553113

In vivo, label-free, three-dimensional quantitative imaging of liver surface using multi-photon microscopy

NASA Astrophysics Data System (ADS)

Zhuo, Shuangmu; Yan, Jie; Kang, Yuzhan; Xu, Shuoyu; Peng, Qiwen; So, Peter T. C.; Yu, Hanry

2014-07-01

Various structural features on the liver surface reflect functional changes in the liver. The visualization of these surface features with molecular specificity is of particular relevance to understanding the physiology and diseases of the liver. Using multi-photon microscopy (MPM), we have developed a label-free, three-dimensional quantitative and sensitive method to visualize various structural features of liver surface in living rat. MPM could quantitatively image the microstructural features of liver surface with respect to the sinuosity of collagen fiber, the elastic fiber structure, the ratio between elastin and collagen, collagen content, and the metabolic state of the hepatocytes that are correlative with the pathophysiologically induced changes in the regions of interest. This study highlights the potential of this technique as a useful tool for pathophysiological studies and possible diagnosis of the liver diseases with further development.

Tabletop imaging of structural evolutions in chemical reactions demonstrated for the acetylene cation

NASA Astrophysics Data System (ADS)

Ibrahim, Heide; Wales, Benji; Beaulieu, Samuel; Schmidt, Bruno E.; Thiré, Nicolas; Fowe, Emmanuel P.; Bisson, Éric; Hebeisen, Christoph T.; Wanie, Vincent; Giguére, Mathieu; Kieffer, Jean-Claude; Spanner, Michael; Bandrauk, André D.; Sanderson, Joseph; Schuurman, Michael S.; Légaré, François

2014-07-01

The introduction of femto-chemistry has made it a primary goal to follow the nuclear and electronic evolution of a molecule in time and space as it undergoes a chemical reaction. Using Coulomb Explosion Imaging, we have shot the first high-resolution molecular movie of a to and fro isomerization process in the acetylene cation. So far, this kind of phenomenon could only be observed using vacuum ultraviolet light from a free-electron laser. Here we show that 266 nm ultrashort laser pulses are capable of initiating rich dynamics through multiphoton ionization. With our generally applicable tabletop approach that can be used for other small organic molecules, we have investigated two basic chemical reactions simultaneously: proton migration and C=C bond breaking, triggered by multiphoton ionization. The experimental results are in excellent agreement with the timescales and relaxation pathways predicted by new and quantitative ab initio trajectory simulations.

Pancreatic cancer cell detection by targeted lipid microbubbles and multiphoton imaging.

PubMed

Cromey, Benjamin; McDaniel, Ashley; Matsunaga, Terry; Vagner, Josef; Kieu, Khanh Quoc; Banerjee, Bhaskar

2018-04-01

Surgical resection of pancreatic cancer represents the only chance of cure and long-term survival in this common disease. Unfortunately, determination of a cancer-free margin at surgery is based on one or two tiny frozen section biopsies, which is far from ideal. Not surprisingly, cancer is usually left behind and is responsible for metastatic disease. We demonstrate a method of receptor-targeted imaging using peptide ligands, lipid microbubbles, and multiphoton microscopy that could lead to a fast and accurate way of examining the entire cut surface during surgery. Using a plectin-targeted microbubble, we performed a blinded in-vitro study to demonstrate avid binding of targeted microbubbles to pancreatic cancer cells but not noncancerous cell lines. Further work should lead to a much-needed point-of-care diagnostic test for determining clean margins in oncologic surgery. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Watching stem cells at work with a flexible multiphoton tomograph

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Hoffmann, Robert; Weinigel, Martin; König, Karsten

2012-03-01

There is a high demand for non-invasive imaging techniques that allow observation of stem cells in their native environment without significant input on cell metabolism, reproduction, and behavior. Easy accessible hair follicle pluripotent stem cells in the bulge area and dermal papilla are potential sources for stem cell based therapy. It has been shown that these cells are able to generate hair, non-follicle skin cells, nerves, vessels, smooth muscles etc. and may participate in wound healing processes. We report on the finding of nestin-GFP expressing stem cells in their native niche in the bulge of the hair follicle of living mice by using high-resolution in-vivo multiphoton tomography. The 3D imaging with submicron resolution was based on two-photon induced fluorescence and second harmonic generation (SHG) of collagen. Migrating stem cells from the bulge to their microenvironment have been detected inside the skin during optical deep tissue sectioning.

In vivo, label-free, three-dimensional quantitative imaging of liver surface using multi-photon microscopy

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

Zhuo, Shuangmu, E-mail: shuangmuzhuo@gmail.com, E-mail: hanry-yu@nuhs.edu.sg; Institute of Laser and Optoelectronics Technology, Fujian Normal University, Fuzhou 350007; Yan, Jie

2014-07-14

Various structural features on the liver surface reflect functional changes in the liver. The visualization of these surface features with molecular specificity is of particular relevance to understanding the physiology and diseases of the liver. Using multi-photon microscopy (MPM), we have developed a label-free, three-dimensional quantitative and sensitive method to visualize various structural features of liver surface in living rat. MPM could quantitatively image the microstructural features of liver surface with respect to the sinuosity of collagen fiber, the elastic fiber structure, the ratio between elastin and collagen, collagen content, and the metabolic state of the hepatocytes that are correlativemore » with the pathophysiologically induced changes in the regions of interest. This study highlights the potential of this technique as a useful tool for pathophysiological studies and possible diagnosis of the liver diseases with further development.« less

Retinal cell imaging in myopic chickens using adaptive optics multiphoton microscopy.

PubMed

Bueno, Juan M; Palacios, Raquel; Giakoumaki, Anastasia; Gualda, Emilio J; Schaeffel, Frank; Artal, Pablo

2014-03-01

Abnormal eye growth induced by visual deprivation can modify the structure and density of the retinal cells. We have used an adaptive optics multiphoton microscope to image photoreceptors (PRs) and ganglion cells (GCs) at different retinal locations in unstained retinas of chicken eyes with about 10D of myopia and their normal-sighted fellow eyes. In all samples, the local averaged inter-PR distance increased with eccentricity. No significant differences in PR density were found between control and myopic eyes. GC density declined in myopic eyes compared to control eyes and the inter-cell distance increased. In normal eyes, the size of the GC cell bodies increased approximately two-fold between the area centralis and the peripheral retina. In myopic eyes, this trend was preserved but the GC bodies were larger at each retinal location, compared to control eyes. Obviously, GC morphology is changing when the retinal area is enlarged in myopic eyes.

Retinal cell imaging in myopic chickens using adaptive optics multiphoton microscopy

PubMed Central

Bueno, Juan M.; Palacios, Raquel; Giakoumaki, Anastasia; Gualda, Emilio J.; Schaeffel, Frank; Artal, Pablo

2014-01-01

Abnormal eye growth induced by visual deprivation can modify the structure and density of the retinal cells. We have used an adaptive optics multiphoton microscope to image photoreceptors (PRs) and ganglion cells (GCs) at different retinal locations in unstained retinas of chicken eyes with about 10D of myopia and their normal-sighted fellow eyes. In all samples, the local averaged inter-PR distance increased with eccentricity. No significant differences in PR density were found between control and myopic eyes. GC density declined in myopic eyes compared to control eyes and the inter-cell distance increased. In normal eyes, the size of the GC cell bodies increased approximately two-fold between the area centralis and the peripheral retina. In myopic eyes, this trend was preserved but the GC bodies were larger at each retinal location, compared to control eyes. Obviously, GC morphology is changing when the retinal area is enlarged in myopic eyes. PMID:24688804

Lanthanide heterometallic terephthalates: Concentration quenching and the principles of the "multiphotonic emission"

NASA Astrophysics Data System (ADS)

Utochnikova, V. V.; Grishko, A. Yu.; Koshelev, D. S.; Averin, A. A.; Lepnev, L. S.; Kuzmina, N. P.

2017-12-01

The principles of the "multiphotonic emission", i.e. multiple emission from one lanthanide ion, in heterometallic lanthanide terephthalates were determined. Thanks to it, another system with the same effect, namely EuxY1-x(dbm)3(Phen) (Hdbm - dibenzoylmethanate, Phen - o-phenanthroline (mistape)) was found. The criteria for concentration quenching appearance were formulated and demonstrated.

Taple-top imaging of the non-adiabatically driven isomerization in the acetylene cation

NASA Astrophysics Data System (ADS)

Beaulieu, Samuel; Ibrahim, Heide; Wales, Benji; Schmidt, Bruno E.; Thiré, Nicolas; Bisson, Éric; Hebeisen, Christoph T.; Wanie, Vincent; Giguere, Mathieu; Kieffer, Jean-Claude; Sanderson, Joe; Schuurman, Michael S.; Légaré, François

2014-05-01

One of the primary goals of modern ultrafast science is to follow nuclear and electronic evolution of molecules as they undergo a photo-chemical reaction. Most of the interesting dynamics phenomena in molecules occur when an electronically excited state is populated. When the energy difference between electronic ground and excited states is large, Free Electron Laser (FEL) and HHG-based VUV sources were, up to date, the only light sources able to efficiently initiate those non-adiabatic dynamics. We have developed a simple table-top approach to initiate those rich dynamics via multiphoton absorption. As a proof of principle, we studied the ultrafast isomerization of the acetylene cation. We have chosen this model system for isomerization since the internal conversion mechanism which leads to proton migration is still under debate since decades. Using 266 nm multiphoton absorption as a pump and 800 nm induced Coulomb Explosion as a probe, we have shoot the first high-resolution molecular movie of the non-adiabatically driven proton migration in the acetylene cation. The experimental results are in excellent agreement with high level ab initio trajectory simulations.

Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry.

PubMed

Schwertner, M; Booth, M J; Neil, M A A; Wilson, T

2004-01-01

Confocal or multiphoton microscopes, which deliver optical sections and three-dimensional (3D) images of thick specimens, are widely used in biology. These techniques, however, are sensitive to aberrations that may originate from the refractive index structure of the specimen itself. The aberrations cause reduced signal intensity and the 3D resolution of the instrument is compromised. It has been suggested to correct for aberrations in confocal microscopes using adaptive optics. In order to define the design specifications for such adaptive optics systems, one has to know the amount of aberrations present for typical applications such as with biological samples. We have built a phase stepping interferometer microscope that directly measures the aberration of the wavefront. The modal content of the wavefront is extracted by employing Zernike mode decomposition. Results for typical biological specimens are presented. It was found for all samples investigated that higher order Zernike modes give only a small contribution to the overall aberration. Therefore, these higher order modes can be neglected in future adaptive optics sensing and correction schemes implemented into confocal or multiphoton microscopes, leading to more efficient designs.

Hypericin-mediated selective photomodification of connective tissues

NASA Astrophysics Data System (ADS)

Hovhannisyan, V.; Hovhannisyan, A.; Ghukasyan, V.; Guo, H. W.; Lin, Hung-Ming; Chen, S. J.; Chen, Yang-Fang; Dong, Chen-Yuan

2017-02-01

Hypericin (Hyp) has received attention due to its high phototoxicity against viruses and anti-tumor photoactivity. Using two-photon imaging, we demonstrated that Hyp induced photosensitized modification of collagen fibers in native tissues. Dynamics of photo-processes was monitored by time-lapse multiphoton imaging. We showed that Hyp-mediated processes in collagen tissues may be used for the selective modification of collagen fibers.

Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo

NASA Astrophysics Data System (ADS)

Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V.; Roberts, Michael S.

2013-02-01

There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo.

PubMed

Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V; Roberts, Michael S

2013-02-01

There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

Temporal focusing-based widefield multiphoton microscopy with spatially modulated illumination for biotissue imaging.

PubMed

Chang, Chia-Yuan; Lin, Cheng-Han; Lin, Chun-Yu; Sie, Yong-Da; Hu, Yvonne Yuling; Tsai, Sheng-Feng; Chen, Shean-Jen

2018-01-01

A developed temporal focusing-based multiphoton excitation microscope (TFMPEM) has a digital micromirror device (DMD) which is adopted not only as a blazed grating for light spatial dispersion but also for patterned illumination simultaneously. Herein, the TFMPEM has been extended to implement spatially modulated illumination at structured frequency and orientation to increase the beam coverage at the back-focal aperture of the objective lens. The axial excitation confinement (AEC) of TFMPEM can be condensed from 3.0 μm to 1.5 μm for a 50 % improvement. By using the TFMPEM with HiLo technique as two structured illuminations at the same spatial frequency but different orientation, reconstructed biotissue images according to the condensed AEC structured illumination are shown obviously superior in contrast and better scattering suppression. Picture: TPEF images of the eosin-stained mouse cerebellar cortex by conventional TFMPEM (left), and the TFMPEM with HiLo technique as 1.09 μm -1 spatially modulated illumination at 90° (center) and 0° (right) orientations. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Volumetric imaging of fast biological dynamics in deep tissue via wavefront engineering

NASA Astrophysics Data System (ADS)

Kong, Lingjie; Tang, Jianyong; Cui, Meng

2016-03-01

To reveal fast biological dynamics in deep tissue, we combine two wavefront engineering methods that were developed in our laboratory, namely optical phase-locked ultrasound lens (OPLUL) based volumetric imaging and iterative multiphoton adaptive compensation technique (IMPACT). OPLUL is used to generate oscillating defocusing wavefront for fast axial scanning, and IMPACT is used to compensate the wavefront distortions for deep tissue imaging. We show its promising applications in neuroscience and immunology.

Multi-photon transitions and Rabi resonance in continuous wave EPR.

PubMed

Saiko, Alexander P; Fedaruk, Ryhor; Markevich, Siarhei A

2015-10-01

The study of microwave-radiofrequency multi-photon transitions in continuous wave (CW) EPR spectroscopy is extended to a Rabi resonance condition, when the radio frequency of the magnetic-field modulation matches the Rabi frequency of a spin system in the microwave field. Using the non-secular perturbation theory based on the Bogoliubov averaging method, the analytical description of the response of the spin system is derived for all modulation frequency harmonics. When the modulation frequency exceeds the EPR linewidth, multi-photon transitions result in sidebands in absorption EPR spectra measured with phase-sensitive detection at any harmonic. The saturation of different-order multi-photon transitions is shown to be significantly different and to be sensitive to the Rabi resonance. The noticeable frequency shifts of sidebands are found to be the signatures of this resonance. The inversion of two-photon lines in some spectral intervals of the out-of-phase first-harmonic signal is predicted under passage through the Rabi resonance. The inversion indicates the transition from absorption to stimulated emission or vice versa, depending on the sideband. The manifestation of the primary and secondary Rabi resonance is also demonstrated in the time evolution of steady-state EPR signals formed by all harmonics of the modulation frequency. Our results provide a theoretical framework for future developments in multi-photon CW EPR spectroscopy, which can be useful for samples with long spin relaxation times and extremely narrow EPR lines. Copyright © 2015 Elsevier Inc. All rights reserved.

Adaptive optics improves multiphoton super-resolution imaging

NASA Astrophysics Data System (ADS)

Zheng, Wei; Wu, Yicong; Winter, Peter; Shroff, Hari

2018-02-01

Three dimensional (3D) fluorescence microscopy has been essential for biological studies. It allows interrogation of structure and function at spatial scales spanning the macromolecular, cellular, and tissue levels. Critical factors to consider in 3D microscopy include spatial resolution, signal-to-noise (SNR), signal-to-background (SBR), and temporal resolution. Maintaining high quality imaging becomes progressively more difficult at increasing depth (where optical aberrations, induced by inhomogeneities of refractive index in the sample, degrade resolution and SNR), and in thick or densely labeled samples (where out-of-focus background can swamp the valuable, in-focus-signal from each plane). In this report, we introduce our new instrumentation to address these problems. A multiphoton structured illumination microscope was simply modified to integrate an adpative optics system for optical aberrations correction. Firstly, the optical aberrations are determined using direct wavefront sensing with a nonlinear guide star and subsequently corrected using a deformable mirror, restoring super-resolution information. We demonstrate the flexibility of our adaptive optics approach on a variety of semi-transparent samples, including bead phantoms, cultured cells in collagen gels and biological tissues. The performance of our super-resolution microscope is improved in all of these samples, as peak intensity is increased (up to 40-fold) and resolution recovered (up to 176+/-10 nm laterally and 729+/-39 nm axially) at depths up to 250 μm from the coverslip surface.

Tabletop Imaging of Structural Evolutions in Chemical Reactions

NASA Astrophysics Data System (ADS)

Ibrahim, Heide; Wales, Benji; Beaulieu, Samuel; Schmidt, Bruno E.; Thiré, Nicolas; Fowe, Emmanuel P.; Bisson, Éric; Hebeisen, Christoph T.; Wanie, Vincent; Giguére, Mathieu; Kieffer, Jean-Claude; Spanner, Michael; Bandrauk, André D.; Sanderson, Joseph; Schuurman, Michael S.; Légaré, François

The first high-resolution molecular movie of proton migration in the acetylene cation is obtained using a tabletop multiphoton pump-probe approach—an alternative to demanding free-electron-lasers and other VUV light sources when ionizing from the HOMO-1.

Multiphoton microscopy as a diagnostic imaging modality for pancreatic neoplasms without hematoxylin and eosin stains

NASA Astrophysics Data System (ADS)

Chen, Youting; Chen, Jing; Chen, Hong; Hong, Zhipeng; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Yanling; Chen, Jianxin

2014-09-01

Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear-cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.

In vivo time-serial multi-modality optical imaging in a mouse model of ovarian tumorigenesis

PubMed Central

Watson, Jennifer M; Marion, Samuel L; Rice, Photini F; Bentley, David L; Besselsen, David G; Utzinger, Urs; Hoyer, Patricia B; Barton, Jennifer K

2014-01-01

Identification of the early microscopic changes associated with ovarian cancer may lead to development of a diagnostic test for high-risk women. In this study we use optical coherence tomography (OCT) and multiphoton microscopy (MPM) (collecting both two photon excited fluorescence [TPEF] and second harmonic generation [SHG]) to image mouse ovaries in vivo at multiple time points. We demonstrate the feasibility of imaging mouse ovaries in vivo during a long-term survival study and identify microscopic changes associated with early tumor development. These changes include alterations in tissue microstructure, as seen by OCT, alterations in cellular fluorescence and morphology, as seen by TPEF, and remodeling of collagen structure, as seen by SHG. These results suggest that a combined OCT-MPM system may be useful for early detection of ovarian cancer. PMID:24145178

Intravital imaging of cutaneous immune responses.

PubMed

Nakamizo, Satoshi; Egawa, Gyohei; Bing, Jasmine Tan Kah; Kabashima, Kenji

2018-05-25

Various immune cells are present in the skin and modulate the cutaneous immune response. In order to capture such dynamic phenomena, intravital imaging is an important technique and there is a possibility to provide substantial information that is not available using conventional histological analysis. Multiphoton microscope enable direct, three-dimensional, minimally invasive imaging of biological samples with high spatiotemporal resolution, and now become the main method for intravital imaging studies. Here, we will introduce the latest knowledge obtained by intravital imaging of the skin. Copyright © 2018 Elsevier Inc. All rights reserved.

Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries

NASA Astrophysics Data System (ADS)

McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

2015-02-01

Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe-/-Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP+ macrophages "dancing on the spot" and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells.

Phototoxicity free quantum dot-based niosome formulation for controlled drug release and its monitoring

NASA Astrophysics Data System (ADS)

Kumar, Sunil; Kang, T. W.; Bala, Suman; Kamboj, Sunil; Jeon, H. C.

2018-04-01

A novel niosomes-based system composed of Hypromellose (HPMC) functionalized fluorescent, biocompatible ZnS:Mn quantum dots (QDs), and anti-HIV drug Tenofovir disoproxil fumarate (TDF) was designed. An appropriate ratio of surfactant Sorbitan Monostearate (SPAN-60) and cholesterol was used to obtain an optimal entrapment efficiency. Initially, after observing the successful interaction of HPMC with SPAN-60, the noisome formulation including (QDs + drug) and HPMC-coated QDs was synthesized by a wet chemical route and characterized by X-ray diffraction (XRD), Transmission electron microscope (TEM) and Selected Electron Diffraction (SAED). Secondly, (QDs + drug) loaded niosome formulations were studied by varying the ratio of SPAN-60 and cholesterol. Multiple studies were done to characterize the shape, size, viscosity, colloidal stability, and entrapment efficiency of (QDs + drug) loaded niosomes. Lastly, pH-dependent (QDs + drug) release profiles were studied by a spectroscopic technique considering the pH of the human gastrointestinal region to obtain the formulation stability of (QDs + drug) release from the niosome vesicles. These studies also include pH-dependent photo-stability measurements based on laser-induced multiphoton excitation technique in the Infrared region. The multiphoton time-resolved studies were completed to avoid the UV induced phototoxicity in the drug delivery modules. Current studies on the formulation of niosomes-based (QDs + drug) system laid a foundation to make a complete phototoxicity free system for tracking controlled drug release and its imaging.

Signal improvement in multiphoton microscopy by reflection with simple mirrors near the sample

NASA Astrophysics Data System (ADS)

Rehberg, Markus; Krombach, Fritz; Pohl, Ulrich; Dietzel, Steffen

2010-03-01

In conventional fluorescence or confocal microscopy, emitted light is generated not only in the focal plane but also above and below. The situation is different in multiphoton-induced fluorescence and multiphoton-induced higher harmonic generation. Here, restriction of signal generation to a single focal point permits that all emitted photons can contribute to image formation if collected, regardless of their path through the specimen. Often, the intensity of the emitted light is rather low in biological specimens. We present a method to significantly increase the fraction of photons collected by an epi (backward) detector by placing a simple mirror, an aluminum-coated coverslip, directly under the sample. Samples investigated include fluorescent test slides, collagen gels, and thin-layered, intact mouse skeletal muscles. Quantitative analysis revealed an intensity increase of second- and third-harmonic generated signal in skeletal muscle of nine- and sevenfold respectively, and of fluorescent signal in test slides of up to twofold. Our approach thus allows significant signal improvement also for situations were a forward detection is impossible, e.g., due to the anatomy of animals in intravital microscopy.

Label-free in vivo in situ diagnostic imaging by cellular metabolism quantification with a flexible multiphoton endomicroscope (Conference Presentation)

NASA Astrophysics Data System (ADS)

Leclerc, Pierre; Hage, Charles-Henri; Fabert, Marc; Brevier, Julien; O'Connor, Rodney P.; Bardet-Coste, Sylvia M.; Habert, Rémi; Braud, Flavie; Kudlinski, Alexandre; Louradour, Frederic

2017-02-01

Multiphoton microscopy is a cutting edge imaging modality leading to increasing advances in biology and also in the clinical field. To use it at its full potential and at the very heart of clinical practice, there have been several developments of fiber-based multiphoton microendoscopes. The application for those probes is now limited by few major restrictions, such as the difficulty to collect autofluorescence signals from tissues and cells theses being inherently weak (e.g. the ones from intracellular NADH or FAD metabolites). This limitation reduces the usefulness of microendoscopy in general, effectively restraining it to morphological imaging modality requiring staining of the tissues. Our aim is to go beyond this limitation, showing for the first time label-free cellular metabolism monitoring, in vivo in situ in real time. The experimental setup is an upgrade of a recently published one (Ducourthial et.al, Scientific Reports, 2016) where femtosecond pulse fiber delivery is further optimized thank's to a new transmissive-GRISM-based pulse stretcher permitting high energy throughput and wide bandwidth. This device allows fast sequential operation with two different excitation wavelengths for efficient two-photon excited NADH and FAD autofluorescence endoscopic detection (i.e. 860 nm for FAD and 760 nm for NADH), enabling cellular optical redox ratio quantification at 8 frames/s. The obtained results on cell models in vitro and also on animal models in vivo (e.g. neurons of a living mouse) prove that we accurately assess the level of NADH and FAD at subcellular resolution through a 3-meters-long fiber with our miniaturized probe (O.D. =2.2 mm).

Spin Multiphoton Antiresonance at Finite Temperatures

NASA Astrophysics Data System (ADS)

Hicke, Christian; Dykman, Mark

2007-03-01

Weakly anisotropic S>1 spin systems display multiphoton antiresonance. It occurs when an Nth overtone of the radiation frequency coincides with the distance between the ground and the Nth excited energy level (divided by ). The coherent response of the spin displays a sharp minimum or maximum as a function of frequency, depending on which state was initially occupied. We find the spectral shape of the response dips/peaks. We also study the stationary response for zero and finite temperatures. The response changes dramatically with increasing temperature, when excited states become occupied even in the absence of radiation. The change is due primarily to the increasing role of single-photon resonances between excited states, which occur at the same frequencies as multiphoton resonances. Single-photon resonances are broad, because the single-photon Rabi frequencies largely exceed the multi-photon ones. This allows us to separate different resonances and to study their spectral shape. We also study the change of the spectrum due to relaxational broadening of the peaks, with account taken of both decay and phase modulation.

Fiber bundle based endomicroscopy prototype with two collection channels for simultaneous coherent anti-Stokes Raman scattering and second harmonic generation imaging

NASA Astrophysics Data System (ADS)

Liu, Zhengfan; Satira, Zachary A.; Wang, Xi; Xu, Xiaoyun; Chen, Xu; Wong, Kelvin; Chen, Shufen; Xin, Jianguo; Wong, Stephen T. C.

2014-02-01

Label-free multiphoton imaging is promising for replacing biopsy and could offer new strategies for intraoperative or surgical applications. Coherent anti-Stokes Raman scattering (CARS) imaging could provide lipid-band contrast, and second harmonic generation (SHG) imaging is useful for imaging collagen, tendon and muscle fibers. A combination of these two imaging modalities could provide rich information and this combination has been studied by researchers to investigate diseases through microscopy imaging. The combination of these two imaging modalities in endomicroscopy imaging has been rarely investigated. In this research, a fiber bundle consisted of one excitation fiber and 18 collection fibers was developed in our endomicroscopy prototype. The 18 collection fibers were divided into two collection channels with 9 fibers in each channel. These two channels could be used together as one channel for effective signal collection or used separately for simplifying detection part of the system. Differences of collection pattern of these two channels were investigated. Collection difference of central excitation fiber and surrounding 18 fibers was also investigated, which reveals the potential ability of this system to measure forward to backward (F/B) ratio in SHG imaging. CARS imaging of mouse adipocyte and SHG imaging of mouse tail tendon were performed to demonstrate the CARS and SHG tissue imaging performance of this system. Simultaneous CARS and SHG imaging ability of this system was demonstrated by mouse tail imaging. This fiber bundle based endomicroscopy imaging prototype, offers a promising platform for constructing efficient fiber-based CARS and SHG multimodal endomicroscopes for label free intraoperative imaging applications.

Multiphoton, confocal, and lifetime microscopy for molecular imaging in cartilage

NASA Astrophysics Data System (ADS)

Wachsmann-Hogiu, Sebastian; Krakow, Deborah; Kirilova, Veneta T.; Cohn, Daniel H.; Bertolotto, Cristina; Acuna, Dora; Fang, Qiyin; Krivorov, Nikola; Farkas, Daniel L.

2005-03-01

It has recently been shown that mutations in Filamin A and B genes produce a large spectrum of skeletal disorders in developing fetuses. However, high-resolution optical microscopy in cartilage growth plate using fluorescent antibody assays, which should elucidate molecular aspects of these disorders, is extremely difficult due to the high level of autofluoresce in this tissue. We apply multiphoton, confocal, lifetime and spectral microscopy to (i) image and characterize autofluorophores in chondrocytes and subtract their contributions to obtain a corrected antibody-marker fluorescence signal, and (ii) measure the interaction between Filamin A and B proteins by detecting the fluorescence resonance energy transfer (FRET) between markers of the two proteins. Taking advantage of the different fluorescence spectra of the endogenous and exogenous markers, we can significantly reduce the autofluorescence background. Preliminary results of the FRET experiments suggest no interaction between Filamin A and B proteins. However, developing of new antibodies targeting the carboxy-terminal immunoglobulin-like domain may be necessary to confirm this result.

Wavelength dependent photoelectron circular dichroism of limonene studied by femtosecond multiphoton laser ionization and electron-ion coincidence imaging

NASA Astrophysics Data System (ADS)

Rafiee Fanood, Mohammad M.; Janssen, Maurice H. M.; Powis, Ivan

2016-09-01

Enantiomers of the monoterpene limonene have been investigated by (2 + 1) resonance enhanced multiphoton ionization and photoelectron circular dichroism employing tuneable, circularly polarized femtosecond laser pulses. Electron imaging detection provides 3D momentum measurement while electron-ion coincidence detection can be used to mass-tag individual electrons. Additional filtering, by accepting only parent ion tagged electrons, can be then used to provide discrimination against higher energy dissociative ionization mechanisms where more than three photons are absorbed to better delineate the two photon resonant, one photon ionization pathway. The promotion of different vibrational levels and, tentatively, different electronic ion core configurations in the intermediate Rydberg states can be achieved with different laser excitation wavelengths (420 nm, 412 nm, and 392 nm), in turn producing different state distributions in the resulting cations. Strong chiral asymmetries in the lab frame photoelectron angular distributions are quantified, and a comparison made with a single photon (synchrotron radiation) measurement at an equivalent photon energy.

A velocity-map imaging study of methyl non-resonant multiphoton ionization from the photodissociation of CH3I in the A-band

PubMed Central

Poullain, Sonia Marggi; Chicharro, David V.; Rubio-Lago, Luis; García-Vela, Alberto

2017-01-01

Chemical reaction dynamics and, particularly, photodissociation in the gas phase are generally studied using pump–probe schemes where a first laser pulse induces the process under study and a second one detects the produced fragments. Providing an efficient detection of ro-vibrationally state-selected photofragments, the resonance enhanced multiphoton ionization (REMPI) technique is, without question, the most popular approach used for the probe step, while non-resonant multiphoton ionization (NRMPI) detection of the products is scarce. The main goal of this work is to test the sensitivity of the NRMPI technique to fragment vibrational distributions arising from molecular photodissociation processes. We revisit the well-known process of methyl iodide photodissociation in the A-band at around 280 nm, using the velocity-map imaging technique in conjunction with NRMPI of the methyl fragment. The detection wavelength, carefully selected to avoid any REMPI transition, was scanned between 325 and 335 nm seeking correlations between the different observables—the product vibrational, translational and angular distributions—and the excitation wavelength of the probe laser pulse. The experimental results have been discussed on the base of quantum dynamics calculations of photofragment vibrational populations carried out on available ab initio potential-energy surfaces using a four-dimensional model. This article is part of the themed issue ‘Theoretical and computational studies of non-equilibrium and non-statistical dynamics in the gas phase, in the condensed phase and at interfaces’. PMID:28320907

Two-photon autofluorescence/FLIM/SHG endoscopy to study the oral cavity and wound healing in humans (Conference Presentation)

NASA Astrophysics Data System (ADS)

König, Karsten

2016-03-01

Monitoring the oral cavity noninvasively with superior 3D resolution is realized by clinical multiphoton tomography and high NA two-photon endoscopy without the need of additional contrast agents. The technology behind this investigation is based on nonlinear optical contrast of the multiphoton tomograph MPTflex®. Furthermore, the miniaturized GRIN endoscope was used to realize more accessibility for more demanding wound conditions in skin. The MPTflex® distinguishes autofluorescence (AF) signals from second harmonic generation (SHG) signals simultaneously. Fluorescence lifetime imaging (FLIM) based on time correlated single photon counting (TCSPC) technology offers additional information on the functional level of the intratissue fluorophores, their binding status, and the contribution of SHG signals in chronic wounds.

Generation of multiphoton entangled quantum states by means of integrated frequency combs.

PubMed

Reimer, Christian; Kues, Michael; Roztocki, Piotr; Wetzel, Benjamin; Grazioso, Fabio; Little, Brent E; Chu, Sai T; Johnston, Tudor; Bromberg, Yaron; Caspani, Lucia; Moss, David J; Morandotti, Roberto

2016-03-11

Complex optical photon states with entanglement shared among several modes are critical to improving our fundamental understanding of quantum mechanics and have applications for quantum information processing, imaging, and microscopy. We demonstrate that optical integrated Kerr frequency combs can be used to generate several bi- and multiphoton entangled qubits, with direct applications for quantum communication and computation. Our method is compatible with contemporary fiber and quantum memory infrastructures and with chip-scale semiconductor technology, enabling compact, low-cost, and scalable implementations. The exploitation of integrated Kerr frequency combs, with their ability to generate multiple, customizable, and complex quantum states, can provide a scalable, practical, and compact platform for quantum technologies. Copyright © 2016, American Association for the Advancement of Science.

An Automated System for the Control of, and Data Acquisition from Multiphoton Ionization and Fluorescence Lifetime Measurements.

DTIC Science & Technology

1986-09-01

Quanta- Ray company , which also supplied the laser used for the multiphoton work. The, burner was mounted on a translator stage from Velmex, Inc...and no longer exists as a process in the system. When the user analysis program has completed, the lifetime program is again automatically re-started...KCHAR) RETURN 100 FORMAT(I3) 101 FORMAT(F7.2) END SUBROUTINE LAB4 FODA SE"oteD C This routine puts the label "INTEGRAL FROM DATA SET" on the MDP C screen

Photon-number-resolving SSPDs with system detection efficiency over 50% at telecom range

NASA Astrophysics Data System (ADS)

Zolotov, P.; Divochiy, A.; Vakhtomin, Yu.; Moshkova, M.; Morozov, P.; Seleznev, V.; Smirnov, K.

2018-02-01

We used technology of making high-efficiency superconducting single-photon detectors as a basis for improvement of photon-number-resolving devices. By adding optical cavity and using an improved NbN superconducting film, we enhanced previously reported system detection efficiency at telecom range for such detectors. Our results show that implementation of optical cavity helps to develop four-section device with quantum efficiency over 50% at 1.55 µm. Performed experimental studies of detecting multi-photon optical pulses showed irregularities over defining multi-photon through single-photon quantum efficiency.

Weak-field multiphoton femtosecond coherent control in the single-cycle regime.

PubMed

Chuntonov, Lev; Fleischer, Avner; Amitay, Zohar

2011-03-28

Weak-field coherent phase control of atomic non-resonant multiphoton excitation induced by shaped femtosecond pulses is studied theoretically in the single-cycle regime. The carrier-envelope phase (CEP) of the pulse, which in the multi-cycle regime does not play any control role, is shown here to be a new effective control parameter that its effect is highly sensitive to the spectral position of the ultrabroad spectrum. Rationally chosen position of the ultrabroadband spectrum coherently induces several groups of multiphoton transitions from the ground state to the excited state of the system: transitions involving only absorbed photons as well as Raman transitions involving both absorbed and emitted photons. The intra-group interference is controlled by the relative spectral phase of the different frequency components of the pulse, while the inter-group interference is controlled jointly by the CEP and the relative spectral phase. Specifically, non-resonant two- and three-photon excitation is studied in a simple model system within the perturbative frequency-domain framework. The developed intuition is then applied to weak-field multiphoton excitation of atomic cesium (Cs), where the simplified model is verified by non-perturbative numerical solution of the time-dependent Schrödinger equation. We expect this work to serve as a basis for a new line of femtosecond coherent control experiments.

Mechanisms of Radiation-Induced Bone Loss and Effect on Prostate Cancer Bone Metastases

DTIC Science & Technology

2012-06-01

Develop intravital multiphoton fluorescence microscopy (IVFM) for real-time imaging of osteocytes in calvariae of transgenic mice using i) GFP to...OT, OB counting) and in vivo bone imaging (months 6-10) 8 20 week old female C57Bl/6 mice (n=30) were used in this experiment. The mice were...divided into 2 groups. One group (group A, n=15) was imaged twice by microCT during the experiment that included a baseline microCT that was given 2 days

A novel clinical multimodal multiphoton tomograph for AF, SHG, CARS imaging, and FLIM

NASA Astrophysics Data System (ADS)

Weinigel, Martin; Breunig, Hans Georg; König, Karsten

2014-02-01

We report on a flexible nonlinear medical tomograph with multiple miniaturized detectors for simultaneous acquisition of two-photon autofluorescence (AF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) images. The simultaneous visualization of the distribution of endogenous fluorophores NAD(P)H, melanin and elastin, SHG-active collagen and as well as non-fluorescent lipids within human skin in vivo is possible. Furthermore, fluorescence lifetime images (FLIM) can be generated using time-correlated single photon counting.

2012 MULTIPHOTON PROCESSES GRC, JUNE 3-8, 2012

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

Walker, Barry

2012-03-08

The sessions will focus on:  Attosecond science;  Strong-field processes in molecules and solids;  Generation of harmonics and attosecond pulses;  Free-electron laser experiments and theory;  Ultrafast imaging;  Applications of very high intensity lasers;  Propagation of intense laser fields.

The development of novel Ytterbium fiber lasers and their applications

NASA Astrophysics Data System (ADS)

Nie, Bai

The aim of my Ph.D. research is to push the fundamental limits holding back the development of novel Yb fiber lasers with high pulse energy and short pulse duration. The purpose of developing these lasers is to use them for important applications such as multiphoton microscopy and laser-induced breakdown spectroscopy. My first project was to develop a short-pulse high-energy ultrafast fiber laser for multiphoton microscopy. To achieve high multiphoton efficiency and depth resolved tissue imaging, ultrashort pulse duration and high pulse energy are required. In order to achieve this, an all-normal dispersion cavity design was adopted. Output performances of the built lasers were investigated by varying several cavity parameters, such as pump laser power, fiber length and intra-cavity spectral filter bandwidth. It was found that the length of the fiber preceding the gain fiber is critical to the laser performance. Generally, the shorter the fiber is, the broader the output spectrum is. The more interesting parameter is the intra-cavity spectral filter bandwidth. Counter intuitively, laser cavities using narrower bandwidth spectral filters generated much broader spectra. It was also found that fiber lasers with very narrow spectral filters produced laser pulses with parabolic profile, which are referred to as self-similar pulses or similaritons. This type of pulse can avoid wave-breaking and is an optimal approach to generate pulses with high pulse energy and ultrashort pulse duration. With a 3nm intra-cavity spectral filter, output pulses with about 20 nJ pulse energy were produced and compressed to about 41 fs full-width-at-half-maximum (FWHM) pulse duration. Due to the loss in the compression device, the peak power of the compressed pulses is about 250 kW. It was the highest peak power generated from a fiber oscillator when this work was published. This laser was used for multiphoton microscopy on living tissues like Drosophila larva and fruit fly wings. Several imaging methods, such as two-photon-excited fluorescence, second harmonic generation, and third harmonic generation, were performed. Not only were single layers of thin tissue imaged, but also depth resolved imaging of thick samples was tested, and three-dimensional image reconstruction was demonstrated. The other project was to develop a simple fiber oscillator for laser-induced breakdown spectroscopy (LIBS). Laser pulses with high energy, high ablation efficiency and low ablation threshold are desirable for this application. We built a fiber laser using up to 200 m long fiber and scaled the output pulse energy up to 450 nJ. This laser was operated in an unusual mode-locking regime and produced noise-like pulses, which have a picosecond long pulse envelope containing multiple irregular femtosecond sub-pulses. This type of pulse was mostly ignored by many earlier researchers. Intra-cavity spectral filters did not affect the laser performance as much as in the similariton lasers and were removed from the laser cavity. Characteristics of our noise-like laser, such as MHz repetition rate, broad spectrum, and picosecond-long pulse envelope containing multiple femtosecond sub-pulses, were found to meet the requirement of an ideal laser source for LIBS. A simple LIBS setup using our laser was demonstrated and atomic emission spectra with very good signal-to-noise ratio were obtained. Composition detection, qualitative concentration determination, and trace detection were also tested. These tests show that our noise-like fiber laser is an ideal laser source for a low-cost and portable LIBS system.

Four-Dimensional Imaging of T Cells in Kidney Transplant Rejection.

PubMed

Hughes, Andrew D; Lakkis, Fadi G; Oberbarnscheidt, Martin H

2018-06-01

Kidney transplantation is the treatment of choice for ESRD but is complicated by the response of the recipient's immune system to nonself histocompatibility antigens on the graft, resulting in rejection. Multiphoton intravital microscopy, referred to as four-dimensional imaging because it records dynamic events in three-dimensional tissue volumes, has emerged as a powerful tool to study immunologic processes in living animals. Here, we will review advances in understanding the complex mechanisms of T cell-mediated rejection made possible by four-dimensional imaging of mouse renal allografts. We will summarize recent data showing that activated (effector) T cell migration to the graft is driven by cognate antigen presented by dendritic cells that surround and penetrate peritubular capillaries, and that T cell-dendritic cell interactions persist in the graft over time, maintaining the immune response in the tissue. Copyright © 2018 by the American Society of Nephrology.

Real-time visualization of melanin granules in normal human skin using combined multiphoton and reflectance confocal microscopy.

PubMed

Majdzadeh, Ali; Lee, Anthony M D; Wang, Hequn; Lui, Harvey; McLean, David I; Crawford, Richard I; Zloty, David; Zeng, Haishan

2015-05-01

Recent advances in biomedical optics have enabled dermal and epidermal components to be visualized at subcellular resolution and assessed noninvasively. Multiphoton microscopy (MPM) and reflectance confocal microscopy (RCM) are noninvasive imaging modalities that have demonstrated promising results in imaging skin micromorphology, and which provide complementary information regarding skin components. This study assesses whether combined MPM/RCM can visualize intracellular and extracellular melanin granules in the epidermis and dermis of normal human skin. We perform MPM and RCM imaging of in vivo and ex vivo skin in the infrared domain. The inherent three-dimensional optical sectioning capability of MPM/RCM is used to image high-contrast granular features across skin depths ranging from 50 to 90 μm. The optical images thus obtained were correlated with conventional histologic examination including melanin-specific staining of ex vivo specimens. MPM revealed highly fluorescent granular structures below the dermal-epidermal junction (DEJ) region. Histochemical staining also demonstrated melanin-containing granules that correlate well in size and location with the granular fluorescent structures observed in MPM. Furthermore, the MPM fluorescence excitation wavelength and RCM reflectance of cell culture-derived melanin were equivalent to those of the granules. This study suggests that MPM can noninvasively visualize and quantify subepidermal melanin in situ. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

In Vivo Multiphoton Microscopy for Investigating Biomechanical Properties of Human Skin.

PubMed

Liang, Xing; Graf, Benedikt W; Boppart, Stephen A

2011-06-01

The biomechanical properties of living cells depend on their molecular building blocks, and are important for maintaining structure and function in cells, the extracellular matrix, and tissues. These biomechanical properties and forces also shape and modify the cellular and extracellular structures under stress. While many studies have investigated the biomechanics of single cells or small populations of cells in culture, or the properties of organs and tissues, few studies have investigated the biomechanics of complex cell populations in vivo. With the use of advanced multiphoton microscopy to visualize in vivo cell populations in human skin, the biomechanical properties are investigated in a depth-dependent manner in the stratum corneum and epidermis using quasi-static mechanical deformations. A 2D elastic registration algorithm was used to analyze the images before and after deformation to determine displacements in different skin layers. In this feasibility study, the images and results from one human subject demonstrate the potential of the technique for revealing differences in elastic properties between the stratum corneum and the rest of the epidermis. This interrogational imaging methodology has the potential to enable a wide range of investigations for understanding how the biomechanical properties of in vivo cell populations influence function in health and disease.

In vivo multiphoton and second harmonic generation microscopy of epithelial carcinogenesis

NASA Astrophysics Data System (ADS)

Vargas, Gracie; Shilagard, Tuya; Sun, Ju; Motamedi, Massoud

2006-02-01

Multiphoton microscopy and second harmonic generation microscopy were used to image epithelial changes in a hamster model for oral malignant transformation. In vivo imaging was performed to characterize morphometric alterations in normal and precancerous regions. Morphometric measurements such as cell nucleus area and epithelial thicknesses obtained from MPM-SHGM were in excellent agreement with histology obtained after in vivo imaging. MPM-SHGM was highly sensitive to spectroscopic and architectural alterations throughout carcinogenesis, showing statistically significant changes in morphology. MPM revealed hyperkeratosis, nuclear enlargement/crowding in dysplasia, and immune cell infiltration. SHGM revealed alterations in submucosal architecture, with a decrease in SHG density evident during early stages of precancer. By combining MPM with SHGM, the basement membrane could be identified in normal, hyperplasia, and dysplasia samples and in some cases of early invasion. The combined technique of MPM-SHGM has the potential to serve as an adjunct to biopsy for assessing precancerous changes and will be investigated further for that purpose. Additionally, the method can provide spatiotemporal assessment of early neoplastic changes in order to elucidate the stages of transformation in vivo and could be used to assess therapeutic efficacy of agents being tested for the treatment of epithelial precancers/cancer.

Singlet gradient index lens for deep in vivo multiphoton microscopy

NASA Astrophysics Data System (ADS)

Murray, Teresa A.; Levene, Michael J.

2012-02-01

Micro-optical probes, including gradient index (GRIN) lenses and microprisms, have expanded the range of in vivo multiphoton microscopy to reach previously inaccessible deep brain structures such as deep cortical layers and the underlying hippocampus in mice. Yet imaging with GRIN lenses has been fundamentally limited by large amounts of spherical aberration and the need to construct compound lenses that limit the field-of-view. Here, we demonstrate the use of 0.5-mm-diameter, 1.7-mm-long GRIN lens singlets with 0.6 numerical aperture in conjunction with a cover glass and a conventional microscope objective correction collar to balance spherical aberrations. The resulting system achieves a lateral resolution of 618 nm and an axial resolution of 5.5 μm, compared to lateral and axial resolutions of ~1 μm and ~15 μm, respectively, for compound GRIN lenses of similar diameter. Furthermore, the GRIN lens singlets display fields-of-view in excess of 150 μm, compared with a few tens of microns for compound GRIN lenses. The GRIN lens/cover glass combination presented here is easy to assemble and inexpensive enough for use as a disposable device, enabling ready adoption by the neuroscience community.

Identifying and quantifying the stromal fibrosis in muscularis propria of colorectal carcinoma by multiphoton microscopy

NASA Astrophysics Data System (ADS)

Chen, Sijia; Yang, Yinghong; Jiang, Weizhong; Feng, Changyin; Chen, Zhifen; Zhuo, Shuangmu; Zhu, Xiaoqin; Guan, Guoxian; Chen, Jianxin

2014-10-01

The examination of stromal fibrosis within colorectal cancer is overlooked, not only because the routine pathological examinations seem to focus more on tumour staging and precise surgical margins, but also because of the lack of efficient diagnostic methods. Multiphoton microscopy (MPM) can be used to study the muscularis stroma of normal and colorectal carcinoma tissue at the molecular level. In this work, we attempt to show the feasibility of MPM for discerning the microstructure of the normal human rectal muscle layer and fibrosis colorectal carcinoma tissue practicably. Three types of muscularis propria stromal fibrosis beneath the colorectal cancer infiltration were first observed through the MPM imaging system by providing intercellular microstructural details in fresh, unstained tissue samples. Our approach also presents the capability of quantifying the extent of stromal fibrosis from both amount and orientation of collagen, which may further characterize the severity of fibrosis. By comparing with the pathology analysis, these results show that the MPM has potential advantages in becoming a histological tool for detecting the stromal fibrosis and collecting prognosis evidence, which may guide subsequent therapy procedures for patients into good prognosis.

Recent Developments in Vascular Imaging Techniques in Tissue Engineering and Regenerative Medicine

PubMed Central

Upputuri, Paul Kumar; Sivasubramanian, Kathyayini; Mark, Chong Seow Khoon; Pramanik, Manojit

2015-01-01

Adequate vascularisation is key in determining the clinical outcome of stem cells and engineered tissue in regenerative medicine. Numerous imaging modalities have been developed and used for the visualization of vascularisation in tissue engineering. In this review, we briefly discuss the very recent advances aiming at high performance imaging of vasculature. We classify the vascular imaging modalities into three major groups: nonoptical methods (X-ray, magnetic resonance, ultrasound, and positron emission imaging), optical methods (optical coherence, fluorescence, multiphoton, and laser speckle imaging), and hybrid methods (photoacoustic imaging). We then summarize the strengths and challenges of these methods for preclinical and clinical applications. PMID:25821821

Transfer, Imaging, and Analysis Plate for Facile Handling of 384 Hanging Drop 3D Tissue Spheroids

PubMed Central

Cavnar, Stephen P.; Salomonsson, Emma; Luker, Kathryn E.; Luker, Gary D.; Takayama, Shuichi

2014-01-01

Three-dimensional culture systems bridge the experimental gap between in vivo and in vitro physiology. However, nonstandardized formation and limited downstream adaptability of 3D cultures have hindered mainstream adoption of these systems for biological applications, especially for low- and moderate-throughput assays commonly used in biomedical research. Here we build on our recent development of a 384-well hanging drop plate for spheroid culture to design a complementary spheroid transfer and imaging (TRIM) plate. The low-aspect ratio wells of the TRIM plate facilitated highfidelity, user-independent, contact-based collection of hanging drop spheroids. Using the TRIM plate, we demonstrated several downstream analyses, including bulk tissue collection for flow cytometry, high-resolution low working-distance immersion imaging, and timely reagent delivery for enzymatic studies. Low working-distance multiphoton imaging revealed a cell type–dependent, macroscopic spheroid structure. Unlike ovarian cancer spheroids, which formed loose, disk-shaped spheroids, human mammary fibroblasts formed tight, spherical, and nutrient-limited spheroids. Beyond the applications we describe here, we expect the hanging drop spheroid plate and complementary TRIM plate to facilitate analyses of spheroids across the spectrum of throughput, particularly for bulk collection of spheroids and high-content imaging. PMID:24051516

Transfer, imaging, and analysis plate for facile handling of 384 hanging drop 3D tissue spheroids.

PubMed

Cavnar, Stephen P; Salomonsson, Emma; Luker, Kathryn E; Luker, Gary D; Takayama, Shuichi

2014-04-01

Three-dimensional culture systems bridge the experimental gap between in vivo and in vitro physiology. However, nonstandardized formation and limited downstream adaptability of 3D cultures have hindered mainstream adoption of these systems for biological applications, especially for low- and moderate-throughput assays commonly used in biomedical research. Here we build on our recent development of a 384-well hanging drop plate for spheroid culture to design a complementary spheroid transfer and imaging (TRIM) plate. The low-aspect ratio wells of the TRIM plate facilitated high-fidelity, user-independent, contact-based collection of hanging drop spheroids. Using the TRIM plate, we demonstrated several downstream analyses, including bulk tissue collection for flow cytometry, high-resolution low working-distance immersion imaging, and timely reagent delivery for enzymatic studies. Low working-distance multiphoton imaging revealed a cell type-dependent, macroscopic spheroid structure. Unlike ovarian cancer spheroids, which formed loose, disk-shaped spheroids, human mammary fibroblasts formed tight, spherical, and nutrient-limited spheroids. Beyond the applications we describe here, we expect the hanging drop spheroid plate and complementary TRIM plate to facilitate analyses of spheroids across the spectrum of throughput, particularly for bulk collection of spheroids and high-content imaging.

Three-photon imaging of ovarian cancer

NASA Astrophysics Data System (ADS)

Barton, Jennifer K.; Amirsolaimani, Babak; Rice, Photini; Hatch, Kenneth; Kieu, Khanh

2016-02-01

Optical imaging methods have the potential to detect ovarian cancer at an early, curable stage. Optical imaging has the disadvantage that high resolution techniques require access to the tissue of interest, but miniature endoscopes that traverse the natural orifice of the reproductive tract, or access the ovaries and fallopian tubes through a small incision in the vagina wall, can provide a minimally-invasive solution. We have imaged both rodent and human ovaries and fallopian tubes with a variety of endoscope-compatible modalities. The recent development of fiber-coupled femtosecond lasers will enable endoscopic multiphoton microscopy (MPM). We demonstrated two- and three-photon excited fluorescence (2PEF, 3PEF), and second- and third-harmonic generation microscopy (SHG, THG) in human ovarian and fallopian tube tissue. A study was undertaken to understand the mechanisms of contrast in these images. Six patients (normal, cystadenoma, and ovarian adenocarcinoma) provided ovarian and fallopian tube biopsies. The tissue was imaged with three-dimensional optical coherence tomography, multiphoton microscopy, and frozen for histological sectioning. Tissue sections were stained with hematoxylin and eosin, Masson's trichrome, and Sudan black. Approximately 1 μm resolution images were obtained with an excitation source at 1550 nm. 2PEF signal was absent. SHG signal was mainly from collagen. 3PEF and THG signal came from a variety of sources, including a strong signal from fatty connective tissue and red blood cells. Adenocarcinoma was characterized by loss of SHG signal, whereas cystic abnormalities showed strong SHG. There was limited overlap of two- and three- photon signals, suggesting that three-photon imaging can provide additional information for early diagnosis of ovarian cancer.

High-resolution imaging of basal cell carcinoma: a comparison between multiphoton microscopy with fluorescence lifetime imaging and reflectance confocal microscopy.

PubMed

Manfredini, Marco; Arginelli, Federica; Dunsby, Christopher; French, Paul; Talbot, Clifford; König, Karsten; Pellacani, Giovanni; Ponti, Giovanni; Seidenari, Stefania

2013-02-01

The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM). The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher's exact test and Spearman's rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores. MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPT-FLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPT-FLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM). According to our data, both methods are suitable to image BCC's features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view. © 2012 John Wiley & Sons A/S.

Single Photon Counting Performance and Noise Analysis of CMOS SPAD-Based Image Sensors.

PubMed

Dutton, Neale A W; Gyongy, Istvan; Parmesan, Luca; Henderson, Robert K

2016-07-20

SPAD-based solid state CMOS image sensors utilising analogue integrators have attained deep sub-electron read noise (DSERN) permitting single photon counting (SPC) imaging. A new method is proposed to determine the read noise in DSERN image sensors by evaluating the peak separation and width (PSW) of single photon peaks in a photon counting histogram (PCH). The technique is used to identify and analyse cumulative noise in analogue integrating SPC SPAD-based pixels. The DSERN of our SPAD image sensor is exploited to confirm recent multi-photon threshold quanta image sensor (QIS) theory. Finally, various single and multiple photon spatio-temporal oversampling techniques are reviewed.

Wavefront correction in two-photon microscopy with a multi-actuator adaptive lens.

PubMed

Bueno, Juan M; Skorsetz, Martin; Bonora, Stefano; Artal, Pablo

2018-05-28

A multi-actuator adaptive lens (AL) was incorporated into a multi-photon (MP) microscope to improve the quality of images of thick samples. Through a hill-climbing procedure the AL corrected for the specimen-induced aberrations enhancing MP images. The final images hardly differed when two different metrics were used, although the sets of Zernike coefficients were not identical. The optimized MP images acquired with the AL were also compared with those obtained with a liquid-crystal-on-silicon spatial light modulator. Results have shown that both devices lead to similar images, which corroborates the usefulness of this AL for MP imaging.

Laser scanning saturated structured illumination microscopy based on phase modulation

NASA Astrophysics Data System (ADS)

Huang, Yujia; Zhu, Dazhao; Jin, Luhong; Kuang, Cuifang; Xu, Yingke; Liu, Xu

2017-08-01

Wide-field saturated structured illumination microscopy has not been widely used due to the requirement of high laser power. We propose a novel method called laser scanning saturated structured illumination microscopy (LS-SSIM), which introduces high order of harmonics frequency and greatly reduces the required laser power for SSIM imaging. To accomplish that, an excitation PSF with two peaks is generated and scanned along different directions on the sample. Raw images are recorded cumulatively by a CCD detector and then reconstructed to form a high-resolution image with extended optical transfer function (OTF). Our theoretical analysis and simulation results show that LS-SSIM method reaches a resolution of 0.16 λ, equivalent to 2.7-fold resolution than conventional wide-field microscopy. In addition, LS-SSIM greatly improves the optical sectioning capability of conventional wide-field illumination system by diminishing our-of-focus light. Furthermore, this modality has the advantage of implementation in multi-photon microscopy with point scanning excitation to image samples in greater depths.

Delivery of cyclodextrin polymers to bacterial biofilms - An exploratory study using rhodamine labelled cyclodextrins and multiphoton microscopy.

PubMed

Thomsen, Hanna; Benkovics, Gábor; Fenyvesi, Éva; Farewell, Anne; Malanga, Milo; Ericson, Marica B

2017-10-15

Cyclodextrin (CD) polymers are interesting nanoparticulate systems for pharmaceutical delivery; however, knowledge regarding their applications towards delivery into complex microbial biofilm structures is so far limited. The challenge is to demonstrate penetration and transport through the biofilm and its exopolysaccharide matrix. The ideal functionalization for penetration into mature biofilms is unexplored. In this paper, we present a novel set of rhodamine labelled βCD-polymers, with different charge moieties, i.e., neutral, anionic, and cationic, and explore their potential delivery into mature Staphylococcus epidermidis biofilms using multiphoton laser scanning microscopy (MPM). The S. epidermidis biofilms, being a medically relevant model organism, were stained with SYTO9. By using MPM, three-dimensional imaging and spectral investigation of the distribution of the βCD-polymers could be obtained. It was found that the cationic βCD-polymers showed significantly higher integration into the biofilms, compared to neutral and anionic functionalized βCDs. None of the carriers presented any inherent toxicity to the biofilms, meaning that the addition of rhodamine moiety does not affect the inertness of the delivery system. Taken together, this study demonstrates a novel approach by which delivery of fluorescently labelled CD nanoparticles to bacterial biofilms can be explored using MPM. Future studies should be undertaken investigating the potential in using cationic functionalization of CD based delivery systems for targeting anti-microbial effects in biofilms. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Monitoring the effect of mechanical stress on mesenchymal stem cell collagen production by multiphoton microscopy

NASA Astrophysics Data System (ADS)

Chen, Wei-Liang; Chang, Chia-Cheng; Chiou, Ling-Ling; Li, Tsung-Hsien; Liu, Yuan; Lee, Hsuan-Shu; Dong, Chen-Yuan

2008-02-01

Tissue engineering is emerging as a promising method for repairing damaged tissues. Due to cartilage's common wear and injury, in vitro production of cartilage replacements have been an active area of research. Finding the optimal condition for the generation of the collagen matrix is crucial in reproducing cartilages that closely match those found in human. Using multiphoton autofluorescence and second-harmonic generation (SHG) microscopy we monitored the effect of mechanical stress on mesenchymal stem cell collagen production. Bone marrow mesenchymal stem cells in the form of pellets were cultured and periodically placed under different mechanical stress by centrifugation over a period of four weeks. The differently stressed samples were imaged several times during the four week period, and the collagen production under different mechanical stress is characterized.

In vivo three-dimensional optical coherence tomography and multiphoton microscopy in a mouse model of ovarian neoplasia

NASA Astrophysics Data System (ADS)

Watson, Jennifer M.; Marion, Samuel L.; Rice, Photini Faith; Bentley, David L.; Besselsen, David; Utzinger, Urs; Hoyer, Patricia B.; Barton, Jennifer K.

2013-03-01

Our goal is to use optical coherence tomography (OCT) and multiphoton microscopy (MPM) to detect early tumor development in a mouse model of ovarian neoplasia. We hope to use information regarding early tumor development to create a diagnostic test for high-risk patients. In this study we collect in vivo images using OCT, second harmonic generation and two-photon excited fluorescence from non-vinylcyclohexene diepoxide (VCD)-dosed and VCD-dosed mice. VCD causes follicular apoptosis (simulating menopause) and leads to tumor development. Using OCT and MPM we visualized the ovarian microstructure and were able to see differences between non-VCD-dosed and VCD-dosed animals. This leads us to believe that OCT and MPM may be useful for detecting changes due to early tumor development.

Evolution of the three-dimensional collagen structure in vascular walls during deformation: an in situ mechanical testing under multiphoton microscopy observation.

PubMed

Nierenberger, Mathieu; Fargier, Guillaume; Ahzi, Saïd; Rémond, Yves

2015-08-01

The collagen fibers' three-dimensional architecture has a strong influence on the mechanical behavior of biological tissues. To accurately model this behavior, it is necessary to get some knowledge about the structure of the collagen network. In the present paper, we focus on the in situ characterization of the collagenous structure, which is present in porcine jugular vein walls. An observation of the vessel wall is first proposed in an unloaded configuration. The vein is then put into a mechanical tensile testing device. As the vein is stretched, three-dimensional images of its collagenous structure are acquired using multiphoton microscopy. Orientation analyses are provided for the multiple images recorded during the mechanical test. From these analyses, the reorientation of the two families of collagen fibers existing in the vein wall is quantified. We noticed that the reorientation of the fibers stops as the tissue stiffness starts decreasing, corresponding to the onset of damage. Besides, no relevant evolutions of the out of plane collagen orientations were observed. Due to the applied loading, our analysis also allowed for linking the stress relaxation within the tissue to its internal collagenous structure. Finally, this analysis constitutes the first mechanical test performed under a multiphoton microscope with a continuous three-dimensional observation of the tissue structure all along the test. It allows for a quantitative evaluation of microstructural parameters combined with a measure of the global mechanical behavior. Such data are useful for the development of structural mechanical models for living tissues.

Non-invasive in vivo characterization of skin wound healing using label-free multiphoton microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Jones, Jake D.; Majid, Fariah; Ramser, Hallie; Quinn, Kyle P.

2017-02-01

Non-healing ulcerative wounds, such as diabetic foot ulcers, are challenging to diagnose and treat due to their numerous possible etiologies and the variable efficacy of advanced wound care products. Thus, there is a critical need to develop new quantitative biomarkers and diagnostic technologies that are sensitive to wound status in order to guide care. The objective of this study was to evaluate the utility of label-free multiphoton microscopy for characterizing wound healing dynamics in vivo and identifying potential differences in diabetic wounds. We isolated and measured an optical redox ratio of FAD/(NADH+FAD) autofluorescence to provide three-dimensional maps of local cellular metabolism. Using a mouse model of wound healing, in vivo imaging at the wound edge identified a significant decrease in the optical redox ratio of the epidermis (p≤0.0103) between Days 3 through 14 compared to Day 1. This decrease in redox ratio coincided with a decrease in NADH fluorescence lifetime and thickening of the epithelium, collectively suggesting a sensitivity to keratinocyte hyperproliferation. In contrast to normal wounds, we have found that keratinocytes from diabetic wounds remain in a proliferative state at later time points with a lower redox ratio at the wound edge. Microstructural organization and composition was also measured from second harmonic generation imaging of collagen and revealed differences between diabetic and non-diabetic wounds. Our work demonstrates label-free multiphoton microscopy offers potential to provide non-invasive structural and functional biomarkers associated with different stages of skin wound healing, which may be used to detect delayed healing and guide treatment.

Novel microfabrication stage allowing for one-photon and multi-photon light assisted molecular immobilization and for multi-photon microscope

NASA Astrophysics Data System (ADS)

Gonçalves, Odete; Snider, Scott; Zadoyan, Ruben; Nguyen, Quoc-Thang; Vorum, Henrik; Petersen, Steffen B.; Neves-Petersen, Maria Teresa

2017-02-01

Light Assisted Molecular Immobilization (LAMI) results in spatially oriented and localized covalent coupling of biomolecules onto thiol reactive surfaces. LAMI is possible due to the conserved spatial proximity between aromatic residues and disulfide bridges in proteins. When aromatic residues are excited with UV light (275-295nm), disulphide bridges are disrupted and the formed thiol groups covalently bind to surfaces. Immobilization hereby reported is achieved in a microfabrication stage coupled to a fs-laser, through one- or multi-photon excitation. The fundamental 840nm output is tripled to 280nm and focused onto the sample, leading to one-photon excitation and molecular immobilization. The sample rests on a xyz-stage with micrometer step resolution and is illuminated according to a pattern uploaded to the software controlling the stage and the shutter. Molecules are immobilized according to such pattern, with micrometer spatial resolution. Spatial masks inserted in the light path lead to light diffraction patterns used to immobilize biomolecules with submicrometer spatial resolution. Light diffraction patterns are imaged by an inbuilt microscope. Two-photon microscopy and imaging of the fluorescent microbeads is shown. Immobilization of proteins, e.g. C-reactive protein, and of an engineered molecular beacon has been successfully achieved. The beacon was coupled to a peptide containing a disulfide bridge neighboring a tryptophan residue, being this way possible to immobilize the beacon on a surface using one-photon LAMI. This technology is being implemented in the creation of point-of-care biosensors aiming at the detection of cancer and cardiovascular disease markers.

Geiger-mode avalanche photodiode focal plane arrays for three-dimensional imaging LADAR

NASA Astrophysics Data System (ADS)

Itzler, Mark A.; Entwistle, Mark; Owens, Mark; Patel, Ketan; Jiang, Xudong; Slomkowski, Krystyna; Rangwala, Sabbir; Zalud, Peter F.; Senko, Tom; Tower, John; Ferraro, Joseph

2010-09-01

We report on the development of focal plane arrays (FPAs) employing two-dimensional arrays of InGaAsP-based Geiger-mode avalanche photodiodes (GmAPDs). These FPAs incorporate InP/InGaAs(P) Geiger-mode avalanche photodiodes (GmAPDs) to create pixels that detect single photons at shortwave infrared wavelengths with high efficiency and low dark count rates. GmAPD arrays are hybridized to CMOS read-out integrated circuits (ROICs) that enable independent laser radar (LADAR) time-of-flight measurements for each pixel, providing three-dimensional image data at frame rates approaching 200 kHz. Microlens arrays are used to maintain high fill factor of greater than 70%. We present full-array performance maps for two different types of sensors optimized for operation at 1.06 μm and 1.55 μm, respectively. For the 1.06 μm FPAs, overall photon detection efficiency of >40% is achieved at <20 kHz dark count rates with modest cooling to ~250 K using integrated thermoelectric coolers. We also describe the first evalution of these FPAs when multi-photon pulses are incident on single pixels. The effective detection efficiency for multi-photon pulses shows excellent agreement with predictions based on Poisson statistics. We also characterize the crosstalk as a function of pulse mean photon number. Relative to the intrinsic crosstalk contribution from hot carrier luminescence that occurs during avalanche current flows resulting from single incident photons, we find a modest rise in crosstalk for multi-photon incident pulses that can be accurately explained by direct optical scattering.

From morphology to clinical pathophysiology: multiphoton fluorescence lifetime imaging at patients' bedside

NASA Astrophysics Data System (ADS)

Mess, Christian; Zens, Katharina; Gorzelanny, Christian; Metze, Dieter; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.; Huck, Volker

2017-02-01

Application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of skin diseases. By means of multiphoton excitation, endogenous biomolecules like NADH, collagen or elastin show autofluorescence or second harmonic generation. Thus, these molecules provide information about the subcellular morphology, epidermal architecture and physiological condition of the skin. To gain a deeper understanding of the linkage between cellular structure and physiological processes, non-invasive multiphotonbased intravital tomography (MPT) and fluorescence lifetime imaging (FLIM) were combined within the scopes of inflammatory skin, chronic wounds and drug delivery in clinical application. The optical biopsies generated via MPT were morphologically analyzed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Independent morphometric algorithms reliably showed a perinuclear accumulation in lesional skin in contrast to an even distribution in healthy skin. Confirmatively, MPT-FLIM showed an obvious metabolic shift in lesions. Moreover, detection of the onset and progression of inflammatory processes could be achieved. The feasibility of primary in vivo tracking of applied therapeutic agents further broadened our scope: We examined the permeation and subsequent distribution of agents directly visualized in patientś skin in short-term repetitive measurements. Furthermore, we performed MPT-FLIM follow-up investigations in the long-term course of therapy. Therefore, clinical MPT-FLIM application offers new insights into the pathophysiology and the individual therapeutic course of skin diseases, facilitating a better understanding of the processes of inflammation and wound healing.

A novel model for ectopic, chronic, intravital multiphoton imaging of bone marrow vasculature and architecture in split femurs

PubMed Central

Bălan, Mirela; Kiefer, Friedemann

2015-01-01

Creating a model for intravital visualization of femoral bone marrow, a major site of hematopoiesis in adult mammalian organisms, poses a serious challenge, in that it needs to overcome bone opacity and the inaccessibility of marrow. Furthermore, meaningful analysis of bone marrow developmental and differentiation processes requires the repetitive observation of the same site over long periods of time, which we refer to as chronic imaging. To surmount these issues, we developed a chronic intravital imaging model that allows the observation of split femurs, ectopically transplanted into a dorsal skinfold chamber of a host mouse. Repeated, long term observations are facilitated by multiphoton microscopy, an imaging technique that combines superior imaging capacity at greater tissue depth with low phototoxicity. The transplanted, ectopic femur was stabilized by its sterile environment and rapidly connected to the host vasculature, allowing further development and observation of extended processes. After optimizing transplant age and grafting procedure, we observed the development of new woven bone and maturation of secondary ossification centers in the transplanted femurs, preceded by the sprouting of a sinusoidal-like vascular network, which was almost entirely composed of femoral endothelial cells. After two weeks, the transplant was still populated with stromal and haematopoietic cells belonging both to donor and host. Over this time frame, the transplant partially retained myeloid progenitor cells with single and multi-lineage differentiation capacity. In summary, our model allowed repeated intravital imaging of bone marrow angiogenesis and hematopoiesis. It represents a promising starting point for the development of improved chronic optical imaging models for femoral bone marrow. PMID:28243515

In vivo multimodal nonlinear optical imaging of mucosal tissue

NASA Astrophysics Data System (ADS)

Sun, Ju; Shilagard, Tuya; Bell, Brent; Motamedi, Massoud; Vargas, Gracie

2004-05-01

We present a multimodal nonlinear imaging approach to elucidate microstructures and spectroscopic features of oral mucosa and submucosa in vivo. The hamster buccal pouch was imaged using 3-D high resolution multiphoton and second harmonic generation microscopy. The multimodal imaging approach enables colocalization and differentiation of prominent known spectroscopic and structural features such as keratin, epithelial cells, and submucosal collagen at various depths in tissue. Visualization of cellular morphology and epithelial thickness are in excellent agreement with histological observations. These results suggest that multimodal nonlinear optical microscopy can be an effective tool for studying the physiology and pathology of mucosal tissue.

Multiphoton Coherent Manipulation in Large-Spin Qubits

NASA Astrophysics Data System (ADS)

Bertaina, S.; Chen, L.; Groll, N.; van Tol, J.; Dalal, N. S.; Chiorescu, I.

2009-02-01

Large-spin Mn2+ ions (S=5/2) diluted in a nonmagnetic MgO matrix of high crystalline symmetry are used to realize a six-level system that can be operated by means of multiphoton coherent Rabi oscillations. This spin system has a very small anisotropy which can be tuned in situ to reversibly transform the system between harmonic and nonharmonic level configurations. Decoherence effects are strongly suppressed as a result of the quasi-isotropic electron interaction with the crystal field and with the Mn55 nuclear spins. These results suggest new ways of manipulating, reading, and resetting spin quantum states which can be applied to encode a qubit across several quantum levels.

The 700-1500 cm{sup −1} region of the S{sub 1} (A{sup ~1}B{sub 2}) state of toluene studied with resonance-enhanced multiphoton ionization (REMPI), zero-kinetic-energy (ZEKE) spectroscopy, and time-resolved slow-electron velocity-map imaging (tr-SEVI) spectroscopy

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

Gardner, Adrian M.; Green, Alistair M.; Tamé-Reyes, Victor M.

We report (nanosecond) resonance-enhanced multiphoton ionization (REMPI), (nanosecond) zero-kinetic-energy (ZEKE) and (picosecond) time-resolved slow-electron velocity map imaging (tr-SEVI) spectra of fully hydrogenated toluene (Tol-h{sub 8}) and the deuterated-methyl group isotopologue (α{sub 3}-Tol-d{sub 3}). Vibrational assignments are made making use of the activity observed in the ZEKE and tr-SEVI spectra, together with the results from quantum chemical and previous experimental results. Here, we examine the 700–1500 cm{sup −1} region of the REMPI spectrum, extending our previous work on the region ≤700 cm{sup −1}. We provide assignments for the majority of the S{sub 1} and cation bands observed, and in particular wemore » gain insight regarding a number of regions where vibrations are coupled via Fermi resonance. We also gain insight into intramolecular vibrational redistribution in this molecule.« less

Identification of intramural metastasis in esophageal cancer using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Xu, Jian; Kang, Deyong; Zhuo, Shuangmu; Zhu, Xiaoqin; Lin, jiangbo; Chen, Jianxin

2017-02-01

Intramural metastasis (IM) of esophageal cancer is defined as metastasis from a primary lesion to the esophageal wall without intraepithelial cancer extension. Esophageal cancer with IM is more common and such cases indicate a poor prognosis. In esophageal surgery, if curative resection is possible, the complete removal of both primary tumor and associated IMs is required. Therefore, accurate diagnosis of IMs in esophageal cancer prior to surgery is of particular importance. Multiphoton microscopy (MPM) with subcellular resolution is well-suited for deep tissue imaging since many endogenous fluorophores of fresh biological tissues are excited through two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). Here, a study to identify IM in fresh tissue section using MPM is reported. In this study, the morphological and spectral differences between IM and surrounding tissue are described. These results show that MPM has the ability to accurately identify IM in esophageal tissues. With improvement of the penetration depth of MPM and the development of multiphton microendoscope, MPM may be a promising imaging technique for preoperative diagnosis of IMs in esophageal cancer in the future.

Ex vivo applications of multiphoton microscopy in urology

NASA Astrophysics Data System (ADS)

Jain, Manu; Mukherjee, Sushmita

2016-03-01

Background: Routine urological surgery frequently requires rapid on-site histopathological tissue evaluation either during biopsy or intra-operative procedure. However, resected tissue needs to undergo processing, which is not only time consuming but may also create artifacts hindering real-time tissue assessment. Likewise, pathologist often relies on several ancillary methods, in addition to H&E to arrive at a definitive diagnosis. Although, helpful these techniques are tedious and time consuming and often show overlapping results. Therefore, there is a need for an imaging tool that can rapidly assess tissue in real-time at cellular level. Multiphoton microscopy (MPM) is one such technique that can generate histology-quality images from fresh and fixed tissue solely based on their intrinsic autofluorescence emission, without the need for tissue processing or staining. Design: Fresh tissue sections (neoplastic and non-neoplastic) from biopsy and surgical specimens of bladder and kidney were obtained. Unstained deparaffinized slides from biopsy of medical kidney disease and oncocytic renal neoplasms were also obtained. MPM images were acquired using with an Olympus FluoView FV1000MPE system. After imaging, fresh tissues were submitted for routine histopathology. Results: Based on the architectural and cellular details of the tissue, MPM could characterize normal components of bladder and kidney. Neoplastic tissue could be differentiated from non-neoplastic tissue and could be further classified as per histopathological convention. Some of the tumors had unique MPM signatures not otherwise seen on H&E sections. Various subtypes of glomerular lesions were identified as well as renal oncocytic neoplasms were differentiated on unstained deparaffinized slides. Conclusions: We envision MPM to become an integral part of regular diagnostic workflow for rapid assessment of tissue. MPM can be used to evaluate the adequacy of biopsies and triage tissues for ancillary studies. It can also be used as an adjunct to frozen section analysis for intra-operative margin assessment. Further, it can play an important role for pathologist for guiding specimen grossing, selecting tissue for tumor banking and as a rapid ancillary diagnostic tool.

Absorption characterization of immersion medium for multiphoton microscopy at the 1700nm window

NASA Astrophysics Data System (ADS)

Wen, Wenhui; Qiu, Ping

2017-02-01

Larger imaging depth is the quest of almost all the imaging modalities, including multiphoton microscopy (MPM). Recently, it has been domonstrated that excitation at the 1700-nm helps extending imaging depth in MPM, optical coherence tomography, as well as photoacoustic imaging compared with excitation at other wavelengths. In MPM, immersion objective lenses with high numerical aperture (NA) are typically used to achieve better signal resolution, higer signal collection efficiency, and stronger signal generation. Although physically short ( mm), this extra optical path length traversed by the excitation light inevitably introduces absorption of the excitation light, and as a result leads to a decrease in the signal generation. Here we demonstrate experimental characterization of absorption spectrum of various immersion media at the 1700-nm window, including water (H2O), deuterium oxide (D2O), and several brands of immersion oil. Our results identify either the best immersion medium for a specific wavelength, or the best wavelength for a specific immersion medium at the 1700-nm window. Furthermore, through quantitative MPM experiments comparing different immersion media, we show that the MPM signal levels can be enhanced by more than ten fold simply by selecting the proper immersion medium, in good agreement with theoretical expectation based on the absorption measurement. Our results will offer guidelines for signal optimization in MPM at the 1700-nm window.

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.

Collective Autoionization in Multiply-Excited Systems: A novel ionization process observed in Helium Nanodroplets

PubMed Central

LaForge, A. C.; Drabbels, M.; Brauer, N. B.; Coreno, M.; Devetta, M.; Di Fraia, M.; Finetti, P.; Grazioli, C.; Katzy, R.; Lyamayev, V.; Mazza, T.; Mudrich, M.; O'Keeffe, P.; Ovcharenko, Y.; Piseri, P.; Plekan, O.; Prince, K. C.; Richter, R.; Stranges, S.; Callegari, C.; Möller, T.; Stienkemeier, F.

2014-01-01

Free electron lasers (FELs) offer the unprecedented capability to study reaction dynamics and image the structure of complex systems. When multiple photons are absorbed in complex systems, a plasma-like state is formed where many atoms are ionized on a femtosecond timescale. If multiphoton absorption is resonantly-enhanced, the system becomes electronically-excited prior to plasma formation, with subsequent decay paths which have been scarcely investigated to date. Here, we show using helium nanodroplets as an example that these systems can decay by a new type of process, named collective autoionization. In addition, we show that this process is surprisingly efficient, leading to ion abundances much greater than that of direct single-photon ionization. This novel collective ionization process is expected to be important in many other complex systems, e.g. macromolecules and nanoparticles, exposed to high intensity radiation fields. PMID:24406316

Identification of non-neoplastic and neoplastic gastric polyps using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Jiang, Shanghai; Kang, Deyong; Xu, Meifang; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Jianxin

2012-12-01

Gastric polyps can be broadly defined as luminal lesions projecting above the plane of the mucosal surface. They are generally divided into non-neoplastic and neoplastic polyps. Accurate diagnosis of neoplastic polyps is important because of their well-known relationship with gastric cancer. Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) is one of the most important recent inventions in biological imaging. In this study, we used MPM to image the microstructure of gastric polyps, including fundic gland polyps, hyperplastic polyps, inflammatory fibroid polyps and adenomas, then compared with gold-standard hematoxylin- eosin(H-E)-stained histopathology. MPM images showed that different gastric polyps have different gland architecture and cell morphology. Dilated, elongated or branch-like hyperplastic polyps are arranged by columnar epithelial cells. Inflammatory fibroid polyps are composed of small, thin-walled blood vessels surrounded by short spindle cells. Fundic glands polyps are lined by parietal cells and chief cells, admixed with normal glands. Gastric adenomas are generally composed of tubules or villi of dysplastic epithelium, which usually show some degree of intestinal-type differentiation toward absorptive cells, goblet cells, endocrine cells. Our results demonstrated that MPM can be used to identify non- neoplastic and neoplastic gastric polyps without the need of any staining procedure.

A graph-theoretical representation of multiphoton resonance processes in superconducting quantum circuits

DOE PAGES

Jooya, Hossein Z.; Reihani, Kamran; Chu, Shih-I

2016-11-21

We propose a graph-theoretical formalism to study generic circuit quantum electrodynamics systems consisting of a two level qubit coupled with a single-mode resonator in arbitrary coupling strength regimes beyond rotating-wave approximation. We define colored-weighted graphs, and introduce different products between them to investigate the dynamics of superconducting qubits in transverse, longitudinal, and bidirectional coupling schemes. In conclusion, the intuitive and predictive picture provided by this method, and the simplicity of the mathematical construction, are demonstrated with some numerical studies of the multiphoton resonance processes and quantum interference phenomena for the superconducting qubit systems driven by intense ac fields.

Single Photon Counting Performance and Noise Analysis of CMOS SPAD-Based Image Sensors

PubMed Central

Dutton, Neale A. W.; Gyongy, Istvan; Parmesan, Luca; Henderson, Robert K.

2016-01-01

SPAD-based solid state CMOS image sensors utilising analogue integrators have attained deep sub-electron read noise (DSERN) permitting single photon counting (SPC) imaging. A new method is proposed to determine the read noise in DSERN image sensors by evaluating the peak separation and width (PSW) of single photon peaks in a photon counting histogram (PCH). The technique is used to identify and analyse cumulative noise in analogue integrating SPC SPAD-based pixels. The DSERN of our SPAD image sensor is exploited to confirm recent multi-photon threshold quanta image sensor (QIS) theory. Finally, various single and multiple photon spatio-temporal oversampling techniques are reviewed. PMID:27447643

Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device.

PubMed

Cheng, Li-Chung; Lien, Chi-Hsiang; Da Sie, Yong; Hu, Yvonne Yuling; Lin, Chun-Yu; Chien, Fan-Ching; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen

2014-08-01

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.

Flexible digital signal processing architecture for narrowband and spread-spectrum lock-in detection in multiphoton microscopy and time-resolved spectroscopy

PubMed Central

Wilson, Jesse W.; Park, Jong Kang; Warren, Warren S.

2015-01-01

The lock-in amplifier is a critical component in many different types of experiments, because of its ability to reduce spurious or environmental noise components by restricting detection to a single frequency and phase. One example application is pump-probe microscopy, a multiphoton technique that leverages excited-state dynamics for imaging contrast. With this application in mind, we present here the design and implementation of a high-speed lock-in amplifier on the field-programmable gate array (FPGA) coprocessor of a data acquisition board. The most important advantage is the inherent ability to filter signals based on more complex modulation patterns. As an example, we use the flexibility of the FPGA approach to enable a novel pump-probe detection scheme based on spread-spectrum communications techniques. PMID:25832238

Flexible digital signal processing architecture for narrowband and spread-spectrum lock-in detection in multiphoton microscopy and time-resolved spectroscopy.

PubMed

Wilson, Jesse W; Park, Jong Kang; Warren, Warren S; Fischer, Martin C

2015-03-01

The lock-in amplifier is a critical component in many different types of experiments, because of its ability to reduce spurious or environmental noise components by restricting detection to a single frequency and phase. One example application is pump-probe microscopy, a multiphoton technique that leverages excited-state dynamics for imaging contrast. With this application in mind, we present here the design and implementation of a high-speed lock-in amplifier on the field-programmable gate array (FPGA) coprocessor of a data acquisition board. The most important advantage is the inherent ability to filter signals based on more complex modulation patterns. As an example, we use the flexibility of the FPGA approach to enable a novel pump-probe detection scheme based on spread-spectrum communications techniques.

Ultrathin endoscopes based on multicore fibers and adaptive optics: a status review and perspectives.

PubMed

Andresen, Esben Ravn; Sivankutty, Siddharth; Tsvirkun, Viktor; Bouwmans, Géraud; Rigneault, Hervé

2016-12-01

We take stock of the progress that has been made into developing ultrathin endoscopes assisted by wave front shaping. We focus our review on multicore fiber-based lensless endoscopes intended for multiphoton imaging applications. We put the work into perspective by comparing with alternative approaches and by outlining the challenges that lie ahead.

Cellular organization and spectral diversity of GFP-like proteins in live coral cells studied by single and multiphoton imaging and microspectroscopy

NASA Astrophysics Data System (ADS)

Salih, Anya; Cox, Guy C.; Larkum, Anthony W.

2003-07-01

Tissues of many marine invertebrates of class Anthozoa contain intensely fluorescent or brightly coloured pigments. These pigments belong to a family of photoactive proteins closely related to Green Fluorescent Protein (GFP), and their emissions range from blue to red wavelengths. The great diversity of these pigments has only recently been realised. To investigate the role of these proteins in corals, we have performed an in vivo fluorescent pigment (FP) spectral and cellular distribution analyses in live coral cells using single and multi-photon laser scanning imaging and microspectroscopy. These analyses revealed that even single colour corals contain spectroscopically heterogeneous pigment mixtures, with 2-5 major colour types in the same area of tissue. They were typically arranged in step-wise light emission energy gradients (e.g. blue, green, yellow, red). The successive overlapping emission-excitation spectral profiles of differently coloured FPs suggested that they were suited for sequential energy coupling. Traces of red FPs (emission = 570-660 nm) were present, even in non-red corals. We confirmed that radiative energy transfer could occur between separate granules of blue and green FPs and that energy transfer was inversely proportional to the square of the distance between them. Multi-photon micro-spectrofluorometric analysis gave significantly improved spectral resolution by restricting FP excitation to a single point in the focal plane of the sample. Pigment heterogeneity at small scales within granules suggested that fluorescence resonance energy transfer (FRET) might be occurring, and we confirmed that this was the case. Thus, energy transfer can take place both radiatively and by FRET, probably functioning in photoprotection by dissipation of excessive solar radiation.

A method of extracting speed-dependent vector correlations from 2 + 1 REMPI ion images.

PubMed

Wei, Wei; Wallace, Colin J; Grubb, Michael P; North, Simon W

2017-07-07

We present analytical expressions for extracting Dixon's bipolar moments in the semi-classical limit from experimental anisotropy parameters of sliced or reconstructed non-sliced images. The current method focuses on images generated by 2 + 1 REMPI (Resonance Enhanced Multi-photon Ionization) and is a necessary extension of our previously published 1 + 1 REMPI equations. Two approaches for applying the new equations, direct inversion and forward convolution, are presented. As demonstration of the new method, bipolar moments were extracted from images of carbonyl sulfide (OCS) photodissociation at 230 nm and NO 2 photodissociation at 355 nm, and the results are consistent with previous publications.

Potential of ultraviolet wide-field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes.

PubMed

Wüstner, Daniel; Brewer, Jonathan R; Bagatolli, Luis; Sage, Daniel

2011-01-01

Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP) excitation microscopy to monitor DHE in living cells. Significantly reduced photobleaching in MP microscopy of DHE enabled us to acquire three-dimensional z-stacks of DHE-stained cells and to obtain high-resolution maps of DHE in surface ruffles, nanotubes, and the apical membrane of epithelial cells. We found that the lateral resolution of MP microscopy is ∼1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 μm persisting over several minutes could be resolved by MP time-lapse microscopy. Diffusion coefficients of 0.25-μm-diameter endocytic vesicles containing DHE were determined by MP spatiotemporal image correlation spectroscopy. The requirement of extremely high laser power for visualization of DHE by MP microscopy made this method less potent for multicolor applications with organelle markers like green fluorescent protein-tagged proteins. The signal-to-noise ratio obtainable by UV-WF imaging could be significantly improved by pixelwise bleach rate fitting and calculation of an amplitude image from the decay model and by frame averaging after pixelwise bleaching correction of the image stacks. We conclude that UV-WF imaging and MP microscopy of DHE provide complementary information regarding membrane distribution and intracellular targeting of sterols. © 2010 Wiley-Liss, Inc.

System design for 3D wound imaging using low-cost mobile devices

NASA Astrophysics Data System (ADS)

Sirazitdinova, Ekaterina; Deserno, Thomas M.

2017-03-01

The state-of-the art method of wound assessment is a manual, imprecise and time-consuming procedure. Per- formed by clinicians, it has limited reproducibility and accuracy, large time consumption and high costs. Novel technologies such as laser scanning microscopy, multi-photon microscopy, optical coherence tomography and hyper-spectral imaging, as well as devices relying on the structured light sensors, make accurate wound assessment possible. However, such methods have limitations due to high costs and may lack portability and availability. In this paper, we present a low-cost wound assessment system and architecture for fast and accurate cutaneous wound assessment using inexpensive consumer smartphone devices. Computer vision techniques are applied either on the device or the server to reconstruct wounds in 3D as dense models, which are generated from images taken with a built-in single camera of a smartphone device. The system architecture includes imaging (smartphone), processing (smartphone or PACS) and storage (PACS) devices. It supports tracking over time by alignment of 3D models, color correction using a reference color card placed into the scene and automatic segmentation of wound regions. Using our system, we are able to detect and document quantitative characteristics of chronic wounds, including size, depth, volume, rate of healing, as well as qualitative characteristics as color, presence of necrosis and type of involved tissue.

Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components.

PubMed

Vogel, Martin; Wingert, Axel; Fink, Rainer H A; Hagl, Christian; Ganikhanov, Feruz; Pfeffer, Christian P

2015-10-01

Using an optical system made from fused silica catalogue optical components, third-order nonlinear microscopy has been enabled on conventional Ti:sapphire laser-based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of ∼300 nm and in multimodal nonlinear optical imaging experiments using third-order sum frequency generation and coherent anti-Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from ∼300 nm to ∼660 nm. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Time-Correlated Single-Photon Counting Fluorescence Imaging of Lipid Domains In Raft-Mimicking Giant Unilamellar Vesicles

NASA Astrophysics Data System (ADS)

Clarke, James; Cheng, Kwan; Shindell, Orrin; Wang, Exing

We have designed and constructed a high-throughput electrofusion chamber and an incubator to fabricate Giant Unilamellar Vesicles (GUVs) consisting of high-melting lipids, low-melting lipids, cholesterol and both ordered and disordered phase sensitive fluorescent probes (DiIC12, dehydroergosterol and BODIPY-Cholesterol). GUVs were formed in a 3 stage pulse sequence electrofusion process with voltages ranging from 50mVpp to 2.2Vpp and frequencies from 5Hz to 10Hz. Steady state and time-correlated single-photon counting (TCSPC) fluorescence lifetime (FLIM) based confocal and/or multi-photon microscopic techniques were used to characterize phase separated lipid domains in GUVs. Confocal imaging measures the probe concentration and the chemical environment of the system. TCSPC techniques determine the chemical environment through the perturbation of fluorescent lifetimes of the probes in the system. The above techniques will be applied to investigate the protein-lipid interactions involving domain formation. Specifically, the mechanisms governing lipid domain formations in the above systems that mimic the lipid rafts in cells will be explored. Murchison Fellowship at Trinity University.

Multimodal fiber source for nonlinear microscopy based on a dissipative soliton laser

PubMed Central

Lamb, Erin S.; Wise, Frank W.

2015-01-01

Recent developments in high energy femtosecond fiber lasers have enabled robust and lower-cost sources for multiphoton-fluorescence and harmonic-generation imaging. However, picosecond pulses are better suited for Raman scattering microscopy, so the ideal multimodal source for nonlinear microcopy needs to provide both durations. Here we present spectral compression of a high-power femtosecond fiber laser as a route to producing transform-limited picosecond pulses. These pulses pump a fiber optical parametric oscillator to yield a robust fiber source capable of providing the synchronized picosecond pulse trains needed for Raman scattering microscopy. Thus, this system can be used as a multimodal platform for nonlinear microscopy techniques. PMID:26417497

Multiphoton Microscopy of Prostate and Periprostatic Neural Tissue: A Promising Imaging Technique for Improving Nerve-Sparing Prostatectomy

PubMed Central

Yadav, Rajiv; Mukherjee, Sushmita; Hermen, Michael; Tan, Gerald; Maxfield, Frederick R.; Webb, Watt W.

2009-01-01

Abstract Background and Purpose Various imaging modalities are under investigation for real-time tissue imaging of periprostatic nerves with the idea of improving the results of nerve-sparing radical prostatectomy. We explored multiphoton microscopy (MPM) for real-time tissue imaging of the prostate and periprostatic neural tissue in a male Sprague-Dawley rat model. The unique advantage of this technique is the acquisition of high-resolution images without necessitating any extrinsic labeling agent and with minimal phototoxic effect on tissue. Materials and Methods The prostate and cavernous nerves were surgically excised from male Sprague-Dawley rats. The imaging was carried out using intrinsic fluorescence and scattering properties of the tissues without any exogenous dye or contrast agent. A custom-built MPM, consisting of an Olympus BX61WI upright frame and a modified MRC 1024 scanhead, was used. A femtosecond pulsed titanium/sapphire laser at 780-nm wavelength was used to excite the tissue; laser power under the objective was modulated via a Pockels cell. Second harmonic generation (SHG) signals were collected at 390 (±35 nm), and broadband autofluorescence was collected at 380 to 530 nm. The images obtained from SHG and from tissue fluorescence were then merged and color coded during postprocessing for better appreciation of details. The corresponding tissues were subjected to hematoxylin and eosin staining for histologic confirmation of the structures. Results High-resolution images of the prostate capsule, underlying acini, and individual cells outlining the glands were obtained at varying magnifications. MPM images of adipose tissue and the neural tissues were also obtained. Histologic confirmation and correlation of the prostate gland, fat, cavernous nerve, and major pelvic ganglion validated the findings of MPM. Conclusion Real-time imaging and microscopic resolution of prostate and periprostatic neural tissue using MPM is feasible without the need for any extrinsic labeling agents. Integration of this imaging modality with operative technique has the potential to improve the precision of nerve-sparing prostatectomy. PMID:19425823

Preparation of wholemount mouse intestine for high-resolution three-dimensional imaging using two-photon microscopy.

PubMed

Appleton, P L; Quyn, A J; Swift, S; Näthke, I

2009-05-01

Visualizing overall tissue architecture in three dimensions is fundamental for validating and integrating biochemical, cell biological and visual data from less complex systems such as cultured cells. Here, we describe a method to generate high-resolution three-dimensional image data of intact mouse gut tissue. Regions of highest interest lie between 50 and 200 mum within this tissue. The quality and usefulness of three-dimensional image data of tissue with such depth is limited owing to problems associated with scattered light, photobleaching and spherical aberration. Furthermore, the highest-quality oil-immersion lenses are designed to work at a maximum distance of

High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope.

PubMed

Li, Yongxiao; Montague, Samantha J; Brüstle, Anne; He, Xuefei; Gillespie, Cathy; Gaus, Katharina; Gardiner, Elizabeth E; Lee, Woei Ming

2018-02-28

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Compact Multiphoton 3D Imaging System for Recording Fast Neuronal Activity

PubMed Central

Vučinić, Dejan; Sejnowski, Terrence J.

2007-01-01

We constructed a simple and compact imaging system designed specifically for the recording of fast neuronal activity in a 3D volume. The system uses an Yb:KYW femtosecond laser we designed for use with acousto-optic deflection. An integrated two-axis acousto-optic deflector, driven by digitally synthesized signals, can target locations in three dimensions. Data acquisition and the control of scanning are performed by a LeCroy digital oscilloscope. The total cost of construction was one order of magnitude lower than that of a typical Ti:sapphire system. The entire imaging apparatus, including the laser, fits comfortably onto a small rig for electrophysiology. Despite the low cost and simplicity, the convergence of several new technologies allowed us to achieve the following capabilities: i) full-frame acquisition at video rates suitable for patch clamping; ii) random access in under ten microseconds with dwelling ability in the nominal focal plane; iii) three-dimensional random access with the ability to perform fast volume sweeps at kilohertz rates; and iv) fluorescence lifetime imaging. We demonstrate the ability to record action potentials with high temporal resolution using intracellularly loaded potentiometric dye di-2-ANEPEQ. Our design proffers easy integration with electrophysiology and promises a more widespread adoption of functional two-photon imaging as a tool for the study of neuronal activity. The software and firmware we developed is available for download at http://neurospy.org/ under an open source license. PMID:17684546

Multimodal optical workstation for simultaneous linear, nonlinear microscopy and nanomanipulation: upgrading a commercial confocal inverted microscope.

PubMed

Mathew, Manoj; Santos, Susana I C O; Zalvidea, Dobryna; Loza-Alvarez, Pablo

2009-07-01

In this work we propose and build a multimodal optical workstation that extends a commercially available confocal microscope (Nikon Confocal C1-Si) to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools such as nanosurgery. The setup allows both subsystems (confocal and nonlinear) to work independently and simultaneously. The workstation enables, for instance, nanosurgery along with simultaneous confocal and brightfield imaging. The nonlinear microscopy capabilities are added around the commercial confocal microscope by exploiting all the flexibility offered by this microscope and without need for any mechanical or electronic modification of the confocal microscope systems. As an example, the standard differential interference contrast condenser and diascopic detector in the confocal microscope are readily used as a forward detection mount for second harmonic generation imaging. The various capabilities of this workstation, as applied directly to biology, are demonstrated using the model organism Caenorhabditis elegans.

Intravital assessment of myelin molecular order with polarimetric multiphoton microscopy

NASA Astrophysics Data System (ADS)

Turcotte, Raphaël; Rutledge, Danette J.; Bélanger, Erik; Dill, Dorothy; Macklin, Wendy B.; Côté, Daniel C.

2016-08-01

Myelin plays an essential role in the nervous system and its disruption in diseases such as multiple sclerosis may lead to neuronal death, thus causing irreversible functional impairments. Understanding myelin biology is therefore of fundamental and clinical importance, but no tools currently exist to describe the fine spatial organization of myelin sheaths in vivo. Here we demonstrate intravital quantification of the myelin molecular structure using a microscopy method based on polarization-resolved coherent Raman scattering. Developmental myelination was imaged noninvasively in live zebrafish. Longitudinal imaging of individual axons revealed changes in myelin organization beyond the diffraction limit. Applied to promyelination drug screening, the method uniquely enabled the identification of focal myelin regions with differential architectures. These observations indicate that the study of myelin biology and the identification of therapeutic compounds will largely benefit from a method to quantify the myelin molecular organization in vivo.

Combining large area fluorescence with multiphoton microscopy for improved detection of oral epithelial neoplasia (Conference Presentation)

NASA Astrophysics Data System (ADS)

Pal, Rahul; Yang, Jinping; Qiu, Suimin; McCammon, Susan; Resto, Vicente; Vargas, Gracie

2016-03-01

Volumetric Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) show promise for revealing indicators of neoplasia representing the complex microstructural organization of mucosa, potentially providing high specificity for detection of neoplasia, but is limited by small imaging area. Large area fluorescence methods on the other hand show high sensitivity appropriate for screening but are hampered by low specificity. In this study, we apply MPAM-SHGM following guidance from large area fluorescence, by either autofluorescence or a targeted metabolic fluorophore, as a potentially clinically viable approach for detection of oral neoplasia. Sites of high neoplastic potentially were identified by large area red/green autofluorescence or by a fluorescently labelled deoxy-glucose analog, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) to highlight areas of high glucose uptake across the buccal pouch of a hamster model for OSCC. Follow-up MPAM-SHGM was conducted on regions of interests (ROIs) to assess whether microscopy would reveal microscopic features associated with neoplasia to confirm or exclude large area fluorescence findings. Parameters for analysis included cytologic metrics, 3D epithelial connective tissue interface metrics (MPAM-SHGM) and intensity of fluorescence (widefield). Imaged sites were biopsied and processed for histology and graded by a pathologist. A small sample of human ex vivo tissues were also imaged. A generalized linear model combining image metrics from large area fluorescence and volumetric MPAM-SHGM indicated the ability to delineate normal and inflammation from neoplasia.

Assessment of liver steatosis and fibrosis in rats using integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique

NASA Astrophysics Data System (ADS)

Lin, Jian; Lu, Fake; Zheng, Wei; Xu, Shuoyu; Tai, Dean; Yu, Hanry; Huang, Zhiwei

2011-11-01

We report the implementation of a unique integrated coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and two-photon excitation fluorescence (TPEF) microscopy imaging technique developed for label-free monitoring of the progression of liver steatosis and fibrosis generated in a bile duct ligation (BDL) rat model. Among the 21 adult rats used in this study, 18 rats were performed with BDL surgery and sacrificed each week from weeks 1 to 6 (n = 3 per week), respectively; whereas 3 rats as control were sacrificed at week 0. Colocalized imaging of the aggregated hepatic fats, collagen fibrils, and hepatocyte morphologies in liver tissue is realized by using the integrated CARS, SHG, and TPEF technique. The results show that there are significant accumulations of hepatic lipid droplets and collagen fibrils associated with severe hepatocyte necrosis in BDL rat liver as compared to a normal liver tissue. The volume of normal hepatocytes keeps decreasing and the fiber collagen content in BDL rat liver follows a growing trend until week 6; whereas the hepatic fat content reaches a maximum in week 4 and then appears to stop growing in week 6, indicating that liver steatosis and fibrosis induced in a BDL rat liver model may develop at different rates. This work demonstrates that the integrated CARS and multiphoton microscopy imaging technique has the potential to provide an effective means for early diagnosis and detection of liver steatosis and fibrosis without labeling.

Assessment of liver steatosis and fibrosis in rats using integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique.

PubMed

Lin, Jian; Lu, Fake; Zheng, Wei; Xu, Shuoyu; Tai, Dean; Yu, Hanry; Huang, Zhiwei

2011-11-01

We report the implementation of a unique integrated coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and two-photon excitation fluorescence (TPEF) microscopy imaging technique developed for label-free monitoring of the progression of liver steatosis and fibrosis generated in a bile duct ligation (BDL) rat model. Among the 21 adult rats used in this study, 18 rats were performed with BDL surgery and sacrificed each week from weeks 1 to 6 (n = 3 per week), respectively; whereas 3 rats as control were sacrificed at week 0. Colocalized imaging of the aggregated hepatic fats, collagen fibrils, and hepatocyte morphologies in liver tissue is realized by using the integrated CARS, SHG, and TPEF technique. The results show that there are significant accumulations of hepatic lipid droplets and collagen fibrils associated with severe hepatocyte necrosis in BDL rat liver as compared to a normal liver tissue. The volume of normal hepatocytes keeps decreasing and the fiber collagen content in BDL rat liver follows a growing trend until week 6; whereas the hepatic fat content reaches a maximum in week 4 and then appears to stop growing in week 6, indicating that liver steatosis and fibrosis induced in a BDL rat liver model may develop at different rates. This work demonstrates that the integrated CARS and multiphoton microscopy imaging technique has the potential to provide an effective means for early diagnosis and detection of liver steatosis and fibrosis without labeling.

ACQ4: an open-source software platform for data acquisition and analysis in neurophysiology research.

PubMed

Campagnola, Luke; Kratz, Megan B; Manis, Paul B

2014-01-01

The complexity of modern neurophysiology experiments requires specialized software to coordinate multiple acquisition devices and analyze the collected data. We have developed ACQ4, an open-source software platform for performing data acquisition and analysis in experimental neurophysiology. This software integrates the tasks of acquiring, managing, and analyzing experimental data. ACQ4 has been used primarily for standard patch-clamp electrophysiology, laser scanning photostimulation, multiphoton microscopy, intrinsic imaging, and calcium imaging. The system is highly modular, which facilitates the addition of new devices and functionality. The modules included with ACQ4 provide for rapid construction of acquisition protocols, live video display, and customizable analysis tools. Position-aware data collection allows automated construction of image mosaics and registration of images with 3-dimensional anatomical atlases. ACQ4 uses free and open-source tools including Python, NumPy/SciPy for numerical computation, PyQt for the user interface, and PyQtGraph for scientific graphics. Supported hardware includes cameras, patch clamp amplifiers, scanning mirrors, lasers, shutters, Pockels cells, motorized stages, and more. ACQ4 is available for download at http://www.acq4.org.

Advances in imaging the innate and adaptive immune response to Toxoplasma gondii

PubMed Central

John, Beena; Weninger, Wolfgang; Hunter, Christopher A

2011-01-01

Toxoplasma gondii is an intracellular protozoan parasite that infects a wide variety of warm-blooded hosts and can have devastating effects in the developing fetus as well as the immunocompromised host. An appreciation of how this organism interacts with the host immune system is crucial to understanding the pathogenesis of this disease. The last decade has been marked by the application of various imaging techniques, such as bioluminescent imaging as well as confocal and multiphoton microscopy to study toxoplasmosis. The ability to manipulate parasites to express fluorescent/bioluminescent markers or model antigens/enzymes combined with the development of reporter mice that allow the detection of distinct immune populations have been crucial to the success of many of these studies. These approaches have permitted the visualization of parasites and immune cells in real-time and provided new insights into the nature of host–pathogen interactions. This article highlights some of the advances in imaging techniques, their strengths and weaknesses, and how these techniques have impacted our understanding of the interaction between parasites and various immune populations during toxoplasmosis. PMID:20860479

Second harmonic generation microscopy of the living human cornea

NASA Astrophysics Data System (ADS)

Artal, Pablo; Ávila, Francisco; Bueno, Juan

2018-02-01

Second Harmonic Generation (SHG) microscopy provides high-resolution structural imaging of the corneal stroma without the need of labelling techniques. This powerful tool has never been applied to living human eyes so far. Here, we present a new compact SHG microscope specifically developed to image the structural organization of the corneal lamellae in living healthy human volunteers. The research prototype incorporates a long-working distance dry objective that allows non-contact three-dimensional SHG imaging of the cornea. Safety assessment and effectiveness of the system were firstly tested in ex-vivo fresh eyes. The maximum average power of the used illumination laser was 20 mW, more than 10 times below the maximum permissible exposure (according to ANSI Z136.1-2000). The instrument was successfully employed to obtain non-contact and non-invasive SHG of the living human eye within well-established light safety limits. This represents the first recording of in vivo SHG images of the human cornea using a compact multiphoton microscope. This might become an important tool in Ophthalmology for early diagnosis and tracking ocular pathologies.

Multiphoton imaging reveals that nanosecond pulsed electric fields collapse tumor and normal vascular perfusion in human glioblastoma xenografts.

PubMed

Bardet, Sylvia M; Carr, Lynn; Soueid, Malak; Arnaud-Cormos, Delia; Leveque, Philippe; O'Connor, Rodney P

2016-10-04

Despite the biomedical advances of the last century, many cancers including glioblastoma are still resistant to existing therapies leaving patients with poor prognoses. Nanosecond pulsed electric fields (nsPEF) are a promising technology for the treatment of cancer that have thus far been evaluated in vitro and in superficial malignancies. In this paper, we develop a tumor organoid model of glioblastoma and apply intravital multiphoton microscopy to assess their response to nsPEFs. We demonstrate for the first time that a single 10 ns, high voltage electric pulse (35-45 kV/cm), collapses the perfusion of neovasculature, and also alters the diameter of capillaries and larger vessels in normal tissue. These results contribute to the fundamental understanding of nsPEF effects in complex tissue environments, and confirm the potential of nsPEFs to disrupt the microenvironment of solid tumors such as glioblastoma.

Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device

PubMed Central

Cheng, Li-Chung; Lien, Chi-Hsiang; Da Sie, Yong; Hu, Yvonne Yuling; Lin, Chun-Yu; Chien, Fan-Ching; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen

2014-01-01

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished. PMID:25136483

Improvement of axial excitation confinement in temporal focusing-based multiphoton microscopy via spatially modulated illumination

NASA Astrophysics Data System (ADS)

Chang, Chia-Yuan; Chen, Shean-Jen

2017-02-01

Conventional temporal focusing-based multiphoton excitation microscopy (TFMPEM) can offer widefield optical sectioning with an axial excitation confinement (AEC) of a few microns. Herein, a developed TFMPEM with a digital micromirror device (DMD), acting as the blazed grating for light spatial dispersion and simultaneous patterned illumination, has been extended to implement spatially modulated illumination at structured frequency and orientation. By implementing the spatially modulated illumination, the beam coverage at the back-focal aperture of the objective lens can be increased. As a result, the AEC can be condensed from 3.0 μm to 1.5 μm in full width at half maximum for a 2-fold enhancement. Furthermore, by using HiLo microscopy with two structured illuminations at the same spatial frequency but different orientation, biotissue images according to the structured illumination with condensed AEC is obviously superior in contrast and scattering suppression.

2010 MULTIPHOTON PROCESSES GORDON RESEARCH CONFERENCE, JUNE 6-11, 2010, TILTON, NH

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

Mette Gaarde

2010-06-11

The Gordon Research Conference on Multiphoton Processes will be held for the 15th time in 2010. The meeting continues to evolve as it embraces both the rapid technological and intellectual growth in the field as well as the multi-disciplinary expertise of the participants. This time the sessions will focus on: (1) Ultrafast coherent control; (2) Free-electron laser experiments and theory; (3) Generation of harmonics and attosecond pulses; (4) Ultrafast imaging; (5) Applications of very high intensity laser fields; (6) Strong-field processes in molecules and solids; (7) Attosecond science; and (8) Controlling light. The scientific program will blur traditional disciplinary boundariesmore » as the presenters and discussion leaders involve chemists, physicists, and optical engineers, representing both experiment and theory. The broad range of expertise and different perspectives of attendees should provide a stimulating and unique environment for solving problems and developing new ideas in this rapidly evolving field.« less

Application of Negative Curvature Hollow-Core Fiber in an Optical Fiber Sensor Setup for Multiphoton Spectroscopy

PubMed Central

Stawska, Hanna Izabela; Mazur, Leszek Mateusz; Kosolapov, Alexey; Kolyadin, Anton; Bereś-Pawlik, Elżbieta

2017-01-01

In this paper, an application of negative curvature hollow core fiber (NCHCF) in an all-fiber, multiphoton fluorescence sensor setup is presented. The dispersion parameter (D) of this fiber does not exceed the value of 5 ps/nm × km across the optical spectrum of (680–750) nm, making it well suited for the purpose of multiphoton excitation of biological fluorophores. Employing 1.5 m of this fiber in a simple, all-fiber sensor setup allows us to perform multiphoton experiments without any dispersion compensation methods. Multiphoton excitation of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) with this fiber shows a 6- and 9-fold increase, respectively, in the total fluorescence signal collected when compared with the commercial solution in the form of a hollow-core photonic band gap fiber (HCPBF). To the author’s best knowledge, this is the first time an NCHCF was used in an optical-fiber sensor setup for multiphoton fluorescence experiments. PMID:28984838

Application of Negative Curvature Hollow-Core Fiber in an Optical Fiber Sensor Setup for Multiphoton Spectroscopy.

PubMed

Popenda, Maciej Andrzej; Stawska, Hanna Izabela; Mazur, Leszek Mateusz; Jakubowski, Konrad; Kosolapov, Alexey; Kolyadin, Anton; Bereś-Pawlik, Elżbieta

2017-10-06

In this paper, an application of negative curvature hollow core fiber (NCHCF) in an all-fiber, multiphoton fluorescence sensor setup is presented. The dispersion parameter (D) of this fiber does not exceed the value of 5 ps/nm × km across the optical spectrum of (680-750) nm, making it well suited for the purpose of multiphoton excitation of biological fluorophores. Employing 1.5 m of this fiber in a simple, all-fiber sensor setup allows us to perform multiphoton experiments without any dispersion compensation methods. Multiphoton excitation of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) with this fiber shows a 6- and 9-fold increase, respectively, in the total fluorescence signal collected when compared with the commercial solution in the form of a hollow-core photonic band gap fiber (HCPBF). To the author's best knowledge, this is the first time an NCHCF was used in an optical-fiber sensor setup for multiphoton fluorescence experiments.

Multiphoton minimal inertia scanning for fast acquisition of neural activity signals

NASA Astrophysics Data System (ADS)

Schuck, Renaud; Go, Mary Ann; Garasto, Stefania; Reynolds, Stephanie; Dragotti, Pier Luigi; Schultz, Simon R.

2018-04-01

Objective. Multi-photon laser scanning microscopy provides a powerful tool for monitoring the spatiotemporal dynamics of neural circuit activity. It is, however, intrinsically a point scanning technique. Standard raster scanning enables imaging at subcellular resolution; however, acquisition rates are limited by the size of the field of view to be scanned. Recently developed scanning strategies such as travelling salesman scanning (TSS) have been developed to maximize cellular sampling rate by scanning only select regions in the field of view corresponding to locations of interest such as somata. However, such strategies are not optimized for the mechanical properties of galvanometric scanners. We thus aimed to develop a new scanning algorithm which produces minimal inertia trajectories, and compare its performance with existing scanning algorithms. Approach. We describe here the adaptive spiral scanning (SSA) algorithm, which fits a set of near-circular trajectories to the cellular distribution to avoid inertial drifts of galvanometer position. We compare its performance to raster scanning and TSS in terms of cellular sampling frequency and signal-to-noise ratio (SNR). Main Results. Using surrogate neuron spatial position data, we show that SSA acquisition rates are an order of magnitude higher than those for raster scanning and generally exceed those achieved by TSS for neural densities comparable with those found in the cortex. We show that this result also holds true for in vitro hippocampal mouse brain slices bath loaded with the synthetic calcium dye Cal-520 AM. The ability of TSS to ‘park’ the laser on each neuron along the scanning trajectory, however, enables higher SNR than SSA when all targets are precisely scanned. Raster scanning has the highest SNR but at a substantial cost in number of cells scanned. To understand the impact of sampling rate and SNR on functional calcium imaging, we used the Cramér-Rao Bound on evoked calcium traces recorded simultaneously with electrophysiology traces to calculate the lower bound estimate of the spike timing occurrence. Significance. The results show that TSS and SSA achieve comparable accuracy in spike time estimates compared to raster scanning, despite lower SNR. SSA is an easily implementable way for standard multi-photon laser scanning systems to gain temporal precision in the detection of action potentials while scanning hundreds of active cells.

High resolution diagnosis of common nevi by multiphoton laser tomography and fluorescence lifetime imaging.

PubMed

Arginelli, Federica; Manfredini, Marco; Bassoli, Sara; Dunsby, Christopher; French, Paul; König, Karsten; Magnoni, Cristina; Ponti, Giovanni; Talbot, Clifford; Seidenari, Stefania

2013-05-01

Multiphoton Laser Tomography (MPT) has developed as a non-invasive tool that allows real-time observation of the skin with subcellular resolution. MPT is readily combined with time resolved detectors to achieve fluorescence lifetime imaging (FLIM). The aim of our study was to identify morphologic MPT/FLIM descriptors of melanocytic nevi, referring to cellular and architectural features. In the preliminary study, MPT/FLIM images referring to 16 ex vivo nevi were simultaneously evaluated by 3 observers for the identification of morphologic descriptors characteristic of melanocytic nevi. Proposed descriptors were discussed and the parameters referring to epidermal keratinocytes, epidermal melanocytes, dermo-epidermal junction, papillary dermis and overall architecture were selected. In the main study, the presence/absence of the specified criteria were blindly evaluated on a test set, comprising 102 ex vivo samples (51 melanocytic nevi, 51 miscellaneous skin lesions) by 2 observers. Twelve descriptors were identified: "short-lifetime cells in the stratum corneum", "melanin-containing keratinocytes", "dendritic cells", "small short-lifetime cells" in the upper and lower layers", "edged papillae", "non-edged papillae", "junctional nests of short-lifetime cells", "dermal cell clusters", "short-lifetime cells in the papilla", "monomorphic and regular histoarchitecture", "architectural disarray". Identified descriptors for benign melanocytic lesions proved sensitive and specific, enabling the differentiation between melanocytic nevi and non-melanocytic lesions. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Pulse-Shaping-Based Nonlinear Microscopy: Development and Applications

NASA Astrophysics Data System (ADS)

Flynn, Daniel Christopher

The combination of optical microscopy and ultrafast spectroscopy make the spatial characterization of chemical kinetics on the femtosecond time scale possible. Commercially available octave-spanning Ti:Sapphire oscillators with sub-8 fs pulse durations can drive a multitude of nonlinear transitions across a significant portion of the visible spectrum with minimal average power. Unfortunately, dispersion from microscope objectives broadens pulse durations, decreases temporal resolution and lowers the peak intensities required for driving nonlinear transitions. In this dissertation, pulse shaping is used to compress laser pulses after the microscope objective. By using a binary genetic algorithm, pulse-shapes are designed to enable selective two-photon excitation. The pulse-shapes are demonstrated in two-photon fluorescence of live COS-7 cells expressing GFP-variants mAmetrine and tdTomato. The pulse-shaping approach is applied to a new multiphoton fluorescence resonance energy transfer (FRET) stoichiometry method that quantifies donor and acceptor molecules in complex, as well as the ratio of total donor to acceptor molecules. Compared to conventional multi-photon imaging techniques that require laser tuning or multiple laser systems to selectively excite individual fluorophores, the pulse-shaping approach offers rapid selective multifluorphore imaging at biologically relevant time scales. By splitting the laser beam into two beams and building a second pulse shaper, a pulse-shaping-based pump-probe microscope is developed. The technique offers multiple imaging modalities, such as excited state absorption (ESA), ground state bleach (GSB), and stimulated emission (SE), enhancing contrast of structures via their unique quantum pathways without the addition of contrast agents. Pulse-shaping based pump-probe microscopy is demonstrated for endogenous chemical-contrast imaging of red blood cells. In the second section of this dissertation, ultrafast spectroscopic techniques are used to characterize structure-function relationships of two-photon absorbing GFP-type probes and optical limiting materials. Fluorescence lifetimes of GFP-type probes are shown to depend on functional group substitution position, therefore, enabling the synthesis of designer probes for the possible study of conformation changes and aggregation in biological systems. Similarly, it is determined that small differences in the structure and dimensionality of organometallic macrocycles result in a diverse set of optical properties, which serves as a basis for the molecular level design of nonlinear optical materials.

High-resolution distributed temperature sensing with the multiphoton-timing technique

NASA Astrophysics Data System (ADS)

Höbel, M.; Ricka, J.; Wüthrich, M.; Binkert, Th.

1995-06-01

We report on a multiphoton-timing distributed temperature sensor (DTS) based on the concept of distributed anti-Stokes Raman thermometry. The sensor combines the advantage of very high spatial resolution (40 cm) with moderate measurement times. In 5 min it is possible to determine the temperature of as many as 4000 points along an optical fiber with an accuracy Delta T less than 2 deg C. The new feature of the DTS system is the combination of a fast single-photon avalanche diode with specially designed real-time signal-processing electronics. We discuss various parameters that affect the operation of analog and photon-timing DTS systems. Particular emphasis is put on the consequences of the nonideal behavior of sensor components and the corresponding correction procedures.

Robust Distant Entanglement Generation Using Coherent Multiphoton Scattering

NASA Astrophysics Data System (ADS)

Chan, Ching-Kit; Sham, L. J.

2013-02-01

We describe a protocol to entangle two qubits at a distance by using resonance fluorescence. The scheme makes use of the postselection of large and distinguishable fluorescence signals corresponding to entangled and unentangled qubit states and has the merits of both high success probability and high entanglement fidelity owing to the multiphoton nature. Our result shows that the entanglement generation is robust against photon fluctuations in the fluorescence signals for a wide range of driving fields. We also demonstrate that this new protocol has an average entanglement duration within the decoherence time of corresponding qubit systems, based on current experimental photon efficiency.

Multiphoton Scattering Tomography with Coherent States.

PubMed

Ramos, Tomás; García-Ripoll, Juan José

2017-10-13

In this work we develop an experimental procedure to interrogate the single- and multiphoton scattering matrices of an unknown quantum system interacting with propagating photons. Our proposal requires coherent state laser or microwave inputs and homodyne detection at the scatterer's output, and provides simultaneous information about multiple-elastic and inelastic-segments of the scattering matrix. The method is resilient to detector noise and its errors can be made arbitrarily small by combining experiments at various laser powers. Finally, we show that the tomography of scattering has to be performed using pulsed lasers to efficiently gather information about the nonlinear processes in the scatterer.

Robust distant entanglement generation using coherent multiphoton scattering.

PubMed

Chan, Ching-Kit; Sham, L J

2013-02-15

We describe a protocol to entangle two qubits at a distance by using resonance fluorescence. The scheme makes use of the postselection of large and distinguishable fluorescence signals corresponding to entangled and unentangled qubit states and has the merits of both high success probability and high entanglement fidelity owing to the multiphoton nature. Our result shows that the entanglement generation is robust against photon fluctuations in the fluorescence signals for a wide range of driving fields. We also demonstrate that this new protocol has an average entanglement duration within the decoherence time of corresponding qubit systems, based on current experimental photon efficiency.

Amplitudes for multiphoton quantum processes in linear optics

NASA Astrophysics Data System (ADS)

Urías, Jesús

2011-07-01

The prominent role that linear optical networks have acquired in the engineering of photon states calls for physically intuitive and automatic methods to compute the probability amplitudes for the multiphoton quantum processes occurring in linear optics. A version of Wick's theorem for the expectation value, on any vector state, of products of linear operators, in general, is proved. We use it to extract the combinatorics of any multiphoton quantum processes in linear optics. The result is presented as a concise rule to write down directly explicit formulae for the probability amplitude of any multiphoton process in linear optics. The rule achieves a considerable simplification and provides an intuitive physical insight about quantum multiphoton processes. The methodology is applied to the generation of high-photon-number entangled states by interferometrically mixing coherent light with spontaneously down-converted light.

Adaptive compensation of aberrations in ultrafast 3D microscopy using a deformable mirror

NASA Astrophysics Data System (ADS)

Sherman, Leah R.; Albert, O.; Schmidt, Christoph F.; Vdovin, Gleb V.; Mourou, Gerard A.; Norris, Theodore B.

2000-05-01

3D imaging using a multiphoton scanning confocal microscope is ultimately limited by aberrations of the system. We describe a system to adaptively compensate the aberrations with a deformable mirror. We have increased the transverse scanning range of the microscope by three with compensation of off-axis aberrations.We have also significantly increased the longitudinal scanning depth with compensation of spherical aberrations from the penetration into the sample. Our correction is based on a genetic algorithm that uses second harmonic or two-photon fluorescence signal excited by femtosecond pulses from the sample as the enhancement parameter. This allows us to globally optimize the wavefront without a wavefront measurement. To improve the speed of the optimization we use Zernike polynomials as the basis for correction. Corrections can be stored in a database for look-up with future samples.

Optical skin biopsies by clinical CARS and multiphoton fluorescence/SHG tomography

NASA Astrophysics Data System (ADS)

König, K.; Breunig, H. G.; Bückle, R.; Kellner-Höfer, M.; Weinigel, M.; Büttner, E.; Sterry, W.; Lademann, J.

2011-06-01

The ultimate challenge for early diagnostics is label-free high-resolution intratissue imaging without taking physical biopsies. A novel hybrid femtosecond laser tomograph provides in vivo optical biopsies of human skin based on non-linear excitation of autofluorescence and the detection of lipids and water by CARS. Applications include skin cancer detection, biosafety tests of intradermal nanoparticles, and the testing of anti-aging products.

Real-time high dynamic range laser scanning microscopy

NASA Astrophysics Data System (ADS)

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-04-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

Development of a multi-scale and multi-modality imaging system to characterize tumours and their microenvironment in vivo

NASA Astrophysics Data System (ADS)

Rouffiac, Valérie; Ser-Leroux, Karine; Dugon, Emilie; Leguerney, Ingrid; Polrot, Mélanie; Robin, Sandra; Salomé-Desnoulez, Sophie; Ginefri, Jean-Christophe; Sebrié, Catherine; Laplace-Builhé, Corinne

2015-03-01

In vivo high-resolution imaging of tumor development is possible through dorsal skinfold chamber implantable on mice model. However, current intravital imaging systems are weakly tolerated along time by mice and do not allow multimodality imaging. Our project aims to develop a new chamber for: 1- long-term micro/macroscopic visualization of tumor (vascular and cellular compartments) and tissue microenvironment; and 2- multimodality imaging (photonic, MRI and sonography). Our new experimental device was patented in March 2014 and was primarily assessed on 75 mouse engrafted with 4T1-Luc tumor cell line, and validated in confocal and multiphoton imaging after staining the mice vasculature using Dextran 155KDa-TRITC or Dextran 2000kDa-FITC. Simultaneously, a universal stage was designed for optimal removal of respiratory and cardiac artifacts during microscopy assays. Experimental results from optical, ultrasound (Bmode and pulse subtraction mode) and MRI imaging (anatomic sequences) showed that our patented design, unlike commercial devices, improves longitudinal monitoring over several weeks (35 days on average against 12 for the commercial chamber) and allows for a better characterization of the early and late tissue alterations due to tumour development. We also demonstrated the compatibility for multimodality imaging and the increase of mice survival was by a factor of 2.9, with our new skinfold chamber. Current developments include: 1- defining new procedures for multi-labelling of cells and tissue (screening of fluorescent molecules and imaging protocols); 2- developing ultrasound and MRI imaging procedures with specific probes; 3- correlating optical/ultrasound/MRI data for a complete mapping of tumour development and microenvironment.

Photodynamic dye adsorption and release performance of natural zeolite

NASA Astrophysics Data System (ADS)

Hovhannisyan, Vladimir; Dong, Chen-Yuan; Chen, Shean-Jen

2017-03-01

Clinoptilolite type of zeolite (CZ) is a promising material for biomedicine and pharmaceutics due to its non-toxicity, thermal stability, expanded surface area, and exceptional ability to adsorb various atoms and organic molecules into micropores. Using multiphoton microscopy, we demonstrated that individual CZ particles produce two-photon excited luminescence and second harmonic generation signal at femtosecond laser excitation, and adsorb photo-dynamically active dyes such as hypericin and methylene blue. Furthermore, the release of hypericin from CZ pores in the presence of biomolecules is shown, and CZ can be considered as an effective material for drug delivery and controlled release in biological systems. The results may open new perspectives in application of CZ in biomedical imaging, and introducing of the optical approaches into the clinical environment.

Photodynamic dye adsorption and release performance of natural zeolite.

PubMed

Hovhannisyan, Vladimir; Dong, Chen-Yuan; Chen, Shean-Jen

2017-03-31

Clinoptilolite type of zeolite (CZ) is a promising material for biomedicine and pharmaceutics due to its non-toxicity, thermal stability, expanded surface area, and exceptional ability to adsorb various atoms and organic molecules into micropores. Using multiphoton microscopy, we demonstrated that individual CZ particles produce two-photon excited luminescence and second harmonic generation signal at femtosecond laser excitation, and adsorb photo-dynamically active dyes such as hypericin and methylene blue. Furthermore, the release of hypericin from CZ pores in the presence of biomolecules is shown, and CZ can be considered as an effective material for drug delivery and controlled release in biological systems. The results may open new perspectives in application of CZ in biomedical imaging, and introducing of the optical approaches into the clinical environment.

Culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy

PubMed Central

Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

2017-01-01

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina i.e. they migrate between the basal inner nuclear layer (INL) and the outer nuclear layer (ONL), respectively, in a process described as interkinetic nuclear migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM. PMID:28287581

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy.

PubMed

Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

2017-02-24

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP] mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Quantum Dot-Based Hybrid Nanostructures and Energy Transfer on the Nanoscale for Single- and Multi-Photon Imaging and Cancer Diagnostics

NASA Astrophysics Data System (ADS)

Nabiev, Igor

2017-01-01

An ideal single-photon (1P) or multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible 1P or two-photon (2P) absorption cross section and the smallest possible highly specific recognition molecules conjugated with the nanoparticle in an oriented manner. However, the conditions used for conjugation of typical recognition molecules (conventional antibodies, Abs) with nanoparticles often provoke their unfolding and/or yield nanoprobes with irregular orientation of Abs on the nanoparticle surface. Conjugation of smaller Ab fragments, such as single-domain antibodies (sdAbs), with quantum dots (QDs) in an oriented manner can be considered as an attractive approach to engineering of ultrasmall diagnostic nanoprobes. QDs conjugated to 13-kDa sdAbs derived from camelid IgG or streptavidin have been used as efficient 1P or 2P excitation probes for imaging of cancer markers. The 2P absorption cross sections (TPACSs) for some conjugates are higher than 49,000 GM (Goeppert-Mayer units), which is close to the theoretical value calculated for CdSe QDs and considerably exceeds that of organic dyes. A further step in advanced QD-based cancer diagnostics has been made through implementation of efficient FRET-based imaging with 2P excitation, which has been demonstrated for double immunostaining complexes formed on the surface of cancer cells from sdAb-QD conjugates (donor) and a combination of monoclonal Abs and secondary antibodies labeled with the AlexaFluor dye (acceptor). The proposed approach permits obtaining an exceptional contrast of 2P imaging of cancer biomarkers without any contribution of cell and tissue autofluorescence in the recorded images.

A new method using multiphoton imaging and morphometric analysis for differentiating chromophobe renal cell carcinoma and oncocytoma kidney tumors

NASA Astrophysics Data System (ADS)

Wu, Binlin; Mukherjee, Sushmita; Jain, Manu

2016-03-01

Distinguishing chromophobe renal cell carcinoma (chRCC) from oncocytoma on hematoxylin and eosin images may be difficult and require time-consuming ancillary procedures. Multiphoton microscopy (MPM), an optical imaging modality, was used to rapidly generate sub-cellular histological resolution images from formalin-fixed unstained tissue sections from chRCC and oncocytoma.Tissues were excited using 780nm wavelength and emission signals (including second harmonic generation and autofluorescence) were collected in different channels between 390 nm and 650 nm. Granular structure in the cell cytoplasm was observed in both chRCC and oncocytoma. Quantitative morphometric analysis was conducted to distinguish chRCC and oncocytoma. To perform the analysis, cytoplasm and granules in tumor cells were segmented from the images. Their area and fluorescence intensity were found in different channels. Multiple features were measured to quantify the morphological and fluorescence properties. Linear support vector machine (SVM) was used for classification. Re-substitution validation, cross validation and receiver operating characteristic (ROC) curve were implemented to evaluate the efficacy of the SVM classifier. A wrapper feature algorithm was used to select the optimal features which provided the best predictive performance in separating the two tissue types (classes). Statistical measures such as sensitivity, specificity, accuracy and area under curve (AUC) of ROC were calculated to evaluate the efficacy of the classification. Over 80% accuracy was achieved as the predictive performance. This method, if validated on a larger and more diverse sample set, may serve as an automated rapid diagnostic tool to differentiate between chRCC and oncocytoma. An advantage of such automated methods are that they are free from investigator bias and variability.

Optical Drug Monitoring: Photoacoustic Imaging of Nanosensors to Monitor Therapeutic Lithium In Vivo

PubMed Central

Cash, Kevin J.; Li, Chiye; Xia, Jun; Wang, Lihong V.; Clark, Heather A.

2015-01-01

Personalized medicine could revolutionize how primary care physicians treat chronic disease and how researchers study fundamental biological questions. To realize this goal we need to develop more robust, modular tools and imaging approaches for in vivo monitoring of analytes. In this report, we demonstrate that synthetic nanosensors can measure physiologic parameters with photoacoustic contrast, and we apply that platform to continuously track lithium levels in vivo. Photoacoustic imaging achieves imaging depths that are unattainable with fluorescence or multiphoton microscopy. We validated the photoacoustic results that illustrate the superior imaging depth and quality of photoacoustic imaging with optical measurements. This powerful combination of techniques will unlock the ability to measure analyte changes in deep tissue and will open up photoacoustic imaging as a diagnostic tool for continuous physiological tracking of a wide range of analytes. PMID:25588028

Optical drug monitoring: photoacoustic imaging of nanosensors to monitor therapeutic lithium in vivo.

PubMed

Cash, Kevin J; Li, Chiye; Xia, Jun; Wang, Lihong V; Clark, Heather A

2015-02-24

Personalized medicine could revolutionize how primary care physicians treat chronic disease and how researchers study fundamental biological questions. To realize this goal, we need to develop more robust, modular tools and imaging approaches for in vivo monitoring of analytes. In this report, we demonstrate that synthetic nanosensors can measure physiologic parameters with photoacoustic contrast, and we apply that platform to continuously track lithium levels in vivo. Photoacoustic imaging achieves imaging depths that are unattainable with fluorescence or multiphoton microscopy. We validated the photoacoustic results that illustrate the superior imaging depth and quality of photoacoustic imaging with optical measurements. This powerful combination of techniques will unlock the ability to measure analyte changes in deep tissue and will open up photoacoustic imaging as a diagnostic tool for continuous physiological tracking of a wide range of analytes.

TRIO Platform: A Novel Low Profile In vivo Imaging Support and Restraint System for Mice.

PubMed

Voziyanov, Vladislav; Kemp, Benjamin S; Dressel, Chelsea A; Ponder, Kayla; Murray, Teresa A

2016-01-01

High resolution, in vivo optical imaging of the mouse brain over time often requires anesthesia, which necessitates maintaining the animal's body temperature and level of anesthesia, as well as securing the head in an optimal, stable position. Controlling each parameter usually requires using multiple systems. Assembling multiple components into the small space on a standard microscope stage can be difficult and some commercially available parts simply do not fit. Furthermore, it is time-consuming to position an animal in the identical position over multiple imaging sessions for longitudinal studies. This is especially true when using an implanted gradient index (GRIN) lens for deep brain imaging. The multiphoton laser beam must be parallel with the shaft of the lens because even a slight tilt of the lens can degrade image quality. In response to these challenges, we have designed a compact, integrated in vivo imaging support system to overcome the problems created by using separate systems during optical imaging in mice. It is a single platform that provides (1) sturdy head fixation, (2) an integrated gas anesthesia mask, and (3) safe warm water heating. This THREE-IN-ONE (TRIO) Platform has a small footprint and a low profile that positions a mouse's head only 20 mm above the microscope stage. This height is about one half to one third the height of most commercially available immobilization devices. We have successfully employed this system, using isoflurane in over 40 imaging sessions with an average of 2 h per session with no leaks or other malfunctions. Due to its smaller size, the TRIO Platform can be used with a wider range of upright microscopes and stages. Most of the components were designed in SOLIDWORKS® and fabricated using a 3D printer. This additive manufacturing approach also readily permits size modifications for creating systems for other small animals.

TRIO Platform: A Novel Low Profile In vivo Imaging Support and Restraint System for Mice

PubMed Central

Voziyanov, Vladislav; Kemp, Benjamin S.; Dressel, Chelsea A.; Ponder, Kayla; Murray, Teresa A.

2016-01-01

High resolution, in vivo optical imaging of the mouse brain over time often requires anesthesia, which necessitates maintaining the animal's body temperature and level of anesthesia, as well as securing the head in an optimal, stable position. Controlling each parameter usually requires using multiple systems. Assembling multiple components into the small space on a standard microscope stage can be difficult and some commercially available parts simply do not fit. Furthermore, it is time-consuming to position an animal in the identical position over multiple imaging sessions for longitudinal studies. This is especially true when using an implanted gradient index (GRIN) lens for deep brain imaging. The multiphoton laser beam must be parallel with the shaft of the lens because even a slight tilt of the lens can degrade image quality. In response to these challenges, we have designed a compact, integrated in vivo imaging support system to overcome the problems created by using separate systems during optical imaging in mice. It is a single platform that provides (1) sturdy head fixation, (2) an integrated gas anesthesia mask, and (3) safe warm water heating. This THREE-IN-ONE (TRIO) Platform has a small footprint and a low profile that positions a mouse's head only 20 mm above the microscope stage. This height is about one half to one third the height of most commercially available immobilization devices. We have successfully employed this system, using isoflurane in over 40 imaging sessions with an average of 2 h per session with no leaks or other malfunctions. Due to its smaller size, the TRIO Platform can be used with a wider range of upright microscopes and stages. Most of the components were designed in SOLIDWORKS® and fabricated using a 3D printer. This additive manufacturing approach also readily permits size modifications for creating systems for other small animals. PMID:27199633

Multiphoton excitation and high-harmonics generation in topological insulator.

PubMed

Avetissian, H K; Avetissian, A K; Avchyan, B R; Mkrtchian, G F

2018-05-10

Multiphoton interaction of coherent electromagnetic radiation with 2D metallic carriers confined on the surface of the 3D topological insulator is considered. A microscopic theory describing the nonlinear interaction of a strong wave and metallic carriers with many-body Coulomb interaction is developed. The set of integrodifferential equations for the interband polarization and carrier occupation distribution is solved numerically. Multiphoton excitation of Fermi-Dirac sea of 2D massless carriers is considered for a THz pump wave. It is shown that in the moderately strong pump wave field along with multiphoton interband/intraband transitions the intense radiation of high harmonics takes place.

Multiphoton excitation and high-harmonics generation in topological insulator

NASA Astrophysics Data System (ADS)

Avetissian, H. K.; Avetissian, A. K.; Avchyan, B. R.; Mkrtchian, G. F.

2018-05-01

Multiphoton interaction of coherent electromagnetic radiation with 2D metallic carriers confined on the surface of the 3D topological insulator is considered. A microscopic theory describing the nonlinear interaction of a strong wave and metallic carriers with many-body Coulomb interaction is developed. The set of integrodifferential equations for the interband polarization and carrier occupation distribution is solved numerically. Multiphoton excitation of Fermi–Dirac sea of 2D massless carriers is considered for a THz pump wave. It is shown that in the moderately strong pump wave field along with multiphoton interband/intraband transitions the intense radiation of high harmonics takes place.

Nonlinear adaptive optics: aberration correction in three photon fluorescence microscopy for mouse brain imaging

NASA Astrophysics Data System (ADS)

Sinefeld, David; Paudel, Hari P.; Wang, Tianyu; Wang, Mengran; Ouzounov, Dimitre G.; Bifano, Thomas G.; Xu, Chris

2017-02-01

Multiphoton fluorescence microscopy is a well-established technique for deep-tissue imaging with subcellular resolution. Three-photon microscopy (3PM) when combined with long wavelength excitation was shown to allow deeper imaging than two-photon microscopy (2PM) in biological tissues, such as mouse brain, because out-of-focus background light can be further reduced due to the higher order nonlinear excitation. As was demonstrated in 2PM systems, imaging depth and resolution can be improved by aberration correction using adaptive optics (AO) techniques which are based on shaping the scanning beam using a spatial light modulator (SLM). In this way, it is possible to compensate for tissue low order aberration and to some extent, to compensate for tissue scattering. Here, we present a 3PM AO microscopy system for brain imaging. Soliton self-frequency shift is used to create a femtosecond source at 1675 nm and a microelectromechanical (MEMS) SLM serves as the wavefront shaping device. We perturb the 1020 segment SLM using a modified nonlinear version of three-point phase shifting interferometry. The nonlinearity of the fluorescence signal used for feedback ensures that the signal is increasing when the spot size decreases, allowing compensation of phase errors in an iterative optimization process without direct phase measurement. We compare the performance for different orders of nonlinear feedback, showing an exponential growth in signal improvement as the nonlinear order increases. We demonstrate the impact of the method by applying the 3PM AO system for in-vivo mouse brain imaging, showing improvement in signal at 1-mm depth inside the brain.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair.

PubMed

Li, Jian; Jahr, Holger; Zheng, Wei; Ren, Pei-Gen

2017-09-07

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Energy-Looping Nanoparticles: Harnessing Excited-State Absorption for Deep-Tissue Imaging.

PubMed

Levy, Elizabeth S; Tajon, Cheryl A; Bischof, Thomas S; Iafrati, Jillian; Fernandez-Bravo, Angel; Garfield, David J; Chamanzar, Maysamreza; Maharbiz, Michel M; Sohal, Vikaas S; Schuck, P James; Cohen, Bruce E; Chan, Emory M

2016-09-27

Near infrared (NIR) microscopy enables noninvasive imaging in tissue, particularly in the NIR-II spectral range (1000-1400 nm) where attenuation due to tissue scattering and absorption is minimized. Lanthanide-doped upconverting nanocrystals are promising deep-tissue imaging probes due to their photostable emission in the visible and NIR, but these materials are not efficiently excited at NIR-II wavelengths due to the dearth of lanthanide ground-state absorption transitions in this window. Here, we develop a class of lanthanide-doped imaging probes that harness an energy-looping mechanism that facilitates excitation at NIR-II wavelengths, such as 1064 nm, that are resonant with excited-state absorption transitions but not ground-state absorption. Using computational methods and combinatorial screening, we have identified Tm(3+)-doped NaYF4 nanoparticles as efficient looping systems that emit at 800 nm under continuous-wave excitation at 1064 nm. Using this benign excitation with standard confocal microscopy, energy-looping nanoparticles (ELNPs) are imaged in cultured mammalian cells and through brain tissue without autofluorescence. The 1 mm imaging depths and 2 μm feature sizes are comparable to those demonstrated by state-of-the-art multiphoton techniques, illustrating that ELNPs are a promising class of NIR probes for high-fidelity visualization in cells and tissue.

Live Imaging of the Lung

PubMed Central

Looney, Mark R.; Bhattacharya, Jahar

2015-01-01

Live lung imaging has spanned the discovery of capillaries in the frog lung by Malpighi to the current use of single and multiphoton imaging of intravital and isolated perfused lung preparations incorporating fluorescent molecular probes and transgenic reporter mice. Along the way, much has been learned about the unique microcirculation of the lung, including immune cell migration and the mechanisms by which cells at the alveolar-capillary interface communicate with each other. In this review, we highlight live lung imaging techniques as applied to the role of mitochondria in lung immunity, mechanisms of signal transduction in lung compartments, studies on the composition of alveolar wall liquid, and neutrophil and platelet trafficking in the lung under homeostatic and inflammatory conditions. New applications of live lung imaging and the limitations of current techniques are discussed. PMID:24245941

Platinum Acetylide Two-Photon Chromophores (Preprint)

DTIC Science & Technology

2007-04-01

nonlinear photonics,6-s microfabrication,9,10 fluorescence imaging, II and photodynamic therapy.12Instantaneous absorption of two lower energy photons...results in initiation of the same photophysical processes as one-photon absorption (lP A) of one high- energy photon. This is advantageous for two...reasons. The first is that because of the use of a lower energy photon a material will be guarded from ionization effects from multiphoton absorption in

Upconversion microparticles as time-resolved luminescent probes for multiphoton microscopy: desired signal extraction from the streaking effect

NASA Astrophysics Data System (ADS)

Pominova, Daria V.; Ryabova, Anastasia V.; Grachev, Pavel V.; Romanishkin, Igor D.; Kuznetsov, Sergei V.; Rozhnova, Julia A.; Yasyrkina, Daria S.; Fedorov, Pavel P.; Loschenov, Victor B.

2016-09-01

The great interest in upconversion nanoparticles exists due to their high efficiency under multiphoton excitation. However, when these particles are used in scanning microscopy, the upconversion luminescence causes a streaking effect due to the long lifetime. This article describes a method of upconversion microparticle luminescence lifetime determination with help of modified Lucy-Richardson deconvolution of laser scanning microscope (LSM) image obtained under near-IR excitation using nondescanned detectors. Determination of the upconversion luminescence intensity and the decay time of separate microparticles was done by intensity profile along the image fast scan axis approximation. We studied upconversion submicroparticles based on fluoride hosts doped with Yb3+-Er3+ and Yb3+-Tm3+ rare earth ion pairs, and the characteristic decay times were 0.1 to 1.5 ms. We also compared the results of LSM measurements with the photon counting method results; the spread of values was about 13% and was associated with the approximation error. Data obtained from live cells showed the possibility of distinguishing the position of upconversion submicroparticles inside and outside the cells by the difference of their lifetime. The proposed technique allows using the upconversion microparticles without shells as probes for the presence of OH- ions and CO2 molecules.

Carcinogenic damage to deoxyribonucleic acid is induced by near-infrared laser pulses in multiphoton microscopy via combination of two- and three-photon absorption

NASA Astrophysics Data System (ADS)

Nadiarnykh, Oleg; Thomas, Giju; Van Voskuilen, Johan; Sterenborg, Henricus J. C. M.; Gerritsen, Hans C.

2012-11-01

Nonlinear optical imaging modalities (multiphoton excited fluorescence, second and third harmonic generation) applied in vivo are increasingly promising for clinical diagnostics and the monitoring of cancer and other disorders, as they can probe tissue with high diffraction-limited resolution at near-infrared (IR) wavelengths. However, high peak intensity of femtosecond laser pulses required for two-photon processes causes formation of cyclobutane-pyrimidine-dimers (CPDs) in cellular deoxyribonucleic acid (DNA) similar to damage from exposure to solar ultraviolet (UV) light. Inaccurate repair of subsequent mutations increases the risk of carcinogenesis. In this study, we investigate CPD damage that results in Chinese hamster ovary cells in vitro from imaging them with two-photon excited autofluorescence. The CPD levels are quantified by immunofluorescent staining. We further evaluate the extent of CPD damage with respect to varied wavelength, pulse width at focal plane, and pixel dwell time as compared with more pronounced damage from UV sources. While CPD damage has been expected to result from three-photon absorption, our results reveal that CPDs are induced by competing two- and three-photon absorption processes, where the former accesses UVA absorption band. This finding is independently confirmed by nonlinear dependencies of damage on laser power, wavelength, and pulse width.

Collagenous extracellular matrix of cartilage submitted to mechanical forces studied by second harmonic generation microscopy.

PubMed

Werkmeister, Elisabeth; de Isla, Natalia; Netter, Patrick; Stoltz, Jean-François; Dumas, Dominique

2010-01-01

Osteoarthritis is a degenerative pathology leading to degradation of the extracellular matrix (ECM). Similar effects can be visualized when applying mechanical or biochemical constraints on cartilaginous tissue. Here, we characterized modification of the ECM appearing under mechanical compression and/or biochemical action (hypoxia environment, nitric oxide and collagenase action). In recent decades, multiphoton microscopy has proved its interest for observing living, thick and opaque biological tissues. Thus, the main components of the cartilaginous ECM can be observed without fluorescent labeling. In particular, the collagen network emits strong second harmonic generation (SHG) signal which could be collected at half of the excitation wavelength. Combining autofluorescence and SHG signal detection enables to obtain complementary structural information. Here, we proved that multiphoton microscopy represents an appropriate tool for ex vitro cartilage imaging. First, we showed that SHG signal specifically comes from collagen (collagenase digestion). Further, we verified that the use of an appropriate band-pass filter enables to reject the autofluorescence from the ECM. Once this specificity was shown, we followed modification of the cartilage ECM submitted to mechanical or biochemical constraints (compression, enzymatic digestion). By performing textural analysis of SHG images (Haralick's method), we showed the restructuration of the collagen network according to constraints.

In-vivo morphologic and spectroscopic investigation of Psoriasis

NASA Astrophysics Data System (ADS)

Kapsokalyvas, Dimitrios; Cicchi, Riccardo; Bruscino, Nicola; Alfieri, Domenico; Massi, Daniela; Lotti, Torello; Pavone, Francesco S.

2011-07-01

Psoriasis is an autoimmune disease of the skin characterized by hyperkeratosis, hyperproliferation of the epidermis, inflammatory cell accumulation and increased dilatation of dermal papillary blood vessels. Cases of psoriasis were investigated in vivo with optical means in order to evaluate the potential of in vivo optical biopsy. A Polarization Multispectral Dermoscope was employed for the macroscopic observation. Features such as the 'dotted' blood vessels pattern was observed with high contrast. High resolution image sections of the epidermis and the dermis were produced with a custom made Multiphoton Microscope. Imaging extended from the surface of the lesion down to the papillary dermis, at a depth of 200 μm. In the epidermis, a characteristic morphology of the stratum corneum found only in Psoriasis was revealed. Additionally, the cytoplasmic area of the cells in the stratum spinosum layer was found to be smaller than normal. In the dermis the morphological features were more pronounced, where the elongated dermal papillae dominated the papillary layer. Their length exceeds 100μm, which is a far greater value compared to that of healthy skin. These in vivo observations are consistent with the ex vivo histopathological observations, supporting both the applicability and potentiality of multispectral dermoscopy and multiphoton microscopy in the field of in vivo optical investigation and biopsy of skin.

Spin noise spectroscopy beyond thermal equilibrium and linear response.

PubMed

Glasenapp, P; Sinitsyn, N A; Yang, Luyi; Rickel, D G; Roy, D; Greilich, A; Bayer, M; Crooker, S A

2014-10-10

Per the fluctuation-dissipation theorem, the information obtained from spin fluctuation studies in thermal equilibrium is necessarily constrained by the system's linear response functions. However, by including weak radio frequency magnetic fields, we demonstrate that intrinsic and random spin fluctuations even in strictly unpolarized ensembles can reveal underlying patterns of correlation and coupling beyond linear response, and can be used to study nonequilibrium and even multiphoton coherent spin phenomena. We demonstrate this capability in a classical vapor of (41)K alkali atoms, where spin fluctuations alone directly reveal Rabi splittings, the formation of Mollow triplets and Autler-Townes doublets, ac Zeeman shifts, and even nonlinear multiphoton coherences.

Holographic storage of biphoton entanglement.

PubMed

Dai, Han-Ning; Zhang, Han; Yang, Sheng-Jun; Zhao, Tian-Ming; Rui, Jun; Deng, You-Jin; Li, Li; Liu, Nai-Le; Chen, Shuai; Bao, Xiao-Hui; Jin, Xian-Min; Zhao, Bo; Pan, Jian-Wei

2012-05-25

Coherent and reversible storage of multiphoton entanglement with a multimode quantum memory is essential for scalable all-optical quantum information processing. Although a single photon has been successfully stored in different quantum systems, storage of multiphoton entanglement remains challenging because of the critical requirement for coherent control of the photonic entanglement source, multimode quantum memory, and quantum interface between them. Here we demonstrate a coherent and reversible storage of biphoton Bell-type entanglement with a holographic multimode atomic-ensemble-based quantum memory. The retrieved biphoton entanglement violates the Bell inequality for 1 μs storage time and a memory-process fidelity of 98% is demonstrated by quantum state tomography.

Multiphoton entanglement concentration and quantum cryptography.

PubMed

Durkin, Gabriel A; Simon, Christoph; Bouwmeester, Dik

2002-05-06

Multiphoton states from parametric down-conversion can be entangled both in polarization and photon number. Maximal high-dimensional entanglement can be concentrated postselectively from these states via photon counting. This makes them natural candidates for quantum key distribution, where the presence of more than one photon per detection interval has up to now been considered undesirable. We propose a simple multiphoton cryptography protocol for the case of low losses.

Roles of Tunneling, Multiphoton Ionization, and Cascade Ionization for Femtosecond Optical Breakdown in Aqueous Media

DTIC Science & Technology

2009-09-01

observed in the wavelength dependence of femtosecond breakdown would indicate a significant role of multiphoton ionization compared to tunneling ...relevant for femtosecond breakdown, and tunnel ionization featuring no Ith() dependence becomes ever more with decreasing pulse duration. However, it...c) Figure 4.22 Wavelength dependence of ionization probabilities by a) avalanche, b) multiphoton, and c) tunneling ionization. 1

Approach to quantify human dermal skin aging using multiphoton laser scanning microscopy

NASA Astrophysics Data System (ADS)

Puschmann, Stefan; Rahn, Christian-Dennis; Wenck, Horst; Gallinat, Stefan; Fischer, Frank

2012-03-01

Extracellular skin structures in human skin are impaired during intrinsic and extrinsic aging. Assessment of these dermal changes is conducted by subjective clinical evaluation and histological and molecular analysis. We aimed to develop a new parameter for the noninvasive quantitative determination of dermal skin alterations utilizing the high-resolution three-dimensional multiphoton laser scanning microscopy (MPLSM) technique. To quantify structural differences between chronically sun-exposed and sun-protected human skin, the respective collagen-specific second harmonic generation and the elastin-specific autofluorescence signals were recorded in young and elderly volunteers using the MPLSM technique. After image processing, the elastin-to-collagen ratio (ELCOR) was calculated. Results show that the ELCOR parameter of volar forearm skin significantly increases with age. For elderly volunteers, the ELCOR value calculated for the chronically sun-exposed temple area is significantly augmented compared to the sun-protected upper arm area. Based on the MPLSM technology, we introduce the ELCOR parameter as a new means to quantify accurately age-associated alterations in the extracellular matrix.

Visualizing liver anatomy, physiology and pharmacology using multiphoton microscopy.

PubMed

Wang, Haolu; Liang, Xiaowen; Gravot, Germain; Thorling, Camilla A; Crawford, Darrell H G; Xu, Zhi Ping; Liu, Xin; Roberts, Michael S

2017-01-01

Multiphoton microscopy (MPM) has become increasingly popular and widely used in both basic and clinical liver studies over the past few years. This technology provides insights into deep live tissues with less photobleaching and phototoxicity, which helps us to better understand the cellular morphology, microenvironment, immune responses and spatiotemporal dynamics of drugs and therapeutic cells in the healthy and diseased liver. This review summarizes the principles, opportunities, applications and limitations of MPM in hepatology. A key emphasis is on the use of fluorescence lifetime imaging (FLIM) to add additional quantification and specificity to the detection of endogenous fluorescent species in the liver as well as exogenous molecules and nanoparticles that are applied to the liver in vivo. We anticipate that in the near future MPM-FLIM will advance our understanding of the cellular and molecular mechanisms of liver diseases, and will be evaluated from bench to bedside, leading to real-time histology of human liver diseases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparing in vivo pump–probe and multiphoton fluorescence microscopy of melanoma and pigmented lesions

PubMed Central

Wilson, Jesse W.; Degan, Simone; Gainey, Christina S.; Mitropoulos, Tanya; Simpson, Mary Jane; Zhang, Jennifer Y.; Warren, Warren S.

2014-01-01

Abstract. We demonstrate a multimodal approach that combines a pump–probe with confocal reflectance and multiphoton autofluorescence microscopy. Pump–probe microscopy has been proven to be of great value in analyzing thin tissue sections of pigmented lesions, as it produces molecular contrast which is inaccessible by other means. However, the higher optical intensity required to overcome scattering in thick tissue leads to higher-order nonlinearities in the optical response of melanin (e.g., two-photon pump and one-photon probe) that present additional challenges for interpreting the data. We show that analysis of pigment composition in vivo must carefully account for signal terms that are nonlinear with respect to the pump and probe intensities. We find that pump–probe imaging gives useful contrast for pigmented structures over a large range of spatial scales (100  μm to 1 cm), making it a potentially useful tool for tracking the progression of pigmented lesions without the need to introduce exogenous contrast agents. PMID:25415567

Real-time CARS imaging reveals a calpain-dependent pathway for paranodal myelin retraction during high-frequency stimulation.

PubMed

Huff, Terry B; Shi, Yunzhou; Sun, Wenjing; Wu, Wei; Shi, Riyi; Cheng, Ji-Xin

2011-03-03

High-frequency electrical stimulation is becoming a promising therapy for neurological disorders, however the response of the central nervous system to stimulation remains poorly understood. The current work investigates the response of myelin to electrical stimulation by laser-scanning coherent anti-Stokes Raman scattering (CARS) imaging of myelin in live spinal tissues in real time. Paranodal myelin retraction at the nodes of Ranvier was observed during 200 Hz electrical stimulation. Retraction was seen to begin minutes after the onset of stimulation and continue for up to 10 min after stimulation was ceased, but was found to reverse after a 2 h recovery period. The myelin retraction resulted in exposure of Kv 1.2 potassium channels visualized by immunofluorescence. Accordingly, treating the stimulated tissue with a potassium channel blocker, 4-aminopyridine, led to the appearance of a shoulder peak in the compound action potential curve. Label-free CARS imaging of myelin coupled with multiphoton fluorescence imaging of immuno-labeled proteins at the nodes of Ranvier revealed that high-frequency stimulation induced paranodal myelin retraction via pathologic calcium influx into axons, calpain activation, and cytoskeleton degradation through spectrin break-down.

ACQ4: an open-source software platform for data acquisition and analysis in neurophysiology research

PubMed Central

Campagnola, Luke; Kratz, Megan B.; Manis, Paul B.

2014-01-01

The complexity of modern neurophysiology experiments requires specialized software to coordinate multiple acquisition devices and analyze the collected data. We have developed ACQ4, an open-source software platform for performing data acquisition and analysis in experimental neurophysiology. This software integrates the tasks of acquiring, managing, and analyzing experimental data. ACQ4 has been used primarily for standard patch-clamp electrophysiology, laser scanning photostimulation, multiphoton microscopy, intrinsic imaging, and calcium imaging. The system is highly modular, which facilitates the addition of new devices and functionality. The modules included with ACQ4 provide for rapid construction of acquisition protocols, live video display, and customizable analysis tools. Position-aware data collection allows automated construction of image mosaics and registration of images with 3-dimensional anatomical atlases. ACQ4 uses free and open-source tools including Python, NumPy/SciPy for numerical computation, PyQt for the user interface, and PyQtGraph for scientific graphics. Supported hardware includes cameras, patch clamp amplifiers, scanning mirrors, lasers, shutters, Pockels cells, motorized stages, and more. ACQ4 is available for download at http://www.acq4.org. PMID:24523692

Automated motion artifact removal for intravital microscopy, without a priori information.

PubMed

Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

2014-03-28

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.

Automated motion artifact removal for intravital microscopy, without a priori information

PubMed Central

Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

2014-01-01

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber. PMID:24676021

Real-time high dynamic range laser scanning microscopy

PubMed Central

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-01-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging. PMID:27032979

Cardiovascular Imaging Using Two-Photon Microscopy

PubMed Central

Scherschel, John A.; Rubart, Michael

2008-01-01

Two-photon excitation microscopy has become the standard technique for high resolution deep tissue and intravital imaging. It provides intrinsic three-dimensional resolution in combination with increased penetration depth compared to single-photon confocal microscopy. This article will describe the basic physical principles of two-photon excitation and will review its multiple applications to cardiovascular imaging, including second harmonic generation and fluorescence laser scanning microscopy. In particular, the capability and limitations of multiphoton microscopy to assess functional heterogeneity on a cellular scale deep within intact, Langendorff-perfused hearts are demonstrated. It will also discuss the use of two-photon excitation-induced release of caged compounds for the study of intracellular calcium signaling and intercellular dye transfer. PMID:18986603

Contextual analysis of immunological response through whole-organ fluorescent imaging.

PubMed

Woodruff, Matthew C; Herndon, Caroline N; Heesters, B A; Carroll, Michael C

2013-09-01

As fluorescent microscopy has developed, significant insights have been gained into the establishment of immune response within secondary lymphoid organs, particularly in draining lymph nodes. While established techniques such as confocal imaging and intravital multi-photon microscopy have proven invaluable, they provide limited insight into the architectural and structural context in which these responses occur. To interrogate the role of the lymph node environment in immune response effectively, a new set of imaging tools taking into account broader architectural context must be implemented into emerging immunological questions. Using two different methods of whole-organ imaging, optical clearing and three-dimensional reconstruction of serially sectioned lymph nodes, fluorescent representations of whole lymph nodes can be acquired at cellular resolution. Using freely available post-processing tools, images of unlimited size and depth can be assembled into cohesive, contextual snapshots of immunological response. Through the implementation of robust iterative analysis techniques, these highly complex three-dimensional images can be objectified into sortable object data sets. These data can then be used to interrogate complex questions at the cellular level within the broader context of lymph node biology. By combining existing imaging technology with complex methods of sample preparation and capture, we have developed efficient systems for contextualizing immunological phenomena within lymphatic architecture. In combination with robust approaches to image analysis, these advances provide a path to integrating scientific understanding of basic lymphatic biology into the complex nature of immunological response.

Bio-optic signatures for advanced glycation end products in the skin in streptozotocin (STZ) Induced Diabetes (Conference Presentation)

NASA Astrophysics Data System (ADS)

Saidian, Mayer; Ponticorvo, Adrien; Rowland, Rebecca A.; Balbado, Melisa L.; Lentsch, Griffin; Balu, Mihaela; Alexander, Micheal; Shiri, Li; Lakey, Jonathan R. T.; Durkin, Anthony J.; Kohen, Roni; Tromberg, Bruce J.

2017-02-01

Type 1diabetes (T1D) is an autoimmune disorder that occurs due to the rapid destruction of insulin-producing beta cells, leading to insulin deficiency and the inability to regulate blood glucose levels and leads to destructive secondary complications. Advanced glycation end (AGEs) products, the result of the cross-linking of reducing sugars and proteins within the tissues, are one of the key causes of major complications associated with diabetes such as renal failure, blindness, nerve damage and vascular changes. Non-invasive techniques to detect AGEs are important for preventing the harmful effects of AGEs during diabetes mellitus. In this study, we utilized multiphoton microscopy to image biopsies taken from control rats and compared them to biopsies taken from streptozotocin (STZ) induced adult male diabetic rats. This was done at two and four weeks after the induction of hyperglycemia (>400 mg/dL) specifically to evaluate the effects of glycation on collagen. We chose to use an in-situ multiphoton microscopy method that combines multiphoton auto-florescence (AF) and second harmonic generation (SHG) to detect the microscopic influence of glycation. Initial results show high auto-florescence levels were present on the collagen, as a result of the accumulation of AGEs only two weeks after the STZ injection and considerably higher levels were present four weeks after the STZ injection. Future projects could involve evaluating advanced glycation end products in a clinical trial of diabetic patients.

Investigating multiphoton phenomena using nonlinear dynamics

NASA Astrophysics Data System (ADS)

Huang, Shu

Many seemingly simple systems can display extraordinarily complex dynamics which has been studied and uncovered through nonlinear dynamical theory. The leitmotif of this thesis is changing phase-space structures and their (linear or non-linear) stabilities by adding control functions (which act on the system as external perturbations) to the relevant Hamiltonians. These phase-space structures may be periodic orbits, invariant tori or their stable and unstable manifolds. One-electron systems and diatomic molecules are fundamental and important staging ground for new discoveries in nonlinear dynamics. In past years, increasing emphasis and effort has been put on the control or manipulation of these systems. Recent developments of nonlinear dynamical tools can provide efficient ways of doing so. In the first subtopic of the thesis, we are adding a control function to restore tori at prescribed locations in phase space. In the remainder of the thesis, a control function with parameters is used to change the linear stability of the periodic orbits which govern the processes in question. In this thesis, we report our theoretical analyses on multiphoton ionization of Rydberg atoms exposed to strong microwave fields and the dissociation of diatomic molecules exposed to bichromatic lasers using nonlinear dynamical tools. This thesis is composed of three subtopics. In the first subtopic, we employ local control theory to reduce the stochastic ionization of hydrogen atom in a strong microwave field by adding a relatively small control term to the original Hamiltonian. In the second subtopic, we perform periodic orbit analysis to investigate multiphoton ionization driven by a bichromatic microwave field. Our results show quantitative and qualitative agreement with previous studies, and hence identify the mechanism through which short periodic orbits organize the dynamics in multiphoton ionization. In addition, we achieve substantial time savings with this approach. In the third subtopic we extend our periodic orbit analysis to the dissociation of diatomic molecules driven by a bichromatic laser. In this problem, our results based on periodic orbit analysis again show good agreement with previous work, and hence promise more potential applications of this approach in molecular physics.

Ultrafast Laser System for Producing on-Demand Single-and Multi-Photon Quantum States

DTIC Science & Technology

2015-09-20

14-Mar-2015 Approved for Public Release; Distribution Unlimited Final Report: Ultrafast laser system for producing on-demand single- and multi...Champaign, IL 61820 -7406 14-Mar-2015 ABSTRACT Number of Papers published in peer-reviewed journals: Final Report: Ultrafast laser system for producing

Microscanners for optical endomicroscopic applications

NASA Astrophysics Data System (ADS)

Hwang, Kyungmin; Seo, Yeong-Hyeon; Jeong, Ki-Hun

2017-12-01

MEMS laser scanning enables the miniaturization of endoscopic catheters for advanced endomicroscopy such as confocal microscopy, multiphoton microscopy, optical coherence tomography, and many other laser scanning microscopy. These advanced biomedical imaging modalities open a great potential for in vivo optical biopsy without surgical excision. They have huge capabilities for detecting on-demand early stage cancer with non-invasiveness. In this article, the scanning arrangement, trajectory, and actuation mechanism of endoscopic microscanners and their endomicroscopic applications will be overviewed.

Photodynamic dye adsorption and release performance of natural zeolite

PubMed Central

Hovhannisyan, Vladimir; Dong, Chen-Yuan; Chen, Shean-Jen

2017-01-01

Clinoptilolite type of zeolite (CZ) is a promising material for biomedicine and pharmaceutics due to its non-toxicity, thermal stability, expanded surface area, and exceptional ability to adsorb various atoms and organic molecules into micropores. Using multiphoton microscopy, we demonstrated that individual CZ particles produce two-photon excited luminescence and second harmonic generation signal at femtosecond laser excitation, and adsorb photo-dynamically active dyes such as hypericin and methylene blue. Furthermore, the release of hypericin from CZ pores in the presence of biomolecules is shown, and CZ can be considered as an effective material for drug delivery and controlled release in biological systems. The results may open new perspectives in application of CZ in biomedical imaging, and introducing of the optical approaches into the clinical environment. PMID:28361968

Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment.

PubMed

Ricard, Clément; Debarbieux, Franck Christian

2014-01-01

The majority of intravital studies on brain tumor in living animal so far rely on dual color imaging. We describe here a multiphoton imaging protocol to dynamically characterize the interactions between six cellular components in a living mouse. We applied this methodology to a clinically relevant glioblastoma multiforme (GBM) model designed in reporter mice with targeted cell populations labeled by fluorescent proteins of different colors. This model permitted us to make non-invasive longitudinal and multi-scale observations of cell-to-cell interactions. We provide examples of such 5D (x,y,z,t,color) images acquired on a daily basis from volumes of interest, covering most of the mouse parietal cortex at subcellular resolution. Spectral deconvolution allowed us to accurately separate each cell population as well as some components of the extracellular matrix. The technique represents a powerful tool for investigating how tumor progression is influenced by the interactions of tumor cells with host cells and the extracellular matrix micro-environment. It will be especially valuable for evaluating neuro-oncological drug efficacy and target specificity. The imaging protocol provided here can be easily translated to other mouse models of neuropathologies, and should also be of fundamental interest for investigations in other areas of systems biology.

Laser separation of lithium isotopes by double resonance enhanced multiphoton ionization of Li/sub 2/

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

Balz, J.G.; Bernheim, R.A.; Gold, L.P.

1987-01-01

Multiphoton ionization spectra of /sup 7/Li/sub 2/, /sup 6/Li/sub 2/, and /sup 7/Li/sup 6/Li vapors have been measured in the 570--650 nm region using a single, low resolution, multimode cw dye laser. A number of wavelengths provide selective multiphoton ionization of one isotopic species demonstrating the possibility of efficient laser-driven isotopic separation in lithium in this wavelength region.

Generation of single- and two-mode multiphoton states in waveguide QED

NASA Astrophysics Data System (ADS)

Paulisch, V.; Kimble, H. J.; Cirac, J. I.; González-Tudela, A.

2018-05-01

Single- and two-mode multiphoton states are the cornerstone of many quantum technologies, e.g., metrology. In the optical regime, these states are generally obtained combining heralded single photons with linear optics tools and post-selection, leading to inherent low success probabilities. In a recent paper [A. González-Tudela et al., Phys. Rev. Lett. 118, 213601 (2017), 10.1103/PhysRevLett.118.213601], we design several protocols that harness the long-range atomic interactions induced in waveguide QED to improve fidelities and protocols of single-mode multiphoton emission. Here, we give full details of these protocols, revisit them to simplify some of their requirements, and also extend them to generate two-mode multiphoton states, such as Yurke or NOON states.

Two-photon quantum walk in a multimode fiber

PubMed Central

Defienne, Hugo; Barbieri, Marco; Walmsley, Ian A.; Smith, Brian J.; Gigan, Sylvain

2016-01-01

Multiphoton propagation in connected structures—a quantum walk—offers the potential of simulating complex physical systems and provides a route to universal quantum computation. Increasing the complexity of quantum photonic networks where the walk occurs is essential for many applications. We implement a quantum walk of indistinguishable photon pairs in a multimode fiber supporting 380 modes. Using wavefront shaping, we control the propagation of the two-photon state through the fiber in which all modes are coupled. Excitation of arbitrary output modes of the system is realized by controlling classical and quantum interferences. This report demonstrates a highly multimode platform for multiphoton interference experiments and provides a powerful method to program a general high-dimensional multiport optical circuit. This work paves the way for the next generation of photonic devices for quantum simulation, computing, and communication. PMID:27152325

Examination of diagnostic features in multiphoton microscopy and optical coherence tomography images of ovarian tumorigenesis in a mouse model

NASA Astrophysics Data System (ADS)

Watson, Jennifer M.

Ovarian cancer is a deadly disease owing to the non-specific symptoms and suspected rapid progression, leading to frequent late stage detection and poor prognosis. Medical imaging methods such as CT, MRI and ultrasound as well as serum testing for cancer markers have had extremely poor performance for early disease detection. Due to the poor performance of available screening methods, and the impracticality and ineffectiveness of taking tissue biopsies from the ovary, women at high risk for developing ovarian cancer are often advised to undergo prophylactic salpingo-oophorectomy. This surgery results in many side effects and is most often unnecessary since only a fraction of high risk women go on to develop ovarian cancer. Better understanding of the early development of ovarian cancer and characterization of morphological changes associated with early disease could lead to the development of an effective screening test for women at high risk. Optical imaging methods including optical coherence tomography (OCT) and multiphoton microscopy (MPM) are excellent tools for studying disease progression owing to the high resolution and depth sectioning capabilities. Further, these techniques are excellent for optical biopsy because they can image in situ non-destructively. In the studies described in this dissertation OCT and MPM are used to identify cellular and tissue morphological changes associated with early tumor development in a mouse model of ovarian cancer. This work is organized into three specific aims. The first aim is to use the images from the MPM phenomenon of second harmonic generation to quantitatively examine the morphological differences in collagen structure in normal mouse ovarian tissue and mouse ovarian tumors. The second aim is to examine the differences in endogenous two-photon excited fluorescence in normal mouse ovarian tissue and mouse ovarian tumors. The third and final aim is to identify changes in ovarian microstructure resulting from early disease development by imaging animals in vivo at three time points during a long-term survival study.

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.

Brain plasticity and functionality explored by nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Sacconi, L.; Allegra, L.; Buffelli, M.; Cesare, P.; D'Angelo, E.; Gandolfi, D.; Grasselli, G.; Lotti, J.; Mapelli, J.; Strata, P.; Pavone, F. S.

2010-02-01

In combination with fluorescent protein (XFP) expression techniques, two-photon microscopy has become an indispensable tool to image cortical plasticity in living mice. In parallel to its application in imaging, multi-photon absorption has also been used as a tool for the dissection of single neurites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. In this work, multi-photon nanosurgery is applied to dissect single climbing fibers expressing GFP in the cerebellar cortex. The morphological consequences are then characterized with time lapse 3-dimensional two-photon imaging over a period of minutes to days after the procedure. Preliminary investigations show that the laser induced fiber dissection recalls a regenerative process in the fiber itself over a period of days. These results show the possibility of this innovative technique to investigate regenerative processes in adult brain. In parallel with imaging and manipulation technique, non-linear microscopy offers the opportunity to optically record electrical activity in intact neuronal networks. In this work, we combined the advantages of second-harmonic generation (SHG) with a random access (RA) excitation scheme to realize a new microscope (RASH) capable of optically recording fast membrane potential events occurring in a wide-field of view. The RASH microscope, in combination with bulk loading of tissue with FM4-64 dye, was used to simultaneously record electrical activity from clusters of Purkinje cells in acute cerebellar slices. Complex spikes, both synchronous and asynchronous, were optically recorded simultaneously across a given population of neurons. Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where action potentials were recorded without averaging across trials. These results show the strength of this technique in describing the temporal dynamics of neuronal assemblies, opening promising perspectives in understanding the computations of neuronal networks.

Apertureless near-field/far-field CW two-photon microscope for biological and material imaging and spectroscopic applications.

PubMed

Nowak, Derek B; Lawrence, A J; Sánchez, Erik J

2010-12-10

We present the development of a versatile spectroscopic imaging tool to allow for imaging with single-molecule sensitivity and high spatial resolution. The microscope allows for near-field and subdiffraction-limited far-field imaging by integrating a shear-force microscope on top of a custom inverted microscope design. The instrument has the ability to image in ambient conditions with optical resolutions on the order of tens of nanometers in the near field. A single low-cost computer controls the microscope with a field programmable gate array data acquisition card. High spatial resolution imaging is achieved with an inexpensive CW multiphoton excitation source, using an apertureless probe and simplified optical pathways. The high-resolution, combined with high collection efficiency and single-molecule sensitive optical capabilities of the microscope, are demonstrated with a low-cost CW laser source as well as a mode-locked laser source.

Optimizing ultrafast illumination for multiphoton-excited fluorescence imaging

PubMed Central

Stoltzfus, Caleb R.; Rebane, Aleksander

2016-01-01

We study the optimal conditions for high throughput two-photon excited fluorescence (2PEF) and three-photon excited fluorescence (3PEF) imaging using femtosecond lasers. We derive relations that allow maximization of the rate of imaging depending on the average power, pulse repetition rate, and noise characteristics of the laser, as well as on the size and structure of the sample. We perform our analysis using ~100 MHz, ~1 MHz and 1 kHz pulse rates and using both a tightly-focused illumination beam with diffraction-limited image resolution, as well loosely focused illumination with a relatively low image resolution, where the latter utilizes separate illumination and fluorescence detection beam paths. Our theoretical estimates agree with the experiments, which makes our approach especially useful for optimizing high throughput imaging of large samples with a field-of-view up to 10x10 cm2. PMID:27231620

Ultrafast optical pulse delivery with fibers for nonlinear microscopy

PubMed Central

Kim, Daekeun; Choi, Heejin; Yazdanfar, Siavash; So, Peter T. C.

2008-01-01

Nonlinear microscopies including multiphoton excitation fluorescence microscopy and multiple-harmonic generation microscopy have recently gained popularity for cellular and tissue imaging. The optimization of these imaging methods for minimally invasive use will require optical fibers to conduct light into tight space where free space delivery is difficult. The delivery of high peak power laser pulses with optical fibers is limited by dispersion resulting from nonlinear refractive index responses. In this paper, we characterize a variety of commonly used optical fibers in terms of how they affect pulse profile and imaging performance of nonlinear microscopy; the following parameters are quantified: spectral bandwidth and temporal pulse width, two-photon excitation efficiency, and optical resolution. A theoretical explanation for the measured performance of these is also provided. PMID:18816597

High-Resolution Methods for Diagnosing Cartilage Damage In Vivo

PubMed Central

Novakofski, Kira D.; Pownder, Sarah L.; Koff, Matthew F.; Williams, Rebecca M.; Potter, Hollis G.; Fortier, Lisa A.

2016-01-01

Advances in current clinical modalities, including magnetic resonance imaging and computed tomography, allow for earlier diagnoses of cartilage damage that could mitigate progression to osteoarthritis. However, current imaging modalities do not detect submicrometer damage. Developments in in vivo or arthroscopic techniques, including optical coherence tomography, ultrasonography, bioelectricity including streaming potential measurement, noninvasive electroarthrography, and multiphoton microscopy can detect damage at an earlier time point, but they are limited by a lack of penetration and the ability to assess an entire joint. This article reviews current advancements in clinical and developing modalities that can aid in the early diagnosis of cartilage injury and facilitate studies of interventional therapeutics. PMID:26958316

In vivo multiphoton microscopy of deep tissue with gradient index lenses

NASA Astrophysics Data System (ADS)

Levene, Michael J.; Dombeck, Daniel A.; Williams, Rebecca M.; Skoch, Jesse; Hickey, Gregory A.; Kasischke, Karl A.; Molloy, Raymond P.; Ingelsson, Martin; Stern, Edward A.; Klucken, Jochen; Bacskai, Brian J.; Zipfel, Warren R.; Hyman, Bradley T.; Webb, Watt W.

2004-06-01

Gradient index lenses enable multiphoton microscopy of deep tissues in the intact animal. In order to assess their applicability to clinical research, we present in vivo multiphoton microscopy with gradient index lenses in brain regions associated with Alzheimer's disease and Parkinson's disease in both transgenic and wild-type mice. We also demonstrate microscopy of ovary in wild type mouse using only intrinsic fluorescence and second harmonic generation, signal sources which may prove useful for both the study and diagnosis of cancer.

An ultrafast nanotip electron gun triggered by grating-coupled surface plasmons

NASA Astrophysics Data System (ADS)

Schröder, Benjamin; Sivis, Murat; Bormann, Reiner; Schäfer, Sascha; Ropers, Claus

2015-12-01

We demonstrate multiphoton photoelectron emission from gold nanotips induced by nanofocusing surface plasmons, resonantly excited on the tip shaft by a grating coupler. The tip is integrated into an electron gun assembly, which facilitates control over the spatial emission sites and allows us to disentangle direct grating emission from plasmon-triggered apex emission. The nanoscale source size of this electron gun concept enables highly coherent electron pulses with applications in ultrafast electron imaging and diffraction.

An ultrafast nanotip electron gun triggered by grating-coupled surface plasmons

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

Schröder, Benjamin; Sivis, Murat; Bormann, Reiner

We demonstrate multiphoton photoelectron emission from gold nanotips induced by nanofocusing surface plasmons, resonantly excited on the tip shaft by a grating coupler. The tip is integrated into an electron gun assembly, which facilitates control over the spatial emission sites and allows us to disentangle direct grating emission from plasmon-triggered apex emission. The nanoscale source size of this electron gun concept enables highly coherent electron pulses with applications in ultrafast electron imaging and diffraction.

In-vivo optical investigation of psoriasis

NASA Astrophysics Data System (ADS)

Kapsokalyvas, Dimitrios; Cicchi, Riccardo; Bruscino, Nicola; Alfieri, Domenico; Massi, Daniela; Lotti, Torello; Pavone, Francesco S.

2011-03-01

Psoriasis is an autoimmune disease of the skin characterized by hyperkeratosis, hyperproliferation of the epidermis, inflammatory cell accumulation and increased dilatation of dermal papillary blood vessels. Cases of psoriasis were investigated in vivo with optical means in order to evaluate the potential of in vivo optical biopsy. A Polarization Multispectral Dermoscope was employed for the macroscopic observation. Features such as the 'dotted' blood vessels pattern was observed with high contrast. The average size of dot vessels in Psoriasis was measured to be 974 μm2 which is much higher compared to healthy skin. High resolution image sections of the epidermis and the dermis were produced with a custom made Multiphoton Microscope. Imaging extended from the surface of the lesion down to the papillary dermis, at a depth of 200 μm. In the epidermis, a characteristic morphology of the stratum corneum found only in Psoriasis was revealed. Additionally, the cytoplasmic area of the cells in the stratum spinosum layer was found to be smaller than normal. In the dermis the morphological features were more pronounced, where the elongated dermal papillae dominated the papillary layer. Their length exceeds 100μm, which is a far greater value compared to that of healthy skin. These in vivo observations are consistent with the ex vivo histopathological observations, supporting both the applicability and potentiality of multispectral dermoscopy and multiphoton microscopy in the field of in vivo optical investigation and biopsy of skin.

Investigation of depilatory mechanism by use of multiphoton fluorescent microscopy

NASA Astrophysics Data System (ADS)

Lin, Chiao-Ying; Lee, Gie-ne; Jee, Shiou-Hwa; Dong, Chen-Yuan; Lin, Sung-Jan

2007-07-01

Transdermal drug delivery provides a non-invasive route of drug administration, and can be a alternative method to oral delivery and injection. The stratum corneum (SC) of skin acts as the main barrier to transdermal drug delivery. Studies suggest that depilatory enhances permeability of drug through the epidermis. However, transdermal delivery pathway and mechanism are not completely understood. Previous studies have found that depilatory changes the keratinocytes of epidermis, and cause the protein in combination with lipid extraction of SC to become disordered. Nevertheless, those studies did not provide images of those processes. The aim of this study is to characterize the penetration enhancing effect of depilatory agent and the associated structural alterations of stratum corneum. Fresh human foreskin is treated by a depilatory agent for 10 minutes and then subjected to the treatment of fluorescent model drugs of hydrophilic rhodamine and hydrophobic rhodamine-RE. The penetration of model drugs is imaged and quantified by multiphoton microscopy. Our results showed that the penetration of both hydrophilic and hydrophobic agents can be enhanced and multifocal detachment of surface corneocytes is revealed. Nile red staining revealed, instead of a regular motar distribution of lipid around the brick of corneocytes, a disorganized and homogenized pattern of lipid distribution. We concluded that depilatory agents enhance drug penetration by disrupting both the cellular integrity of corneocytes and the regular packing of intercellular lipid of stratum corneum.

Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP).

PubMed

Yang, Sunny Y; Amor, Souheila; Laguerre, Aurélien; Wong, Judy M Y; Monchaud, David

2017-05-01

The development of quadruplex-directed molecular diagnostic and therapy rely on mechanistic insights gained at both cellular and tissue levels by fluorescence imaging. This technique is based on fluorescent reporters that label cellular DNA and RNA quadruplexes to spatiotemporally address their complex cell biology. The photophysical characteristics of quadruplex probes usually dictate the modality of cell imaging by governing the selection of the light source (lamp, LED, laser), the optical light filters and the detection modality. Here, we report the characterizations of prototype from a new generation of quadruplex dye termed G4-REP (for quadruplex-specific red-edge probe) that provides fluorescence responses regardless of the excitation wavelength and modality (owing to the versatility gained through the red-edge effect), thus allowing for diverse applications and most imaging facilities. This is demonstrated by cell images (and associated quantifications) collected through confocal and multiphoton microscopy as well as through real-time live-cell imaging system over extended period, monitoring both non-cancerous and cancerous human cell lines. Our results promote a new way of designing versatile, efficient and convenient quadruplex-reporting dyes for tracking these higher-order nucleic acid structures in living human cells. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

Multi-photon UV photolysis of gaseous polycyclic aromatic hydrocarbons: Extinction spectra and dynamics

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

Walsh, A. J.; Gash, E. W.; Mansfield, M. W. D.

The extinction spectra of static naphthalene and static biphenylene vapor, each buffered with a noble gas at room temperature, were measured as a function of time in the region between 390 and 850 nm after UV multi-photon laser photolysis at 308 nm. Employing incoherent broadband cavity enhanced absorption spectroscopy (IBBCEAS), the spectra were found to be unstructured with a general lack of isolated features suggesting that the extinction was not solely based on absorption but was in fact dominated by scattering from particles formed in the photolysis of the respective polycyclic aromatic hydrocarbon. Following UV multi-photon photolysis, the extinction dynamicsmore » of the static (unstirred) closed gas-phase system exhibits extraordinary quasi-periodic and complex oscillations with periods ranging from seconds to many minutes, persisting for up to several hours. Depending on buffer gas type and pressure, several types of dynamical responses could be generated (classified as types I, II, and III). They were studied as a function of temperature and chamber volume for different experimental conditions and possible explanations for the oscillations are discussed. A conclusive model for the observed phenomena has not been established. However, a number of key hypotheses have made based on the measurements in this publication: (a) Following the multi-photon UV photolysis of naphthalene (or biphenylene), particles are formed on a timescale not observable using IBBCEAS. (b) The observed temporal behavior cannot be described on basis of a chemical reaction scheme alone. (c) The pressure dependence of the system's responses is due to transport phenomena of particles in the chamber. (d) The size distribution and the refractive indices of particles are time dependent and evolve on a timescale of minutes to hours. The rate of particle coagulation, involving coalescent growth and particle agglomeration, affects the observed oscillations. (e) The walls of the chamber act as a sink. The wall conditions (which could not be quantitatively characterized) have a profound influence on the dynamics of the system and on its slow return to an equilibrium state.« less

Multi-photon UV photolysis of gaseous polycyclic aromatic hydrocarbons: Extinction spectra and dynamics

NASA Astrophysics Data System (ADS)

Walsh, A. J.; Ruth, A. A.; Gash, E. W.; Mansfield, M. W. D.

2013-08-01

The extinction spectra of static naphthalene and static biphenylene vapor, each buffered with a noble gas at room temperature, were measured as a function of time in the region between 390 and 850 nm after UV multi-photon laser photolysis at 308 nm. Employing incoherent broadband cavity enhanced absorption spectroscopy (IBBCEAS), the spectra were found to be unstructured with a general lack of isolated features suggesting that the extinction was not solely based on absorption but was in fact dominated by scattering from particles formed in the photolysis of the respective polycyclic aromatic hydrocarbon. Following UV multi-photon photolysis, the extinction dynamics of the static (unstirred) closed gas-phase system exhibits extraordinary quasi-periodic and complex oscillations with periods ranging from seconds to many minutes, persisting for up to several hours. Depending on buffer gas type and pressure, several types of dynamical responses could be generated (classified as types I, II, and III). They were studied as a function of temperature and chamber volume for different experimental conditions and possible explanations for the oscillations are discussed. A conclusive model for the observed phenomena has not been established. However, a number of key hypotheses have made based on the measurements in this publication: (a) Following the multi-photon UV photolysis of naphthalene (or biphenylene), particles are formed on a timescale not observable using IBBCEAS. (b) The observed temporal behavior cannot be described on basis of a chemical reaction scheme alone. (c) The pressure dependence of the system's responses is due to transport phenomena of particles in the chamber. (d) The size distribution and the refractive indices of particles are time dependent and evolve on a timescale of minutes to hours. The rate of particle coagulation, involving coalescent growth and particle agglomeration, affects the observed oscillations. (e) The walls of the chamber act as a sink. The wall conditions (which could not be quantitatively characterized) have a profound influence on the dynamics of the system and on its slow return to an equilibrium state.

Modification of emission photon statistics from single quantum dots using metal/SiO2 core/shell nanostructures.

PubMed

Naiki, Hiroyuki; Oikawa, Hidetoshi; Masuo, Sadahiro

2017-04-12

Emission photon statistics, i.e., single-photon and multi-photon emissions, of isolated QDs is required for tailoring optoelectronic applications. In this article, we demonstrate that the emission photon statistics can be modified by the control of the spectral overlap of the QDs with the localized surface plasmon resonance (LSPR) of the metal nanoparticle (metal NP) and by the distance between the QD and the metal NP. Moreover, the contribution to the modification of the emission photon statistics, which is the excitation and emission enhancements and the quenching generated by the spectral overlap and the distance, is elucidated. By fabricating well-defined SiO 2 -coated AgNPs and AuNPs (metal/SiO 2 ), the spectral overlap originated from the metal species of Ag and Au and the distance constituted by the thickness of the SiO 2 shell are controlled. The probability of single-photon emission of single QD was increased by the enhancement of the excitation rate via adjusting the distance using Ag/SiO 2 while the single-photon emission was converted to multi-photon emission by the effect of exciton quenching at a short distance and a small spectral overlap. By contrast, the probability of multi-photon emission was increased by enhancement of the multi-photon emission rate and the quenching via the spectral overlap using Au/SiO 2 . These results indicated the fundamental finding to control emission photon statistics in single QDs by controlling the spectral overlap and the distance, and understand the interaction of plasmonic nanostructures and single QD systems.

Ion, X-ray, UV and Neutron Microbeam Systems for Cell Irradiation.

PubMed

Bigelow, A W; Randers-Pehrson, G; Garty, G; Geard, C R; Xu, Y; Harken, A D; Johnson, G W; Brenner, D J

2010-08-08

The array of microbeam cell-irradiation systems, available to users at the Radiological Research Accelerator Facility (RARAF), Center for Radiological Research, Columbia University, is expanding. The HVE 5MV Singletron particle accelerator at the facility provides particles to two focused ion microbeam lines: the sub-micron microbeam II and the permanent magnetic microbeam (PMM). Both the electrostatic quadrupole lenses on the microbeam II system and the magnetic quadrupole lenses on the PMM system are arranged as compound lenses consisting of two quadrupole triplets with "Russian" symmetry. Also, the RARAF accelerator is a source for a proton-induced x-ray microbeam (undergoing testing) and is projected to supply protons to a neutron microbeam based on the (7)Li(p, n)(7)Be nuclear reaction (under development). Leveraging from the multiphoton microscope technology integrated within the microbeam II endstation, a UV microspot irradiator - based on multiphoton excitation - is available for facility users. Highlights from radiation-biology demonstrations on single living mammalian cells are included in this review of microbeam systems for cell irradiation at RARAF.

Robust distant-entanglement generation using coherent multiphoton scattering

NASA Astrophysics Data System (ADS)

Chan, Ching-Kit; Sham, L. J.

2013-03-01

The generation and controllability of entanglement between distant quantum states have been the heart of quantum computation and quantum information processing. Existing schemes for solid state qubit entanglement are based on the single-photon spectroscopy that has the merit of a high fidelity entanglement creation, but with a very limited efficiency. This severely restricts the scalability for a qubit network system. Here, we describe a new distant entanglement protocol using coherent multiphoton scattering. The scheme makes use of the postselection of large and distinguishable photon signals, and has both a high success probability and a high entanglement fidelity. Our result shows that the entanglement generation is robust against photon fluctuations, and has an average entanglement duration within the decoherence time in various qubit systems, based on existing experimental parameters. This research was supported by the U.S. Army Research Office MURI award W911NF0910406 and by NSF grant PHY-1104446.

Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

NASA Astrophysics Data System (ADS)

Darvin, M. E.; Richter, H.; Zhu, Y. J.; Meinke, M. C.; Knorr, F.; Gonchukov, S. A.; Koenig, K.; Lademann, J.

2014-07-01

Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted.

Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

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

Darvin, M E; Richter, H; Zhu, Y J

Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed bymore » using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted. (laser biophotonics)« less

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

PubMed Central

Vielreicher, M.; Schürmann, S.; Detsch, R.; Schmidt, M. A.; Buttgereit, A.; Boccaccini, A.; Friedrich, O.

2013-01-01

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging. PMID:23864499

Development of an applicator for multiphoton PDT

NASA Astrophysics Data System (ADS)

Graschew, Georgi; Bastian, Matthias; Rakowsky, Stefan; Roelofs, Theo A.; Balanos, Evangelos; Schlag, Peter M.; Steinmeyer, Gunter; Elsaesser, Thomas

2004-09-01

Multiphoton excitation of photosensitizers for laser induced fluorescence diagnosis (LIFD) and photodynamic therapy (PDT) of tumors has the advantage of greater tissue penetration due to the longer wavelength of irradiation. However, multiphoton LIFD and PDT are presently not clinically applicable as there are no applicators available for the delivery of the pulsed laser radiation to the operating room. As an approach, in this contribution the beam delivery through photonic crystal fibers has been investigated. Pulses of a Ti:sapphire laser of 100 fs pulse duration and an average power of 150 mW have been transported through such a fiber of 25 m length and the resulting pulses show the absence of nonlinear contributions but still a broadening of the pulse to 2 ps due to the dispersion of the fiber. It is planned to compensate this broadening by a grating in front of the fiber. Alternatively, the transport of laser radiation of 150 fs and 100 mW through a mirror-joint-arm used for conventional CO2 lasers has been tested showing no broadening of the laser pulses. Two-photon photodynamic activity of mTHPC-CMPEG4 shall serve as a test of the laser light transport system.

Functional enucleation of porcine oocytes for somatic cell nuclear transfer using femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Kuetemeyer, K.; Lucas-Hahn, A.; Petersen, B.; Hassel, P.; Lemme, E.; Niemann, H.; Heisterkamp, A.

2010-02-01

Cloning of several mammalian species has been achieved by somatic cell nuclear transfer over the last decade. However, this method still results in very low efficiencies originating from biological and technical aspects. The highly-invasive mechanical enucleation belongs to the technical aspects and requires considerable micromanipulation skill. In this paper, we present a novel non-invasive method for combined oocyte imaging and automated functional enucleation using femtosecond (fs) laser pulses. After three-dimensional imaging of Hoechst-labeled porcine oocytes by multiphoton microscopy, our self-developed software automatically determined the metaphase plate position and shape. Subsequent irradiation of this volume with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation. We show that functional fs laser-based enucleation of porcine oocytes completely inhibited further embryonic development while maintaining intact oocyte morphology. In contrast, non-irradiated oocytes were able to develop to the blastocyst stage without significant differences to control oocytes. Our results indicate that fs laser systems offer great potential for oocyte imaging and enucleation as a fast, easy to use and reliable tool which may improve the efficiency of somatic cell clone production.

Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery.

PubMed

Jalani, Ghulam; Naccache, Rafik; Rosenzweig, Derek H; Haglund, Lisbet; Vetrone, Fiorenzo; Cerruti, Marta

2016-01-27

Lanthanide-doped upconverting nanoparticles (UCNPs) have emerged as excellent nanotransducers for converting longer wavelength near-infrared (NIR) light to shorter wavelengths spanning the ultraviolet (UV) to the visible (Vis) regions of the spectrum via a multiphoton absorption process, known as upconversion. Here, we report the development of NIR to UV-Vis-NIR UCNPs consisting of LiYF4:Yb(3+)/Tm(3+)@SiO2 individually coated with a 10 ± 2 nm layer of chitosan (CH) hydrogel cross-linked with a photocleavable cross-linker (PhL). We encapsulated fluorescent-bovine serum albumin (FITC-BSA) inside the gel. Under 980 nm excitation, the upconverted UV emission cleaves the PhL cross-links and instantaneously liberates the FITC-BSA under 2 cm thick tissue. The release is immediately arrested if the excitation source is switched off. The upconverted NIR light allows for the tracking of particles under the tissue. Nucleus pulposus (NP) cells cultured with UCNPs are viable both in the presence and in the absence of laser irradiation. Controlled drug delivery of large biomolecules and deep tissue imaging make this system an excellent theranostic platform for tissue engineering, biomapping, and cellular imaging applications.

Enhanced eumelanin emission by stepwise three-photon excitation

NASA Astrophysics Data System (ADS)

Kerimo, Josef; Rajadhyaksha, Milind; DiMarzio, Charles A.

2011-03-01

Eumelanin fluorescence from Sepia officinalis and black human hair was activated with near-infrared radiation and multiphoton excitation. A third order multiphoton absorption by a step-wise process appears to be the underlying mechanism. The activation was caused by a photochemical process since it could not be reproduced by simple heating. Both fluorescence and brightfield imaging indicate the near-infrared irradiation caused photodamage to the eumelanin and the activated emission originated from the photodamaged region. At least two different components with about thousand-fold enhanced fluorescence were activated and could be distinguished by their excitation properties. One component was excited with wavelengths in the visible region and exhibited linear absorption dependence. The second component could be excited with near-infrared wavelengths and had a third order dependence on the laser power. The third order dependence is explained by a step-wise excited state absorption (ESA) process since it could be observed equally with the CW and femtosecond lasers. The new method for photoactivating the eumelanin fluorescence was used to map the melanin content in human hair.

Multiphoton luminescent graphene quantum dots for in vivo tracking of human adipose-derived stem cells

NASA Astrophysics Data System (ADS)

Kim, Jin; Song, Sung Ho; Jin, Yoonhee; Park, Hyun-Ji; Yoon, Hyewon; Jeon, Seokwoo; Cho, Seung-Woo

2016-04-01

The applicability of graphene quantum dots (GQDs) for the in vitro and in vivo live imaging and tracking of different types of human stem cells is investigated. GQDs synthesized by the modified graphite intercalated compound method show efficient cellular uptake with improved biocompatibility and highly sensitive optical properties, indicating their feasibility as a bio-imaging probe for stem cell therapy.The applicability of graphene quantum dots (GQDs) for the in vitro and in vivo live imaging and tracking of different types of human stem cells is investigated. GQDs synthesized by the modified graphite intercalated compound method show efficient cellular uptake with improved biocompatibility and highly sensitive optical properties, indicating their feasibility as a bio-imaging probe for stem cell therapy. Electronic supplementary information (ESI) available: Additional results. See DOI: 10.1039/c6nr02143c

Calculation of multiphoton ionization processes

NASA Technical Reports Server (NTRS)

Chang, T. N.; Poe, R. T.

1976-01-01

We propose an accurate and efficient procedure in the calculation of multiphoton ionization processes. In addition to the calculational advantage, this procedure also enables us to study the relative contributions of the resonant and nonresonant intermediate states.

Quantum cryptography with perfect multiphoton entanglement.

PubMed

Luo, Yuhui; Chan, Kam Tai

2005-05-01

Multiphoton entanglement in the same polarization has been shown theoretically to be obtainable by type-I spontaneous parametric downconversion (SPDC), which can generate bright pulses more easily than type-II SPDC. A new quantum cryptographic protocol utilizing polarization pairs with the detected type-I entangled multiphotons is proposed as quantum key distribution. We calculate the information capacity versus photon number corresponding to polarization after considering the transmission loss inside the optical fiber, the detector efficiency, and intercept-resend attacks at the level of channel error. The result compares favorably with all other schemes employing entanglement.

Multiphoton Absorption is Probably Not the Primary Threshold Damage Mechanism for Femtosecond Laser Pulse Exposures in the Retinal Pigment Epithelium

DTIC Science & Technology

2004-01-01

Tromberg, and E. Gratton, "Two-photon excited lifetime imaging of autofluorescence in cells during UTVA and NIR photostress", J. Micros. 183, pp. 197-204...1996. 4. K. Konig, Y. Liu, G. J. Sonek, M. W. Berns, and B. J. Tromberg, " Autofluorescence spectroscopy of optically trapped cells", Photochem...34, Photochem. Photobiol. 70, pp. 146-151, 1999. 10. R. D. Glickman, "Phototoxicity to the retina : Mechanisms of damage", International Journal of

Fluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Batista, Ana; König, Karsten

2014-02-01

The multiphoton FLIM tomograph MPTflex with its flexible scan head, articulated arm, and the tunable femtosecond laser source was employed to study cell monolayers and 3D cell clusters. FLIM was performed with 250 ps temporal resolution and submicron special resolution using time-correlated single photon counting. The autofluorescence based on NAD(P)H and flavins/flavoproteins has been measured in mouse embryonic fibroblasts, induced pluripotent stem cells (iPS cells) originated from mouse embryonic fibroblasts and non-proliferative mouse embryonic fibroblasts.

Laser-induced radiation microbeam technology and simultaneous real-time fluorescence imaging in live cells.

PubMed

Botchway, Stanley W; Reynolds, Pamela; Parker, Anthony W; O'Neill, Peter

2012-01-01

The use of nano- and microbeam techniques to induce and identify subcellular localized energy deposition within a region of a living cell provides a means to investigate the effects of low radiation doses. Particularly within the nucleus where the propagation and processing of deoxyribonucleic acid (DNA) damage (and repair) in both targeted and nontargeted cells, the latter being able to study cell-cell (bystander) effects. We have pioneered a near infrared (NIR) femtosecond laser microbeam to mimic ionizing radiation through multiphoton absorption within a 3D femtoliter volume of a highly focused Gaussian laser beam. The novel optical microbeam mimics both complex ionizing and UV-radiation-type cell damage including double strand breaks (DSBs). Using the microbeam technology, we have been able to investigate the formation of DNA DSB and subsequent recruitment of repair proteins to the submicrometer size site of damage introduced in viable cells. The use of a phosphorylated H2AX (γ-H2AX a marker for DSBs, visualized by immunofluorescent staining) and real-time imaging of fluorescently labeling proteins, the dynamics of recruitment of repair proteins in viable mammalian cells can be observed. Here we show the recruitment of ATM, p53 binding protein 1 (53BP1), and RAD51, an integral protein of the homologous recombination process in the DNA repair pathway and Ku-80-GFP involved in the nonhomologous end joining (NHEJ) pathway as exemplar repair process to show differences in the repair kinetics of DNA DSBs. The laser NIR multiphoton microbeam technology shows persistent DSBs at later times post laser irradiation which are indicative of DSBs arising at replication presumably from UV photoproducts or clustered damage containing single strand breaks (SSBs) that are also observed. Effects of the cell cycle may also be investigated in real time. Postirradiation and fixed cells studies show that in G1 cells a fraction of multiphoton laser-induced DSBs is persistent for >6h in addition to those induced at replication demonstrating the broad range of timescales taken to repair DNA damage. Copyright © 2012 Elsevier Inc. All rights reserved.

Processing multiphoton states through operation on a single photon: Methods and applications

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

Lin Qing; He Bing; Bergou, Janos A.

2009-10-15

Multiphoton states are widely applied in quantum information technology. By the methods presented in this paper, the structure of a multiphoton state in the form of multiple single-photon qubit products can be mapped to a single-photon qudit, which could also be in a separable product with other photons. This makes possible the manipulation of such multiphoton states by processing single-photon states. The optical realization of unknown qubit discrimination [B. He, J. A. Bergou, and Y.-H. Ren, Phys. Rev. A 76, 032301 (2007)] is simplified with the transformation methods. Another application is the construction of quantum logic gates, where the inversemore » transformations back to the input state spaces are also necessary. We especially show that the modified setups to implement the transformations can realize the deterministic multicontrol gates (including Toffoli gate) operating directly on the products of single-photon qubits.« less

A CANDLE for a deeper in vivo insight

PubMed Central

Coupé, Pierrick; Munz, Martin; Manjón, Jose V; Ruthazer, Edward S; Louis Collins, D.

2012-01-01

A new Collaborative Approach for eNhanced Denoising under Low-light Excitation (CANDLE) is introduced for the processing of 3D laser scanning multiphoton microscopy images. CANDLE is designed to be robust for low signal-to-noise ratio (SNR) conditions typically encountered when imaging deep in scattering biological specimens. Based on an optimized non-local means filter involving the comparison of filtered patches, CANDLE locally adapts the amount of smoothing in order to deal with the noise inhomogeneity inherent to laser scanning fluorescence microscopy images. An extensive validation on synthetic data, images acquired on microspheres and in vivo images is presented. These experiments show that the CANDLE filter obtained competitive results compared to a state-of-the-art method and a locally adaptive optimized nonlocal means filter, especially under low SNR conditions (PSNR<8dB). Finally, the deeper imaging capabilities enabled by the proposed filter are demonstrated on deep tissue in vivo images of neurons and fine axonal processes in the Xenopus tadpole brain. PMID:22341767

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

Breast Cancer Treatment in the Era of Molecular Imaging

PubMed Central

Edelhauser, Gundula; Funovics, Martin

2008-01-01

Summary Molecular imaging employs molecularly targeted probes to visualize and often quantify distinct disease-specific markers and pathways. Modalities like intravital confocal or multiphoton microscopy, near-infrared fluorescence combined with endoscopy, surface reflectance imaging, or fluorescence-mediated tomography, and radionuclide imaging with positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are increasingly used for small animal high-throughput screening, drug development and testing, and monitoring gene therapy experiments. In the clinical treatment of breast cancer, PET and SPECT as well as magnetic resonance-based molecular imaging are already established for the staging of distant disease and intrathoracic nodal status, for patient selection regarding receptor-directed treatments, and to gain early information about treatment efficacy. In the near future, reporter gene imaging during gene therapy and further spatial and qualitative characterization of the disease can become clinically possible with radionuclide and optical methods. Ultimately, it may be expected that every level of breast cancer treatment will be affected by molecular imaging, including screening. PMID:21048912

Exploring infrared neural stimulation with multimodal nonlinear imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Adams, Wilson R.; Mahadevan-Jansen, Anita

2017-02-01

Infrared neural stimulation (INS) provides optical control of neural excitability using near to mid-infrared (mid-IR) light, which allows for spatially selective, artifact-free excitation without the introduction of exogenous agents or genetic modification. Although neural excitability is mediated by a transient temperature increase due to water absorption of IR energy, the molecular nature of IR excitability in neural tissue remains unknown. Current research suggests that transient changes in local tissue temperature give rise to a myriad of cellular responses that have been individually attributed to IR mediated excitability. To further elucidate the underlying biophysical mechanisms, we have begun work towards employing a novel multimodal nonlinear imaging platform to probe the molecular underpinnings of INS. Our imaging system performs coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS), two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG) and thermal imaging into a single platform that allows for unprecedented co-registration of thermal and biochemical information in real-time. Here, we present our work leveraging CARS and SRS in acute thalamocortical brain slice preparations. We observe the evolution of lipid and protein-specific Raman bands during INS and electrically evoked activity in real-time. Combined with two-photon fluorescence and second harmonic generation, we offer insight to cellular metabolism and membrane dynamics during INS. Thermal imaging allows for the coregistration of acquired biochemical information with temperature information. Our work previews the versatility and capabilities of coherent Raman imaging combined with multiphoton imaging to observe biophysical phenomena for neuroscience applications.

Optical tomography of human skin with subcellular spatial and picosecond time resolution using intense near infrared femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Koenig, Karsten; Wollina, Uwe; Riemann, Iris; Peukert, Christiane; Halbhuber, Karl-Juergen; Konrad, Helga; Fischer, Peter; Fuenfstueck, Veronika; Fischer, Tobias W.; Elsner, Peter

2002-06-01

We describe the novel high resolution imaging tool DermaInspect 100 for non-invasive diagnosis of dermatological disorders based on multiphoton autofluorescence imaging (MAI)and second harmonic generation. Femtosecond laser pulses in the spectral range of 750 nm to 850 nm have been used to image in vitro and in vivo human skin with subcellular spatial and picosecond temporal resolution. The non-linear induced autofluorescence originates mainly from naturally endogenous fluorophores/protein structures like NAD(P)H, flavins, keratin, collagen, elastin, porphyrins and melanin. Second harmonic generation was observed in the stratum corneum and in the dermis. The system with a wavelength-tunable compact 80 MHz Ti:sapphire laser, a scan module with galvo scan mirrors, piezoelectric objective positioner, fast photon detector and time-resolved single photon counting unit was used to perform optical sectioning and 3D autofluorescence lifetime imaging (t-mapping). In addition, a modified femtosecond laser scanning microscope was involved in autofluorescence measurements. Tissues of patients with psoriasis, nevi, dermatitis, basalioma and melanoma have been investigated. Individual cells and skin structures could be clearly visualized. Intracellular components and connective tissue structures could be further characterized by tuning the excitation wavelength in the range of 750 nm to 850 nm and by calculation of mean fluorescence lifetimes per pixel and of particular regions of interest. The novel non-invasive imaging system provides 4D (x,y,z,t) optical biopsies with subcellular resolution and offers the possibility to introduce a further optical diagnostic method in dermatology.

Photothermal optical coherence tomography of epidermal growth factor receptor in live cells using immunotargeted gold nanospheres

NASA Astrophysics Data System (ADS)

Skala, Melissa C.; Crow, Matthew J.; Wax, Adam; Izatt, Joseph A.

2009-02-01

Molecular imaging is a powerful tool for investigating disease processes and potential therapies in both in vivo and in vitro systems. However, high resolution molecular imaging has been limited to relatively shallow penetration depths that can be accessed with microscopy. Optical coherence tomography (OCT) is an optical analogue to ultrasound with relatively good penetration depth (1-2 mm) and resolution (~1-10 μm). We have developed and characterized photothermal OCT as a molecular contrast mechanism that allows for high resolution molecular imaging at deeper penetration depths than microscopy. Our photothermal system consists of an amplitude-modulated heating beam that spatially overlaps with the focused spot of the sample arm of a spectral-domain OCT microscope. Validation experiments in tissue-like phantoms containing gold nanospheres that absorb at 532 nm revealed a sensitivity of 14 parts per million nanospheres (weight/weight) in a tissue-like environment. The nanospheres were then conjugated to anti-EGFR, and molecular targeting was confirmed in cells that over-express EGFR (MDA-MB-468) and cells that express low levels of EGFR (MDA-MB-435). Molecular imaging in three-dimensional tissue constructs was confirmed with a significantly lower photothermal signal (p<0.0001) from the constructs composed of cells that express low levels of EGFR compared to the over-expressing cell constructs (300% signal increase). This technique could potentially augment confocal and multiphoton microscopy as a method for deep-tissue, depth-resolved molecular imaging with relatively high resolution and target sensitivity, without photobleaching or cytotoxicity.

Optical metabolic imaging for monitoring tracheal health

NASA Astrophysics Data System (ADS)

Sharick, Joe T.; Gil, Daniel A.; Choma, Michael A.; Skala, Melissa C.

2016-04-01

The health of the tracheal mucosa and submucosa is a vital yet poorly understood component of critical care medicine, and a minimally-invasive method is needed to monitor tracheal health in patients. Of particular interest are the ciliated cells of the tracheal epithelium that move mucus away from the lungs and prevent respiratory infection. Optical metabolic imaging (OMI) allows cellular-level measurement of metabolism, and is a compelling method for assessing tracheal health because ciliary motor proteins require ATP to function. In this pilot study, we apply multiphoton imaging of the fluorescence intensities and lifetimes of metabolic co-enzymes NAD(P)H and FAD to the mucosa and submucosa of ex vivo mouse trachea. We demonstrate the feasibility and potential diagnostic utility of these measurements for assessing tracheal health and pathophysiology at the single-cell level.

Fiber-based tunable repetition rate source for deep tissue two-photon fluorescence microscopy.

PubMed

Charan, Kriti; Li, Bo; Wang, Mengran; Lin, Charles P; Xu, Chris

2018-05-01

Deep tissue multiphoton imaging requires high peak power to enhance signal and low average power to prevent thermal damage. Both goals can be advantageously achieved through laser repetition rate tuning instead of simply adjusting the average power. We show that the ideal repetition rate for deep two-photon imaging in the mouse brain is between 1 and 10 MHz, and we present a fiber-based source with an arbitrarily tunable repetition rate within this range. The performance of the new source is compared to a mode-locked Ti:Sapphire (Ti:S) laser for in vivo imaging of mouse brain vasculature. At 2.5 MHz, the fiber source requires 5.1 times less average power to obtain the same signal as a standard Ti:S laser operating at 80 MHz.

Multimodal Nonlinear Optical Microscopy

PubMed Central

Yue, Shuhua; Slipchenko, Mikhail N.; Cheng, Ji-Xin

2013-01-01

Because each nonlinear optical (NLO) imaging modality is sensitive to specific molecules or structures, multimodal NLO imaging capitalizes the potential of NLO microscopy for studies of complex biological tissues. The coupling of multiphoton fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering (CARS) has allowed investigation of a broad range of biological questions concerning lipid metabolism, cancer development, cardiovascular disease, and skin biology. Moreover, recent research shows the great potential of using CARS microscope as a platform to develop more advanced NLO modalities such as electronic-resonance-enhanced four-wave mixing, stimulated Raman scattering, and pump-probe microscopy. This article reviews the various approaches developed for realization of multimodal NLO imaging as well as developments of new NLO modalities on a CARS microscope. Applications to various aspects of biological and biomedical research are discussed. PMID:24353747

Label-free imaging of atherosclerotic plaques using third-harmonic generation microscopy

PubMed Central

Small, David M.; Jones, Jason S.; Tendler, Irwin I.; Miller, Paul E.; Ghetti, Andre; Nishimura, Nozomi

2017-01-01

Multiphoton microscopy using laser sources in the mid-infrared range (MIR, 1,300 nm and 1,700 nm) was used to image atherosclerotic plaques from murine and human samples. Third harmonic generation (THG) from atherosclerotic plaques revealed morphological details of cellular and extracellular lipid deposits. Simultaneous nonlinear optical signals from the same laser source, including second harmonic generation and endogenous fluorescence, resulted in label-free images of various layers within the diseased vessel wall. The THG signal adds an endogenous contrast mechanism with a practical degree of specificity for atherosclerotic plaques that complements current nonlinear optical methods for the investigation of cardiovascular disease. Our use of whole-mount tissue and backward scattered epi-detection suggests THG could potentially be used in the future as a clinical tool. PMID:29359098

Why Do Silver Trimers Intercalated in DNA Exhibit Unique Nonlinear Properties That Are Promising for Applications?

PubMed

Bonačić-Koutecký, Vlasta; Perić, Martina; Sanader, Željka

2018-05-17

Our investigation of one-photon absorption (OPA) and nonlinear optical (NLO) properties such as two-photon absorption (TPA) of silver trimer intercalated in DNA based on TDDFT approach allowed us to propose a mechanism responsible for large TPA cross sections of such NLO-phores. We present a concept that illustrates the key role of quantum cluster as well as of nucleotide bases from the immediate neighborhood. For this purpose, different surroundings consisting of guanine-cytosine and adenine-thymine such as (GCGC) and (ATAT) have been investigated that are exhibiting substantially different values of TPA cross sections. This has been confirmed by extending the immediate surroundings as well as using the two-layer quantum mechanics/molecular mechanics (QM/MM) approach. We focus on the cationic closed-shell system and illustrate that the neutral open-shell system shifts OPA spectra into the NIR regime, which is suitable for applications. Thus, in this contribution, we propose novel NLO-phores inducing large TPA cross sections, opening the route for multiphoton imaging.

Spatial cell firing during virtual navigation of open arenas by head-restrained mice.

PubMed

Chen, Guifen; King, John Andrew; Lu, Yi; Cacucci, Francesca; Burgess, Neil

2018-06-18

We present a mouse virtual reality (VR) system which restrains head-movements to horizontal rotations, compatible with multi-photon imaging. This system allows expression of the spatial navigation and neuronal firing patterns characteristic of real open arenas (R). Comparing VR to R: place and grid, but not head-direction, cell firing had broader spatial tuning; place, but not grid, cell firing was more directional; theta frequency increased less with running speed; whereas increases in firing rates with running speed and place and grid cells' theta phase precession were similar. These results suggest that the omni-directional place cell firing in R may require local-cues unavailable in VR, and that the scale of grid and place cell firing patterns, and theta frequency, reflect translational motion inferred from both virtual (visual and proprioceptive) and real (vestibular translation and extra-maze) cues. By contrast, firing rates and theta phase precession appear to reflect visual and proprioceptive cues alone. © 2018, Chen et al.

Optimizing Ti:Sapphire laser for quantitative biomedical imaging

NASA Astrophysics Data System (ADS)

James, Jeemol; Thomsen, Hanna; Hanstorp, Dag; Alemán Hérnandez, Felipe Ademir; Rothe, Sebastian; Enger, Jonas; Ericson, Marica B.

2018-02-01

Ti:Sapphire lasers are powerful tools in the field of scientific research and industry for a wide range of applications such as spectroscopic studies and microscopic imaging where tunable near-infrared light is required. To push the limits of the applicability of Ti:Sapphire lasers, fundamental understanding of the construction and operation is required. This paper presents two projects, (i) dealing with the building and characterization of custom built tunable narrow linewidth Ti:Sapphire laser for fundamental spectroscopy studies; and the second project (ii) the implementation of a fs-pulsed commercial Ti:Sapphire laser in an experimental multiphoton microscopy platform. For the narrow linewidth laser, a gold-plated diffraction grating with a Littrow geometry was implemented for highresolution wavelength selection. We demonstrate that the laser is tunable between 700 to 950 nm, operating in a pulsed mode with a repetition rate of 1 kHz and maximum average output power around 350 mW. The output linewidth was reduced from 6 GHz to 1.5 GHz by inserting an additional 6 mm thick etalon. The bandwidth was measured by means of a scanning Fabry Perot interferometer. Future work will focus on using a fs-pulsed commercial Ti:Sapphire laser (Tsunami, Spectra physics), operating at 80 MHz and maximum average output power around 1 W, for implementation in an experimental multiphoton microscopy set up dedicated for biomedical applications. Special focus will be on controlling pulse duration and dispersion in the optical components and biological tissue using pulse compression. Furthermore, time correlated analysis of the biological samples will be performed with the help of time correlated single photon counting module (SPCM, Becker&Hickl) which will give a novel dimension in quantitative biomedical imaging.

EDITORIAL: Theory of Quantum Gases and Quantum Coherence: The Cortona BEC Workshop, 29 October-2 November 2005

NASA Astrophysics Data System (ADS)

Capuzzi, Pablo; Chitra, R.; Menotti, Chiara; Minguzz, Anna; Vignolo, Patrizia

2006-05-01

Nonlinear, or multiphoton, interaction of intense laser radiation with matter has been a key research subject for about four decades. Every three years, the International Conference on Multiphoton Processes (ICOMP) covers the latest advances in the field. Intense-field physics has seen phenomenal progress over the last decade. What looked like dreams in the mid-nineties have become routine today. Major theoretical, experimental and technological advances in fundamental science and applications of multiphoton processes cover such diverse areas as precision measurements, femtosecond and now attosecond metrology, quantum control of atomic and molecular dynamics, laser machining of solid state materials, laser acceleration of electrons and protons, and medical applications. This special issue of Journal of Physics B: Atomic, Molecular and Optical Physics (J. Phys. B) contains a collection of articles originating from the Tenth International Conference on Multiphoton Processes (ICOMP 2005) held on 9-14 October 2005 in Orford, Quebec, Canada (general chair Lou DiMauro, Ohio State University, program co-chairs Paul Corkum and Misha Ivanov, National Research Council of Canada). The conference focused on atoms and molecules in strong fields, femtosecond and attosecond processes, propagation of intense pulses, and of course multiphoton processes which lie at the foundation of all these subjects. Articles presented in this issue cover several key areas of intense-field physics. These include strong field ionization of atoms, molecules and inside transparent dielectric materials, methods of generation and characterization of attosecond XUV pulses and pulse trains, and new approaches to using intense laser fields and/or attosecond pulses for studying entangled systems and imaging electronic and nuclear dynamics with sub-Ångstrom spatial and sub-femtosecond temporal resolution. We have tried to group the papers according to these general areas. We would like to use this opportunity to thank all the participants of ICOMP-X, and in particular the contributors to this issue, for the high quality of science presented at the conference and in this journal. The success of the conference would not have been possible without the program committee which included D Charalambidis, L Cocke, R Freeman, Y Fujimura, S Goreslavsky, A L'Huillier, F Krausz, R Levis, S H Lin, A Maquet, J Marangos, K Midorikawa, G Mourou, P Salieres, W Sandner, K Schafer, A Scrinzi, A M Sergeev, H Stapelfeldt, A Starace, J Ullrich, M Vrakking, and K Yamanouchi. A particularly lively atmosphere in the discussions was ensured by many students who were able to participate in the conference, in part due to generous support of the Canadian Institute for Photonic Innovations (CIPI) to the Canadian, and of the US Department of Energy Office of Basic Energy Sciences to the American students. Additional support to the conference was provided by the Natural Sciences and Engineering Research Council (NSERC), the National Research Council of Canada (NRC), Pfeiffer Vacuum, Femtolasers Produktions GmbH, Roentdek Handels GmbH, Coherent Laser Products, and Amplitude Technologies. Last but not least, the guest editors of this special issue would like to acknowledge the tremendous amount of work done by the staff of J. Phys. B in handling all aspects of the publication process. In particular, we would like to thank Isabelle Auffret-Babak, Alice Malhador and Joanna Dingley from the editorial team, Katie Gerrard in production and the Editor-in-Chief, Professor J-M Rost.

Photoelectron circular dichroism in the multiphoton ionization by short laser pulses. II. Three- and four-photon ionization of fenchone and camphor.

PubMed

Müller, Anne D; Artemyev, Anton N; Demekhin, Philipp V

2018-06-07

Angle-resolved multiphoton ionization of fenchone and camphor by short intense laser pulses is computed by the time-dependent single center method. Thereby, the photoelectron circular dichroism (PECD) in the three-photon resonance enhanced ionization and four-photon above-threshold ionization of these molecules is investigated in detail. The computational results are in satisfactory agreement with the available experimental data, measured for randomly oriented fenchone and camphor molecules at different wavelengths of the exciting pulses. We predict a significant enhancement of the multiphoton PECD for uniaxially oriented fenchone and camphor.

Photoelectron circular dichroism in the multiphoton ionization by short laser pulses. II. Three- and four-photon ionization of fenchone and camphor

NASA Astrophysics Data System (ADS)

Müller, Anne D.; Artemyev, Anton N.; Demekhin, Philipp V.

2018-06-01

Angle-resolved multiphoton ionization of fenchone and camphor by short intense laser pulses is computed by the time-dependent single center method. Thereby, the photoelectron circular dichroism (PECD) in the three-photon resonance enhanced ionization and four-photon above-threshold ionization of these molecules is investigated in detail. The computational results are in satisfactory agreement with the available experimental data, measured for randomly oriented fenchone and camphor molecules at different wavelengths of the exciting pulses. We predict a significant enhancement of the multiphoton PECD for uniaxially oriented fenchone and camphor.

Experimental observation of multiphoton Thomson scattering

NASA Astrophysics Data System (ADS)

Yan, Wenchao; Golovin, Grigory; Fruhling, Colton; Haden, Daniel; Zhang, Ping; Zhang, Jun; Zhao, Baozhen; Liu, Cheng; Chen, Shouyuan; Banerjee, Sudeep; Umstadter, Donald

2016-10-01

With the advent of high-power lasers, several multiphoton processes have been reported involving electrons in strong fields. For electrons that were initially bound to atoms, both multiphoton ionization and scattering have been reported. However, for free electrons, only low-order harmonic generation has been observed until now. This limitation stems from past difficulty in achieving the required ultra-high-field strengths in scattering experiments. Highly relativistic laser intensities are required to reach the multiphoton regime of Thomson scattering, and generate high harmonics from free electrons. The scaling parameter is the normalized vector potential (a0). Previous experiments have observed phenomena in the weakly relativistic case (a0 >> 1). In ultra-intense fields (a0 >>1), the anomalous electron trajectory is predicted to produce a spectrum characterized by the merging of multiple high-order harmonic generation into a continuum. This may be viewed as the multiphoton Thomson scattering regime analogous to the wiggler of a synchrotron. Thus, the light produced reflects the electrons behavior in an ultra-intense lase field. We discuss the first experiments in the highly relativistic case (a0 15). This material is based upon work supported by NSF No. PHY-153700; US DOE, Office of Science, BES, # DE-FG02-05ER15663; AFOSR # FA9550-11-1-0157; and DHS DNDO # HSHQDC-13-C-B0036.

Utilizing nonlinear optical microscopy to investigate the development of early cancer in nude mice in vivo

NASA Astrophysics Data System (ADS)

Wang, Chun-Chin; Li, Feng-Chieh; Lin, Sung-Jan; Lo, Wen; Dong, Chen-Yuan

2007-07-01

In this investigation, we used in vivo nonlinear optical microscopy to image normal and carcinogen DMBA treated skin tissues of nude mice. We acquired two-photon autofluroescence and second harmonic generation (SHG) images of the skin tissue, and applied the ASI (Autofluorescence versus SHG Index) to the resulting image. This allows us to visualize and quantify the interaction between mouse skin cells and the surrounding connective tissue. We found that as the imaging depth increases, ASI has a different distribution in the normal and the treated skin tissues. Since the DMBA treated skin eventually became squamous cell carcinoma (SCC), our results show that the physiological changes to mouse skin en route to become cancer can be effectively tracked by multiphoton microscopy. We envision this approach to be effective in studying tumor biology and tumor treatment procedures.

Light and sound - emerging imaging techniques for inflammatory bowel disease

PubMed Central

Knieling, Ferdinand; Waldner, Maximilian J

2016-01-01

Patients with inflammatory bowel disease are known to have a high demand of recurrent evaluation for therapy and disease activity. Further, the risk of developing cancer during the disease progression is increasing from year to year. New, mostly non-radiant, quick to perform and quantitative methods are challenging, conventional endoscopy with biopsy as gold standard. Especially, new physical imaging approaches utilizing light and sound waves have facilitated the development of advanced functional and molecular modalities. Besides these advantages they hold the promise to predict personalized therapeutic responses and to spare frequent invasive procedures. Within this article we highlight their potential for initial diagnosis, assessment of disease activity and surveillance of cancer development in established techniques and recent advances such as wide-view full-spectrum endoscopy, chromoendoscopy, autofluorescence endoscopy, endocytoscopy, confocal laser endoscopy, multiphoton endoscopy, molecular imaging endoscopy, B-mode and Doppler ultrasound, contrast-enhanced ultrasound, ultrasound molecular imaging, and elastography. PMID:27433080

Multi-modality optical imaging of ovarian cancer in a post-menopausal mouse model

NASA Astrophysics Data System (ADS)

Watson, Jennifer M.; Rice, Photini Faith; Marion, Samuel L.; Bentley, David L.; Brewer, Molly A.; Utzinger, Urs; Hoyer, Patricia B.; Barton, Jennifer K.

2011-03-01

Our goal is to use optical imaging to detect cancer development on the sub cellular scale. By determining the microscopic changes that precede ovarian cancer we hope to develop a minimally invasive screening test for high risk patients. A mouse ovarian cancer model has been developed by treating mice with 4-Vinylcyclohexene Diepoxide to induce ovarian failure and 7, 12-Dimethylbenz[a]anthracene (DMBA) to induce ovarian cancer. Using optical coherence tomography (OCT) and multiphoton microscopy (MPM) we have obtained co-registered en face images of sixty-seven mouse ovaries ex vivo and forty-two ovaries in vivo. Preliminary analysis indicates that OCT and MPM can visualize ovarian microstructure. During the next year we will be completing a long term survival study using post-menopausal mice that have been treated with DMBA to induce cancer and imaged in vivo at time points before and after treatment.

In vivo multiphoton-microscopy of picosecond-laser-induced optical breakdown in human skin.

PubMed

Balu, Mihaela; Lentsch, Griffin; Korta, Dorota Z; König, Karsten; Kelly, Kristen M; Tromberg, Bruce J; Zachary, Christopher B

2017-08-01

Improvements in skin appearance resulting from treatment with fractionated picosecond-lasers have been noted, but optimizing the treatment efficacy depends on a thorough understanding of the specific skin response. The development of non-invasive laser imaging techniques in conjunction with laser therapy can potentially provide feedback for guidance and optimizing clinical outcome. The purpose of this study was to demonstrate the capability of multiphoton microscopy (MPM), a high-resolution, label-free imaging technique, to characterize in vivo the skin response to a fractionated non-ablative picosecond-laser treatment. Two areas on the arm of a volunteer were treated with a fractionated picosecond laser at the Dermatology Clinic, UC Irvine. The skin response to treatment was imaged in vivo with a clinical MPM-based tomograph at 3 hours and 24 hours after treatment and seven additional time points over a 4-week period. MPM revealed micro-injuries present in the epidermis. Pigmented cells were particularly damaged in the process, suggesting that melanin is likely the main absorber for laser induced optical breakdown. Damaged individual cells were distinguished as early as 3 hours post pico-laser treatment with the 532 nm wavelength, and 24 hours post-treatment with both 532 and 1064 nm wavelengths. At later time points, clusters of cellular necrotic debris were imaged across the treated epidermis. After 24 hours of treatment, inflammatory cells were imaged in the proximity of epidermal micro-injuries. The epidermal injuries were exfoliated over a 4-week period. This observational and descriptive pilot study demonstrates that in vivo MPM imaging can be used non-invasively to provide label-free contrast for describing changes in human skin following a fractionated non-ablative laser treatment. The results presented in this study represent the groundwork for future longitudinal investigations on an expanded number of subjects to understand the response to treatment in different skin types with different laser parameters, critical factors in optimizing treatment outcome. Lasers Surg. Med. 49:555-562, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Multimodal imaging of vocal fold scarring in a rabbit model by multiphoton microscopy

NASA Astrophysics Data System (ADS)

Kazarine, Alexei; Bouhabel, Sarah; Douillette, Annie H.; Kost, Karen; Li-Jessen, Nicole Y. K.; Mongeau, Luc; Wiseman, Paul W.

2017-02-01

Vocal fold scarring as a result of injury or disease can lead to voice disorders which can significantly affect the quality of life. During the scarring process, the normally elastic tissue of the vocal fold lamina propria is replaced by a much stiffer collagen-based fibrotic tissue, which impacts the fold's ability to vibrate. Surgical removal of this tissue is often ineffective and can result in further scarring. Injectable biomaterials, a form of tissue engineering, have been proposed as a potential solution to reduce existing scars or prevent scarring altogether. In order to properly evaluate the effectiveness of these new materials, multiphoton microscopy emerges as an effective tool due to its intrinsic multiple label free contrast mechanisms that highlight extracellular matrix elements. In this study, we evaluate the spatial distribution of collagen and elastin fibers in a rabbit model using second harmonic generation (SHG), third harmonic generation (THG) and two photon autofluorescence (TPAF) applied to unlabeled tissue sections. In comparison to traditional methods that rely on histological staining or immunohistochemistry, SHG, THG and TPAF provide a more reliable detection of these native proteins. The evaluation of collagen levels allows us to follow the extent of scarring, while the presence of elastin fibers is thought to be indicative of the level of healing of the injured fold. Using these imaging modalities, we characterize the outcome of injectable biomaterial treatments in order to direct future treatments for tissue engineering.

Raman-based imaging uncovers the effects of alginate hydrogel implants in spinal cord injury

NASA Astrophysics Data System (ADS)

Galli, Roberta; Tamosaityte, Sandra; Koch, Maria; Sitoci-Ficici, Kerim H.; Later, Robert; Uckermann, Ortrud; Beiermeister, Rudolf; Gelinsky, Michael; Schackert, Gabriele; Kirsch, Matthias; Koch, Edmund; Steiner, Gerald

2015-07-01

The treatment of spinal cord injury by using implants that provide a permissive environment for axonal growth is in the focus of the research for regenerative therapies. Here, Raman-based label-free techniques were applied for the characterization of morphochemical properties of surgically induced spinal cord injury in the rat that received an implant of soft unfunctionalized alginate hydrogel. Raman microspectroscopy followed by chemometrics allowed mapping the different degenerative areas, while multimodal multiphoton microscopy (e.g. the combination of coherent anti-Stokes Raman scattering (CARS), endogenous two-photon fluorescence and second harmonic generation on the same platform) enabled to address the morphochemistry of the tissue at cellular level. The regions of injury, characterized by demyelination and scarring, were retrieved and the distribution of key tissue components was evaluated by Raman mapping. The alginate hydrogel was detected in the lesion up to six months after implantation and had positive effects on the nervous tissue. For instance, multimodal multiphoton microscopy complemented the results of Raman mapping, providing the micromorphology of lipid-rich tissue structures by CARS and enabling to discern lipid-rich regions that contained myelinated axons from degenerative regions characterized by myelin fragmentation and presence of foam cells. These findings demonstrate that Raman-based imaging methods provide useful information for the evaluation of alginate implant effects and have therefore the potential to contribute to new strategies for monitoring degenerative and regenerative processes induced in SCI, thereby improving the effectiveness of therapies.

Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in a rat liver cell line

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

Barhoumi, Rola, E-mail: rmouneimne@cvm.tamu.edu; Mouneimne, Youssef; Ramos, Ernesto

2011-05-15

Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an 'advanced unmixing process', identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 h. The application of the advanced unmixing process resulted inmore » the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to the analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.« less

Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.

PubMed

Heymann, Felix; Niemietz, Patricia M; Peusquens, Julia; Ergen, Can; Kohlhepp, Marlene; Mossanen, Jana C; Schneider, Carlo; Vogt, Michael; Tolba, Rene H; Trautwein, Christian; Martin, Christian; Tacke, Frank

2015-03-24

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring. The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange. To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4). CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Diamond-like-carbon nanoparticle production and agglomeration following UV multi-photon excitation of static naphthalene/helium gas mixtures

NASA Astrophysics Data System (ADS)

Walsh, A. J.; Tielens, A. G. G. M.; Ruth, A. A.

2016-07-01

We report the formation of nanoparticles with significant diamond character after UV multi-photon laser excitation of gaseous naphthalene, buffered in static helium gas, at room temperature. The nanoparticles are identified in situ by their absorption and scattering spectra between 400 and 850 nm, which are modeled using Mie theory. Comparisons of the particles' spectroscopic and optical properties with those of carbonaceous materials indicate a sp3/sp2 hybridization ratio of 8:1 of the particles formed. The particle extinction in the closed static (unstirred) gas-phase system exhibits a complex and quasi-oscillatory time dependence for the duration of up to several hours with periods ranging from seconds to many minutes. The extinction dynamics of the system is based on a combination of transport features and particle interaction, predominantly agglomeration. The relatively long period of agglomeration allows for a unique analysis of the agglomeration process of diamond-like carbon nanoparticles in situ.

Femtosecond two-photon Rabi oscillations in excited He driven by ultrashort intense laser fields

NASA Astrophysics Data System (ADS)

Fushitani, M.; Liu, C.-N.; Matsuda, A.; Endo, T.; Toida, Y.; Nagasono, M.; Togashi, T.; Yabashi, M.; Ishikawa, T.; Hikosaka, Y.; Morishita, T.; Hishikawa, A.

2016-02-01

Coherent light-matter interaction provides powerful methods for manipulating quantum systems. Rabi oscillation is one such process. As it enables complete population transfer to a target state, it is thus routinely exploited in a variety of applications in photonics, notably quantum information processing. The extension of coherent control techniques to the multiphoton regime offers wider applicability, and access to highly excited or dipole-forbidden transition states. However, the multiphoton Rabi process is often disrupted by other competing nonlinear effects such as the a.c. Stark shift, especially at the high laser-field intensities necessary to achieve ultrafast Rabi oscillations. Here we demonstrate a new route to drive two-photon Rabi oscillations on timescales as short as tens of femtoseconds, by utilizing the strong-field phenomenon known as Freeman resonance. The scenario is not specific to atomic helium as investigated in the present study, but broadly applicable to other systems, thus opening new prospects for the ultrafast manipulation of Rydberg states.

An engineered CARS substrate with giant field enhancement in crisscross dimer nanostructure.

PubMed

Zhang, Jia; Chen, Shu; Wang, Junqiao; Mu, Kaijun; Fan, Chunzhen; Liang, Erjun; Ding, Pei

2018-01-15

We theoretically investigate the optical properties of a nanostructure consisting of the two identical and symmetrically arranged crisscrosses. A plasmonic Fano resonance is induced by a strong interplay between bright mode and dark modes, where the bright mode is due to electric dipole resonance while dark modes originate from the magnetic dipole induced by LC resonances. In this article, we find that the electric field "hotspots" corresponding to three different wavelengths can be positioned at the same spatial position, and its spectral tunability is achieved by changing geometric parameters. The crisscrosses system can be designed as a plasmonic substrate for enhancing Coherent Anti-Stokes Raman Scattering (CARS) signal. This discovery provides a new method to achieve single molecule detection. At the same time, it also has many important applications for multi-photon imaging and other nonlinear optical processes, such as four-wave mixing and stimulated Raman scattering.

Tuning single-photon sources for telecom multi-photon experiments.

PubMed

Greganti, Chiara; Schiansky, Peter; Calafell, Irati Alonso; Procopio, Lorenzo M; Rozema, Lee A; Walther, Philip

2018-02-05

Multi-photon state generation is of great interest for near-future quantum simulation and quantum computation experiments. To-date spontaneous parametric down-conversion is still the most promising process, even though two major impediments still exist: accidental photon noise (caused by the probabilistic non-linear process) and imperfect single-photon purity (arising from spectral entanglement between the photon pairs). In this work, we overcome both of these difficulties by (1) exploiting a passive temporal multiplexing scheme and (2) carefully optimizing the spectral properties of the down-converted photons using periodically-poled KTP crystals. We construct two down-conversion sources in the telecom wavelength regime, finding spectral purities of > 91%, while maintaining high four-photon count rates. We use single-photon grating spectrometers together with superconducting nanowire single-photon detectors to perform a detailed characterization of our multi-photon source. Our methods provide practical solutions to produce high-quality multi-photon states, which are in demand for many quantum photonics applications.

ARTICLES: Variation of the absorption cross section of high-power infrared laser radiation in homologous series of CnH2n+1OH molecules

NASA Astrophysics Data System (ADS)

Bagratashvili, Viktor N.; Brodskaya, E. A.; Vereshchagina, Lyudmila N.; Kuz'min, M. V.; Osmanov, R. R.; Putilin, F. N.; Stuchebryukhov, A. A.

1984-11-01

An experimental investigation was made of variation of the characteristics of infrared multiphoton absorption in a homologous series of CnH2n+1OH alcohols (n = 1-5) excited with CO2 laser pulses. The dependences of the energy absorbed by the molecules on the frequency and energy density of laser radiation were determined by the optoacoustic method. It was found that the multiphoton absorption cross section decreases on increase in the radiation energy density at a rate which becomes slower on increase in the molecular size. A model is proposed for multiphoton excitation of molecules in a homologous series. This model is based on an analysis of a resonant mode interacting with the infrared radiation field and coupled to a reservoir of modes that do not interact with the field. The model predicts correctly the change in the multiphoton absorption cross section on increase in the number of the degrees of freedom of a molecule.

Mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development.

PubMed

Walton, Katherine D; Kolterud, Asa

2014-09-04

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine(1). Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought(1). The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth(2). Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Fiber-based tunable repetition rate source for deep tissue two-photon fluorescence microscopy

PubMed Central

Charan, Kriti; Li, Bo; Wang, Mengran; Lin, Charles P.; Xu, Chris

2018-01-01

Deep tissue multiphoton imaging requires high peak power to enhance signal and low average power to prevent thermal damage. Both goals can be advantageously achieved through laser repetition rate tuning instead of simply adjusting the average power. We show that the ideal repetition rate for deep two-photon imaging in the mouse brain is between 1 and 10 MHz, and we present a fiber-based source with an arbitrarily tunable repetition rate within this range. The performance of the new source is compared to a mode-locked Ti:Sapphire (Ti:S) laser for in vivo imaging of mouse brain vasculature. At 2.5 MHz, the fiber source requires 5.1 times less average power to obtain the same signal as a standard Ti:S laser operating at 80 MHz. PMID:29760989

Optically based quantification of absolute cerebral metabolic rate of oxygen (CMRO2) with high spatial resolution in rodents

NASA Astrophysics Data System (ADS)

Yaseen, Mohammad A.; Srinivasan, Vivek J.; Sakadžić, Sava; Vinogradov, Sergei A.; Boas, David A.

2010-02-01

Measuring oxygen delivery in brain tissue is important for identifying the pathophysiological changes associated with brain injury and various diseases such as cancer, stroke, and Alzheimer's disease. We have developed a multi-modal imaging system for minimally invasive measurement of cerebral oxygenation and blood flow in small animals with high spatial resolution. The system allows for simultaneous measurement of blood flow using Fourier-domain optical coherence tomography, and oxygen partial pressure (pO2) using either confocal or multiphoton phosphorescence lifetime imaging with exogenous porphyrin-based dyes sensitive to dissolved oxygen. Here we present the changes in pO2 and blood flow in superficial cortical vessels of Sprague Dawley rats in response to conditions such as hypoxia, hyperoxia, and functional stimulation. pO2 measurements display considerable heterogeneity over distances that cannot be resolved with more widely used oxygen-monitoring techniques such as BOLD-fMRI. Large increases in blood flow are observed in response to functional stimulation and hypoxia. Our system allows for quantification of cerebral metabolic rate of oxygen (CMRO2) with high spatial resolution, providing a better understanding of metabolic dynamics during functional stimulation and under various neuropathologies. Ultimately, better insight into the underlying mechanisms of neuropathologies will facilitate the development of improved therapeutic strategies to minimize damage to brain tissue.

Multi-photon microscopy of tobacco-exposed organotypic skin models

NASA Astrophysics Data System (ADS)

Dao, Belinda; Yamazaki, Alissa; Sun, Chung Ho; Wang, Zifu; Pham, Nguyen; Oldham, Michael; Wong, Brian J. F.

2006-02-01

Cigarette smoking is the most preventable cause of death in the United States. Researchers have extensively studied smoking in regards to its association with cancer, cardiovascular, and pulmonary disease. In contrast, the impact of cigarette smoking on skin has received much less attention. To provide a better understanding of the effect of cigarette smoking on the human dermal layer, this study used multi-photon microscopy (MPM) to examine collagen in organotypic skin models exposed to cigarette smoke condensate (CSC). Adult and neonatal organotypic tissue-engineered artificial skin models (RAFTs) were constructed and exposed to varying concentrations of CSC. Imaging of the RAFTs was performed using MPM and second-harmonic generation signals (SHG), which allowed for collagen structure to be viewed and analyzed as well as for collagen density to be assessed from derived depth-dependent decay (DDD) values. RAFT contraction as related to exposure concentration was monitored as well. Results indicated a dose dependent between contraction rates and CSC concentration. Collagen structure showed more preservation of its original structure at a greater depth in RAFTs with higher concentrations of CSC. No clear trends could be drawn from analysis of derived DDD values.

Identification of tumor cells infiltrating into connective tissue in esophageal cancer by multiphoton microscopy

NASA Astrophysics Data System (ADS)

Xu, Jian; Jiang, Liwei; Kang, Deyong; Wu, Xuejing; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Lin, Jiangbo; Chen, Jianxin

2016-10-01

Esophageal cancer is one of the most common malignancies of the gastrointestinal cancers and carries poorer prognosis than other gastrointestinal cancers. In general practice, the depth of tumor infiltration in esophageal wall is crucial to establishing appropriate treatment plan which is established by detecting the tumor infiltration depth. Connective tissue is one of the main structures that form the esophageal wall. So, identification of tumor cells infiltrating into connective tissue is helping for detecting the tumor infiltration depth. Our aim is to evaluate whether multiphoton microscopy (MPM) can be used to detect tumor cells infiltrating into connective tissue in the esophageal cancer. MPM is well-suited for real-time detecting morphologic and cellular changes in fresh tissues since many endogenous fluorophores of fresh tissues are excited through two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). In this work, microstructure of tumor cells and connective tissue are first studied. Then, morphological changes of collagen fibers after the infiltration of tumor cells are shown. These results show that MPM has the ability to detect tumor cells infiltrating into connective tissue in the esophageal cancer. In the future, MPM may be a promising imaging technique for detecting tumor cells in esophageal cancer.

Neural plasticity explored by correlative two-photon and electron/SPIM microscopy

NASA Astrophysics Data System (ADS)

Allegra Mascaro, A. L.; Silvestri, L.; Costantini, I.; Sacconi, L.; Maco, B.; Knott, G. W.; Pavone, F. S.

2013-06-01

Plasticity of the central nervous system is a complex process which involves the remodeling of neuronal processes and synaptic contacts. However, a single imaging technique can reveal only a small part of this complex machinery. To obtain a more complete view, complementary approaches should be combined. Two-photon fluorescence microscopy, combined with multi-photon laser nanosurgery, allow following the real-time dynamics of single neuronal processes in the cerebral cortex of living mice. The structural rearrangement elicited by this highly confined paradigm of injury can be imaged in vivo first, and then the same neuron could be retrieved ex-vivo and characterized in terms of ultrastructural features of the damaged neuronal branch by means of electron microscopy. Afterwards, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, based on the use of major blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from its apical portion, the whole pyramidal neuron can then be segmented and located in the correct cortical layer. With the correlative approach presented here, researchers will be able to place in a three-dimensional anatomic context the neurons whose dynamics have been observed with high detail in vivo.

Modification of microneedles using inkjet printing

NASA Astrophysics Data System (ADS)

Boehm, R. D.; Miller, P. R.; Hayes, S. L.; Monteiro-Riviere, N. A.; Narayan, R. J.

2011-06-01

In this study, biodegradable acid anhydride copolymer microneedles containing quantum dots were fabricated by means of visible light dynamic mask micro-stereolithography-micromolding and inkjet printing. Nanoindentation was performed to obtain the hardness and the Young's modulus of the biodegradable acid anhydride copolymer. Imaging of quantum dots within porcine skin was accomplished by means of multiphoton microscopy. Our results suggest that the combination of visible light dynamic mask micro-stereolithography-micromolding and inkjet printing enables fabrication of solid biodegradable microneedles with a wide range of geometries as well as a wide range of pharmacologic agent compositions.

Recent progress in tissue optical clearing for spectroscopic application

NASA Astrophysics Data System (ADS)

Sdobnov, A. Yu.; Darvin, M. E.; Genina, E. A.; Bashkatov, A. N.; Lademann, J.; Tuchin, V. V.

2018-05-01

This paper aims to review recent progress in optical clearing of the skin and over naturally turbid biological tissues and blood using this technique in vivo and in vitro with multiphoton microscopy, confocal Raman microscopy, confocal microscopy, NIR spectroscopy, optical coherence tomography, and laser speckle contrast imaging. Basic principles of the technique, its safety, advantages and limitations are discussed. The application of optical clearing agent on a tissue allows for controlling the optical properties of tissue. Optical clearing-induced reduction of tissue scattering significantly facilitates the observation of deep-located tissue regions, at the same time improving the resolution and image contrast for a variety of optical imaging methods suitable for clinical applications, such as diagnostics and laser treatment of skin diseases, mucosal tumor imaging, laser disruption of pathological abnormalities, etc. Structural images of different skin layers obtained ex vivo for porcine ear skin samples at application of Omnipaque™ and glycerol solutions during 60 min. Red color corresponds to TPEAF signal channel. Green color corresponds to SHG signal channel.