An ex vivo rat eye model to aid development of high-resolution retina imaging devices for rodents

NASA Astrophysics Data System (ADS)

van Oterendorp, Christian; Martin, Keith R.; Zhong, Jiang Jian; Diaz-Santana, Luis

2010-09-01

High resolution in vivo retinal imaging in rodents is becoming increasingly important in eye research. Development of suitable imaging devices currently requires many lengthy animal procedures. We present an ex vivo rat model eye with fluorescently labelled retinal ganglion cells (RGC) and nerve fibre bundles that reduces the need for animal procedures while preserving key properties of the living rat eye. Optical aberrations and scattering of four model eyes and eight live rat eyes were quantified using a Shack-Hartmann sensor. Fluorescent images from RGCs were obtained using a prototype scanning laser ophthalmoscope. The wavefront aberration root mean square value without defocus did not significantly differ between model and living eyes. Higher order aberrations were slightly higher but RGC image quality was comparable to published in vivo work. Overall, the model allows a large reduction in number and duration of animal procedures required to develop new in vivo retinal imaging devices.

Live dynamic analysis of the developing cardiovascular system in mice

NASA Astrophysics Data System (ADS)

Lopez, Andrew L.; Wang, Shang; Larin, Kirill V.; Larina, Irina V.

2017-02-01

The study of the developing cardiovascular system in mice is important for understanding human cardiogenesis and congenital heart defects. Our research focuses on imaging early development in the mouse embryo to specifically understand cardiovascular development under the regulation of dynamic factors like contractile force and blood flow using optical coherence tomography (OCT). We have previously developed an OCT based approach that combines static embryo culture and advanced image processing with computational modeling to live-image mouse embryos and obtain 4D (3D+time) cardiodynamic datasets. Here we present live 4D dynamic blood flow imaging of the early embryonic mouse heart in correlation with heart wall movement. We are using this approach to understand how specific mutations impact heart wall dynamics, and how this influences flow patterns and cardiogenesis. We perform studies in mutant embryos with cardiac phenotypes such as myosin regulatory light chain 2, atrial isoform (Mlc2a). This work is brings us closer to understanding the connections between dynamic mechanical factors and gene programs responsible for early cardiovascular development.

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.

Long Term Ex Vivo Culture and Live Imaging of Drosophila Larval Imaginal Discs.

PubMed

Tsao, Chia-Kang; Ku, Hui-Yu; Lee, Yuan-Ming; Huang, Yu-Fen; Sun, Yi Henry

Continuous imaging of live tissues provides clear temporal sequence of biological events. The Drosophila imaginal discs have been popular experimental subjects for the study of a wide variety of biological phenomena, but long term culture that allows normal development has not been satisfactory. Here we report a culture method that can sustain normal development for 18 hours and allows live imaging. The method is validated in multiple discs and for cell proliferation, differentiation and migration. However, it does not support disc growth and cannot support cell proliferation for more than 7 to 12 hr. We monitored the cellular behavior of retinal basal glia in the developing eye disc and found that distinct glia type has distinct properties of proliferation and migration. The live imaging provided direct proof that wrapping glia differentiated from existing glia after migrating to the anterior front, and unexpectedly found that they undergo endoreplication before wrapping axons, and their nuclei migrate up and down along the axons. UV-induced specific labeling of a single carpet glia also showed that the two carpet glia membrane do not overlap and suggests a tiling or repulsion mechanism between the two cells. These findings demonstrated the usefulness of an ex vivo culture method and live imaging.

DESIGN, SYNTHESIS, AND APPLICATION OF THE TRIMETHOPRIM-BASED CHEMICAL TAG FOR LIVE CELL IMAGING

PubMed Central

Jing, Chaoran; Cornish, Virginia W.

2013-01-01

Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live cell imaging. Alternative protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

PubMed

Agrawalla, Bikram Keshari; Chandran, Yogeswari; Phue, Wut-Hmone; Lee, Sung-Chan; Jeong, Yun-Mi; Wan, Si Yan Diana; Kang, Nam-Young; Chang, Young-Tae

2015-04-29

Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

Implantable self-reset CMOS image sensor and its application to hemodynamic response detection in living mouse brain

NASA Astrophysics Data System (ADS)

Yamaguchi, Takahiro; Takehara, Hiroaki; Sunaga, Yoshinori; Haruta, Makito; Motoyama, Mayumi; Ohta, Yasumi; Noda, Toshihiko; Sasagawa, Kiyotaka; Tokuda, Takashi; Ohta, Jun

2016-04-01

A self-reset pixel of 15 × 15 µm2 with high signal-to-noise ratio (effective peak SNR ≃64 dB) for an implantable image sensor has been developed for intrinsic signal detection arising from hemodynamic responses in a living mouse brain. For detecting local conversion between oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) in brain tissues, an implantable imaging device was fabricated with our newly designed self-reset image sensor and orange light-emitting diodes (LEDs; λ = 605 nm). We demonstrated imaging of hemodynamic responses in the sensory cortical area accompanied by forelimb stimulation of a living mouse. The implantable imaging device for intrinsic signal detection is expected to be a powerful tool to measure brain activities in living animals used in behavioral analysis.

Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy

PubMed Central

Fu, Qinyi; Martin, Benjamin L.; Matus, David Q.; Gao, Liang

2016-01-01

Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos. PMID:27004937

Live-cell imaging of budding yeast telomerase RNA and TERRA.

PubMed

Laprade, Hadrien; Lalonde, Maxime; Guérit, David; Chartrand, Pascal

2017-02-01

In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

PubMed Central

Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

2016-01-01

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

PubMed

Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Swann, Karl; Borri, Paola

2016-06-15

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. © 2016. Published by The Company of Biologists Ltd.

High-speed adaptive optics line scan confocal retinal imaging for human eye

PubMed Central

Wang, Xiaolin; Zhang, Yuhua

2017-01-01

Purpose Continuous and rapid eye movement causes significant intraframe distortion in adaptive optics high resolution retinal imaging. To minimize this artifact, we developed a high speed adaptive optics line scan confocal retinal imaging system. Methods A high speed line camera was employed to acquire retinal image and custom adaptive optics was developed to compensate the wave aberration of the human eye’s optics. The spatial resolution and signal to noise ratio were assessed in model eye and in living human eye. The improvement of imaging fidelity was estimated by reduction of intra-frame distortion of retinal images acquired in the living human eyes with frame rates at 30 frames/second (FPS), 100 FPS, and 200 FPS. Results The device produced retinal image with cellular level resolution at 200 FPS with a digitization of 512×512 pixels/frame in the living human eye. Cone photoreceptors in the central fovea and rod photoreceptors near the fovea were resolved in three human subjects in normal chorioretinal health. Compared with retinal images acquired at 30 FPS, the intra-frame distortion in images taken at 200 FPS was reduced by 50.9% to 79.7%. Conclusions We demonstrated the feasibility of acquiring high resolution retinal images in the living human eye at a speed that minimizes retinal motion artifact. This device may facilitate research involving subjects with nystagmus or unsteady fixation due to central vision loss. PMID:28257458

High-speed adaptive optics line scan confocal retinal imaging for human eye.

PubMed

Lu, Jing; Gu, Boyu; Wang, Xiaolin; Zhang, Yuhua

2017-01-01

Continuous and rapid eye movement causes significant intraframe distortion in adaptive optics high resolution retinal imaging. To minimize this artifact, we developed a high speed adaptive optics line scan confocal retinal imaging system. A high speed line camera was employed to acquire retinal image and custom adaptive optics was developed to compensate the wave aberration of the human eye's optics. The spatial resolution and signal to noise ratio were assessed in model eye and in living human eye. The improvement of imaging fidelity was estimated by reduction of intra-frame distortion of retinal images acquired in the living human eyes with frame rates at 30 frames/second (FPS), 100 FPS, and 200 FPS. The device produced retinal image with cellular level resolution at 200 FPS with a digitization of 512×512 pixels/frame in the living human eye. Cone photoreceptors in the central fovea and rod photoreceptors near the fovea were resolved in three human subjects in normal chorioretinal health. Compared with retinal images acquired at 30 FPS, the intra-frame distortion in images taken at 200 FPS was reduced by 50.9% to 79.7%. We demonstrated the feasibility of acquiring high resolution retinal images in the living human eye at a speed that minimizes retinal motion artifact. This device may facilitate research involving subjects with nystagmus or unsteady fixation due to central vision loss.

Functional optical coherence tomography for live dynamic analysis of mouse embryonic cardiogenesis

NASA Astrophysics Data System (ADS)

Wang, Shang; Lopez, Andrew L.; Larina, Irina V.

2018-02-01

Blood flow, heart contraction, and tissue stiffness are important regulators of cardiac morphogenesis and function during embryonic development. Defining how these factors are integrated is critically important to advance prevention, diagnostics, and treatment of congenital heart defects. Mammalian embryonic development is taking place deep within the female body, which makes cardiodynamic imaging and analysis during early developmental stages in humans inaccessible. With thousands of mutant lines available and well-established genetic manipulation tools, mouse is a great model to understand how biomechanical factors are integrated with molecular pathways to regulate cardiac function and development. Dynamic imaging and quantitative analysis of the biomechanics of live mouse embryos have become increasingly important, which demands continuous advancements in imaging techniques and live assessment approaches. This has been one of the major drives to keep pushing the frontier of embryonic imaging for better resolution, higher speed, deeper penetration, and more diverse and effective contrasts. Optical coherence tomography (OCT) has played a significant role in addressing such demands, and its features in non-labeling imaging, 3D capability, a large working distance, and various functional derivatives allow OCT to cover a number of specific applications in embryonic imaging. Recently, our group has made several technical improvements in using OCT to probe the biomechanical aspects of live developing mouse embryos at early stages. These include the direct volumetric structural and functional imaging of the cardiodynamics, four-dimensional quantitative Doppler imaging and analysis of the cardiac blood flow, and fourdimensional blood flow separation from the cardiac wall tissue in the beating embryonic heart. Here, we present a short review of these studies together with brief descriptions of the previous work that demonstrate OCT as a valuable and useful imaging tool for the research in developmental cardiology.

Conjugated Polymer with Intrinsic Alkyne Units for Synergistically Enhanced Raman Imaging in Living Cells.

PubMed

Li, Shengliang; Chen, Tao; Wang, Yunxia; Liu, Libing; Lv, Fengting; Li, Zhiliang; Huang, Yanyi; Schanze, Kirk S; Wang, Shu

2017-10-16

Development of Raman-active materials with enhanced and distinctive Raman vibrations in the Raman-silent region (1800-2800 cm -1 ) is highly required for specific molecular imaging of living cells with high spatial resolution. Herein, water-soluble cationic conjugated polymers (CCPs), poly(phenylene ethynylene) (PPE) derivatives, are explored for use as alkyne-state-dependent Raman probes for living cell imaging due to synergetic enhancement effect of alkyne vibrations in Raman-silent region compared to alkyne-containing small molecules. The enhanced alkyne signals result from the integration of alkyne groups into the rigid backbone and the delocalized π-conjugated structure. PPE-based conjugated polymer nanoparticles (CPNs) were also prepared as Raman-responsive nanomaterials for distinct imaging application. This work opens a new way into the development of conjugated polymer materials for enhanced Raman imaging. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Real-time capture and reconstruction system with multiple GPUs for a 3D live scene by a generation from 4K IP images to 8K holograms.

PubMed

Ichihashi, Yasuyuki; Oi, Ryutaro; Senoh, Takanori; Yamamoto, Kenji; Kurita, Taiichiro

2012-09-10

We developed a real-time capture and reconstruction system for three-dimensional (3D) live scenes. In previous research, we used integral photography (IP) to capture 3D images and then generated holograms from the IP images to implement a real-time reconstruction system. In this paper, we use a 4K (3,840 × 2,160) camera to capture IP images and 8K (7,680 × 4,320) liquid crystal display (LCD) panels for the reconstruction of holograms. We investigate two methods for enlarging the 4K images that were captured by integral photography to 8K images. One of the methods increases the number of pixels of each elemental image. The other increases the number of elemental images. In addition, we developed a personal computer (PC) cluster system with graphics processing units (GPUs) for the enlargement of IP images and the generation of holograms from the IP images using fast Fourier transform (FFT). We used the Compute Unified Device Architecture (CUDA) as the development environment for the GPUs. The Fast Fourier transform is performed using the CUFFT (CUDA FFT) library. As a result, we developed an integrated system for performing all processing from the capture to the reconstruction of 3D images by using these components and successfully used this system to reconstruct a 3D live scene at 12 frames per second.

Development of bimolecular fluorescence complementation using rsEGFP2 for detection and super-resolution imaging of protein-protein interactions in live cells

PubMed Central

Wang, Sheng; Ding, Miao; Chen, Xuanze; Chang, Lei; Sun, Yujie

2017-01-01

Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy. Combined with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of a rsEGFP2-based BiFC assay by detecting and visualizing specifically the heterodimerization interactions between Bcl-xL and Bak as well as the dynamics of the complex on mitochondria membrane in live cells. PMID:28663931

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

PubMed

Yang, Yang; Guan, Xiangming

2017-05-01

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.

Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells

PubMed Central

Kelbauskas, Laimonas; Shetty, Rishabh; Cao, Bin; Wang, Kuo-Chen; Smith, Dean; Wang, Hong; Chao, Shi-Hui; Gangaraju, Sandhya; Ashcroft, Brian; Kritzer, Margaret; Glenn, Honor; Johnson, Roger H.; Meldrum, Deirdre R.

2017-01-01

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field. PMID:29226240

Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging.

PubMed

Jing, Chaoran; Cornish, Virginia W

2013-01-01

Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live-cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E. coli dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live-cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live-cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. © 2013 by John Wiley & Sons, Inc.

High content live cell imaging for the discovery of new antimalarial marine natural products

PubMed Central

2012-01-01

Background The human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum. Methods A high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope. Results Image analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasite's phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts. Conclusion Collectively, our results show that high-content live cell-imaging (HCLCI) can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials. PMID:22214291

High content live cell imaging for the discovery of new antimalarial marine natural products.

PubMed

Cervantes, Serena; Stout, Paige E; Prudhomme, Jacques; Engel, Sebastian; Bruton, Matthew; Cervantes, Michael; Carter, David; Tae-Chang, Young; Hay, Mark E; Aalbersberg, William; Kubanek, Julia; Le Roch, Karine G

2012-01-03

The human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum. A high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope. Image analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasite's phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts. Collectively, our results show that high-content live cell-imaging (HCLCI) can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials. © 2011 Cervantes et al; licensee BioMed Central Ltd.

Time-lapse cinematography in living Drosophila tissues: preparation of material.

PubMed

Davis, Ilan; Parton, Richard M

2006-11-01

The fruit fly, Drosophila melanogaster, has been an extraordinarily successful model organism for studying the genetic basis of development and evolution. It is arguably the best-understood complex multicellular model system, owing its success to many factors. Recent developments in imaging techniques, in particular sophisticated fluorescence microscopy methods and equipment, now allow cellular events to be studied at high resolution in living material. This ability has enabled the study of features that tend to be lost or damaged by fixation, such as transient or dynamic events. Although many of the techniques of live cell imaging in Drosophila are shared with the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties in keeping the cells alive, introducing fluorescent probes, and imaging through thick hazy cytoplasm. This protocol outlines the preparation of major tissue types amenable to study by time-lapse cinematography and different methods for keeping them alive.

Simple blood-feeding method for live imaging of gut tube remodeling in regenerating planarians.

PubMed

Hosoda, Kazutaka; Morimoto, Mizuki; Motoishi, Minako; Nishimura, Osamu; Agata, Kiyokazu; Umesono, Yoshihiko

2016-04-01

Live cell imaging is a powerful technique to study cellular dynamics in vivo during animal development and regeneration. However, few live imaging methods have been reported for studying planarian regeneration. Here, we developed a simple method for steady visualization of gut tube remodeling during regeneration of a living freshwater planarian, Dugesia japonica. When planarians were fed blood several times, gut branches were well-visualized in living intact animals under normal bright-field illumination. Interestingly, tail fragments derived from these colored planarians enabled successive observation of the processes of the formation of a single anterior gut branch in the prepharyngeal region from the preexisting two posterior gut branches in the same living animals during head regeneration. Furthermore, we combined this method and RNA interference (RNAi) and thereby showed that a D. japonica raf-related gene (DjrafA) and mek-related gene (DjmekA) we identified both play a major role in the activation of extracellular signal-regulated kinase (ERK) signaling during planarian regeneration, as indicated by their RNAi-induced defects on gut tube remodeling in a time-saving initial screening using blood-feeding without immunohistochemical detection of the gut. Thus, this blood-feeding method is useful for live imaging of gut tube remodeling, and provides an advance for the field of regeneration study in planarians. © 2016 Japanese Society of Developmental Biologists.

Prolonged in vivo imaging of Xenopus laevis.

PubMed

Hamilton, Paul W; Henry, Jonathan J

2014-08-01

While live imaging of embryonic development over long periods of time is a well established method for embryos of the frog Xenopus laevis, once development has progressed to the swimming stages, continuous live imaging becomes more challenging because the tadpoles must be immobilized. Current imaging techniques for these advanced stages generally require bringing the tadpoles in and out of anesthesia for short imaging sessions at selected time points, severely limiting the resolution of the data. Here we demonstrate that creating a constant flow of diluted tricaine methanesulfonate (MS-222) over a tadpole greatly improves their survival under anesthesia. Based on this result, we describe a new method for imaging stage 48 to 65 X. laevis, by circulating the anesthetic using a peristaltic pump. This supports the animal during continuous live imaging sessions for at least 48 hr. The addition of a stable optical window allows for high quality imaging through the anesthetic solution. This automated imaging system provides for the first time a method for continuous observations of developmental and regenerative processes in advanced stages of Xenopus over 2 days. Developmental Dynamics 243:1011-1019, 2014. © 2014 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

Biodynamic Doppler imaging of subcellular motion inside 3D living tissue culture and biopsies (Conference Presentation)

NASA Astrophysics Data System (ADS)

Nolte, David D.

2016-03-01

Biodynamic imaging is an emerging 3D optical imaging technology that probes up to 1 mm deep inside three-dimensional living tissue using short-coherence dynamic light scattering to measure the intracellular motions of cells inside their natural microenvironments. Biodynamic imaging is label-free and non-invasive. The information content of biodynamic imaging is captured through tissue dynamics spectroscopy that displays the changes in the Doppler signatures from intracellular constituents in response to applied compounds. The affected dynamic intracellular mechanisms include organelle transport, membrane undulations, cytoskeletal restructuring, strain at cellular adhesions, cytokinesis, mitosis, exo- and endo-cytosis among others. The development of 3D high-content assays such as biodynamic profiling can become a critical new tool for assessing efficacy of drugs and the suitability of specific types of tissue growth for drug discovery and development. The use of biodynamic profiling to predict clinical outcome of living biopsies to cancer therapeutics can be developed into a phenotypic companion diagnostic, as well as a new tool for therapy selection in personalized medicine. This invited talk will present an overview of the optical, physical and physiological processes involved in biodynamic imaging. Several different biodynamic imaging modalities include motility contrast imaging (MCI), tissue-dynamics spectroscopy (TDS) and tissue-dynamics imaging (TDI). A wide range of potential applications will be described that include process monitoring for 3D tissue culture, drug discovery and development, cancer therapy selection, embryo assessment for in-vitro fertilization and artificial reproductive technologies, among others.

Live CLEM imaging to analyze nuclear structures at high resolution.

PubMed

Haraguchi, Tokuko; Osakada, Hiroko; Koujin, Takako

2015-01-01

Fluorescence microscopy (FM) and electron microscopy (EM) are powerful tools for observing molecular components in cells. FM can provide temporal information about cellular proteins and structures in living cells. EM provides nanometer resolution images of cellular structures in fixed cells. We have combined FM and EM to develop a new method of correlative light and electron microscopy (CLEM), called "Live CLEM." In this method, the dynamic behavior of specific molecules of interest is first observed in living cells using fluorescence microscopy (FM) and then cellular structures in the same cell are observed using electron microscopy (EM). Following image acquisition, FM and EM images are compared to enable the fluorescent images to be correlated with the high-resolution images of cellular structures obtained using EM. As this method enables analysis of dynamic events involving specific molecules of interest in the context of specific cellular structures at high resolution, it is useful for the study of nuclear structures including nuclear bodies. Here we describe Live CLEM that can be applied to the study of nuclear structures in mammalian cells.

Quantitative analysis of phosphoinositide 3-kinase (PI3K) signaling using live-cell total internal reflection fluorescence (TIRF) microscopy.

PubMed

Johnson, Heath E; Haugh, Jason M

2013-12-02

This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3-kinases (PI3Ks) in mammalian cells. The first four protocols cover live-cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live-cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors. Copyright © 2013 John Wiley & Sons, Inc.

Imaging live cells at high spatiotemporal resolution for lab-on-a-chip applications.

PubMed

Chin, Lip Ket; Lee, Chau-Hwang; Chen, Bi-Chang

2016-05-24

Conventional optical imaging techniques are limited by the diffraction limit and difficult-to-image biomolecular and sub-cellular processes in living specimens. Novel optical imaging techniques are constantly evolving with the desire to innovate an imaging tool that is capable of seeing sub-cellular processes in a biological system, especially in three dimensions (3D) over time, i.e. 4D imaging. For fluorescence imaging on live cells, the trade-offs among imaging depth, spatial resolution, temporal resolution and photo-damage are constrained based on the limited photons of the emitters. The fundamental solution to solve this dilemma is to enlarge the photon bank such as the development of photostable and bright fluorophores, leading to the innovation in optical imaging techniques such as super-resolution microscopy and light sheet microscopy. With the synergy of microfluidic technology that is capable of manipulating biological cells and controlling their microenvironments to mimic in vivo physiological environments, studies of sub-cellular processes in various biological systems can be simplified and investigated systematically. In this review, we provide an overview of current state-of-the-art super-resolution and 3D live cell imaging techniques and their lab-on-a-chip applications, and finally discuss future research trends in new and breakthrough research areas of live specimen 4D imaging in controlled 3D microenvironments.

Nanoscale live cell optical imaging of the dynamics of intracellular microvesicles in neural cells.

PubMed

Lee, Sohee; Heo, Chaejeong; Suh, Minah; Lee, Young Hee

2013-11-01

Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

Live visualization of genomic loci with BiFC-TALE

PubMed Central

Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

2017-01-01

Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP. PMID:28074901

Live visualization of genomic loci with BiFC-TALE.

PubMed

Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

2017-01-11

Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP.

WE-DE-BRA-01: SCIENCE COUNCIL JUNIOR INVESTIGATOR COMPETITION WINNER: Acceleration of a Limited-Angle Intrafraction Verification (LIVE) System Using Adaptive Prior Knowledge Based Image Estimation

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

Zhang, Y; Yin, F; Ren, L

Purpose: To develop an adaptive prior knowledge based image estimation method to reduce the scan angle needed in the LIVE system to reconstruct 4D-CBCT for intrafraction verification. Methods: The LIVE system has been previously proposed to reconstructs 4D volumetric images on-the-fly during arc treatment for intrafraction target verification and dose calculation. This system uses limited-angle beam’s eye view (BEV) MV cine images acquired from the treatment beam together with the orthogonally acquired limited-angle kV projections to reconstruct 4D-CBCT images for target verification during treatment. In this study, we developed an adaptive constrained free-form deformation reconstruction technique in LIVE to furthermore » reduce the scanning angle needed to reconstruct the CBCT images. This technique uses free form deformation with energy minimization to deform prior images to estimate 4D-CBCT based on projections acquired in limited angle (orthogonal 6°) during the treatment. Note that the prior images are adaptively updated using the latest CBCT images reconstructed by LIVE during treatment to utilize the continuity of patient motion.The 4D digital extended-cardiac-torso (XCAT) phantom was used to evaluate the efficacy of this technique with LIVE system. A lung patient was simulated with different scenario, including baseline drifts, amplitude change and phase shift. Limited-angle orthogonal kV and beam’s eye view (BEV) MV projections were generated for each scenario. The CBCT reconstructed by these projections were compared with the ground-truth generated in XCAT.Volume-percentage-difference (VPD) and center-of-mass-shift (COMS) were calculated between the reconstructed and the ground-truth tumors to evaluate the reconstruction accuracy. Results: Using orthogonal-view of 6° kV and BEV- MV projections, the VPD/COMS values were 12.7±4.0%/0.7±0.5 mm, 13.0±5.1%/0.8±0.5 mm, and 11.4±5.4%/0.5±0.3 mm for the three scenarios, respectively. Conclusion: The technique enables LIVE to accurately reconstruct 4D-CBCT images using only orthogonal 6° angle, which greatly improves the efficiency and reduces dose of LIVE for intrafraction verification.« less

Coherent Raman Scattering Microscopy in Biology and Medicine.

PubMed

Zhang, Chi; Zhang, Delong; Cheng, Ji-Xin

2015-01-01

Advancements in coherent Raman scattering (CRS) microscopy have enabled label-free visualization and analysis of functional, endogenous biomolecules in living systems. When compared with spontaneous Raman microscopy, a key advantage of CRS microscopy is the dramatic improvement in imaging speed, which gives rise to real-time vibrational imaging of live biological samples. Using molecular vibrational signatures, recently developed hyperspectral CRS microscopy has improved the readout of chemical information available from CRS images. In this article, we review recent achievements in CRS microscopy, focusing on the theory of the CRS signal-to-noise ratio, imaging speed, technical developments, and applications of CRS imaging in bioscience and clinical settings. In addition, we present possible future directions that the use of this technology may take.

Coherent Raman Scattering Microscopy in Biology and Medicine

PubMed Central

Zhang, Chi; Zhang, Delong; Cheng, Ji-Xin

2016-01-01

Advancements in coherent Raman scattering (CRS) microscopy have enabled label-free visualization and analysis of functional, endogenous biomolecules in living systems. When compared with spontaneous Raman microscopy, a key advantage of CRS microscopy is the dramatic improvement in imaging speed, which gives rise to real-time vibrational imaging of live biological samples. Using molecular vibrational signatures, recently developed hyperspectral CRS microscopy has improved the readout of chemical information available from CRS images. In this article, we review recent achievements in CRS microscopy, focusing on the theory of the CRS signal-to-noise ratio, imaging speed, technical developments, and applications of CRS imaging in bioscience and clinical settings. In addition, we present possible future directions that the use of this technology may take. PMID:26514285

Undergraduate Student Perceptions of the Use of Ultrasonography in the Study of "Living Anatomy"

ERIC Educational Resources Information Center

Ivanusic, Jason; Cowie, Brian; Barrington, Michael

2010-01-01

Ultrasonography is a noninvasive imaging modality, and modern ultrasound machines are portable, inexpensive (relative to other imaging modalities), and user friendly. The aim of this study was to explore student perceptions of the use of ultrasound to teach "living anatomy". A module utilizing transthoracic echocardiography was developed and…

A general strategy for developing cell-permeable photo-modulatable organic fluorescent probes for live-cell super-resolution imaging.

PubMed

Pan, Deng; Hu, Zhe; Qiu, Fengwu; Huang, Zhen-Li; Ma, Yilong; Wang, Yina; Qin, Lingsong; Zhang, Zhihong; Zeng, Shaoqun; Zhang, Yu-Hui

2014-11-20

Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes. Here we report a general strategy for constructing cell-permeable photo-modulatable organic fluorescent probes for live-cell SMLM by exploiting the remarkable cytosolic delivery ability of a cell-penetrating peptide (rR)3R2. We develop photo-modulatable organic fluorescent probes consisting of a (rR)3R2 peptide coupled to a cell-impermeable organic fluorophore and a recognition unit. Our results indicate that these organic probes are not only cell permeable but can also specifically and directly label endogenous targeted proteins. Using the probes, we obtain super-resolution images of lysosomes and endogenous F-actin under physiological conditions. We resolve the dynamics of F-actin with 10 s temporal resolution in live cells and discern fine F-actin structures with diameters of ~80 nm. These results open up new avenues in the design of fluorescent probes for live-cell super-resolution imaging.

Live-cell imaging of nuclear-chromosomal dynamics in bovine in vitro fertilised embryos.

PubMed

Yao, Tatsuma; Suzuki, Rie; Furuta, Natsuki; Suzuki, Yuka; Kabe, Kyoko; Tokoro, Mikiko; Sugawara, Atsushi; Yajima, Akira; Nagasawa, Tomohiro; Matoba, Satoko; Yamagata, Kazuo; Sugimura, Satoshi

2018-05-10

Nuclear/chromosomal integrity is an important prerequisite for the assessment of embryo quality in artificial reproductive technology. However, lipid-rich dark cytoplasm in bovine embryos prevents its observation by visible light microscopy. We performed live-cell imaging using confocal laser microscopy that allowed long-term imaging of nuclear/chromosomal dynamics in bovine in vitro fertilised (IVF) embryos. We analysed the relationship between nuclear/chromosomal aberrations and in vitro embryonic development and morphological blastocyst quality. Three-dimensional live-cell imaging of 369 embryos injected with mRNA encoding histone H2B-mCherry and enhanced green fluorescent protein (EGFP)-α-tubulin was performed from single-cell to blastocyst stage for eight days; 17.9% reached the blastocyst stage. Abnormalities in the number of pronuclei (PN), chromosomal segregation, cytokinesis, and blastomere number at first cleavage were observed at frequencies of 48.0%, 30.6%, 8.1%, and 22.2%, respectively, and 13.0%, 6.2%, 3.3%, and 13.4%, respectively, for abnormal embryos developed into blastocysts. A multivariate analysis showed that abnormal chromosome segregation (ACS) and multiple PN correlated with delayed timing and abnormal blastomere number at first cleavage, respectively. In morphologically transferrable blastocysts, 30-40% of embryos underwent ACS and had abnormal PN. Live-cell imaging may be useful for analysing the association between nuclear/chromosomal dynamics and embryonic development in bovine embryos.

Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells.

PubMed

Okuno, Masanari; Hamaguchi, Hiro-o

2010-12-15

We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1 mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

PubMed Central

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-01-01

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass. PMID:23083712

Biological aspects of the development and self-concept in adolescents living in single-parent families.

PubMed

Vecek, Andrea; Vidović, Vesna; Milicić, Jasna; Spoljar-Vrzina, Sanja; Vecek, Nenad; Arch-Vecek, Branka

2009-09-01

In this study we investigate whether there are differences between adolescents who grow up in single-parent families and those who grow up in nucleus families. We have decided that there are no differences in the physical development between the adolescents who are growing up in single parent families and those growing up in nucleus families. There is no difference in the self-concept between these two groups, except in the ethical and moral self-image of adolescents living with one parent. Adolescents living in single-parent families have a weaker moral self-image. It can thus be concluded that physical development and positive self-concept (a favorable image of oneself) in adolescents do not depend on whether an adolescent is growing up in a single-parent or a nucleus family, but on the different characteristics of parents and their relationship with children, whether they are married or not. For the children development the best is healthy marriage of their parents.

A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging.

PubMed

Specht, Elizabeth A; Braselmann, Esther; Palmer, Amy E

2017-02-10

Fluorescent tools have revolutionized our ability to probe biological dynamics, particularly at the cellular level. Fluorescent sensors have been developed on several platforms, utilizing either small-molecule dyes or fluorescent proteins, to monitor proteins, RNA, DNA, small molecules, and even cellular properties, such as pH and membrane potential. We briefly summarize the impressive history of tool development for these various applications and then discuss the most recent noteworthy developments in more detail. Particular emphasis is placed on tools suitable for single-cell analysis and especially live-cell imaging applications. Finally, we discuss prominent areas of need in future fluorescent tool development-specifically, advancing our capability to analyze and integrate the plethora of high-content data generated by fluorescence imaging.

High-Sensitivity Real-Time Imaging of Dual Protein-Protein Interactions in Living Subjects Using Multicolor Luciferases

PubMed Central

Hida, Naoki; Awais, Muhammad; Takeuchi, Masaki; Ueno, Naoto; Tashiro, Mayuri; Takagi, Chiyo; Singh, Tanuja; Hayashi, Makoto; Ohmiya, Yoshihiro; Ozawa, Takeaki

2009-01-01

Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects. PMID:19536355

High-speed atomic force microscopy imaging of live mammalian cells

PubMed Central

Shibata, Mikihiro; Watanabe, Hiroki; Uchihashi, Takayuki; Ando, Toshio; Yasuda, Ryohei

2017-01-01

Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons. PMID:28900590

Real-Time Confocal Imaging Of The Living Eye

NASA Astrophysics Data System (ADS)

Jester, James V.; Cavanagh, H. Dwight; Essepian, John; Shields, William J.; Lemp, Michael A.

1989-12-01

In 1986, we adapted the Tandem Scanning Reflected Light Microscope of Petran and Hadraysky to permit non-invasive, confocal imaging of the living eye in real-time. We were first to obtain stable, confocal optical sections in vivo, from human and animal eyes. Using confocal imaging systems we have now studied living, normal volunteers, rabbits, cats and primates sequentially, non-invasively, and in real-time. The continued development of real-time confocal imaging systems will unlock the door to a new field of cell biology involving for the first time the study of dynamic cellular processes in living organ systems. Towards this end we have concentrated our initial studies on three areas (1) evaluation of confocal microscope systems for real-time image acquisition, (2) studies of the living normal cornea (epithelium, stroma, endothelium) in human and other species; and (3) sequential wound-healing responses in the cornea in single animals to lamellar-keratectomy injury (cellular migration, inflammation, scarring). We believe that this instrument represents an important, new paradigm for research in cell biology and pathology and that it will fundamentally alter all experimental and clinical approaches in future years.

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

PubMed Central

Almassalha, Luay M.; Bauer, Greta M.; Chandler, John E.; Gladstein, Scott; Cherkezyan, Lusik; Stypula-Cyrus, Yolanda; Weinberg, Samuel; Zhang, Di; Thusgaard Ruhoff, Peder; Roy, Hemant K.; Subramanian, Hariharan; Chandel, Navdeep S.; Szleifer, Igal; Backman, Vadim

2016-01-01

The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure–function relationship in live cells. PMID:27702891

Intranucleus Single-Molecule Imaging in Living Cells.

PubMed

Shao, Shipeng; Xue, Boxin; Sun, Yujie

2018-06-01

Many critical processes occurring in mammalian cells are stochastic and can be directly observed at the single-molecule level within their physiological environment, which would otherwise be obscured in an ensemble measurement. There are various fundamental processes in the nucleus, such as transcription, replication, and DNA repair, the study of which can greatly benefit from intranuclear single-molecule imaging. However, the number of such studies is relatively small mainly because of lack of proper labeling and imaging methods. In the past decade, tremendous efforts have been devoted to developing tools for intranuclear imaging. Here, we mainly describe the recent methodological developments of single-molecule imaging and their emerging applications in the live nucleus. We also discuss the remaining issues and provide a perspective on future developments and applications of this field. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Live small-animal X-ray lung velocimetry and lung micro-tomography at the Australian Synchrotron Imaging and Medical Beamline.

PubMed

Murrie, Rhiannon P; Morgan, Kaye S; Maksimenko, Anton; Fouras, Andreas; Paganin, David M; Hall, Chris; Siu, Karen K W; Parsons, David W; Donnelley, Martin

2015-07-01

The high flux and coherence produced at long synchrotron beamlines makes them well suited to performing phase-contrast X-ray imaging of the airways and lungs of live small animals. Here, findings of the first live-animal imaging on the Imaging and Medical Beamline (IMBL) at the Australian Synchrotron are reported, demonstrating the feasibility of performing dynamic lung motion measurement and high-resolution micro-tomography. Live anaesthetized mice were imaged using 30 keV monochromatic X-rays at a range of sample-to-detector propagation distances. A frame rate of 100 frames s(-1) allowed lung motion to be determined using X-ray velocimetry. A separate group of humanely killed mice and rats were imaged by computed tomography at high resolution. Images were reconstructed and rendered to demonstrate the capacity for detailed, user-directed display of relevant respiratory anatomy. The ability to perform X-ray velocimetry on live mice at the IMBL was successfully demonstrated. High-quality renderings of the head and lungs visualized both large structures and fine details of the nasal and respiratory anatomy. The effect of sample-to-detector propagation distance on contrast and resolution was also investigated, demonstrating that soft tissue contrast increases, and resolution decreases, with increasing propagation distance. This new capability to perform live-animal imaging and high-resolution micro-tomography at the IMBL enhances the capability for investigation of respiratory diseases and the acceleration of treatment development in Australia.

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Ra, Hyejun; Gonzalez-Gonzalez, Emilio; Smith, Bryan R.; Gambhir, Sanjiv S.; Kino, Gordon S.; Solgaard, Olav; Kaspar, Roger L.; Contag, Christopher H.

2010-05-01

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

[Development of a Fluorescence Probe for Live Cell Imaging].

PubMed

Shibata, Aya

2017-01-01

 Probes that detect specific biological materials are indispensable tools for deepening our understanding of various cellular phenomena. In live cell imaging, the probe must emit fluorescence only when a specific substance is detected. In this paper, we introduce a new probe we developed for live cell imaging. Glutathione S-transferase (GST) activity is higher in tumor cells than in normal cells and is involved in the development of resistance to various anticancer drugs. We previously reported the development of a general strategy for the synthesis of probes for detection of GST enzymes, including fluorogenic, bioluminogenic, and 19 F-NMR probes. Arylsulfonyl groups were used as caging groups during probe design. The fluorogenic probes were successfully used to quantitate very low levels of GST activity in cell extracts and were also successfully applied to the imaging of microsomal MGST1 activity in living cells. The bioluminogenic and 19 F-NMR probes were able to detect GST activity in Escherichia coli cells. Oligonucleotide-templated reactions are powerful tools for nucleic acid sensing. This strategy exploits the target strand as a template for two functionalized probes and provides a simple molecular mechanism for multiple turnover reactions. We developed a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe completed its reaction within 30 s of initiation and amplified the fluorescence signal from 0.5 pM target oligonucleotide by 1500 fold under isothermal conditions. Additionally, we applied the oligonucleotide-templated reaction for molecular releasing and peptide detection.

Live cell imaging of in vitro human trophoblast syncytialization.

PubMed

Wang, Rui; Dang, Yan-Li; Zheng, Ru; Li, Yue; Li, Weiwei; Lu, Xiaoyin; Wang, Li-Juan; Zhu, Cheng; Lin, Hai-Yan; Wang, Hongmei

2014-06-01

Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live- cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development. © 2014 by the Society for the Study of Reproduction, Inc.

Near-infrared fluorescent proteins for multicolor in vivo imaging

PubMed Central

Shcherbakova, Daria M.; Verkhusha, Vladislav V.

2013-01-01

Near-infrared fluorescent proteins are in high demand for in vivo imaging. We developed four spectrally distinct fluorescent proteins, iRFP670, iRFP682, iRFP702, and iRFP720, from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging in living cells and mice using standard approaches. Five iRFPs including previously engineered iRFP713 allow multicolor imaging in living mice with spectral unmixing. PMID:23770755

Intravital microscopy

PubMed Central

Masedunskas, Andrius; Milberg, Oleg; Porat-Shliom, Natalie; Sramkova, Monika; Wigand, Tim; Amornphimoltham, Panomwat; Weigert, Roberto

2012-01-01

Intravital microscopy is an extremely powerful tool that enables imaging several biological processes in live animals. Recently, the ability to image subcellular structures in several organs combined with the development of sophisticated genetic tools has made possible extending this approach to investigate several aspects of cell biology. Here we provide a general overview of intravital microscopy with the goal of highlighting its potential and challenges. Specifically, this review is geared toward researchers that are new to intravital microscopy and focuses on practical aspects of carrying out imaging in live animals. Here we share the know-how that comes from first-hand experience, including topics such as choosing the right imaging platform and modality, surgery and stabilization techniques, anesthesia and temperature control. Moreover, we highlight some of the approaches that facilitate subcellular imaging in live animals by providing numerous examples of imaging selected organelles and the actin cytoskeleton in multiple organs. PMID:22992750

In vivo 3D PIXE-micron-CT imaging of Drosophila melanogaster using a contrast agent

NASA Astrophysics Data System (ADS)

Matsuyama, Shigeo; Hamada, Naoki; Ishii, Keizo; Nozawa, Yuichiro; Ohkura, Satoru; Terakawa, Atsuki; Hatori, Yoshinobu; Fujiki, Kota; Fujiwara, Mitsuhiro; Toyama, Sho

2015-04-01

In this study, we developed a three-dimensional (3D) computed tomography (CT) in vivo imaging system for imaging small insects with micrometer resolution. The 3D CT imaging system, referred to as 3D PIXE-micron-CT (PIXEμCT), uses characteristic X-rays produced by ion microbeam bombardment of a metal target. PIXEμCT was used to observe the body organs and internal structure of a living Drosophila melanogaster. Although the organs of the thorax were clearly imaged, the digestive organs in the abdominal cavity could not be clearly discerned initially, with the exception of the rectum and the Malpighian tubule. To enhance the abdominal images, a barium sulfate powder radiocontrast agent was added. For the first time, 3D images of the ventriculus of a living D. melanogaster were obtained. Our results showed that PIXEμCT can provide in vivo 3D-CT images that reflect correctly the structure of individual living organs, which is expected to be very useful in biological research.

Ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration.

PubMed

Mort, Richard L; Keighren, Margaret; Hay, Leonard; Jackson, Ian J

2014-05-19

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles(1,2). Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture(3-6). Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue(3,6). High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture(6). By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel.

A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

PubMed Central

Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

2014-01-01

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

Normal development of the tomato clownfish Amphiprion frenatus: live imaging and in situ hybridization analyses of mesodermal and neurectodermal development.

PubMed

Ghosh, J; Wilson, R W; Kudoh, T

2009-12-01

The normal embryonic development of the tomato clownfish Amphiprion frenatus was analysed using live imaging and by in situ hybridization for detection of mesodermal and neurectodermal development. Both morphology of live embryos and tissue-specific staining revealed significant differences in the gross developmental programme of A. frenatus compared with better-known teleost fish models, in particular, initiation of somitogenesis before complete epiboly, initiation of narrowing of the neurectoderm (neurulation) before somitogenesis, relatively early pigmentation of melanophores at the 10-15 somite stage and a distinctive pattern of melanophore distribution. These results suggest evolutionary adaptability of the teleost developmental programme. The ease of obtaining eggs, in vitro culture of the embryo, in situ staining analyses and these reported characteristics make A. frenatus a potentially important model marine fish species for studying embryonic development, physiology, ecology and evolution.

Introduction to Modern Methods in Light Microscopy.

PubMed

Ryan, Joel; Gerhold, Abby R; Boudreau, Vincent; Smith, Lydia; Maddox, Paul S

2017-01-01

For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.

Development of bioluminescence imaging of respiratory syncytial virus (RSV) in virus-infected live mice and its use for evaluation of therapeutics and vaccines.

PubMed

Fuentes, Sandra; Arenas, Diego; Moore, Martin M; Golding, Hana; Khurana, Surender

2017-01-23

Respiratory Syncytial virus (RSV) is one of the leading causes of pneumonia among infants with no human vaccine or efficient curative treatments. Efforts are underway to develop new RSV vaccines and therapeutics. There is a dire need for animal models for preclinical evaluation and selection of products against RSV. Herein, we developed a whole body bioluminescence imaging to follow replication of RSV A2 virus strain expressing firefly luciferase (RSVA2-line19-FFL) in live BALB/c mice that can be used as an extremely sensitive readout for studying effects of antiviral and vaccines in living mice. Strong bioluminescence signal was detected in the nasal cavity and in the lungs following intranasal infection of mice with RSVA2-line19-FFL. The kinetics of viral replication in lungs quantified by daily live imaging strongly correlated with viral titers measured by ex-vivo plaque assay and by assessing viral RNA by qRT-PCR. Vaccination of mice with a pre-fusion F protein elicited high neutralizing antibody titers conferring strong protective immunity against virus replication in the nasal cavity and lungs. In contrast, post-challenge treatment of mice with the monoclonal antibody Palivizumab two days after infection reduced viral replication in the nasal cavity at day 4, but only modestly reduced virus loads in the lungs by day 5. In contrast to RSV bioluminescence, plaque assay did not detect viral titers in lungs on day 5 in Palivizumab-treated animals. This difference between viral loads measured by the two assays was found to be due to coating of virions with the Palivizumab that blocked infection of target cells in vitro and shows importance of live imaging in evaluation of RSV therapeutics. This recombinant RSV based live imaging animal model is convenient and valuable tool that can be used to study host dissemination of RSV and evaluation of antiviral compounds and vaccines against RSV. Published by Elsevier Ltd.

Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

PubMed

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-10-17

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Activatable Fluorescence Probe via Self-Immolative Intramolecular Cyclization for Histone Deacetylase Imaging in Live Cells and Tissues.

PubMed

Liu, Xianjun; Xiang, Meihao; Tong, Zongxuan; Luo, Fengyan; Chen, Wen; Liu, Feng; Wang, Fenglin; Yu, Ru-Qin; Jiang, Jian-Hui

2018-05-01

Histone deacetylases (HDACs) play essential roles in transcription regulation and are valuable theranostic targets. However, there are no activatable fluorescent probes for imaging of HDAC activity in live cells. Here, we develop for the first time a novel activatable two-photon fluorescence probe that enables in situ imaging of HDAC activity in living cells and tissues. The probe is designed by conjugating an acetyl-lysine mimic substrate to a masked aldehyde-containing fluorophore via a cyanoester linker. Upon deacetylation by HDAC, the probe undergoes a rapid self-immolative intramolecular cyclization reaction, producing a cyanohydrin intermediate that is spontaneously rapidly decomposed into the highly fluorescent aldehyde-containing two-photon fluorophore. The probe is shown to exhibit high sensitivity, high specificity, and fast response for HDAC detection in vitro. Imaging studies reveal that the probe is able to directly visualize and monitor HDAC activity in living cells. Moreover, the probe is demonstrated to have the capability of two-photon imaging of HDAC activity in deep tissue slices up to 130 μm. This activatable fluorescent probe affords a useful tool for evaluating HDAC activity and screening HDAC-targeting drugs in both live cell and tissue assays.

Time-lapse imaging of neural development: zebrafish lead the way into the fourth dimension.

PubMed

Rieger, Sandra; Wang, Fang; Sagasti, Alvaro

2011-07-01

Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults. Copyright © 2011 Wiley-Liss, Inc.

Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells

PubMed Central

Fruhwirth, Gilbert O.; Ameer-Beg, Simon; Cook, Richard; Watson, Timothy; Ng, Tony; Festy, Frederic

2010-01-01

Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications. PMID:20588974

Biologically inspired robots elicit a robust fear response in zebrafish

NASA Astrophysics Data System (ADS)

Ladu, Fabrizio; Bartolini, Tiziana; Panitz, Sarah G.; Butail, Sachit; Macrı, Simone; Porfiri, Maurizio

2015-03-01

We investigate the behavioral response of zebrafish to three fear-evoking stimuli. In a binary choice test, zebrafish are exposed to a live allopatric predator, a biologically-inspired robot, and a computer-animated image of the live predator. A target tracking algorithm is developed to score zebrafish behavior. Unlike computer-animated images, the robotic and live predator elicit a robust avoidance response. Importantly, the robotic stimulus elicits more consistent inter-individual responses than the live predator. Results from this effort are expected to aid in hypothesis-driven studies on zebrafish fear response, by offering a valuable approach to maximize data-throughput and minimize animal subjects.

A new xanthene-based two-photon fluorescent probe for the imaging of 1,4-dithiothreitol (DTT) in living cells.

PubMed

Wang, Chao; Dong, Baoli; Kong, Xiuqi; Zhang, Nan; Song, Wenhui; Lin, Weiying

2018-06-21

1,4-Dithiothreitol (DTT) has wide applications in cell biology and biochemistry. Development of effective methods for monitoring DTT in biological systems is important for the safe handling and study of toxicity to humans. Herein, we describe a two-photon fluorescence probe (Rh-DTT) to detect DTT in living systems for the first time. Rh-DTT showed high selectivity and sensitivity to DTT. Rh-DTT can be successfully used for the two-photon imaging of DTT in living cells, and also can detect DTT in living tissues and mice. © 2018 John Wiley & Sons, Ltd.

Live-cell imaging of multiple endogenous mRNAs permits the direct observation of RNA granule dynamics.

PubMed

Yatsuzuka, Kenji; Sato, Shin-Ichi; Pe, Kathleen Beverly; Katsuda, Yousuke; Takashima, Ippei; Watanabe, Mizuki; Uesugi, Motonari

2018-06-08

Here, we developed two pairs of high-contrast chemical probes and their RNA aptamers with distinct readout channels that permitted simultaneous live-cell imaging of endogenous β-actin and cortactin mRNAs. Application of this technology allowed the direct observation of the formation process of stress granules, protein-RNA assemblies essential for cellular response to the environment.

Live face detection based on the analysis of Fourier spectra

NASA Astrophysics Data System (ADS)

Li, Jiangwei; Wang, Yunhong; Tan, Tieniu; Jain, Anil K.

2004-08-01

Biometrics is a rapidly developing technology that is to identify a person based on his or her physiological or behavioral characteristics. To ensure the correction of authentication, the biometric system must be able to detect and reject the use of a copy of a biometric instead of the live biometric. This function is usually termed "liveness detection". This paper describes a new method for live face detection. Using structure and movement information of live face, an effective live face detection algorithm is presented. Compared to existing approaches, which concentrate on the measurement of 3D depth information, this method is based on the analysis of Fourier spectra of a single face image or face image sequences. Experimental results show that the proposed method has an encouraging performance.

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

PubMed Central

Andersen, Erica; Asuri, Namrata; Clay, Matthew; Halloran, Mary

2010-01-01

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons. Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages. Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1. Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work. PMID:20130524

High-resolution imaging of living mammalian cells bound by nanobeads-connected antibodies in a medium using scanning electron-assisted dielectric microscopy

NASA Astrophysics Data System (ADS)

Okada, Tomoko; Ogura, Toshihiko

2017-02-01

Nanometre-scale-resolution imaging technologies for liquid-phase specimens are indispensable tools in various scientific fields. In biology, observing untreated living cells in a medium is essential for analysing cellular functions. However, nanoparticles that bind living cells in a medium are hard to detect directly using traditional optical or electron microscopy. Therefore, we previously developed a novel scanning electron-assisted dielectric microscope (SE-ADM) capable of nanoscale observations. This method enables observation of intact cells in aqueous conditions. Here, we use this SE-ADM system to clearly observe antibody-binding nanobeads in liquid-phase. We also report the successful direct detection of streptavidin-conjugated nanobeads binding to untreated cells in a medium via a biotin-conjugated anti-CD44 antibody. Our system is capable of obtaining clear images of cellular organelles and beads on the cells at the same time. The direct observation of living cells with nanoparticles in a medium allowed by our system may contribute the development of carriers for drug delivery systems (DDS).

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

PubMed Central

Cafferty, Patrick; Xie, Xiaojun; Browne, Kristen; Auld, Vanessa J.

2009-01-01

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals. PMID:19590493

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

PubMed Central

Taylor, Matthew P.; Kratchmarov, Radomir; Enquist, Lynn W.

2013-01-01

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread. PMID:23978901

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

PubMed Central

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-01-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

PubMed

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-03

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

NASA Astrophysics Data System (ADS)

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

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.

Bessel beam fluorescence lifetime tomography of live embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Peng, Leilei

2016-03-01

Optical tomography allows isotropic 3D imaging of embryos. Scanning-laser optical tomography (SLOT) has superior light collecting efficiency than wide-field optical tomography, making it ideal for fluorescence imaging of live embryos. We previously reported an imaging system that combines SLOT with a novel Fourier-multiplexed fluorescence lifetime imaging (FmFLIM) technique named FmFLIM-SLOT. FmFLIM-SLOT performs multiplexed FLIM-FRET readout of multiple FRET sensors in live embryos. Here we report a recent effort on improving the spatial resolution of the FmFLIM-SLOT system in order to image complex biochemical processes in live embryos at the cellular level. Optical tomography has to compromise between resolution and the depth of view. In SLOT, the commonly-used focused Gaussian beam diverges quickly from the focal plane, making it impossible to achieve high resolution imaging in a large volume specimen. We thus introduce Bessel beam laser-scanning tomography, which illuminates the sample with a spatial-light-modulator-generated Bessel beam that has an extended focal depth. The Bessel beam is scanned across the whole specimen. Fluorescence projection images are acquired at equal angular intervals as the sample rotates. Reconstruction artifacts due to annular-rings of the Bessel beam are removed by a modified 3D filtered back projection algorithm. Furthermore, in combination of Fourier-multiplexing fluorescence lifetime imaging (FmFLIM) method, the Bessel FmFLIM-SLOT system is capable of perform 3D lifetime imaging of live embryos at cellular resolution. The system is applied to in-vivo imaging of transgenic Zebrafish embryos. Results prove that Bessel FmFLIM-SLOT is a promising imaging method in development biology research.

In Vitro Culturing and Live Imaging of Drosophila Egg Chambers: A History and Adaptable Method.

PubMed

Peters, Nathaniel C; Berg, Celeste A

2016-01-01

The development of the Drosophila egg chamber encompasses a myriad of diverse germline and somatic events, and as such, the egg chamber has become a widely used and influential developmental model. Advantages of this system include physical accessibility, genetic tractability, and amenability to microscopy and live culturing, the last of which is the focus of this chapter. To provide adequate context, we summarize the structure of the Drosophila ovary and egg chamber, the morphogenetic events of oogenesis, the history of egg-chamber live culturing, and many of the important discoveries that this culturing has afforded. Subsequently, we discuss various culturing methods that have facilitated analyses of different stages of egg-chamber development and different types of cells within the egg chamber, and we present an optimized protocol for live culturing Drosophila egg chambers.We designed this protocol for culturing late-stage Drosophila egg chambers and live imaging epithelial tube morphogenesis, but with appropriate modifications, it can be used to culture egg chambers of any stage. The protocol employs a liquid-permeable, weighted "blanket" to gently hold egg chambers against the coverslip in a glass-bottomed culture dish so the egg chambers can be imaged on an inverted microscope. This setup provides a more buffered, stable, culturing environment than previously published methods by using a larger volume of culture media, but the setup is also compatible with small volumes. This chapter should aid researchers in their efforts to culture and live-image Drosophila egg chambers, further augmenting the impressive power of this model system.

In vitro culturing and live imaging of Drosophila egg chambers: A history and adaptable method

PubMed Central

Peters, Nathaniel C.; Berg, Celeste A.

2017-01-01

Summary/Abstract The development of the Drosophila egg chamber encompasses a myriad of diverse germline and somatic events, and as such, the egg chamber has become a widely used and influential developmental model. Advantages of this system include physical accessibility, genetic tractability, and amenability to microscopy and live culturing, the last of which is the focus of this chapter. To provide adequate context, we summarize the structure of the Drosophila ovary and egg chamber, the morphogenetic events of oogenesis, the history of egg-chamber live culturing, and many of the important discoveries that this culturing has afforded. Subsequently, we discuss various culturing methods that have facilitated analyses of different stages of egg-chamber development and different types of cells within the egg chamber, and we present an optimized protocol for live culturing Drosophila egg chambers. We designed this protocol for culturing late-stage Drosophila egg chambers and live imaging epithelial tube morphogenesis, but with appropriate modifications it can be used to culture egg chambers of any stage. The protocol employs a liquid-permeable, weighted, “blanket” to gently hold egg chambers against the coverslip in a glass-bottomed culture dish so the egg chambers can be imaged on an inverted microscope. This setup provides a more buffered, stable culturing environment than previously published methods by using a larger volume of culture media, but the setup is also compatible with small volumes. This chapter should aid researchers in their efforts to culture and live image Drosophila egg chambers, further augmenting the impressive power of this model system. PMID:27557572

Dynamic views of living cell fine structure revealed by birefringence imaging

NASA Astrophysics Data System (ADS)

Oldenbourg, Rudolf

2001-11-01

We have been developing and applying a new type of polarized light microscope, the new Pol-Scope, which dramatically enhances the unique capabilities of the traditional polarizing microscope. In living cells, without applying exogenous dyes or florescent labels, we have studied the dynamic organization of filamentous actin in neuronal growth cones and improved the efficiency of spindle imaging for in-vitro fertilization and enucleation procedures.

Ultrastructural imaging and molecular modeling of live bacteria using soft x-ray contact microscopy with nanoseconds laser-plasma radiation

NASA Astrophysics Data System (ADS)

Kado, Masataka; Richardson, Martin C.; Gaebel, Kai; Torres, David S.; Rajyaguru, Jayshree; Muszynski, Michael J.

1995-09-01

X-ray images of the various live bacteria, such as Staphylococcus and Streptococcus, and micromolecule such as chromosomal DNA from Escherichis coli, and Lipopolysacchride from Burkholderia cepacia, are obtained with soft x-ray contact microscopy. A compact tabletop type glass laser system is used to produce x-rays from Al, Si, and Au targets. The PMMA photoresists are used to record x-ray images. An AFM (atomic force microscope) is used to reproduce the x-ray images from the developed photoresists. The performance of the 50nm spatial resolutions are achieved and images are able to be discussed on the biological view.

Cell Migration in Tissues: Explant Culture and Live Imaging.

PubMed

Staneva, Ralitza; Barbazan, Jorge; Simon, Anthony; Vignjevic, Danijela Matic; Krndija, Denis

2018-01-01

Cell migration is a process that ensures correct cell localization and function in development and homeostasis. In disease such as cancer, cells acquire an upregulated migratory capacity that leads to their dissemination throughout the body. Live imaging of cell migration allows for better understanding of cell behaviors in development, adult tissue homeostasis and disease. We have optimized live imaging procedures to track cell migration in adult murine tissue explants derived from: (1) healthy gut; (2) primary intestinal carcinoma; and (3) the liver, a common metastatic site. To track epithelial cell migration in the gut, we generated an inducible fluorescent reporter mouse, enabling us to visualize and track individual cells in unperturbed gut epithelium. To image intratumoral cancer cells, we use a spontaneous intestinal cancer model based on the activation of Notch1 and deletion of p53 in the mouse intestinal epithelium, which gives rise to aggressive carcinoma. Interaction of cancer cells with a metastatic niche, the mouse liver, is addressed using a liver colonization model. In summary, we describe a method for long-term 3D imaging of tissue explants by two-photon excitation microscopy. Explant culturing and imaging can help understand dynamic behavior of cells in homeostasis and disease, and would be applicable to various tissues.

Fast and background-free three-dimensional (3D) live-cell imaging with lanthanide-doped upconverting nanoparticles.

PubMed

Jo, Hong Li; Song, Yo Han; Park, Jinho; Jo, Eun-Jung; Goh, Yeongchang; Shin, Kyujin; Kim, Min-Gon; Lee, Kang Taek

2015-12-14

We report on the development of a three-dimensional (3D) live-cell imaging technique with high spatiotemporal resolution using lanthanide-doped upconverting nanoparticles (UCNPs). It employs the sectioning capability of confocal microscopy except that the two-dimensional (2D) section images are acquired by wide-field epi-fluorescence microscopy. Although epi-fluorescence images are contaminated with the out-of-focus background in general, the near-infrared (NIR) excitation used for the excitation of UCNPs does not generate any autofluorescence, which helps to lower the background. Moreover, the image blurring due to defocusing was naturally eliminated in the image reconstruction process. The 3D images were used to investigate the cellular dynamics such as nuclear uptake and single-particle tracking that require 3D description.

Live-cell imaging of cell signaling using genetically encoded fluorescent reporters.

PubMed

Ni, Qiang; Mehta, Sohum; Zhang, Jin

2018-01-01

Synergistic advances in fluorescent protein engineering and live-cell imaging techniques in recent years have fueled the concurrent development and application of genetically encoded fluorescent reporters that are tailored for tracking signaling dynamics in living systems over multiple length and time scales. These biosensors are uniquely suited for this challenging task, owing to their specificity, sensitivity, and versatility, as well as to the noninvasive and nondestructive nature of fluorescence and the power of genetic encoding. Over the past 10 years, a growing number of fluorescent reporters have been developed for tracking a wide range of biological signals in living cells and animals, including second messenger and metabolite dynamics, enzyme activation and activity, and cell cycle progression and neuronal activity. Many of these biosensors are gaining wide use and are proving to be indispensable for unraveling the complex biological functions of individual signaling molecules in their native environment, the living cell, shedding new light on the structural and molecular underpinnings of cell signaling. In this review, we highlight recent advances in protein engineering that are likely to help expand and improve the design and application of these valuable tools. We then turn our focus to specific examples of live-cell imaging using genetically encoded fluorescent reporters as an important platform for advancing our understanding of G protein-coupled receptor signaling and neuronal activity. © 2017 Federation of European Biochemical Societies.

Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging.

PubMed

Ploemen, Ivo H J; Prudêncio, Miguel; Douradinha, Bruno G; Ramesar, Jai; Fonager, Jannik; van Gemert, Geert-Jan; Luty, Adrian J F; Hermsen, Cornelus C; Sauerwein, Robert W; Baptista, Fernanda G; Mota, Maria M; Waters, Andrew P; Que, Ivo; Lowik, Clemens W G M; Khan, Shahid M; Janse, Chris J; Franke-Fayard, Blandine M D

2009-11-18

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc(con), expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

PubMed Central

Douradinha, Bruno G.; Ramesar, Jai; Fonager, Jannik; van Gemert, Geert-Jan; Luty, Adrian J. F.; Hermsen, Cornelus C.; Sauerwein, Robert W.; Baptista, Fernanda G.; Mota, Maria M.; Waters, Andrew P.; Que, Ivo; Lowik, Clemens W. G. M.; Khan, Shahid M.; Janse, Chris J.; Franke-Fayard, Blandine M. D.

2009-01-01

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium. PMID:19924309

Use of sonic tomography to detect and quantify wood decay in living trees1

PubMed Central

Gilbert, Gregory S.; Ballesteros, Javier O.; Barrios-Rodriguez, Cesar A.; Bonadies, Ernesto F.; Cedeño-Sánchez, Marjorie L.; Fossatti-Caballero, Nohely J.; Trejos-Rodríguez, Mariam M.; Pérez-Suñiga, José Moises; Holub-Young, Katharine S.; Henn, Laura A. W.; Thompson, Jennifer B.; García-López, Cesar G.; Romo, Amanda C.; Johnston, Daniel C.; Barrick, Pablo P.; Jordan, Fulvia A.; Hershcovich, Shiran; Russo, Natalie; Sánchez, Juan David; Fábrega, Juan Pablo; Lumpkin, Raleigh; McWilliams, Hunter A.; Chester, Kathleen N.; Burgos, Alana C.; Wong, E. Beatriz; Diab, Jonathan H.; Renteria, Sonia A.; Harrower, Jennifer T.; Hooton, Douglas A.; Glenn, Travis C.; Faircloth, Brant C.; Hubbell, Stephen P.

2016-01-01

Premise of the study: Field methodology and image analysis protocols using acoustic tomography were developed and evaluated as a tool to estimate the amount of internal decay and damage of living trees, with special attention to tropical rainforest trees with irregular trunk shapes. Methods and Results: Living trunks of a diversity of tree species in tropical rainforests in the Republic of Panama were scanned using an Argus Electronic PiCUS 3 Sonic Tomograph and evaluated for the amount and patterns of internal decay. A protocol using ImageJ analysis software was used to quantify the proportions of intact and compromised wood. The protocols provide replicable estimates of internal decay and cavities for trees of varying shapes, wood density, and bark thickness. Conclusions: Sonic tomography, coupled with image analysis, provides an efficient, noninvasive approach to evaluate decay patterns and structural integrity of even irregularly shaped living trees. PMID:28101433

Three-Dimensional, Live-Cell Imaging of Chromatin Dynamics in Plant Nuclei Using Chromatin Tagging Systems.

PubMed

Hirakawa, Takeshi; Matsunaga, Sachihiro

2016-01-01

In plants, chromatin dynamics spatiotemporally change in response to various environmental stimuli. However, little is known about chromatin dynamics in the nuclei of plants. Here, we introduce a three-dimensional, live-cell imaging method that can monitor chromatin dynamics in nuclei via a chromatin tagging system that can visualize specific genomic loci in living plant cells. The chromatin tagging system is based on a bacterial operator/repressor system in which the repressor is fused to fluorescent proteins. A recent refinement of promoters for the system solved the problem of gene silencing and abnormal pairing frequencies between operators. Using this system, we can detect the spatiotemporal dynamics of two homologous loci as two fluorescent signals within a nucleus and monitor the distance between homologous loci. These live-cell imaging methods will provide new insights into genome organization, development processes, and subnuclear responses to environmental stimuli in plants.

Morpholine Derivative-Functionalized Carbon Dots-Based Fluorescent Probe for Highly Selective Lysosomal Imaging in Living Cells.

PubMed

Wu, Luling; Li, Xiaolin; Ling, Yifei; Huang, Chusen; Jia, Nengqin

2017-08-30

The development of a suitable fluorescent probe for the specific labeling and imaging of lysosomes through the direct visual fluorescent signal is extremely important for understanding the dysfunction of lysosomes, which might induce various pathologies, including neurodegenerative diseases, cancer, and Alzheimer's disease. Herein, a new carbon dot-based fluorescent probe (CDs-PEI-ML) was designed and synthesized for highly selective imaging of lysosomes in live cells. In this probe, PEI (polyethylenimine) is introduced to improve water solubility and provide abundant amine groups for the as-prepared CDs-PEI, and the morpholine group (ML) serves as a targeting unit for lysosomes. More importantly, passivation with PEI could dramatically increase the fluorescence quantum yield of CDs-PEI-ML as well as their stability in fluorescence emission under different excitation wavelength. Consequently, experimental data demonstrated that the target probe CDs-PEI-ML has low cytotoxicity and excellent photostability. Additionally, further live cell imaging experiment indicated that CDs-PEI-ML is a highly selective fluorescent probe for lysosomes. We speculate the mechanism for selective staining of lysosomes that CDs-PEI-ML was initially taken up by lysosomes through the endocytic pathway and then accumulated in acidic lysosomes. It is notable that there was less diffusion of CDs-PEI-ML into cytoplasm, which could be ascribed to the presence of lysosome target group morpholine on surface of CDs-PEI-ML. The blue emission wavelength combined with the high photo stability and ability of long-lasting cell imaging makes CDs-PEI-ML become an alternative fluorescent probe for multicolor labeling and long-term tracking of lysosomes in live cells and the potential application in super-resolution imaging. To best of our knowledge, there are still limited carbon dots-based fluorescent probes that have been studied for specific lysosomal imaging in live cells. The concept of surface functionality of carbon dots will also pave a new avenue for developing carbon dots-based fluorescent probes for subcellular labeling.

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

PubMed

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

2018-05-18

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

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis

PubMed Central

Masuda, Isao; Igarashi, Takao; Sakaguchi, Reiko; Nitharwal, Ram G.; Takase, Ryuichi; Han, Kyu Young; Leslie, Benjamin J.; Liu, Cuiping; Gamper, Howard; Ha, Taekjip; Sanyal, Suparna

2017-01-01

Abstract Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering. PMID:27956502

Hologlyphics: volumetric image synthesis performance system

NASA Astrophysics Data System (ADS)

Funk, Walter

2008-02-01

This paper describes a novel volumetric image synthesis system and artistic technique, which generate moving volumetric images in real-time, integrated with music. The system, called the Hologlyphic Funkalizer, is performance based, wherein the images and sound are controlled by a live performer, for the purposes of entertaining a live audience and creating a performance art form unique to volumetric and autostereoscopic images. While currently configured for a specific parallax barrier display, the Hologlyphic Funkalizer's architecture is completely adaptable to various volumetric and autostereoscopic display technologies. Sound is distributed through a multi-channel audio system; currently a quadraphonic speaker setup is implemented. The system controls volumetric image synthesis, production of music and spatial sound via acoustic analysis and human gestural control, using a dedicated control panel, motion sensors, and multiple musical keyboards. Music can be produced by external acoustic instruments, pre-recorded sounds or custom audio synthesis integrated with the volumetric image synthesis. Aspects of the sound can control the evolution of images and visa versa. Sounds can be associated and interact with images, for example voice synthesis can be combined with an animated volumetric mouth, where nuances of generated speech modulate the mouth's expressiveness. Different images can be sent to up to 4 separate displays. The system applies many novel volumetric special effects, and extends several film and video special effects into the volumetric realm. Extensive and various content has been developed and shown to live audiences by a live performer. Real world applications will be explored, with feedback on the human factors.

Mechanism of Change in Cognitive Behavioral Therapy for Body Image and Self-Care on ART Adherence Among Sexual Minority Men Living with HIV.

PubMed

Lamb, Kalina M; Nogg, Kelsey A; Safren, Steven A; Blashill, Aaron J

2018-05-11

Body image disturbance is a common problem reported among sexual minority men living with HIV, and is associated with poor antiretroviral therapy (ART) adherence. Recently, a novel integrated intervention (cognitive behavioral therapy for body image and self-care; CBT-BISC) was developed and pilot tested to simultaneously improve body image and ART adherence in this population. Although CBT-BISC has demonstrated preliminary efficacy in improving ART adherence, the mechanisms of change are unknown. Utilizing data from a two-armed randomized controlled trial (N = 44 sexual minority men living with HIV), comparing CBT-BISC to an enhanced treatment as usual (ETAU) condition, sequential process mediation via latent difference scores was assessed, with changes in body image disturbance entered as the mechanism between treatment condition and changes in ART adherence. Participants assigned to CBT-BISC reported statistically significant reductions in body image disturbance post-intervention, which subsequently predicted changes in ART adherence from post-intervention to long term follow-up (b = 20.01, SE = 9.11, t = 2.19, p = 0.028). One pathway in which CBT-BISC positively impacts ART adherence is through reductions in body image disturbance. Body image disturbance represents one, of likely several, mechanism that prospectively predicts ART adherence among sexual minority men living with HIV.

Development of background-free tame fluorescent probes for intracellular live cell imaging

PubMed Central

Alamudi, Samira Husen; Satapathy, Rudrakanta; Kim, Jihyo; Su, Dongdong; Ren, Haiyan; Das, Rajkumar; Hu, Lingna; Alvarado-Martínez, Enrique; Lee, Jung Yeol; Hoppmann, Christian; Peña-Cabrera, Eduardo; Ha, Hyung-Ho; Park, Hee-Sung; Wang, Lei; Chang, Young-Tae

2016-01-01

Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as ‘tame' probes, and novel tools for live cell intracellular imaging. PMID:27321135

Murine fetal echocardiography.

PubMed

Kim, Gene H

2013-02-15

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures.

Intravital microscopy: a novel tool to study cell biology in living animals.

PubMed

Weigert, Roberto; Sramkova, Monika; Parente, Laura; Amornphimoltham, Panomwat; Masedunskas, Andrius

2010-05-01

Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.

Design and validation of a new ratiometric intracellular pH imaging probe using lanthanide-doped upconverting nanoparticles.

PubMed

Du, Shuoren; Hernández-Gil, Javier; Dong, Hao; Zheng, Xiaoyu; Lyu, Guangming; Bañobre-López, Manuel; Gallo, Juan; Sun, Ling-Dong; Yan, Chun-Hua; Long, Nicholas J

2017-10-17

pH homeostasis is strictly controlled at a subcellular level. A deregulation of the intra/extra/subcellular pH environment is associated with a number of diseases and as such, the monitoring of the pH state of cells and tissues is a valuable diagnostic tool. To date, only a few tools have been developed to measure the pH in living cells with the spatial resolution needed for intracellular imaging. Among the techniques available, only optical imaging offers enough resolution and biocompatibility to be proposed for subcellular pH monitoring. We present herein a ratiometric probe based on upconversion nanoparticles modified with a pH sensitive moiety for the quantitative imaging of pH at the subcellular level in living cells. This system provides the properties required for live cell quantitative imaging i.e. positive cellular uptake, biocompatibility, long wavelength excitation, sensitive response to pH within a biologically relevant range, and self-referenced signal.

[Quantitative data analysis for live imaging of bone.

PubMed

Seno, Shigeto

Bone tissue is a hard tissue, it was difficult to observe the interior of the bone tissue alive. With the progress of microscopic technology and fluorescent probe technology in recent years, it becomes possible to observe various activities of various cells forming bone society. On the other hand, the quantitative increase in data and the diversification and complexity of the images makes it difficult to perform quantitative analysis by visual inspection. It has been expected to develop a methodology for processing microscopic images and data analysis. In this article, we introduce the research field of bioimage informatics which is the boundary area of biology and information science, and then outline the basic image processing technology for quantitative analysis of live imaging data of bone.

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

PubMed

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

2013-03-01

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

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

PubMed Central

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

2013-01-01

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

Synthesis and Live-Cell Imaging of Fluorescent Sterols for Analysis of Intracellular Cholesterol Transport.

PubMed

Modzel, Maciej; Lund, Frederik W; Wüstner, Daniel

2017-01-01

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol ® ; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

Intelligent Design of Nano-Scale Molecular Imaging Agents

PubMed Central

Kim, Sung Bae; Hattori, Mitsuru; Ozawa, Takeaki

2012-01-01

Visual representation and quantification of biological processes at the cellular and subcellular levels within living subjects are gaining great interest in life science to address frontier issues in pathology and physiology. As intact living subjects do not emit any optical signature, visual representation usually exploits nano-scale imaging agents as the source of image contrast. Many imaging agents have been developed for this purpose, some of which exert nonspecific, passive, and physical interaction with a target. Current research interest in molecular imaging has mainly shifted to fabrication of smartly integrated, specific, and versatile agents that emit fluorescence or luminescence as an optical readout. These agents include luminescent quantum dots (QDs), biofunctional antibodies, and multifunctional nanoparticles. Furthermore, genetically encoded nano-imaging agents embedding fluorescent proteins or luciferases are now gaining popularity. These agents are generated by integrative design of the components, such as luciferase, flexible linker, and receptor to exert a specific on–off switching in the complex context of living subjects. In the present review, we provide an overview of the basic concepts, smart design, and practical contribution of recent nano-scale imaging agents, especially with respect to genetically encoded imaging agents. PMID:23235326

Intelligent design of nano-scale molecular imaging agents.

PubMed

Kim, Sung Bae; Hattori, Mitsuru; Ozawa, Takeaki

2012-12-12

Visual representation and quantification of biological processes at the cellular and subcellular levels within living subjects are gaining great interest in life science to address frontier issues in pathology and physiology. As intact living subjects do not emit any optical signature, visual representation usually exploits nano-scale imaging agents as the source of image contrast. Many imaging agents have been developed for this purpose, some of which exert nonspecific, passive, and physical interaction with a target. Current research interest in molecular imaging has mainly shifted to fabrication of smartly integrated, specific, and versatile agents that emit fluorescence or luminescence as an optical readout. These agents include luminescent quantum dots (QDs), biofunctional antibodies, and multifunctional nanoparticles. Furthermore, genetically encoded nano-imaging agents embedding fluorescent proteins or luciferases are now gaining popularity. These agents are generated by integrative design of the components, such as luciferase, flexible linker, and receptor to exert a specific on-off switching in the complex context of living subjects. In the present review, we provide an overview of the basic concepts, smart design, and practical contribution of recent nano-scale imaging agents, especially with respect to genetically encoded imaging agents.

Go_LIVE! - Global Near-real-time Land Ice Velocity data from Landsat 8 at NSIDC

NASA Astrophysics Data System (ADS)

Klinger, M. J.; Fahnestock, M. A.; Scambos, T. A.; Gardner, A. S.; Haran, T. M.; Moon, T. A.; Hulbe, C. L.; Berthier, E.

2016-12-01

The National Snow and Ice Data Center (NSIDC) is developing a processing and staging system under NASA funding for near-real-time global ice velocity data derived from Landsat 8 panchromatic imagery: Global Land Ice Velocity Extraction from Landsat (Go_LIVE). The system performs repeat image feature tracking using newly developed Python Correlation (PyCorr) software applied to image pairs covering all glaciers > 5km2 as well as both ice sheets. We correlate each Landsat 8 path-row image with matching path-row images acquired within the previous 400 days. Real-Time (RT) panchromatic Landsat 8 L1T images have geolocation accuracy of 5 meters and high radiometric sensitivity (12-bit), allowing for feature matching over low-contrast snow and ice surfaces. High-pass filters are applied to the imagery to enhance local surface texture and improve correlation returns. Despite the excellent geolocation accuracy of Landsat 8, the remaining error introduces an artificial offset in the velocity returns. To correct this error, we apply a shift to the x and y grids to bring the displacement field to zero over known stationary features such as bedrock. For ice sheet interiors where stationary features do not exist, we use near-zero (<10 ma-1) or slow-moving ice areas (10-25 ma-1) to refine velocities. Go_LIVE will eventually include Landsat 7, 5 and 4 imagery as well. Go_LIVE runs on the University of Colorado's supercomputer and Peta Library storage system to process 10,000 image pairs per hour. We are currently developing a web-based data access site at NSIDC. The data are provided in NetCDF (Network Common Data Format) as geolocated grids of x and y velocity components at 300 m spacing with accompanying error and quality parameters. Extensive data sets currently exist for Alaskan, Antarctic, and Greenlandic ice areas, and are available upon request to NSIDC. Go_LIVE's goal for 2017 is a system that updates global ice velocity at few-day or shorter latency.

Live cell imaging of the HIV-1 life cycle

PubMed Central

Campbell, Edward M.; Hope, Thomas J.

2010-01-01

Technology developed in the past 10 years has dramatically increased the ability of researchers to directly visualize and measure various stages of the HIV type 1 (HIV-1) life cycle. In many cases, imaging-based approaches have filled critical gaps in our understanding of how certain aspects of viral replication occur in cells. Specifically, live cell imaging has allowed a better understanding of dynamic, transient events that occur during HIV-1 replication, including the steps involved in viral fusion, trafficking of the viral nucleoprotein complex in the cytoplasm and even the nucleus during infection and the formation of new virions from an infected cell. In this review, we discuss how researchers have exploited fluorescent microscopy methodologies to observe and quantify these events occurring during the replication of HIV-1 in living cells. PMID:18977142

Recent advancements in structured-illumination microscopy toward live-cell imaging.

PubMed

Hirano, Yasuhiro; Matsuda, Atsushi; Hiraoka, Yasushi

2015-08-01

Fluorescence microscopy allows us to observe fluorescently labeled molecules in diverse biological processes and organelle structures within living cells. However, the diffraction limit restricts its spatial resolution to about half of its wavelength, limiting the capability of biological observation at the molecular level. Structured-illumination microscopy (SIM), a type of super-resolution microscopy, doubles the spatial resolution in all three dimensions by illuminating the sample with a patterned excitation light, followed by computer reconstruction. SIM uses a relatively low illumination power compared with other methods of super-resolution microscopy and is easily available for multicolor imaging. SIM has great potential for meeting the requirements of live-cell imaging. Recent developments in diverse types of SIM have achieved higher spatial (∼50 nm lateral) and temporal (∼100 Hz) resolutions. Here, we review recent advancements in SIM and discuss its application in noninvasive live-cell imaging. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

Three-dimensional spectral-spatial EPR imaging of free radicals in the heart: a technique for imaging tissue metabolism and oxygenation.

PubMed Central

Kuppusamy, P; Chzhan, M; Vij, K; Shteynbuk, M; Lefer, D J; Giannella, E; Zweier, J L

1994-01-01

It has been hypothesized that free radical metabolism and oxygenation in living organs and tissues such as the heart may vary over the spatially defined tissue structure. In an effort to study these spatially defined differences, we have developed electron paramagnetic resonance imaging instrumentation enabling the performance of three-dimensional spectral-spatial images of free radicals infused into the heart and large vessels. Using this instrumentation, high-quality three-dimensional spectral-spatial images of isolated perfused rat hearts and rabbit aortas are obtained. In the isolated aorta, it is shown that spatially and spectrally accurate images of the vessel lumen and wall could be obtained in this living vascular tissue. In the isolated rat heart, imaging experiments were performed to determine the kinetics of radical clearance at different spatial locations within the heart during myocardial ischemia. The kinetic data show the existence of regional and transmural differences in myocardial free radical clearance. It is further demonstrated that EPR imaging can be used to noninvasively measure spatially localized oxygen concentrations in the heart. Thus, the technique of spectral-spatial EPR imaging is shown to be a powerful tool in providing spatial information regarding the free radical distribution, metabolism, and tissue oxygenation in living biological organs and tissues. Images PMID:8159757

Far-field photostable optical nanoscopy (PHOTON) for real-time super-resolution single-molecular imaging of signaling pathways of single live cells

NASA Astrophysics Data System (ADS)

Huang, Tao; Browning, Lauren M.; Xu, Xiao-Hong Nancy

2012-04-01

Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions.Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11739h

Bright fluorescent Streptococcus pneumoniae for live-cell imaging of host-pathogen interactions.

PubMed

Kjos, Morten; Aprianto, Rieza; Fernandes, Vitor E; Andrew, Peter W; van Strijp, Jos A G; Nijland, Reindert; Veening, Jan-Willem

2015-03-01

Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Advanced Motion Compensation Methods for Intravital Optical Microscopy

PubMed Central

Vinegoni, Claudio; Lee, Sungon; Feruglio, Paolo Fumene; Weissleder, Ralph

2013-01-01

Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions. PMID:24273405

DNAzyme sensors for detection of metal ions in the environment and imaging them in living cells

PubMed Central

McGhee, Claire E.; Loh, Kang Yong

2017-01-01

The on-site and real-time detection of metal ions is important for environmental monitoring and for understanding the impact of metal ions on human health. However, developing sensors selective for a wide range of metal ions that can work in the complex matrices of untreated samples and cells presents significant challenges. To meet these challenges, DNAzymes, an emerging class of metal ion-dependent enzymes selective for almost any metal ion, have been functionalized with fluorophores, nanoparticles and other imaging agents and incorporated into sensors for the detection of metal ions in environmental samples and for imaging the metal ions in living cells. Herein, we highlight the recent developments of DNAzyme-based fluorescent, colorimetric, SERS, electrochemical and electrochemiluminscent sensors for metal ions for these applications. PMID:28458112

Highly Sensitive Detection of Caspase-3/7 Activity in Living Mice Using Enzyme-Responsive 19F MRI Nanoprobes.

PubMed

Akazawa, Kazuki; Sugihara, Fuminori; Nakamura, Tatsuya; Mizukami, Shin; Kikuchi, Kazuya

2018-05-16

Highly sensitive imaging of enzymatic activities in the deep tissues of living mammals provides useful information about their biological functions and for developing new drugs; however, such imaging is challenging. 19 F magnetic resonance imaging (MRI) is suitable for noninvasive visualization of enzymatic activities without endogenous background signals. Although various enzyme-responsive 19 F MRI probes have been developed, most cannot be used for in vivo imaging because of their low sensitivity. Recently, we developed unique nanoparticles, called FLAMEs, that are composed of a liquid perfluorocarbon core and a robust silica shell, and demonstrated their outstanding sensitivity in vivo. Here, we report a highly functionalized nanoprobe, FLAME-DEVD 2, with an OFF/ON 19 F MRI switch for detecting caspase-3/7 activity based on the paramagnetic relaxation enhancement effect. To improve the cleavage efficiency of peptides by caspase-3, we designed a novel Gd 3+ complex-conjugated peptide, DEVD X ( X = 1, 2), which is a substrate peptide sequence tandemly repeated X times, and demonstrated that DEVD 2 showed faster cleavage kinetics than DEVD 1. By incorporating this novel concept into a signal activation strategy, FLAME-DEVD 2 showed a high 19 F MRI signal enhancement rate in response to caspase-3 activity. After intravenous injection of FLAME-DEVD 2 and an apoptosis-inducing reagent, caspase-3/7 activity in the spleen of a living mouse was successfully imaged by 19 F MRI. This imaging platform shows great potential for highly sensitive detection of enzymatic activities in vivo.

Observing DNA in live cells.

PubMed

Weiss, Lucien E; Naor, Tal; Shechtman, Yoav

2018-06-19

The structural organization and dynamics of DNA are known to be of paramount importance in countless cellular processes, but capturing these events poses a unique challenge. Fluorescence microscopy is well suited for these live-cell investigations, but requires attaching fluorescent labels to the species under investigation. Over the past several decades, a suite of techniques have been developed for labeling and imaging DNA, each with various advantages and drawbacks. Here, we provide an overview of the labeling and imaging tools currently available for visualizing DNA in live cells, and discuss their suitability for various applications. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Integrated dual-tomography for refractive index analysis of free-floating single living cell with isotropic superresolution.

PubMed

B, Vinoth; Lai, Xin-Ji; Lin, Yu-Chih; Tu, Han-Yen; Cheng, Chau-Jern

2018-04-13

Digital holographic microtomography is a promising technique for three-dimensional (3D) measurement of the refractive index (RI) profiles of biological specimens. Measurement of the RI distribution of a free-floating single living cell with an isotropic superresolution had not previously been accomplished. To the best of our knowledge, this is the first study focusing on the development of an integrated dual-tomographic (IDT) imaging system for RI measurement of an unlabelled free-floating single living cell with an isotropic superresolution by combining the spatial frequencies of full-angle specimen rotation with those of beam rotation. A novel 'UFO' (unidentified flying object) like shaped coherent transfer function is obtained. The IDT imaging system does not require any complex image-processing algorithm for 3D reconstruction. The working principle was successfully demonstrated and a 3D RI profile of a single living cell, Candida rugosa, was obtained with an isotropic superresolution. This technology is expected to set a benchmark for free-floating single live sample measurements without labeling or any special sample preparations for the experiments.

Peering into Cells One Molecule at a Time: Single-molecule and plasmon-enhanced fluorescence super-resolution imaging

NASA Astrophysics Data System (ADS)

Biteen, Julie

2013-03-01

Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale, allowing us to unlock the mysteries of how biomolecules work together to achieve the complexity that is a cell. This high-resolution, non-destructive method for examining subcellular events has opened up an exciting new frontier: the study of macromolecular localization and dynamics in living cells. We have developed methods for single-molecule investigations of live bacterial cells, and have used these techniques to investigate thee important prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each system presents unique challenges, and we will discuss the important methods developed for each system. Furthermore, we use the plasmon modes of bio-compatible metal nanoparticles to enhance the emissivity of single-molecule fluorophores. The resolution of single-molecule imaging in cells is generally limited to 20-40 nm, far worse than the 1.5-nm localization accuracies which have been attained in vitro. We use plasmonics to improve the brightness and stability of single-molecule probes, and in particular fluorescent proteins, which are widely used for bio-imaging. We find that gold-coupled fluorophores demonstrate brighter, longer-lived emission, yielding an overall enhancement in total photons detected. Ultimately, this results in increased localization accuracy for single-molecule imaging. Furthermore, since fluorescence intensity is proportional to local electromagnetic field intensity, these changes in decay intensity and rate serve as a nm-scale read-out of the field intensity. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging, and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

Time-lapse monitoring of TLR2 ligand internalization with newly developed fluorescent probes.

PubMed

Arai, Yohei; Yokoyama, Kouhei; Kawahara, Yuki; Feng, Qi; Ohta, Ippei; Shimoyama, Atsushi; Inuki, Shinsuke; Fukase, Koichi; Kabayama, Kazuya; Fujimoto, Yukari

2018-05-23

As a mammalian toll-like receptor family member protein, TLR2 recognizes lipoproteins from bacteria and modulates the immune response by inducing the expression of various cytokines. We have developed fluorescence-labeled TLR2 ligands with either hydrophilic or hydrophobic fluorescence groups. The labeled ligands maintained the inflammatory IL-6 induction activity and enabled us to observe the internalization and colocalization of the TLR2 ligands using live-cell imaging. The time-lapse monitoring in the live-cell imaging of the fluorescence-labeled TLR2 ligand showed that TLR2/CD14 expression in the host cells enhanced the internalization of TLR2 ligand molecules.

Thermal particle image velocity estimation of fire plume flow

Treesearch

Xiangyang Zhou; Lulu Sun; Shankar Mahalingam; David R. Weise

2003-01-01

For the purpose of studying wildfire spread in living vegetation such as chaparral in California, a thermal particle image velocity (TPIV) algorithm for nonintrusively measuring flame gas velocities through thermal infrared (IR) imagery was developed. By tracing thermal particles in successive digital IR images, the TPIV algorithm can estimate the velocity field in a...

Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

NASA Astrophysics Data System (ADS)

Winckler, Pascale; Lartigue, Lydia; Giannone, Gregory; de Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

2013-08-01

Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

Iris Image Classification Based on Hierarchical Visual Codebook.

PubMed

Zhenan Sun; Hui Zhang; Tieniu Tan; Jianyu Wang

2014-06-01

Iris recognition as a reliable method for personal identification has been well-studied with the objective to assign the class label of each iris image to a unique subject. In contrast, iris image classification aims to classify an iris image to an application specific category, e.g., iris liveness detection (classification of genuine and fake iris images), race classification (e.g., classification of iris images of Asian and non-Asian subjects), coarse-to-fine iris identification (classification of all iris images in the central database into multiple categories). This paper proposes a general framework for iris image classification based on texture analysis. A novel texture pattern representation method called Hierarchical Visual Codebook (HVC) is proposed to encode the texture primitives of iris images. The proposed HVC method is an integration of two existing Bag-of-Words models, namely Vocabulary Tree (VT), and Locality-constrained Linear Coding (LLC). The HVC adopts a coarse-to-fine visual coding strategy and takes advantages of both VT and LLC for accurate and sparse representation of iris texture. Extensive experimental results demonstrate that the proposed iris image classification method achieves state-of-the-art performance for iris liveness detection, race classification, and coarse-to-fine iris identification. A comprehensive fake iris image database simulating four types of iris spoof attacks is developed as the benchmark for research of iris liveness detection.

Advanced imaging techniques for the study of plant growth and development.

PubMed

Sozzani, Rosangela; Busch, Wolfgang; Spalding, Edgar P; Benfey, Philip N

2014-05-01

A variety of imaging methodologies are being used to collect data for quantitative studies of plant growth and development from living plants. Multi-level data, from macroscopic to molecular, and from weeks to seconds, can be acquired. Furthermore, advances in parallelized and automated image acquisition enable the throughput to capture images from large populations of plants under specific growth conditions. Image-processing capabilities allow for 3D or 4D reconstruction of image data and automated quantification of biological features. These advances facilitate the integration of imaging data with genome-wide molecular data to enable systems-level modeling. Copyright © 2013 Elsevier Ltd. All rights reserved.

Live-cell imaging of conidial anastomosis tube fusion during colony initiation in Fusarium oxysporum

PubMed Central

Kurian, Smija M.; Di Pietro, Antonio

2018-01-01

Fusarium oxysporum exhibits conidial anastomosis tube (CAT) fusion during colony initiation to form networks of conidial germlings. Here we determined the optimal culture conditions for this fungus to undergo CAT fusion between microconidia in liquid medium. Extensive high resolution, confocal live-cell imaging was performed to characterise the different stages of CAT fusion, using genetically encoded fluorescent labelling and vital fluorescent organelle stains. CAT homing and fusion were found to be dependent on adhesion to the surface, in contrast to germ tube development which occurs in the absence of adhesion. Staining with fluorescently labelled concanavalin A indicated that the cell wall composition of CATs differs from that of microconidia and germ tubes. The movement of nuclei, mitochondria, vacuoles and lipid droplets through fused germlings was observed by live-cell imaging. PMID:29734342

Imaging of Fluoride Ion in Living Cells and Tissues with a Two-Photon Ratiometric Fluorescence Probe

PubMed Central

Zhu, Xinyue; Wang, Jianxi; Zhang, Jianjian; Chen, Zhenjie; Zhang, Haixia; Zhang, Xiaoyu

2015-01-01

A reaction-based two-photon (TP) ratiometric fluorescence probe Z2 has been developed and successfully applied to detect and image fluoride ion in living cells and tissues. The Z2 probe was designed designed to utilize an ICT mechanism between n-butylnaphthalimide as a fluorophore and tert-butyldiphenylsilane (TBDPS) as a response group. Upon addition of fluoride ion, the Si-O bond in the Z2 would be cleaved, and then a stronger electron-donating group was released. The fluorescent changes at 450 and 540 nm, respectively, made it possible to achieve ratiometric fluorescence detection. The results indicated that the Z2 could ratiometrically detect and image fluoride ion in living cells and tissues in a depth of 250 μm by two-photon microscopy (TPM). PMID:25594597

Live-cell imaging of conidial anastomosis tube fusion during colony initiation in Fusarium oxysporum.

PubMed

Kurian, Smija M; Di Pietro, Antonio; Read, Nick D

2018-01-01

Fusarium oxysporum exhibits conidial anastomosis tube (CAT) fusion during colony initiation to form networks of conidial germlings. Here we determined the optimal culture conditions for this fungus to undergo CAT fusion between microconidia in liquid medium. Extensive high resolution, confocal live-cell imaging was performed to characterise the different stages of CAT fusion, using genetically encoded fluorescent labelling and vital fluorescent organelle stains. CAT homing and fusion were found to be dependent on adhesion to the surface, in contrast to germ tube development which occurs in the absence of adhesion. Staining with fluorescently labelled concanavalin A indicated that the cell wall composition of CATs differs from that of microconidia and germ tubes. The movement of nuclei, mitochondria, vacuoles and lipid droplets through fused germlings was observed by live-cell imaging.

Live imaging of mitosis in the developing mouse embryonic cortex.

PubMed

Pilaz, Louis-Jan; Silver, Debra L

2014-06-04

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Microfluidic Approaches to Synchrotron Radiation-Based Fourier Transform Infrared (SR-FTIR) Spectral Microscopy of Living Biosystems

PubMed Central

Loutherback, Kevin; Birarda, Giovanni; Chen, Liang; Holman, Hoi-Ying N.

2016-01-01

A long-standing desire in biological and biomedical sciences is to be able to probe cellular chemistry as biological processes are happening inside living cells. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal distributions and relative abundances of biomolecules of a specimen by their characteristic vibrational modes. Despite great progress in recent years, SR-FTIR imaging of living biological systems remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. To meet this challenge, microfluidic devices have emerged as a method to control the water thickness while providing a hospitable environment to measure cellular processes and responses over many hours or days. This paper will provide an overview of microfluidic device development for SR-FTIR imaging of living biological systems, provide contrast between the various techniques including closed and open-channel designs, and discuss future directions of development within this area. Even as the fundamental science and technological demonstrations develop, other ongoing issues must be addressed; for example, choosing applications whose experimental requirements closely match device capabilities, and developing strategies to efficiently complete the cycle of development. These will require imagination, ingenuity and collaboration. PMID:26732243

Microfluidic approaches to synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy of living biosystems

DOE PAGES

Loutherback, Kevin; Birarda, Giovanni; Chen, Liang; ...

2016-02-15

A long-standing desire in biological and biomedical sciences is to be able to probe cellular chemistry as biological processes are happening inside living cells. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal distributions and relative abundances of biomolecules of a specimen by their characteristic vibrational modes. Despite great progress in recent years, SR-FTIR imaging of living biological systems remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. To meet this challenge, microfluidic devices have emerged as a method to control the watermore » thickness while providing a hospitable environment to measure cellular processes and responses over many hours or days. This paper will provide an overview of microfluidic device development for SR-FTIR imaging of living biological systems, provide contrast between the various techniques including closed and open-channel designs, and discuss future directions of development within this area. Even as the fundamental science and technological demonstrations develop, other ongoing issues must be addressed; for example, choosing applications whose experimental requirements closely match device capabilities, and developing strategies to efficiently complete the cycle of development. These will require imagination, ingenuity and collaboration.« less

Microfluidic approaches to synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy of living biosystems

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

Loutherback, Kevin; Birarda, Giovanni; Chen, Liang

A long-standing desire in biological and biomedical sciences is to be able to probe cellular chemistry as biological processes are happening inside living cells. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal distributions and relative abundances of biomolecules of a specimen by their characteristic vibrational modes. Despite great progress in recent years, SR-FTIR imaging of living biological systems remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. To meet this challenge, microfluidic devices have emerged as a method to control the watermore » thickness while providing a hospitable environment to measure cellular processes and responses over many hours or days. This paper will provide an overview of microfluidic device development for SR-FTIR imaging of living biological systems, provide contrast between the various techniques including closed and open-channel designs, and discuss future directions of development within this area. Even as the fundamental science and technological demonstrations develop, other ongoing issues must be addressed; for example, choosing applications whose experimental requirements closely match device capabilities, and developing strategies to efficiently complete the cycle of development. These will require imagination, ingenuity and collaboration.« less

Readily Available Fluorescent Probe for Carbon Monoxide Imaging in Living Cells.

PubMed

Feng, Weiyong; Liu, Dandan; Feng, Shumin; Feng, Guoqiang

2016-11-01

Carbon monoxide (CO) is an important gasotransmitter in living systems and its fluorescent detection is of particular interest. However, fluorescent detection of CO in living cells is still challenging due to lack of effective probes. In this paper, a readily available fluorescein-based fluorescent probe was developed for rapid detection of CO. This probe can be used to detect CO in almost wholly aqueous solution under mild conditions and shows high selectivity and sensitivity for CO with colorimetric and remarkable fluorescent turn-on signal changes. The detection limit of this probe for CO is as low as 37 nM with a linear range of 0-30 μM. More importantly, this probe (1 μM dose) can be conveniently used for fluorescent imaging CO in living cells.

Live-cell CRISPR imaging in plants reveals dynamic telomere movements.

PubMed

Dreissig, Steven; Schiml, Simon; Schindele, Patrick; Weiss, Oda; Rutten, Twan; Schubert, Veit; Gladilin, Evgeny; Mette, Michael F; Puchta, Holger; Houben, Andreas

2017-08-01

Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Long Time-lapse Nanoscopy with Spontaneously Blinking Membrane Probes

PubMed Central

Takakura, Hideo; Zhang, Yongdeng; Erdmann, Roman S.; Thompson, Alexander D.; Lin, Yu; McNellis, Brian; Rivera-Molina, Felix; Uno, Shin-nosuke; Kamiya, Mako; Urano, Yasuteru; Rothman, James E.; Bewersdorf, Joerg; Schepartz, Alanna; Toomre, Derek

2017-01-01

Long time-lapse, diffraction-unlimited super-resolution imaging of cellular structures and organelles in living cells is highly challenging, as it requires dense labeling, bright, highly photostable dyes, and non-toxic conditions. We developed a set of high-density, environment-sensitive (HIDE) membrane probes based on HMSiR that assemble in situ and enable long time-lapse, live cell nanoscopy of discrete cellular structures and organelles with high spatio-temporal resolution. HIDE-enabled nanoscopy movies are up to 50x longer than movies obtained with labeled proteins, reveal the 2D dynamics of the mitochondria, plasma membrane, and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum in living cells. These new HIDE probes also facilitate the acquisition of live cell, two-color, super-resolution images, greatly expanding the utility of nanoscopy to visualize processes and structures in living cells. PMID:28671662

Teachable, high-content analytics for live-cell, phase contrast movies.

PubMed

Alworth, Samuel V; Watanabe, Hirotada; Lee, James S J

2010-09-01

CL-Quant is a new solution platform for broad, high-content, live-cell image analysis. Powered by novel machine learning technologies and teach-by-example interfaces, CL-Quant provides a platform for the rapid development and application of scalable, high-performance, and fully automated analytics for a broad range of live-cell microscopy imaging applications, including label-free phase contrast imaging. The authors used CL-Quant to teach off-the-shelf universal analytics, called standard recipes, for cell proliferation, wound healing, cell counting, and cell motility assays using phase contrast movies collected on the BioStation CT and BioStation IM platforms. Similar to application modules, standard recipes are intended to work robustly across a wide range of imaging conditions without requiring customization by the end user. The authors validated the performance of the standard recipes by comparing their performance with truth created manually, or by custom analytics optimized for each individual movie (and therefore yielding the best possible result for the image), and validated by independent review. The validation data show that the standard recipes' performance is comparable with the validated truth with low variation. The data validate that the CL-Quant standard recipes can provide robust results without customization for live-cell assays in broad cell types and laboratory settings.

Detecting and Imaging of γ-Glutamytranspeptidase Activity in Serum, Live Cells, and Pathological Tissues with a High Signal-Stability Probe by Releasing a Precipitating Fluorochrome.

PubMed

Ou-Yang, Juan; Li, Yong-Fei; Wu, Ping; Jiang, Wen-Li; Liu, Hong-Wen; Li, Chun-Yan

2018-06-20

γ-Glutamytranspeptidase (GGT) is a significant tumor-related biomarker that overexpresses in several tumor cells. Accurate detection and imaging of GGT activity in serum, live cells, and pathological tissues hold great significance for cancer diagnosis, treatment, and management. Recently developed small molecule fluorescent probes for GGT tend to diffuse to the whole cytoplasm and then translocate out of live cells after enzymatic reaction, which make them fail to provide high spatial resolution and long-term imaging in biological systems. To address these problems, a novel fluorescent probe (HPQ-PDG) which releases a precipitating fluorochrome upon the catalysis of GGT is designed and synthesized. HPQ-PDG is able to detect GGT activity with high spatial resolution and good signal-stability. The large Stokes shift of the probe enables it to detect the activity of GGT in serum samples with high sensitivity. To our delight, the probe is used for imaging GGT activity in live cells with the ability of discriminating cancer cells from normal cells. What's more, we successfully apply it for pathological tissues imaging, with the results indicating that the potential application of HPQ-PDG in histopathological examination. All these results demonstrate the potential application of HPQ-PDG in the clinic.

Visual Self-Recognition in Mirrors and Live Videos: Evidence for a Developmental Asynchrony

ERIC Educational Resources Information Center

Suddendorf, Thomas; Simcock, Gabrielle; Nielsen, Mark

2007-01-01

Three experiments (N = 123) investigated the development of live-video self-recognition using the traditional mark test. In Experiment 1, 24-, 30- and 36-month-old children saw a live video image of equal size and orientation as a control group saw in a mirror. The video version of the test was more difficult than the mirror version with only the…

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

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

Costantini, Lindsey M.; Irvin, Susan C.; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFPmore » enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.« less

Imaging Complex Protein Metabolism in Live Organisms by Stimulated Raman Scattering Microscopy with Isotope Labeling

PubMed Central

2016-01-01

Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans. PMID:25560305

Micro-magnetic resonance imaging study of live quail embryos during embryonic development.

PubMed

Duce, Suzanne; Morrison, Fiona; Welten, Monique; Baggott, Glenn; Tickle, Cheryll

2011-01-01

Eggs containing live Japanese quail embryos were imaged using micro-magnetic resonance imaging (μMRI) at 24-h intervals from Day 0 to 8, the period during which the main body axis is being laid down and organogenesis is taking place. Considerable detail of non-embryonic structures such as the latebra was revealed at early stages but the embryo could only be visualized around Day 3. Three-dimensional (3D) changes in embryo length and volume were quantified and also changes in volume in the extra- and non-embryonic components. The embryo increased in length by 43% and nearly trebled in volume between Day 4 and Day 5. Although the amount of yolk remained fairly constant over the first 5 days, the amount of albumen decreases significantly and was replaced by extra-embryonic fluid (EEF). ¹H longitudinal (T₁) and transverse (T₂) relaxation times of different regions within the eggs were determined over the first 6 days of development. The T₂ measurements mirrored the changes in image intensity observed, which can be related to the aqueous protein concentrations. In addition, a comparison of the development of Day 0 to 3 quail embryos exposed to radiofrequency (rf) pulses, 7 T static magnetic fields and magnetic field gradients for an average of 7 h with the development of control embryos did not reveal any gross changes, thus confirming that μMRI is a suitable tool for following the development of live avian embryos over time from the earliest stages. Copyright © 2011 Elsevier Inc. All rights reserved.

Live-Cell Imaging of DNA Methylation Based on Synthetic-Molecule/Protein Hybrid Probe.

PubMed

Kumar, Naresh; Hori, Yuichiro; Kikuchi, Kazuya

2018-06-04

The epigenetic modification of DNA involves the conversion of cytosine to 5-methylcytosine, also known as DNA methylation. DNA methylation is important in modulating gene expression and thus, regulating genome and cellular functions. Recent studies have shown that aberrations in DNA methylation are associated with various epigenetic disorders or diseases including cancer. This stimulates great interest in the development of methods that can detect and visualize DNA methylation. For instance, fluorescent proteins (FPs) in conjugation with methyl-CpG-binding domain (MBD) have been employed for live-cell imaging of DNA methylation. However, the FP-based approach showed fluorescence signals for both the DNA-bound and -unbound states and thus differentiation between these states is difficult. Synthetic-molecule/protein hybrid probes can provide an alternative to overcome this restriction. In this article, we discuss the synthetic-molecule/protein hybrid probe that we developed recently for live-cell imaging of DNA methylation, which exhibited fluorescence enhancement only after binding to methylated DNA. © 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Live biospeckle laser imaging of root tissues.

PubMed

Braga, Roberto A; Dupuy, L; Pasqual, M; Cardoso, R R

2009-06-01

Live imaging is now a central component for the study of plant developmental processes. Currently, most techniques are extremely constraining: they rely on the marking of specific cellular structures which generally apply to model species because they require genetic transformations. The biospeckle laser (BSL) system was evaluated as an instrument to measure biological activity in plant tissues. The system allows collecting biospeckle patterns from roots which are grown in gels. Laser illumination has been optimized to obtain the images without undesirable specular reflections from the glass tube. Data on two different plant species were obtained and the ability of three different methods to analyze the biospeckle patterns are presented. The results showed that the biospeckle could provide quantitative indicators of the molecular activity from roots which are grown in gel substrate in tissue culture. We also presented a particular experimental configuration and the optimal approach to analyze the images. This may serve as a basis to further works on live BSL in order to study root development.

A High-Content Live-Cell Viability Assay and Its Validation on a Diverse 12K Compound Screen.

PubMed

Chiaravalli, Jeanne; Glickman, J Fraser

2017-08-01

We have developed a new high-content cytotoxicity assay using live cells, called "ImageTOX." We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

Development of an ultralow-light-level luminescence image analysis system for dynamic measurements of transcriptional activity in living and migrating cells.

PubMed

Maire, E; Lelièvre, E; Brau, D; Lyons, A; Woodward, M; Fafeur, V; Vandenbunder, B

2000-04-10

We have developed an approach to study in single living epithelial cells both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.

Development and characterization of a scintillating cell imaging dish for radioluminescence microscopy.

PubMed

Sengupta, Debanti; Kim, Tae Jin; Almasi, Sepideh; Miller, Stuart; Marton, Zsolt; Nagarkar, Vivek; Pratx, Guillem

2018-04-16

Radioluminescence microscopy is an emerging modality that can be used to image radionuclide probes with micron-scale resolution. This technique is particularly useful as a way to probe the metabolic behavior of single cells and to screen and characterize radiopharmaceuticals, but the quality of the images is critically dependent on the scintillator material used to image the cells. In this paper, we detail the development of a microscopy dish made of a thin-film scintillating material, Lu2O3:Eu, that could be used as the blueprint for a future consumable product. After developing a simple quality control method based on long-lived alpha and beta sources, we characterize the radioluminescence properties of various thin-film scintillator samples. We find consistent performance for most samples, but also identify a few samples that do not meet the specifications, thus stressing the need for routine quality control prior to biological experiments. In addition, we test and quantify the transparency of the material, and demonstrate that transparency correlates with thickness. Finally, we evaluate the biocompatibility of the material and show that the microscopy dish can produce radioluminescent images of live single cells.

Live imaging of apoptotic cells in zebrafish

PubMed Central

van Ham, Tjakko J.; Mapes, James; Kokel, David; Peterson, Randall T.

2010-01-01

Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.—Van Ham, T. J., Mapes, J., Kokel, D., Peterson, R. T. Live imaging of apoptotic cells in zebrafish. PMID:20601526

Live dynamic OCT imaging of cardiac structure and function in mouse embryos with 43 Hz direct volumetric data acquisition

NASA Astrophysics Data System (ADS)

Wang, Shang; Singh, Manmohan; Lopez, Andrew L.; Wu, Chen; Raghunathan, Raksha; Schill, Alexander; Li, Jiasong; Larin, Kirill V.; Larina, Irina V.

2016-03-01

Efficient phenotyping of cardiac dynamics in live mouse embryos has significant implications on understanding of early mammalian heart development and congenital cardiac defects. Recent studies established optical coherence tomography (OCT) as a powerful tool for live embryonic heart imaging in various animal models. However, current four-dimensional (4D) OCT imaging of the beating embryonic heart largely relies on gated data acquisition or postacquisition synchronization, which brings errors when cardiac cycles lack perfect periodicity and is time consuming and computationally expensive. Here, we report direct 4D OCT imaging of the structure and function of cardiac dynamics in live mouse embryos achieved by employing a Fourier domain mode-locking swept laser source that enables ~1.5 MHz A-line rate. Through utilizing both forward and backward scans of a resonant mirror, we obtained a ~6.4 kHz frame rate, which allows for a direct volumetric data acquisition speed of ~43 Hz, around 20 times of the early-stage mouse embryonic heart rate. Our experiments were performed on mouse embryos at embryonic day 9.5. Time-resolved 3D cardiodynamics clearly shows the heart structure in motion. We present analysis of cardiac wall movement and its velocity from the primitive atrium and ventricle. Our results suggest that the combination of ultrahigh-speed OCT imaging with live embryo culture could be a useful embryonic heart phenotyping approach for mouse mutants modeling human congenital heart diseases.

An automated live imaging platform for studying merozoite egress-invasion in malaria cultures.

PubMed

Crick, Alex J; Tiffert, Teresa; Shah, Sheel M; Kotar, Jurij; Lew, Virgilio L; Cicuta, Pietro

2013-03-05

Most cases of severe and fatal malaria are caused by the intraerythrocytic asexual reproduction cycle of Plasmodium falciparum. One of the most intriguing and least understood stages in this cycle is the brief preinvasion period during which dynamic merozoite-red-cell interactions align the merozoite apex in preparation for penetration. Studies of the molecular mechanisms involved in this process face formidable technical challenges, requiring multiple observations of merozoite egress-invasion sequences in live cultures under controlled experimental conditions, using high-resolution microscopy and a variety of fluorescent imaging tools. Here we describe a first successful step in the development of a fully automated, robotic imaging platform to enable such studies. Schizont-enriched live cultures of P. falciparum were set up on an inverted stage microscope with software-controlled motorized functions. By applying a variety of imaging filters and selection criteria, we identified infected red cells that were likely to rupture imminently, and recorded their coordinates. We developed a video-image analysis to detect and automatically record merozoite egress events in 100% of the 40 egress-invasion sequences recorded in this study. We observed a substantial polymorphism of the dynamic condition of pre-egress infected cells, probably reflecting asynchronies in the diversity of confluent processes leading to merozoite release. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Radiolabeled Peptide Scaffolds for PET/SPECT - Optical in Vivo Imaging of Carbohydrate-Lectin Interactions

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

Deutscher, Susan

2014-09-30

The objective of this research is to develop phage display-selected peptides into radio- and fluoresecently- labeled scaffolds for the multimodal imaging of carbohydrate-lectin interactions. While numerous protein and receptor systems are being explored for the development of targeted imaging agents, the targeting and analysis of carbohydrate-lectin complexes in vivo remains relatively unexplored. Antibodies, nanoparticles, and peptides are being developed that target carbohydrate-lectin complexes in living systems. However, antibodies and nanoparticles often suffer from slow clearance and toxicity problems. Peptides are attractive alternative vehicles for the specific delivery of radionuclides or fluorophores to sites of interest in vivo, although, because ofmore » their size, uptake and retention may be less than antibodies. We have selected high affinity peptides that bind a specific carbohydrate-lectin complex involved in cell-cell adhesion and cross-linking using bacteriophage (phage) display technologies (1,2). These peptides have allowed us to probe the role of these antigens in cell adhesion. Fluorescent versions of the peptides have been developed for optical imaging and radiolabeled versions have been used in single photon emission computed tomography (SPECT) and positron emission tomography (PET) in vivo imaging (3-6). A benefit in employing the radiolabeled peptides in SPECT and PET is that these imaging modalities are widely used in living systems and offer deep tissue sensitivity. Radiolabeled peptides, however, often exhibit poor stability and high kidney uptake in vivo. Conversely, optical imaging is sensitive and offers good spatial resolution, but is not useful for deep tissue penetration and is semi-quantitative. Thus, multimodality imaging that relies on the strengths of both radio- and optical- imaging is a current focus for development of new in vivo imaging agents. We propose a novel means to improve the efficacy of radiolabeled and fluorescently labeled peptides, including our lectin/carbohydrate- targeting peptides, by displaying the targeting epitopes on small ~29 amino acid cyclic plant protein scaffolds known as cyclotides. Cyclotides are extremely stable molecules with long serum half-lives and low kidney uptake (7). More than one copy of the peptide can be engineered into the cyclotide loops, thus increasing the avidity of the peptide construct for its target.« less

4-D photoacoustic tomography.

PubMed

Xiang, Liangzhong; Wang, Bo; Ji, Lijun; Jiang, Huabei

2013-01-01

Photoacoustic tomography (PAT) offers three-dimensional (3D) structural and functional imaging of living biological tissue with label-free, optical absorption contrast. These attributes lend PAT imaging to a wide variety of applications in clinical medicine and preclinical research. Despite advances in live animal imaging with PAT, there is still a need for 3D imaging at centimeter depths in real-time. We report the development of four dimensional (4D) PAT, which integrates time resolutions with 3D spatial resolution, obtained using spherical arrays of ultrasonic detectors. The 4D PAT technique generates motion pictures of imaged tissue, enabling real time tracking of dynamic physiological and pathological processes at hundred micrometer-millisecond resolutions. The 4D PAT technique is used here to image needle-based drug delivery and pharmacokinetics. We also use this technique to monitor 1) fast hemodynamic changes during inter-ictal epileptic seizures and 2) temperature variations during tumor thermal therapy.

Theranostic nanoemulsions: codelivery of hydrophobic drug and hydrophilic imaging probe for cancer therapy and imaging.

PubMed

Yang, Xinggang; Wang, Dun; Ma, Yan; Zhao, Qiang; Fallon, John K; Liu, Dan; Xu, Xian Emma; Wang, Yongjun; He, Zhonggui; Liu, Feng

2014-12-01

To develop a theranostic nanoemulsion (TNE) that can codeliver the conjugates of a hydrophobic drug paclitaxel (PTX) and a hydrophilic imaging probe sulforhodamine B (SRB). The TNE was established using core-matched technology, and can achieve high encapsulation efficiency and synchronized release of the loaded cargo. It has been examined for a correlation between the dynamic uptake of PTX and the intensity of SRB imaging signal in different organs. Our data demonstrate that the TNE, with improved circulation time, increases therapeutic efficacy and imaging efficiency in both drug-sensitive and drug-resistant cancer. The TNE could not satisfy the demand of visual diagnosis in the living animal because of interference. We therefore formulated a long-circulating theranostic nanoemulsion (LCTNE). Results showed that the LCTNE can meet imaging requirements in vivo. The LCTNE plays a good therapeutic and diagnostic role for subcutaneous tumors in the living animal.

Education and the Living Image: Reflections on Imagery, Fantasy, and the Art of Recognition.

ERIC Educational Resources Information Center

Abbs, Peter

1981-01-01

The educational role of the artist is close to that of the dreamer in the sense that they are active collaborators in the extraordinary process through which instinct and bodily function are converted into image and fantasy. The development of an image can release powerful flows of intellectual energy. (JN)

[Optimizing histological image data for 3-D reconstruction using an image equalizer].

PubMed

Roth, A; Melzer, K; Annacker, K; Lipinski, H G; Wiemann, M; Bingmann, D

2002-01-01

Bone cells form a wired network within the extracellular bone matrix. To analyse this complex 3D structure, we employed a confocal fluorescence imaging procedure to visualize live bone cells within their native surrounding. By means of newly developed image processing software, the "Image-Equalizer", we aimed to enhanced the contrast and eliminize artefacts in such a way that cell bodies as well as fine interconnecting processes were visible.

Development of 3D imaging technique of reconstructed human epidermis with immortalized human epidermal cell line.

PubMed

Inoue, Yu; Hasegawa, Seiji; Miyachi, Katsuma; Yamada, Takaaki; Nakata, Satoru; Ipponjima, Sari; Hibi, Terumasa; Nemoto, Tomomi; Tanaka, Masahiko; Suzuki, Ryo; Hirashima, Naohide

2018-05-01

The epidermis, the outermost layer of the skin, retains moisture and functions as a physical barrier against the external environment. Epidermal cells are continuously replaced by turnover, and thus to understand in detail the dynamic cellular events in the epidermis, techniques to observe live tissues in 3D are required. Here, we established a live 3D imaging technique for epidermis models. We first obtained immortalized human epidermal cell lines which have a normal differentiation capacity and fluorescence-labelled cytoplasm or nuclei. The reconstituted 3D epidermis was prepared with these lines. Using this culture system, we were able to observe the structure of the reconstituted epidermis live in 3D, which was similar to an in vivo epidermis, and evaluate the effect of a skin irritant. This technique may be useful for dermatological science and drug development. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Semi-automatic central-chest lymph-node definition from 3D MDCT images

NASA Astrophysics Data System (ADS)

Lu, Kongkuo; Higgins, William E.

2010-03-01

Central-chest lymph nodes play a vital role in lung-cancer staging. The three-dimensional (3D) definition of lymph nodes from multidetector computed-tomography (MDCT) images, however, remains an open problem. This is because of the limitations in the MDCT imaging of soft-tissue structures and the complicated phenomena that influence the appearance of a lymph node in an MDCT image. In the past, we have made significant efforts toward developing (1) live-wire-based segmentation methods for defining 2D and 3D chest structures and (2) a computer-based system for automatic definition and interactive visualization of the Mountain central-chest lymph-node stations. Based on these works, we propose new single-click and single-section live-wire methods for segmenting central-chest lymph nodes. The single-click live wire only requires the user to select an object pixel on one 2D MDCT section and is designed for typical lymph nodes. The single-section live wire requires the user to process one selected 2D section using standard 2D live wire, but it is more robust. We applied these methods to the segmentation of 20 lymph nodes from two human MDCT chest scans (10 per scan) drawn from our ground-truth database. The single-click live wire segmented 75% of the selected nodes successfully and reproducibly, while the success rate for the single-section live wire was 85%. We are able to segment the remaining nodes, using our previously derived (but more interaction intense) 2D live-wire method incorporated in our lymph-node analysis system. Both proposed methods are reliable and applicable to a wide range of pulmonary lymph nodes.

SU-E-J-134: An Augmented-Reality Optical Imaging System for Accurate Breast Positioning During Radiotherapy

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

Nazareth, D; Malhotra, H; French, S

Purpose: Breast radiotherapy, particularly electronic compensation, may involve large dose gradients and difficult patient positioning problems. We have developed a simple self-calibrating augmented-reality system, which assists in accurately and reproducibly positioning the patient, by displaying her live image from a single camera superimposed on the correct perspective projection of her 3D CT data. Our method requires only a standard digital camera capable of live-view mode, installed in the treatment suite at an approximately-known orientation and position (rotation R; translation T). Methods: A 10-sphere calibration jig was constructed and CT imaged to provide a 3D model. The (R,T) relating the cameramore » to the CT coordinate system were determined by acquiring a photograph of the jig and optimizing an objective function, which compares the true image points to points calculated with a given candidate R and T geometry. Using this geometric information, 3D CT patient data, viewed from the camera's perspective, is plotted using a Matlab routine. This image data is superimposed onto the real-time patient image, acquired by the camera, and displayed using standard live-view software. This enables the therapists to view both the patient's current and desired positions, and guide the patient into assuming the correct position. The method was evaluated using an in-house developed bolus-like breast phantom, mounted on a supporting platform, which could be tilted at various angles to simulate treatment-like geometries. Results: Our system allowed breast phantom alignment, with an accuracy of about 0.5 cm and 1 ± 0.5 degree. Better resolution could be possible using a camera with higher-zoom capabilities. Conclusion: We have developed an augmented-reality system, which combines a perspective projection of a CT image with a patient's real-time optical image. This system has the potential to improve patient setup accuracy during breast radiotherapy, and could possibly be used for other disease sites as well.« less

Evanescent field microscopy techniques for studying dynamics at the surface of living cells

NASA Astrophysics Data System (ADS)

Sund, Susan E.

This thesis presents two distinct optical microscopy techniques for applications in cell biophysics: (a)the extension to living cells of an established technique, total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) for the first time in imaging mode; and (b)the novel development of polarized total internal reflection fluorescence (p- TIRF) to study membrane orientation in living cells. Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about the relevant chemical kinetic rates in vivo. TIR/FRAP, an established technique which can measure reversible biomolecular kinetic rates at surfaces, is extended here to measure kinetic parameters of microinjected rhodamine actin at the cytofacial surface of the plasma membrane of living cultured smooth muscle cells. For the first time, spatial imaging (with a CCD camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging allows production of spatially resolved images of kinetic data, and calculation of correlation distances, cell-wide gradients, and kinetic parameter dependence on initial fluorescence intensity. In living cells, membrane curvature occurs both in easily imaged large scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method, p-TIRF, is introduced here to visualize such regions. The method is based on fluorescence of the oriented membrane probe diI- C18-(3) (diI) excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane. A theoretical background of the technique and experimental verifications are presented in samples of protein solutions, model lipid bilayers, and living cells. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time- course maps of membrane orientations on diI labeled macrophages from which low visibility membrane structures can be identified and quantified. The TIR images are sharpened and contrast-enhanced by deconvoluting them with an experimentally-measured point spread function.

Improvement of an algorithm for recognition of liveness using perspiration in fingerprint devices

NASA Astrophysics Data System (ADS)

Parthasaradhi, Sujan T.; Derakhshani, Reza; Hornak, Lawrence A.; Schuckers, Stephanie C.

2004-08-01

Previous work in our laboratory and others have demonstrated that spoof fingers made of a variety of materials including silicon, Play-Doh, clay, and gelatin (gummy finger) can be scanned and verified when compared to a live enrolled finger. Liveness, i.e. to determine whether the introduced biometric is coming from a live source, has been suggested as a means to circumvent attacks using spoof fingers. We developed a new liveness method based on perspiration changes in the fingerprint image. Recent results showed approximately 90% classification rate using different classification methods for various technologies including optical, electro-optical, and capacitive DC, a shorter time window and a diverse dataset. This paper focuses on improvement of the live classification rate by using a weight decay method during the training phase in order to improve the generalization and reduce the variance of the neural network based classifier. The dataset included fingerprint images from 33 live subjects, 33 spoofs created with dental impression material and Play-Doh, and fourteen cadaver fingers. 100% live classification was achieved with 81.8 to 100% spoof classification, depending on the device technology. The weight-decay method improves upon past reports by increasing the live and spoof classification rate.

Recent development of nanoparticles for molecular imaging

NASA Astrophysics Data System (ADS)

Kim, Jonghoon; Lee, Nohyun; Hyeon, Taeghwan

2017-10-01

Molecular imaging enables us to non-invasively visualize cellular functions and biological processes in living subjects, allowing accurate diagnosis of diseases at early stages. For successful molecular imaging, a suitable contrast agent with high sensitivity is required. To date, various nanoparticles have been developed as contrast agents for medical imaging modalities. In comparison with conventional probes, nanoparticles offer several advantages, including controllable physical properties, facile surface modification and long circulation time. In addition, they can be integrated with various combinations for multimodal imaging and therapy. In this opinion piece, we highlight recent advances and future perspectives of nanomaterials for molecular imaging. This article is part of the themed issue 'Challenges for chemistry in molecular imaging'.

Construction of an efficient two-photon fluorescent probe for imaging nitroreductase in live cells and tissues

NASA Astrophysics Data System (ADS)

Zhou, Liyi; Gong, Liang; Hu, Shunqin

2018-06-01

Compared with traditional confocal microscopy, two-photon fluorescence microscopy (TPFM), which excites a two-photon (TP) fluorophore by near-infrared light, provides improved three-dimensional image resolution with increased tissue-image depth (>500 μm) and an extended observation time. Therefore, the development of novel functional TP fluorophores has attracted great attention in recent years. Herein, a novel TP fluorophore CM-NH2, which have the donor-π-acceptor (D-π-A)-structure, was designed and synthesized. We further used this dye developed a new type of TP fluorescent probe CM-NO2 for detecting nitroreductase (NTR). Upon incubated with NTR for 15 min, CM-NO2 displayed a 90-fold fluorescence enhancement at 505 nm and the maximal TP action cross-section value after reaction was detected and calculated to be 200 GM at 760 nm. The probe exhibited excellent properties such as high sensitivity, high selectivity, low cytotoxicity, and high photostability. Moreover, the probe was utilized to image the tumor hypoxia in live HeLa cells. Finally, using the CM-NO2 to image NTR in tissues was demonstrated.

Time-resolved optical imaging provides a molecular snapshot of altered metabolic function in living human cancer cell models

NASA Astrophysics Data System (ADS)

Sud, Dhruv; Zhong, Wei; Beer, David G.; Mycek, Mary-Ann

2006-05-01

A fluorescence lifetime imaging microscopy (FLIM) method was developed and applied to investigate metabolic function in living human normal esophageal (HET-1) and Barrett’s adenocarcinoma (SEG-1) cells. In FLIM, image contrast is based on fluorophore excited state lifetimes, which reflect local biochemistry and molecular activity. Unique FLIM system attributes, including variable ultrafast time gating (≥ 200 ps), wide spectral tunability (337.1 - 960 nm), large temporal dynamic range (≥ 600 ps), and short data acquisition and processing times (15 s), enabled the study of two key molecules consumed at the termini of the oxidative phosphorylation pathway, NADH and oxygen, in living cells under controlled and calibrated environmental conditions. NADH is an endogenous cellular fluorophore detectable in living human tissues that has been shown to be a quantitative biomarker of dysplasia in the esophagus. Lifetime calibration of an oxygen-sensitive, ruthenium-based cellular stain enabled in vivo oxygen level measurements with a resolution of 8 μM over the entire physiological range (1 - 300 μM). Starkly higher intracellular oxygen and NADH levels in living SEG-1 vs. HET-1 cells were detected by FLIM and attributed to altered metabolic pathways in malignant cells.

Using Dual Fluorescence Reporting Genes to Establish an In Vivo Imaging Model of Orthotopic Lung Adenocarcinoma in Mice.

PubMed

Lai, Cheng-Wei; Chen, Hsiao-Ling; Yen, Chih-Ching; Wang, Jiun-Long; Yang, Shang-Hsun; Chen, Chuan-Mu

2016-12-01

Lung adenocarcinoma is characterized by a poor prognosis and high mortality worldwide. In this study, we purposed to use the live imaging techniques and a reporter gene that generates highly penetrative near-infrared (NIR) fluorescence to establish a preclinical animal model that allows in vivo monitoring of lung cancer development and provides a non-invasive tool for the research on lung cancer pathogenesis and therapeutic efficacy. A human lung adenocarcinoma cell line (A549), which stably expressed the dual fluorescence reporting gene (pCAG-iRFP-2A-Venus), was used to generate subcutaneous or orthotopic lung cancer in nude mice. Cancer development was evaluated by live imaging via the NIR fluorescent signals from iRFP, and the signals were verified ex vivo by the green fluorescence of Venus from the gross lung. The tumor-bearing mice received miR-16 nucleic acid therapy by intranasal administration to demonstrate therapeutic efficacy in this live imaging system. For the subcutaneous xenografts, the detection of iRFP fluorescent signals revealed delicate changes occurring during tumor growth that are not distinguishable by conventional methods of tumor measurement. For the orthotopic xenografts, the positive correlation between the in vivo iRFP signal from mice chests and the ex vivo green fluorescent signal from gross lung tumors and the results of the suppressed tumorigenesis by miR-16 treatment indicated that lung tumor size can be accurately quantified by the emission of NIR fluorescence. In addition, orthotopic lung tumor localization can be accurately visualized using iRFP fluorescence tomography in vivo, thus revealing the trafficking of lung tumor cells. We introduced a novel dual fluorescence lung cancer model that provides a non-invasive option for preclinical research via the use of NIR fluorescence in live imaging of lung.

The 'Soil Cover App' - a new tool for fast determination of dead and living biomass on soil

NASA Astrophysics Data System (ADS)

Bauer, Thomas; Strauss, Peter; Riegler-Nurscher, Peter; Prankl, Johann; Prankl, Heinrich

2017-04-01

Worldwide many agricultural practices aim on soil protection strategies using living or dead biomass as soil cover. Especially for the case when management practices are focusing on soil erosion mitigation the effectiveness of these practices is directly driven by the amount of soil coverleft on the soil surface. Hence there is a need for quick and reliable methods of soil cover estimation not only for living biomass but particularly for dead biomass (mulch). Available methods for the soil cover measurement are either subjective, depending on an educated guess or time consuming, e.g., if the image is analysed manually at grid points. We therefore developed a mobile application using an algorithm based on entangled forest classification. The final output of the algorithm gives classified labels for each pixel of the input image as well as the percentage of each class which are living biomass, dead biomass, stones and soil. Our training dataset consisted of more than 250 different images and their annotated class information. Images have been taken in a set of different environmental conditions such as light, soil coverages from between 0% to 100%, different materials such as living plants, residues, straw material and stones. We compared the results provided by our mobile application with a data set of 180 images that had been manually annotated A comparison between both methods revealed a regression slope of 0.964 with a coefficient of determination R2 = 0.92, corresponding to an average error of about 4%. While average error of living plant classification was about 3%, dead residue classification resulted in an 8% error. Thus the new mobile application tool offers a fast and easy way to obtain information on the protective potential of a particular agricultural management site.

Image acquisition optimization of a limited-angle intrafraction verification (LIVE) system for lung radiotherapy.

PubMed

Zhang, Yawei; Deng, Xinchen; Yin, Fang-Fang; Ren, Lei

2018-01-01

Limited-angle intrafraction verification (LIVE) has been previously developed for four-dimensional (4D) intrafraction target verification either during arc delivery or between three-dimensional (3D)/IMRT beams. Preliminary studies showed that LIVE can accurately estimate the target volume using kV/MV projections acquired over orthogonal view 30° scan angles. Currently, the LIVE imaging acquisition requires slow gantry rotation and is not clinically optimized. The goal of this study is to optimize the image acquisition parameters of LIVE for different patient respiratory periods and gantry rotation speeds for the effective clinical implementation of the system. Limited-angle intrafraction verification imaging acquisition was optimized using a digital anthropomorphic phantom (XCAT) with simulated respiratory periods varying from 3 s to 6 s and gantry rotation speeds varying from 1°/s to 6°/s. LIVE scanning time was optimized by minimizing the number of respiratory cycles needed for the four-dimensional scan, and imaging dose was optimized by minimizing the number of kV and MV projections needed for four-dimensional estimation. The estimation accuracy was evaluated by calculating both the center-of-mass-shift (COMS) and three-dimensional volume-percentage-difference (VPD) between the tumor in estimated images and the ground truth images. The robustness of LIVE was evaluated with varied respiratory patterns, tumor sizes, and tumor locations in XCAT simulation. A dynamic thoracic phantom (CIRS) was used to further validate the optimized imaging schemes from XCAT study with changes of respiratory patterns, tumor sizes, and imaging scanning directions. Respiratory periods, gantry rotation speeds, number of respiratory cycles scanned and number of kV/MV projections acquired were all positively correlated with the estimation accuracy of LIVE. Faster gantry rotation speed or longer respiratory period allowed less respiratory cycles to be scanned and less kV/MV projections to be acquired to estimate the target volume accurately. Regarding the scanning time minimization, for patient respiratory periods of 3-4 s, gantry rotation speeds of 1°/s, 2°/s, 3-6°/s required scanning of five, four, and three respiratory cycles, respectively. For patient respiratory periods of 5-6 s, the corresponding respiratory cycles required in the scan changed to four, three, and two cycles, respectively. Regarding the imaging dose minimization, for patient respiratory periods of 3-4 s, gantry rotation speeds of 1°/s, 2-4°/s, 5-6°/s required acquiring of 7, 5, 4 kV and MV projections, respectively. For patient respiratory periods of 5-6 s, 5 kV and 5 MV projections are sufficient for all gantry rotation speeds. The optimized LIVE system was robust against breathing pattern, tumor size and tumor location changes. In the CIRS study, the optimized LIVE system achieved the average center-of-mass-shift (COMS)/volume-percentage-difference (VPD) of 0.3 ± 0.1 mm/7.7 ± 2.0% for the scanning time priority case, 0.2 ± 0.1 mm/6.1 ± 1.2% for the imaging dose priority case, respectively, among all gantry rotation speeds tested. LIVE was robust against different scanning directions investigated. The LIVE system has been preliminarily optimized for different patient respiratory periods and treatment gantry rotation speeds using digital and physical phantoms. The optimized imaging parameters, including number of respiratory cycles scanned and kV/MV projection numbers acquired, provide guidelines for optimizing the scanning time and imaging dose of the LIVE system for its future evaluations and clinical implementations through patient studies. © 2017 American Association of Physicists in Medicine.

Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques.

PubMed

Paddock, Stephen W; Eliceiri, Kevin W

2014-01-01

Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells. The laser scanning confocal microscope continues to be chosen for most routine work although a number of instruments have been developed for more specific applications. Significant improvements have been made to all areas of the confocal approach, not only to the instruments themselves, but also to the protocols of specimen preparation, to the analysis, the display, the reproduction, sharing and management of confocal images using bioinformatics techniques.

Selective loss of verbal imagery.

PubMed

Mehta, Z; Newcombe, F

1996-05-01

This single case study of the ability to generate verbal and non-verbal imagery in a woman who sustained a gunshot wound to the brain reports a significant difficulty in generating images of word shapes but not a significant problem in generating object images. Further dissociation, however, was observed in her ability to generate images of living vs non-living material. She made more errors in imagery and factual information tasks for non-living items than for living items. This pattern contrasts with our previous report of the agnosic patient, M.S., who had severe difficulty in generating images of living material, whereas his ability to image the shape of words was comparable to that of normal control subjects. Furthermore, with regard to the generation of images of living compared with non-living material, M.S. shows more errors with living than nonliving items. In contrast, the present patient, S.M., made significantly more errors with non-living relative to living items. There appear to be two types of double dissociation which reinforce the growing evidence of dissociable impairments in the ability to generate images for different types of verbal and non-verbal material. Such dissociations, presumably related to sensory and cognitive processing demands, address the problem of the neural basis of imagery.

Comparison of intravital thinned skull and cranial window approaches to study CNS immunobiology in the mouse cortex

PubMed Central

Dorand, R Dixon; Barkauskas, Deborah S; Evans, Teresa A; Petrosiute, Agne; Huang, Alex Y

2014-01-01

Fluorescent imaging coupled with high-resolution femtosecond pulsed infrared lasers allows for interrogation of cellular interactions deeper in living tissues than ever imagined. Intravital imaging of the central nervous system (CNS) has provided insights into neuronal development, synaptic transmission, and even immune interactions. In this review we will discuss the two most common intravital approaches for studying the cerebral cortex in the live mouse brain for pre-clinical studies, the thinned skull and cranial window techniques, and focus on the advantages and drawbacks of each approach. In addition, we will discuss the use of neuronal physiologic parameters as determinants of successful surgical and imaging preparation. PMID:25568834

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana.

PubMed

Kurihara, Daisuke; Kimata, Yusuke; Higashiyama, Tetsuya; Ueda, Minako

2017-09-11

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

Optical coherence microscopy as a novel, non-invasive method for the 4D live imaging of early mammalian embryos.

PubMed

Karnowski, Karol; Ajduk, Anna; Wieloch, Bartosz; Tamborski, Szymon; Krawiec, Krzysztof; Wojtkowski, Maciej; Szkulmowski, Maciej

2017-06-23

Imaging of living cells based on traditional fluorescence and confocal laser scanning microscopy has delivered an enormous amount of information critical for understanding biological processes in single cells. However, the requirement for a high numerical aperture and fluorescent markers still limits researchers' ability to visualize the cellular architecture without causing short- and long-term photodamage. Optical coherence microscopy (OCM) is a promising alternative that circumvents the technical limitations of fluorescence imaging techniques and provides unique access to fundamental aspects of early embryonic development, without the requirement for sample pre-processing or labeling. In the present paper, we utilized the internal motion of cytoplasm, as well as custom scanning and signal processing protocols, to effectively reduce the speckle noise typical for standard OCM and enable high-resolution intracellular time-lapse imaging. To test our imaging system we used mouse and pig oocytes and embryos and visualized them through fertilization and the first embryonic division, as well as at selected stages of oogenesis and preimplantation development. Because all morphological and morphokinetic properties recorded by OCM are believed to be biomarkers of oocyte/embryo quality, OCM may represent a new chapter in imaging-based preimplantation embryo diagnostics.

Optical Electrophysiology in the Developing Heart.

PubMed

Thomas, Kandace; Goudy, Julie; Henley, Trevor; Bressan, Michael

2018-05-11

The heart is the first organ system to form in the embryo. Over the course of development, cardiomyocytes with differing morphogenetic, molecular, and physiological characteristics are specified and differentiate and integrate with one another to assemble a coordinated electromechanical pumping system that can function independently of any external stimulus. As congenital malformation of the heart presents the leading class of birth defects seen in humans, the molecular genetics of heart development have garnered much attention over the last half century. However, understanding how genetic perturbations manifest at the level of the individual cell function remains challenging to investigate. Some of the barriers that have limited our capacity to construct high-resolution, comprehensive models of cardiac physiological maturation are rapidly being removed by advancements in the reagents and instrumentation available for high-speed live imaging. In this review, we briefly introduce the history of imaging approaches for assessing cardiac development, describe some of the reagents and tools required to perform live imaging in the developing heart, and discuss how the combination of modern imaging modalities and physiological probes can be used to scale from subcellular to whole-organ analysis. Through these types of imaging approaches, critical insights into the processes of cardiac physiological development can be directly examined in real-time. Moving forward, the synthesis of modern molecular biology and imaging approaches will open novel avenues to investigate the mechanisms of cardiomyocyte maturation, providing insight into the etiology of congenital heart defects, as well as serving to direct approaches for designing stem-cell or regenerative medicine protocols for clinical application.

JPRS Report China QIUSHISeeking Truth No, 1 December 1989

DTIC Science & Technology

1990-02-06

two here. When you read those vivid and catchy titles and lyrical comments with strong political content, the image of a victor and a revolutionary...In my essay "Love for Green Hills," published in 1980,1 suggested "letting those living on a mountain live off the mountain by developing the

High speed multiphoton imaging

NASA Astrophysics Data System (ADS)

Li, Yongxiao; Brustle, Anne; Gautam, Vini; Cockburn, Ian; Gillespie, Cathy; Gaus, Katharina; Lee, Woei Ming

2016-12-01

Intravital multiphoton microscopy has emerged as a powerful technique to visualize cellular processes in-vivo. Real time processes revealed through live imaging provided many opportunities to capture cellular activities in living animals. The typical parameters that determine the performance of multiphoton microscopy are speed, field of view, 3D imaging and imaging depth; many of these are important to achieving data from in-vivo. Here, we provide a full exposition of the flexible polygon mirror based high speed laser scanning multiphoton imaging system, PCI-6110 card (National Instruments) and high speed analog frame grabber card (Matrox Solios eA/XA), which allows for rapid adjustments between frame rates i.e. 5 Hz to 50 Hz with 512 × 512 pixels. Furthermore, a motion correction algorithm is also used to mitigate motion artifacts. A customized control software called Pscan 1.0 is developed for the system. This is then followed by calibration of the imaging performance of the system and a series of quantitative in-vitro and in-vivo imaging in neuronal tissues and mice.

The Effects of Immigration and Media Influence on Body Image Among Pakistani Men

PubMed Central

Saghir, Sheeba; Hyland, Lynda

2017-01-01

This study examined the role of media influence and immigration on body image among Pakistani men. Attitudes toward the body were compared between those living in Pakistan (n = 56) and those who had immigrated to the United Arab Emirates (n = 58). Results of a factorial analysis of variance demonstrated a significant main effect of immigrant status. Pakistani men living in the United Arab Emirates displayed poorer body image than those in the Pakistan sample. Results also indicated a second main effect of media influence.Those highly influenced by the media displayed poorer body image. No interaction effect was observed between immigrant status and media influence on body image. These findings suggest that media influence and immigration are among important risk factors for the development of negative body image among non-Western men. Interventions designed to address the negative effects of the media and immigration may be effective at reducing body image disorders and other related health problems in this population. PMID:28625116

The Effects of Immigration and Media Influence on Body Image Among Pakistani Men.

PubMed

Saghir, Sheeba; Hyland, Lynda

2017-07-01

This study examined the role of media influence and immigration on body image among Pakistani men. Attitudes toward the body were compared between those living in Pakistan ( n = 56) and those who had immigrated to the United Arab Emirates ( n = 58). Results of a factorial analysis of variance demonstrated a significant main effect of immigrant status. Pakistani men living in the United Arab Emirates displayed poorer body image than those in the Pakistan sample. Results also indicated a second main effect of media influence.Those highly influenced by the media displayed poorer body image. No interaction effect was observed between immigrant status and media influence on body image. These findings suggest that media influence and immigration are among important risk factors for the development of negative body image among non-Western men. Interventions designed to address the negative effects of the media and immigration may be effective at reducing body image disorders and other related health problems in this population.

Cell biochemistry studied by single-molecule imaging.

PubMed

Mashanov, G I; Nenasheva, T A; Peckham, M; Molloy, J E

2006-11-01

Over the last decade, there have been remarkable developments in live-cell imaging. We can now readily observe individual protein molecules within living cells and this should contribute to a systems level understanding of biological pathways. Direct observation of single fluorophores enables several types of molecular information to be gathered. Temporal and spatial trajectories enable diffusion constants and binding kinetics to be deduced, while analyses of fluorescence lifetime, intensity, polarization or spectra give chemical and conformational information about molecules in their cellular context. By recording the spatial trajectories of pairs of interacting molecules, formation of larger molecular complexes can be studied. In the future, multicolour and multiparameter imaging of single molecules in live cells will be a powerful analytical tool for systems biology. Here, we discuss measurements of single-molecule mobility and residency at the plasma membrane of live cells. Analysis of diffusional paths at the plasma membrane gives information about its physical properties and measurement of temporal trajectories enables rates of binding and dissociation to be derived. Meanwhile, close scrutiny of individual fluorophore trajectories enables ideas about molecular dimerization and oligomerization related to function to be tested directly.

SERS imaging of cell-surface biomolecules metabolically labeled with bioorthogonal Raman reporters.

PubMed

Xiao, Ming; Lin, Liang; Li, Zefan; Liu, Jie; Hong, Senlian; Li, Yaya; Zheng, Meiling; Duan, Xuanming; Chen, Xing

2014-08-01

Live imaging of biomolecules with high specificity and sensitivity as well as minimal perturbation is essential for studying cellular processes. Here, we report the development of a bioorthogonal surface-enhanced Raman scattering (SERS) imaging approach that exploits small Raman reporters for visualizing cell-surface biomolecules. The cells were cultured and imaged by SERS microscopy on arrays of Raman-enhancing nanoparticles coated on silicon wafers or glass slides. The Raman reporters including azides, alkynes, and carbondeuterium bonds are small in size and spectroscopically bioorthogonal (background-free). We demonstrated that various cell-surface biomolecules including proteins, glycans, and lipids were metabolically incorporated with the corresponding precursors bearing a Raman reporter and visualized by SERS microscopy. The coupling of SERS microscopy with bioorthogonal Raman reporters expands the capabilities of live-cell microscopy beyond the modalities of fluorescence and label-free imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy

NASA Astrophysics Data System (ADS)

Ahmed, Syeed Ehsan

Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance change in nanometer scale. In this study we have investigated the effect of dopamine in cAMP dynamics in vivo. In our study two-photon fluorescence microscope was used for imaging mushroom bodies inside live Drosophila melanogaster brain and we developed a method for studying the change in cyclic AMP level.

Imaging of oxygenation in 3D tissue models with multi-modal phosphorescent probes

NASA Astrophysics Data System (ADS)

Papkovsky, Dmitri B.; Dmitriev, Ruslan I.; Borisov, Sergei

2015-03-01

Cell-penetrating phosphorescence based probes allow real-time, high-resolution imaging of O2 concentration in respiring cells and 3D tissue models. We have developed a panel of such probes, small molecule and nanoparticle structures, which have different spectral characteristics, cell penetrating and tissue staining behavior. The probes are compatible with conventional live cell imaging platforms and can be used in different detection modalities, including ratiometric intensity and PLIM (Phosphorescence Lifetime IMaging) under one- or two-photon excitation. Analytical performance of these probes and utility of the O2 imaging method have been demonstrated with different types of samples: 2D cell cultures, multi-cellular spheroids from cancer cell lines and primary neurons, excised slices from mouse brain, colon and bladder tissue, and live animals. They are particularly useful for hypoxia research, ex-vivo studies of tissue physiology, cell metabolism, cancer, inflammation, and multiplexing with many conventional fluorophors and markers of cellular function.

Imaging the environment of green fluorescent protein.

PubMed Central

Suhling, Klaus; Siegel, Jan; Phillips, David; French, Paul M W; Lévêque-Fort, Sandrine; Webb, Stephen E D; Davis, Daniel M

2002-01-01

An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also probe their environment. Such a technique is demonstrated here using fluorescence lifetime imaging of green fluorescent protein (GFP). We first show, by time-correlated single-photon counting, that the fluorescence decay of GFP depends on the local refractive index. This is in agreement with the Strickler Berg formula, relating the Einstein A and B coefficients for absorption and spontaneous emission in molecules. We then quantitatively image, by wide-field time-gated fluorescence lifetime imaging, the refractive index of the environment of GFP. This novel approach paves the way for imaging the biophysical environment of specific GFP-tagged proteins in live cells. PMID:12496126

Magnetic Resonance Microscopy of the Lung

NASA Astrophysics Data System (ADS)

Johnson, G. Allan

1999-11-01

The lung presents both challenges and opportunities for study by magnetic resonance imaging (MRI). The technical challenges arise from respiratory and cardiac motion, limited signal from the tissues, and unique physical structure of the lung. These challenges are heightened in magnetic resonance microscopy (MRM) where the spatial resolution may be up to a million times higher than that of conventional MRI. The development of successful techniques for MRM of the lung present enormous opportunities for basic studies of lung structure and function, toxicology, environmental stress, and drug discovery by permitting investigators to study this most essential organ nondestructively in the live animal. Over the last 15 years, scientists at the Duke Center for In Vivo Microscopy have developed techniques for MRM in the live animal through an interdisciplinary program of biology, physics, chemistry, electrical engineering, and computer science. This talk will focus on the development of specialized radiofrequency coils for lung imaging, projection encoding methods to limit susceptibility losses, specialized support structures to control and monitor physiologic motion, and the most recent development of hyperpolarized gas imaging with ^3He and ^129Xe.

SU-E-I-56: Scan Angle Reduction for a Limited-Angle Intrafraction Verification (LIVE) System

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

Ren, L; Zhang, Y; Yin, F

Purpose: To develop a novel adaptive reconstruction strategy to further reduce the scanning angle required by the limited-angle intrafraction verification (LIVE) system for intrafraction verification. Methods: LIVE acquires limited angle MV projections from the exit fluence of the arc treatment beam or during gantry rotation between static beams. Orthogonal limited-angle kV projections are also acquired simultaneously to provide additional information. LIVE considers the on-board 4D-CBCT images as a deformation of the prior 4D-CT images, and solves the deformation field based on deformation models and data fidelity constraint. LIVE reaches a checkpoint after a limited-angle scan, and reconstructs 4D-CBCT for intrafractionmore » verification at the checkpoint. In adaptive reconstruction strategy, a larger scanning angle of 30° is used for the first checkpoint, and smaller scanning angles of 15° are used for subsequent checkpoints. The onboard images reconstructed at the previous adjacent checkpoint are used as the prior images for reconstruction at the current checkpoint. As the algorithm only needs to reconstruct the small deformation occurred between adjacent checkpoints, projections from a smaller scan angle provide enough information for the reconstruction. XCAT was used to simulate tumor motion baseline drift of 2mm along sup-inf direction at every subsequent checkpoint, which are 15° apart. Adaptive reconstruction strategy was used to reconstruct the images at each checkpoint using orthogonal 15° kV and MV projections. Results: Results showed that LIVE reconstructed the tumor volumes accurately using orthogonal 15° kV-MV projections. Volume percentage differences (VPDs) were within 5% and center of mass shifts (COMS) were within 1mm for reconstruction at all checkpoints. Conclusion: It's feasible to use an adaptive reconstruction strategy to further reduce the scan angle needed by LIVE to allow faster and more frequent intrafraction verification to minimize the treatment errors in lung cancer treatments. Grant from Varian Medical System.« less

Exploring Shop Window Displays

ERIC Educational Resources Information Center

Christopoulou, Martha

2011-01-01

Using visual resources from everyday life in art lessons can enrich students' knowledge about the creation of visual images, artifacts, and sites, and develop their critical understanding about the cultural impact of these images and their effects on people's lives. Through examining an exhibition in the windows of Selfridges department store in…

Halo-free phase contrast microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Nguyen, Tan H.; Kandel, Mikhail E.; Shakir, Haadi M.; Best, Catherine; Do, Minh N.; Popescu, Gabriel

2017-02-01

The phase contrast (PC) method is one of the most impactful developments in the four-century long history of microscopy. It allows for intrinsic, nondestructive contrast of transparent specimens, such as live cells. However, PC is plagued by the halo artifact, a result of insufficient spatial coherence in the illumination field, which limits its applicability. We present a new approach for retrieving halo-free phase contrast microscopy (hfPC) images by upgrading the conventional PC microscope with an external interferometric module, which generates sufficient data for reversing the halo artifact. Measuring four independent intensity images, our approach first measures haloed phase maps of the sample. We solve for the halo-free sample transmission function by using a physical model of the image formation under partial spatial coherence. Using this halo-free sample transmission, we can numerically generate artifact-free PC images. Furthermore, this transmission can be further used to obtain quantitative information about the sample, e.g., the thickness with known refractive indices, dry mass of live cells during their cycles. We tested our hfPC method on various control samples, e.g., beads, pillars and validated its potential for biological investigation by imaging live HeLa cells, red blood cells, and neurons.

Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.

PubMed

Subach, Fedor V; Patterson, George H; Renz, Malte; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V

2010-05-12

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

Synthetic-Molecule/Protein Hybrid Probe with Fluorogenic Switch for Live-Cell Imaging of DNA Methylation.

PubMed

Hori, Yuichiro; Otomura, Norimichi; Nishida, Ayuko; Nishiura, Miyako; Umeno, Maho; Suetake, Isao; Kikuchi, Kazuya

2018-02-07

Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.

Sulfur and nitrogen binary doped carbon dots derived from ammonium thiocyanate for selective probing doxycycline in living cells and multicolor cell imaging.

PubMed

Xue, Mingyue; Zhang, Liangliang; Zhan, Zhihua; Zou, Mengbing; Huang, Yong; Zhao, Shulin

2016-04-01

A novel sulfur and nitrogen binary doped carbon dots (S,N-CDs) was synthesized by one-step manner through the hydrothermal treatment of citric acid (CA) and ammonium thiocyanate, and the procedures for biomedical applications, including probing doxycycline in living cells and multicolor cell imaging were developed. The obtained S,N-CDs are stable in aqueous solution, possess a very high quantum yield (QY, 74.15%) and good photostability. The fluorescence of S,N-CDs can be specifically quenched by doxycycline, providing a convenient turn-off assay of doxycycline. This assay shows a wide linear detection range from 0.08 to 60 μM with a low detection limit of 20 nM. The present method also displays a good selectivity. More importantly, the S,N-CDs have an excellent biocompatibility and low cytotoxicity, allowing the multicolor cell imaging and doxycycline detection in living cells. Consequently, the developed doxycycline methods is facile, low-cost, biocompatible, sensitive and selective, which may hold the potential applications in the fields of food safety and environmental monitoring, as well as cancer therapy and related mechanism research. Copyright © 2015 Elsevier B.V. All rights reserved.

Live Imaging of Adult Neural Stem Cells in Rodents

PubMed Central

Ortega, Felipe; Costa, Marcos R.

2016-01-01

The generation of cells of the neural lineage within the brain is not restricted to early development. New neurons, oligodendrocytes, and astrocytes are produced in the adult brain throughout the entire murine life. However, despite the extensive research performed in the field of adult neurogenesis during the past years, fundamental questions regarding the cell biology of adult neural stem cells (aNSCs) remain to be uncovered. For instance, it is crucial to elucidate whether a single aNSC is capable of differentiating into all three different macroglial cell types in vivo or these distinct progenies constitute entirely separate lineages. Similarly, the cell cycle length, the time and mode of division (symmetric vs. asymmetric) that these cells undergo within their lineage progression are interesting questions under current investigation. In this sense, live imaging constitutes a valuable ally in the search of reliable answers to the previous questions. In spite of the current limitations of technology new approaches are being developed and outstanding amount of knowledge is being piled up providing interesting insights in the behavior of aNSCs. Here, we will review the state of the art of live imaging as well as the alternative models that currently offer new answers to critical questions. PMID:27013941

Vibrational imaging of glucose uptake activity in live cells and tissues by stimulated Raman scattering microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hu, Fanghao; Chen, Zhixing; Zhang, Luyuan; Shen, Yihui; Wei, Lu; Min, Wei

2016-03-01

Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.

A drug-compatible and temperature-controlled microfluidic device for live-cell imaging.

PubMed

Chen, Tong; Gomez-Escoda, Blanca; Munoz-Garcia, Javier; Babic, Julien; Griscom, Laurent; Wu, Pei-Yun Jenny; Coudreuse, Damien

2016-08-01

Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging. © 2016 The Authors.

A drug-compatible and temperature-controlled microfluidic device for live-cell imaging

PubMed Central

Chen, Tong; Gomez-Escoda, Blanca; Munoz-Garcia, Javier; Babic, Julien; Griscom, Laurent; Wu, Pei-Yun Jenny

2016-01-01

Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging. PMID:27512142

All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide.

PubMed

Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

2017-02-10

While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

PubMed Central

Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

2017-01-01

While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab. PMID:28186188

All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

NASA Astrophysics Data System (ADS)

Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

2017-02-01

While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

Reducing scan angle using adaptive prior knowledge for a limited-angle intrafraction verification (LIVE) system for conformal arc radiotherapy.

PubMed

Zhang, Yawei; Yin, Fang-Fang; Zhang, You; Ren, Lei

2017-05-07

The purpose of this study is to develop an adaptive prior knowledge guided image estimation technique to reduce the scan angle needed in the limited-angle intrafraction verification (LIVE) system for 4D-CBCT reconstruction. The LIVE system has been previously developed to reconstruct 4D volumetric images on-the-fly during arc treatment for intrafraction target verification and dose calculation. In this study, we developed an adaptive constrained free-form deformation reconstruction technique in LIVE to further reduce the scanning angle needed to reconstruct the 4D-CBCT images for faster intrafraction verification. This technique uses free form deformation with energy minimization to deform prior images to estimate 4D-CBCT based on kV-MV projections acquired in extremely limited angle (orthogonal 3°) during the treatment. Note that the prior images are adaptively updated using the latest CBCT images reconstructed by LIVE during treatment to utilize the continuity of the respiratory motion. The 4D digital extended-cardiac-torso (XCAT) phantom and a CIRS 008A dynamic thoracic phantom were used to evaluate the effectiveness of this technique. The reconstruction accuracy of the technique was evaluated by calculating both the center-of-mass-shift (COMS) and 3D volume-percentage-difference (VPD) of the tumor in reconstructed images and the true on-board images. The performance of the technique was also assessed with varied breathing signals against scanning angle, lesion size, lesion location, projection sampling interval, and scanning direction. In the XCAT study, using orthogonal-view of 3° kV and portal MV projections, this technique achieved an average tumor COMS/VPD of 0.4  ±  0.1 mm/5.5  ±  2.2%, 0.6  ±  0.3 mm/7.2  ±  2.8%, 0.5  ±  0.2 mm/7.1  ±  2.6%, 0.6  ±  0.2 mm/8.3  ±  2.4%, for baseline drift, amplitude variation, phase shift, and patient breathing signal variation, respectively. In the CIRS phantom study, this technique achieved an average tumor COMS/VPD of 0.7  ±  0.1 mm/7.5  ±  1.3% for a 3 cm lesion and 0.6  ±  0.2 mm/11.4  ±  1.5% for a 2 cm lesion in the baseline drift case. The average tumor COMS/VPD were 0.5  ±  0.2 mm/10.8  ±  1.4%, 0.4  ±  0.3 mm/7.3  ±  2.9%, 0.4  ±  0.2 mm/7.4  ±  2.5%, 0.4  ±  0.2 mm/7.3  ±  2.8% for the four real patient breathing signals, respectively. Results demonstrated that the adaptive prior knowledge guided image estimation technique with LIVE system is robust against scanning angle, lesion size, location and scanning direction. It can estimate on-board images accurately with as little as 6 projections in orthogonal-view 3° angle. In conclusion, adaptive prior knowledge guided image reconstruction technique accurately estimates 4D-CBCT images using extremely-limited angle and projections. This technique greatly improves the efficiency and accuracy of LIVE system for ultrafast 4D intrafraction verification of lung SBRT treatments.

Imaging live humans through smoke and flames using far-infrared digital holography.

PubMed

Locatelli, M; Pugliese, E; Paturzo, M; Bianco, V; Finizio, A; Pelagotti, A; Poggi, P; Miccio, L; Meucci, R; Ferraro, P

2013-03-11

The ability to see behind flames is a key challenge for the industrial field and particularly for the safety field. Development of new technologies to detect live people through smoke and flames in fire scenes is an extremely desirable goal since it can save human lives. The latest technologies, including equipment adopted by fire departments, use infrared bolometers for infrared digital cameras that allow users to see through smoke. However, such detectors are blinded by flame-emitted radiation. Here we show a completely different approach that makes use of lensless digital holography technology in the infrared range for successful imaging through smoke and flames. Notably, we demonstrate that digital holography with a cw laser allows the recording of dynamic human-size targets. In this work, easy detection of live, moving people is achieved through both smoke and flames, thus demonstrating the capability of digital holography at 10.6 μm.

Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

PubMed

Balbach, Sebastian T; Boiani, Michele

2015-01-01

Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.

Non-invasive assessment of otolith formation during development of the Japanese red-bellied newt, Cynops pyrrhogaster

NASA Technical Reports Server (NTRS)

Koike, H.; Nakamura, K.; Nishimura, K.; Kashima, I.; Wiederhold, M. L.; Asashima, M.

1995-01-01

Pre-mated adult female newts and embryos have been flown on the International Microgravity Laboratory-2 (IML-2) Space Shuttle flight in 1994 (Wiederhold et al., 1992b). With the specimens available from this flight, the calcification of otoliths, ulna, radius and backbone of the flown larvae and adult newts were analyzed. The experiments presented here studied the development of the otoliths on the ground. Otoliths of living newts, from embryo to adult, were observed in situ with the application of a new X-ray and bio-imaging analyzer system. For the establishment of this method, newts at different developmental stages were used. An imaging plate temporarily stores the X-ray energy pattern at the bio-imaging analyzer. A latent image on the imaging plate was transformed into a digital time series signal with an image reader. Acquired digital information was computed with the image processor. The processed information was recorded on film with an image recorder, in order to visualize it on an enlargement computed radiograph. To analyze development of the otoliths, photo-stimulated luminescence level was detected by an image analyzer, using transmitted X-ray photons. A single clump of otoconia could first be seen at stage 33. Stage-36 embryos first have distinguishable otoliths, with the utricle in front and saccule behind. Our results show that this X-ray method detects the otoliths equally as well as sectioning. In the newt, the mandibular/maxillary bone formed before the spine. It is suspected that for the newt embryo, living in water, feeding becomes necessary prior to support of the body.

Phage display and molecular imaging: expanding fields of vision in living subjects.

PubMed

Cochran, R; Cochran, Frank

2010-01-01

In vivo molecular imaging enables non-invasive visualization of biological processes within living subjects, and holds great promise for diagnosis and monitoring of disease. The ability to create new agents that bind to molecular targets and deliver imaging probes to desired locations in the body is critically important to further advance this field. To address this need, phage display, an established technology for the discovery and development of novel binding agents, is increasingly becoming a key component of many molecular imaging research programs. This review discusses the expanding role played by phage display in the field of molecular imaging with a focus on in vivo applications. Furthermore, new methodological advances in phage display that can be directly applied to the discovery and development of molecular imaging agents are described. Various phage library selection strategies are summarized and compared, including selections against purified target, intact cells, and ex vivo tissue, plus in vivo homing strategies. An outline of the process for converting polypeptides obtained from phage display library selections into successful in vivo imaging agents is provided, including strategies to optimize in vivo performance. Additionally, the use of phage particles as imaging agents is also described. In the latter part of the review, a survey of phage-derived in vivo imaging agents is presented, and important recent examples are highlighted. Other imaging applications are also discussed, such as the development of peptide tags for site-specific protein labeling and the use of phage as delivery agents for reporter genes. The review concludes with a discussion of how phage display technology will continue to impact both basic science and clinical applications in the field of molecular imaging.

Development and Validation of a Photonumeric Scale for Assessment of Chin Retrusion.

PubMed

Sykes, Jonathan M; Carruthers, Alastair; Hardas, Bhushan; Murphy, Diane K; Jones, Derek; Carruthers, Jean; Donofrio, Lisa; Creutz, Lela; Marx, Ann; Dill, Sara

2016-10-01

A validated scale is needed for objective and reproducible comparisons of chin appearance before and after chin augmentation in practice and clinical studies. To describe the development and validation of the 5-point photonumeric Allergan Chin Retrusion Scale. The Allergan Chin Retrusion Scale was developed to include an assessment guide, verbal descriptors, morphed images, and real subject images for each scale grade. The clinical significance of a 1-point score difference was evaluated in a review of multiple image pairs representing varying differences in severity. Interrater and intrarater reliability was evaluated in a live-subject validation study (N = 298) completed during 2 sessions occurring 3 weeks apart. A difference of ≥1 point on the scale was shown to reflect a clinically meaningful difference (mean [95% confidence interval] absolute score difference, 1.07 [0.94-1.20] for clinically different image pairs and 0.51 [0.39-0.63] for not clinically different pairs). Intrarater agreement between the 2 live-subject validation sessions was substantial (mean weighted kappa = 0.79). Interrater agreement was substantial during the second rating session (0.68, primary end point). The Allergan Chin Retrusion Scale is a validated and reliable scale for physician rating of severity of chin retrusion.

Adult stem cell lineage tracing and deep tissue imaging

PubMed Central

Fink, Juergen; Andersson-Rolf, Amanda; Koo, Bon-Kyoung

2015-01-01

Lineage tracing is a widely used method for understanding cellular dynamics in multicellular organisms during processes such as development, adult tissue maintenance, injury repair and tumorigenesis. Advances in tracing or tracking methods, from light microscopy-based live cell tracking to fluorescent label-tracing with two-photon microscopy, together with emerging tissue clearing strategies and intravital imaging approaches have enabled scientists to decipher adult stem and progenitor cell properties in various tissues and in a wide variety of biological processes. Although technical advances have enabled time-controlled genetic labeling and simultaneous live imaging, a number of obstacles still need to be overcome. In this review, we aim to provide an in-depth description of the traditional use of lineage tracing as well as current strategies and upcoming new methods of labeling and imaging. [BMB Reports 2015; 48(12): 655-667] PMID:26634741

Some uses of wavelets for imaging dynamic processes in live cochlear structures

NASA Astrophysics Data System (ADS)

Boutet de Monvel, J.

2007-09-01

A variety of image and signal processing algorithms based on wavelet filtering tools have been developed during the last few decades, that are well adapted to the experimental variability typically encountered in live biological microscopy. A number of processing tools are reviewed, that use wavelets for adaptive image restoration and for motion or brightness variation analysis by optical flow computation. The usefulness of these tools for biological imaging is illustrated in the context of the restoration of images of the inner ear and the analysis of cochlear motion patterns in two and three dimensions. I also report on recent work that aims at capturing fluorescence intensity changes associated with vesicle dynamics at synaptic zones of sensory hair cells. This latest application requires one to separate the intensity variations associated with the physiological process under study from the variations caused by motion of the observed structures. A wavelet optical flow algorithm for doing this is presented, and its effectiveness is demonstrated on artificial and experimental image sequences.

Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.

PubMed

Evans, Conor L; Potma, Eric O; Puoris'haag, Mehron; Côté, Daniel; Lin, Charles P; Xie, X Sunney

2005-11-15

Imaging living organisms with molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine, however, would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with molecular specificity. We have developed a sensitive technique for vibrational imaging of tissues by combining coherent anti-Stokes Raman scattering (CARS) with video-rate microscopy. Backscattering of the intense forward-propagating CARS radiation in tissue gives rise to a strong epi-CARS signal that makes in vivo imaging possible. This substantially large signal allows for real-time monitoring of dynamic processes, such as the diffusion of chemical compounds, in tissues. By tuning into the CH(2) stretching vibrational band, we demonstrate CARS imaging and spectroscopy of lipid-rich tissue structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes, with unprecedented contrast at subcellular resolution.

Fast live cell imaging at nanometer scale using annihilating filter-based low-rank Hankel matrix approach

NASA Astrophysics Data System (ADS)

Min, Junhong; Carlini, Lina; Unser, Michael; Manley, Suliana; Ye, Jong Chul

2015-09-01

Localization microscopy such as STORM/PALM can achieve a nanometer scale spatial resolution by iteratively localizing fluorescence molecules. It was shown that imaging of densely activated molecules can accelerate temporal resolution which was considered as major limitation of localization microscopy. However, this higher density imaging needs to incorporate advanced localization algorithms to deal with overlapping point spread functions (PSFs). In order to address this technical challenges, previously we developed a localization algorithm called FALCON1, 2 using a quasi-continuous localization model with sparsity prior on image space. It was demonstrated in both 2D/3D live cell imaging. However, it has several disadvantages to be further improved. Here, we proposed a new localization algorithm using annihilating filter-based low rank Hankel structured matrix approach (ALOHA). According to ALOHA principle, sparsity in image domain implies the existence of rank-deficient Hankel structured matrix in Fourier space. Thanks to this fundamental duality, our new algorithm can perform data-adaptive PSF estimation and deconvolution of Fourier spectrum, followed by truly grid-free localization using spectral estimation technique. Furthermore, all these optimizations are conducted on Fourier space only. We validated the performance of the new method with numerical experiments and live cell imaging experiment. The results confirmed that it has the higher localization performances in both experiments in terms of accuracy and detection rate.

Precision of Readout at the hunchback Gene: Analyzing Short Transcription Time Traces in Living Fly Embryos

PubMed Central

Tran, Huy; Ferraro, Teresa; Lucas, Tanguy; Guillou, Aurelien; Coppey, Mathieu; Dostatni, Nathalie

2016-01-01

The simultaneous expression of the hunchback gene in the numerous nuclei of the developing fly embryo gives us a unique opportunity to study how transcription is regulated in living organisms. A recently developed MS2-MCP technique for imaging nascent messenger RNA in living Drosophila embryos allows us to quantify the dynamics of the developmental transcription process. The initial measurement of the morphogens by the hunchback promoter takes place during very short cell cycles, not only giving each nucleus little time for a precise readout, but also resulting in short time traces of transcription. Additionally, the relationship between the measured signal and the promoter state depends on the molecular design of the reporting probe. We develop an analysis approach based on tailor made autocorrelation functions that overcomes the short trace problems and quantifies the dynamics of transcription initiation. Based on live imaging data, we identify signatures of bursty transcription initiation from the hunchback promoter. We show that the precision of the expression of the hunchback gene to measure its position along the anterior-posterior axis is low both at the boundary and in the anterior even at cycle 13, suggesting additional post-transcriptional averaging mechanisms to provide the precision observed in fixed embryos. PMID:27942043

Live-cell imaging: new avenues to investigate retinal regeneration

PubMed Central

Lahne, Manuela; Hyde, David R.

2017-01-01

Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish (Danio rerio) possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration. PMID:28966629

Live-cell imaging: new avenues to investigate retinal regeneration.

PubMed

Lahne, Manuela; Hyde, David R

2017-08-01

Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish ( Danio rerio ) possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.

HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

NASA Astrophysics Data System (ADS)

Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

2016-03-01

Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

Improvements in low-cost label-free QPI microscope for live cell imaging

NASA Astrophysics Data System (ADS)

Seniya, C.; Towers, C. E.; Towers, D. P.

2017-07-01

This paper reports an improvement in the development of a low-cost QPI microscope offering new capabilities in term of phase measurement accuracy for label-free live samples in the longer term (i.e., hours to days). The spatially separated scattered and non-scattered image light fields are reshaped in the Fourier plane and modulated to form an interference image at a CCD camera. The apertures that enable these two beams to be generated have been optimised by means of laser-cut apertures placed on the mirrors of a Michelson interferometer and has improved the phase measuring and reconstruction capability of the QPI microscope. The microscope was tested with transparent onion cells as an object of interest.

A Simple BODIPY-Based Viscosity Probe for Imaging of Cellular Viscosity in Live Cells

PubMed Central

Su, Dongdong; Teoh, Chai Lean; Gao, Nengyue; Xu, Qing-Hua; Chang, Young-Tae

2016-01-01

Intracellular viscosity is a fundamental physical parameter that indicates the functioning of cells. In this work, we developed a simple boron-dipyrromethene (BODIPY)-based probe, BTV, for cellular mitochondria viscosity imaging by coupling a simple BODIPY rotor with a mitochondria-targeting unit. The BTV exhibited a significant fluorescence intensity enhancement of more than 100-fold as the solvent viscosity increased. Also, the probe showed a direct linear relationship between the fluorescence lifetime and the media viscosity, which makes it possible to trace the change of the medium viscosity. Furthermore, it was demonstrated that BTV could achieve practical applicability in the monitoring of mitochondrial viscosity changes in live cells through fluorescence lifetime imaging microscopy (FLIM). PMID:27589762

A Simple BODIPY-Based Viscosity Probe for Imaging of Cellular Viscosity in Live Cells.

PubMed

Su, Dongdong; Teoh, Chai Lean; Gao, Nengyue; Xu, Qing-Hua; Chang, Young-Tae

2016-08-31

Intracellular viscosity is a fundamental physical parameter that indicates the functioning of cells. In this work, we developed a simple boron-dipyrromethene (BODIPY)-based probe, BTV, for cellular mitochondria viscosity imaging by coupling a simple BODIPY rotor with a mitochondria-targeting unit. The BTV exhibited a significant fluorescence intensity enhancement of more than 100-fold as the solvent viscosity increased. Also, the probe showed a direct linear relationship between the fluorescence lifetime and the media viscosity, which makes it possible to trace the change of the medium viscosity. Furthermore, it was demonstrated that BTV could achieve practical applicability in the monitoring of mitochondrial viscosity changes in live cells through fluorescence lifetime imaging microscopy (FLIM).

A benzothiazole-based fluorescent probe for hypochlorous acid detection and imaging in living cells

NASA Astrophysics Data System (ADS)

Nguyen, Khac Hong; Hao, Yuanqiang; Zeng, Ke; Fan, Shengnan; Li, Fen; Yuan, Suke; Ding, Xuejing; Xu, Maotian; Liu, You-Nian

2018-06-01

A benzothiazole-based turn-on fluorescent probe with a large Stokes shift (190 nm) has been developed for hypochlorous acid detection. The probe displays prompt fluorescence response for HClO with excellent selectivity over other reactive oxygen species as well as a low detection limit of 0.08 μM. The sensing mechanism involves the HClO-induced specific oxidation of oxime moiety of the probe to nitrile oxide, which was confirmed by HPLC-MS technique. Furthermore, imaging studies demonstrated that the probe is cell permeable and can be applied to detect HClO in living cells.

Stimulated Emission Depletion Live-Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction

PubMed Central

Wagner, Eva; Lauterbach, Marcel A.; Kohl, Tobias; Westphal, Volker; Williams, George S.B.; Steinbrecher, Julia H.; Streich, Jan-Hendrik; Korff, Brigitte; Tuan, Hoang-Trong M.; Hagen, Brian; Luther, Stefan; Hasenfuss, Gerd; Parlitz, Ulrich; Jafri, M. Saleet; Hell, Stefan W.; Lederer, W. Jonathan; Lehnart, Stephan E.

2014-01-01

Rationale Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing. PMID:22723297

Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors

PubMed Central

Danhier, Pierre; Krishnamachary, Balaji; Bharti, Santosh; Kakkad, Samata; Mironchik, Yelena; Bhujwalla, Zaver M.

2015-01-01

Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF) activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc) and a variant of the enhanced green fluorescent protein (EGFP). Both ODD-luciferase and EGFP were under the promotion of a poly-hypoxia-response element sequence (5xHRE). The cells constitutively expressed tdTomato red fluorescent protein. For validating the imaging strategy, cells were incubated under hypoxia (1% O2) for 48 hours and then reoxygenated. The luciferase activity of PC3-HRE-EGFP/HRE-ODD-luc/tdtomato cells detected by bioluminescent imaging rapidly decreased after reoxygenation, whereas EGFP levels in these cells remained stable for several hours. After in vitro validation, PC3-HRE-EGFP/HRE-ODD-luc/tdtomato tumors were implanted subcutaneously and orthotopically in nude male mice and imaged in vivo and ex vivo using optical imaging in proof-of-principle studies to demonstrate differences in optical patterns between EGFP expression and bioluminescence. This novel "timer" imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP can provide a time frame of HRE activation in PC3 prostate cancer cells and will be useful to understand the temporal changes in hypoxia and HIF activity during cancer progression and following treatments including HIF targeting strategies. PMID:26696369

Norcyanine dyes with benzo[c,d]indolium moiety: Spectral sensitivity with pH change for fluorescence pH imaging in living cells.

PubMed

Guan, Li; Liu, Qi; Zhang, Borui; Wang, Lanying

2017-01-01

Fluorescence pH imaging in living cells is a rapidly expanding research direction, however, it relies on the development of pH-sensitive fluorescent imaging agents. Here four norcyanine dyes with benzo[c,d]indolium moiety, exhibiting high spectral sensitivity with pH changes, were synthesized for fluorescence pH imaging in living cells, and characterized by 1 H NMR, 13 C NMR, IR, UV-Vis and HRMS. The investigation of their spectral properties in methanol and water showed that the absorption and emission maxima were in the region 488-618nm and 583-651nm, respectively, and four dyes exhibited high photostability. The pH spectral titrations showed that selective dye D1 had pH-dependent absorption spectral changes within the pH range of 2.4 to 9.4, and high fluorescent spectral sensitivity at pH5.0-8.0, with a pK a of 5.0. A cell association study indicated that dye D1 exhibited no or mild cytotoxicity at the application dose and duration, and could be accumulated in cells and mainly distributed in the cytoplasm, giving red fluorescence imaging. In particular, dye D1 could achieve pH-dependent fluorescence imaging in living cells with the increase of pH from 3.0 to 8.0, at excitation wavelength of 543nm and receiving wavelength of 655-755nm, which was valuable for studying the weak acidic, neutral and weak alkaline biological tissue compartments. Copyright © 2016 Elsevier B.V. All rights reserved.

Construction of an efficient two-photon fluorescent probe for imaging nitroreductase in live cells and tissues.

PubMed

Zhou, Liyi; Gong, Liang; Hu, Shunqin

2018-06-15

Compared with traditional confocal microscopy, two-photon fluorescence microscopy (TPFM), which excites a two-photon (TP) fluorophore by near-infrared light, provides improved three-dimensional image resolution with increased tissue-image depth (>500μm) and an extended observation time. Therefore, the development of novel functional TP fluorophores has attracted great attention in recent years. Herein, a novel TP fluorophore CM-NH 2 , which have the donor-π-acceptor (D-π-A)-structure, was designed and synthesized. We further used this dye developed a new type of TP fluorescent probe CM-NO 2 for detecting nitroreductase (NTR). Upon incubated with NTR for 15min, CM-NO 2 displayed a ~90-fold fluorescence enhancement at 505nm and the maximal TP action cross-section value after reaction was detected and calculated to be 200 GM at 760nm. The probe exhibited excellent properties such as high sensitivity, high selectivity, low cytotoxicity, and high photostability. Moreover, the probe was utilized to image the tumor hypoxia in live HeLa cells. Finally, using the CM-NO 2 to image NTR in tissues was demonstrated. Copyright © 2018 Elsevier B.V. All rights reserved.

Functional Imaging of Retinal Photoreceptors and Inner Neurons Using Stimulus-Evoked Intrinsic Optical Signals

PubMed Central

Yao, Xin-Cheng; Li, Yi-Chao

2013-01-01

Retinal development is a dynamic process both anatomically and functionally. High-resolution imaging and dynamic monitoring of photoreceptors and inner neurons can provide important information regarding the structure and function of the developing retina. In this chapter, we describe intrinsic optical signal (IOS) imaging as a high spatiotemporal resolution method for functional study of living retinal tissues. IOS imaging is based on near infrared (NIR) light detection of stimulus-evoked transient change of inherent optical characteristics of the cells. With no requirement for exogenous biomarkers, IOS imaging is totally noninvasive for functional mapping of stimulus-evoked spatiotemporal dynamics of the photoreceptors and inner retinal neurons. PMID:22688714

[Health-related images and concepts among adolescents living in rural areas of Brazil].

PubMed

Costa, Anny Giselly Milhome da; Vieira, Neiva Francenely Cunha; Gubert, Fabiane do Amaral; Ferreira, Adriana Gomes Nogueira; Scopacasa, Lígia Fernandes; Pinheiro, Patrícia Neyva da Costa

2013-08-01

The objective of this study was to describe health-related images and concepts among adolescents living in rural areas of Brazil, using photography. This was a qualitative community-based participatory study that used the photovoice method for data collection with groups of teenagers. Over a four-month period, 26 participants identified health problems in the rural community, took photographs, and reflected critically on the local reality. The adolescents presented pictures and stories that they organized into research themes and categories, representing inadequate living conditions for appropriate socioeconomic and cultural development and limiting the opportunities for change in this community. The study proved to be a positive health education strategy, involving young people in the community's health and maximizing the voice of teenagers as protagonists in their own history.

Simultaneous live imaging of the transcription and nuclear position of specific genes

PubMed Central

Ochiai, Hiroshi; Sugawara, Takeshi; Yamamoto, Takashi

2015-01-01

The relationship between genome organization and gene expression has recently been established. However, the relationships between spatial organization, dynamics, and transcriptional regulation of the genome remain unknown. In this study, we developed a live-imaging method for simultaneous measurements of the transcriptional activity and nuclear position of endogenous genes, which we termed the ‘Real-time Observation of Localization and EXpression (ROLEX)’ system. We demonstrated that ROLEX is highly specific and does not affect the expression level of the target gene. ROLEX enabled detection of sub-genome-wide mobility changes that depended on the state of Nanog transactivation in embryonic stem cells. We believe that the ROLEX system will become a powerful tool for exploring the relationship between transcription and nuclear dynamics in living cells. PMID:26092696

Quantitative dispersion microscopy

PubMed Central

Fu, Dan; Choi, Wonshik; Sung, Yongjin; Yaqoob, Zahid; Dasari, Ramachandra R.; Feld, Michael

2010-01-01

Refractive index dispersion is an intrinsic optical property and a useful source of contrast in biological imaging studies. In this report, we present the first dispersion phase imaging of living eukaryotic cells. We have developed quantitative dispersion microscopy based on the principle of quantitative phase microscopy. The dual-wavelength quantitative phase microscope makes phase measurements at 310 nm and 400 nm wavelengths to quantify dispersion (refractive index increment ratio) of live cells. The measured dispersion of living HeLa cells is found to be around 1.088, which agrees well with that measured directly for protein solutions using total internal reflection. This technique, together with the dry mass and morphology measurements provided by quantitative phase microscopy, could prove to be a useful tool for distinguishing different types of biomaterials and studying spatial inhomogeneities of biological samples. PMID:21113234

Recent advances in live cell imaging of hepatoma cells

PubMed Central

2014-01-01

Live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. With the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. The success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. This review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. Moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. PMID:25005127

Live cell imaging at the Munich ion microbeam SNAKE - a status report.

PubMed

Drexler, Guido A; Siebenwirth, Christian; Drexler, Sophie E; Girst, Stefanie; Greubel, Christoph; Dollinger, Günther; Friedl, Anna A

2015-02-18

Ion microbeams are important tools in radiobiological research. Still, the worldwide number of ion microbeam facilities where biological experiments can be performed is limited. Even fewer facilities combine ion microirradiation with live-cell imaging to allow microscopic observation of cellular response reactions starting very fast after irradiation and continuing for many hours. At SNAKE, the ion microbeam facility at the Munich 14 MV tandem accelerator, a large variety of biological experiments are performed on a regular basis. Here, recent developments and ongoing research projects at the ion microbeam SNAKE are presented with specific emphasis on live-cell imaging experiments. An overview of the technical details of the setup is given, including examples of suitable biological samples. By ion beam focusing to submicrometer beam spot size and single ion detection it is possible to target subcellular structures with defined numbers of ions. Focusing of high numbers of ions to single spots allows studying the influence of high local damage density on recruitment of damage response proteins.

Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics.

PubMed

Umezawa, Keitaro; Yoshida, Masafumi; Kamiya, Mako; Yamasoba, Tatsuya; Urano, Yasuteru

2017-03-01

Alterations in glutathione (GSH) homeostasis are associated with a variety of diseases and cellular functions, and therefore, real-time live-cell imaging and quantification of GSH dynamics are important for understanding pathophysiological processes. However, existing fluorescent probes are unsuitable for these purposes due to their irreversible fluorogenic mechanisms or slow reaction rates. In this work, we have successfully overcome these problems by establishing a design strategy inspired by Mayr's work on nucleophilic reaction kinetics. The synthesized probes exhibit concentration-dependent, reversible and rapid absorption/fluorescence changes (t 1/2  = 620 ms at [GSH] = 1 mM), as well as appropriate K d values (1-10 mM: within the range of intracellular GSH concentrations). We also developed FRET-based ratiometric probes, and demonstrated that they are useful for quantifying GSH concentration in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds.

Imaging enzyme-triggered self-assembly of small molecules inside live cells

PubMed Central

Gao, Yuan; Shi, Junfeng; Yuan, Dan; Xu, Bing

2012-01-01

Self-assembly of small molecules in water to form nanofibers, besides generating sophisticated biomaterials, promises a simple system inside cells for regulating cellular processes. But lack of a convenient approach for studying the self-assembly of small molecules inside cells hinders the development of such systems. Here we report a method to image enzyme-triggered self-assembly of small molecules inside live cells. After linking a fluorophore to a self-assembly motif to make a precursor, we confirmed by 31P NMR and rheology that enzyme-triggered conversion of the precursor to a hydrogelator results in the formation of a hydrogel via self-assembly. The imaging contrast conferred by the nanofibers of the hydrogelators allowed the evaluation of intracellular self-assembly; the dynamics, and the localization of the nanofibers of the hydrogelators in live cells. This approach explores supramolecular chemistry inside cells and may lead to new insights, processes, or materials at the interface of chemistry and biology. PMID:22929790

Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics

NASA Astrophysics Data System (ADS)

Umezawa, Keitaro; Yoshida, Masafumi; Kamiya, Mako; Yamasoba, Tatsuya; Urano, Yasuteru

2017-03-01

Alterations in glutathione (GSH) homeostasis are associated with a variety of diseases and cellular functions, and therefore, real-time live-cell imaging and quantification of GSH dynamics are important for understanding pathophysiological processes. However, existing fluorescent probes are unsuitable for these purposes due to their irreversible fluorogenic mechanisms or slow reaction rates. In this work, we have successfully overcome these problems by establishing a design strategy inspired by Mayr's work on nucleophilic reaction kinetics. The synthesized probes exhibit concentration-dependent, reversible and rapid absorption/fluorescence changes (t1/2 = 620 ms at [GSH] = 1 mM), as well as appropriate Kd values (1-10 mM: within the range of intracellular GSH concentrations). We also developed FRET-based ratiometric probes, and demonstrated that they are useful for quantifying GSH concentration in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds.

Bacterial infection imaging with [18F]fluoropropyl-trimethoprim

PubMed Central

Lee, Iljung; Hou, Catherine; Weng, Chi-Chang; Li, Shihong; Lieberman, Brian P.; Zeng, Chenbo; Mankoff, David A.; Mach, Robert H.

2017-01-01

There is often overlap in the diagnostic features of common pathologic processes such as infection, sterile inflammation, and cancer both clinically and using conventional imaging techniques. Here, we report the development of a positron emission tomography probe for live bacterial infection based on the small-molecule antibiotic trimethoprim (TMP). [18F]fluoropropyl-trimethoprim, or [18F]FPTMP, shows a greater than 100-fold increased uptake in vitro in live bacteria (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) relative to controls. In a rodent myositis model, [18F]FPTMP identified live bacterial infection without demonstrating confounding increased signal in the same animal from other etiologies including chemical inflammation (turpentine) and cancer (breast carcinoma). Additionally, the biodistribution of [18F]FPTMP in a nonhuman primate shows low background in many important tissues that may be sites of infection such as the lungs and soft tissues. These results suggest that [18F]FPTMP could be a broadly useful agent for the sensitive and specific imaging of bacterial infection with strong translational potential. PMID:28716936

Surface plasmon resonance sensing: from purified biomolecules to intact cells.

PubMed

Su, Yu-Wen; Wang, Wei

2018-04-12

Surface plasmon resonance (SPR) has become a well-recognized label-free technique for measuring the binding kinetics between biomolecules since the invention of the first SPR-based immunosensor in 1980s. The most popular and traditional format for SPR analysis is to monitor the real-time optical signals when a solution containing ligand molecules is flowing over a sensor substrate functionalized with purified receptor molecules. In recent years, rapid development of several kinds of SPR imaging techniques have allowed for mapping the dynamic distribution of local mass density within single living cells with high spatial and temporal resolutions and reliable sensitivity. Such capability immediately enabled one to investigate the interaction between important biomolecules and intact cells in a label-free, quantitative, and single cell manner, leading to an exciting new trend of cell-based SPR bioanalysis. In this Trend Article, we first describe the principle and technical features of two types of SPR imaging techniques based on prism and objective, respectively. Then we survey the intact cell-based applications in both fundamental cell biology and drug discovery. We conclude the article with comments and perspectives on the future developments. Graphical abstract Recent developments in surface plasmon resonance (SPR) imaging techniques allow for label-free mapping the mass-distribution within single living cells, leading to great expansions in biomolecular interactions studies from homogeneous substrates functionalized with purified biomolecules to heterogeneous substrates containing individual living cells.

Wireless live streaming video of laparoscopic surgery: a bandwidth analysis for handheld computers.

PubMed

Gandsas, Alex; McIntire, Katherine; George, Ivan M; Witzke, Wayne; Hoskins, James D; Park, Adrian

2002-01-01

Over the last six years, streaming media has emerged as a powerful tool for delivering multimedia content over networks. Concurrently, wireless technology has evolved, freeing users from desktop boundaries and wired infrastructures. At the University of Kentucky Medical Center, we have integrated these technologies to develop a system that can wirelessly transmit live surgery from the operating room to a handheld computer. This study establishes the feasibility of using our system to view surgeries and describes the effect of bandwidth on image quality. A live laparoscopic ventral hernia repair was transmitted to a single handheld computer using five encoding speeds at a constant frame rate, and the quality of the resulting streaming images was evaluated. No video images were rendered when video data were encoded at 28.8 kilobytes per second (Kbps), the slowest encoding bitrate studied. The highest quality images were rendered at encoding speeds greater than or equal to 150 Kbps. Of note, a 15 second transmission delay was experienced using all four encoding schemes that rendered video images. We believe that the wireless transmission of streaming video to handheld computers has tremendous potential to enhance surgical education. For medical students and residents, the ability to view live surgeries, lectures, courses and seminars on handheld computers means a larger number of learning opportunities. In addition, we envision that wireless enabled devices may be used to telemonitor surgical procedures. However, bandwidth availability and streaming delay are major issues that must be addressed before wireless telementoring becomes a reality.

Non-invasive molecular imaging for preclinical cancer therapeutic development

PubMed Central

O'Farrell, AC; Shnyder, SD; Marston, G; Coletta, PL; Gill, JH

2013-01-01

Molecular and non-invasive imaging are rapidly emerging fields in preclinical cancer drug discovery. This is driven by the need to develop more efficacious and safer treatments, the advent of molecular-targeted therapeutics, and the requirements to reduce and refine current preclinical in vivo models. Such bioimaging strategies include MRI, PET, single positron emission computed tomography, ultrasound, and optical approaches such as bioluminescence and fluorescence imaging. These molecular imaging modalities have several advantages over traditional screening methods, not least the ability to quantitatively monitor pharmacodynamic changes at the cellular and molecular level in living animals non-invasively in real time. This review aims to provide an overview of non-invasive molecular imaging techniques, highlighting the strengths, limitations and versatility of these approaches in preclinical cancer drug discovery and development. PMID:23488622

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel.

PubMed

Bolgioni, Amanda F; Vittoria, Marc A; Ganem, Neil J

2018-05-14

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 °C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material1

PubMed Central

Bell, Karen; Mitchell, Steve; Paultre, Danae; Posch, Markus; Oparka, Karl

2013-01-01

Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 μm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology. PMID:23457228

Visualizing dopamine released from living cells using a nanoplasmonic probe

NASA Astrophysics Data System (ADS)

Qin, W. W.; Wang, S. P.; Li, J.; Peng, T. H.; Xu, Y.; Wang, K.; Shi, J. Y.; Fan, C. H.; Li, D.

2015-09-01

We report the development of an ultrasensitive nanoplasmonic probe for discriminative detection and imaging of dopamine released from living cells. The sensing mechanism is based on the dopamine-induced seeded-growth of Au nanoparticles (Au NPs) that leads to the shift of the plasmon band. This platform allows for the detection of dopamine with a detection limit down to 0.25 pM within 1 min. This nanoplasmonic assay is further applied to visualize the release of dopamine from living rat pheochromocytoma (PC12) cells under ATP-stimulation with dark-field microscopy (DFM). The DFM results together with real time fluorescence imaging of PC12 cells stained with the Fluo calcium indicator, suggested that ATP stimulated-release of dopamine is concomitant with the Ca2+ influx, and the influx of Ca2+ is through ATP-activated channels instead of the voltage-gated Ca2+ channel (VGC).We report the development of an ultrasensitive nanoplasmonic probe for discriminative detection and imaging of dopamine released from living cells. The sensing mechanism is based on the dopamine-induced seeded-growth of Au nanoparticles (Au NPs) that leads to the shift of the plasmon band. This platform allows for the detection of dopamine with a detection limit down to 0.25 pM within 1 min. This nanoplasmonic assay is further applied to visualize the release of dopamine from living rat pheochromocytoma (PC12) cells under ATP-stimulation with dark-field microscopy (DFM). The DFM results together with real time fluorescence imaging of PC12 cells stained with the Fluo calcium indicator, suggested that ATP stimulated-release of dopamine is concomitant with the Ca2+ influx, and the influx of Ca2+ is through ATP-activated channels instead of the voltage-gated Ca2+ channel (VGC). Electronic supplementary information (ESI) available: Fig. S1-S4 and Table S1. See DOI: 10.1039/c5nr04433b

An automated microfluidic platform for C. elegans embryo arraying, phenotyping, and long-term live imaging

NASA Astrophysics Data System (ADS)

Cornaglia, Matteo; Mouchiroud, Laurent; Marette, Alexis; Narasimhan, Shreya; Lehnert, Thomas; Jovaisaite, Virginija; Auwerx, Johan; Gijs, Martin A. M.

2015-05-01

Studies of the real-time dynamics of embryonic development require a gentle embryo handling method, the possibility of long-term live imaging during the complete embryogenesis, as well as of parallelization providing a population’s statistics, while keeping single embryo resolution. We describe an automated approach that fully accomplishes these requirements for embryos of Caenorhabditis elegans, one of the most employed model organisms in biomedical research. We developed a microfluidic platform which makes use of pure passive hydrodynamics to run on-chip worm cultures, from which we obtain synchronized embryo populations, and to immobilize these embryos in incubator microarrays for long-term high-resolution optical imaging. We successfully employ our platform to investigate morphogenesis and mitochondrial biogenesis during the full embryonic development and elucidate the role of the mitochondrial unfolded protein response (UPRmt) within C. elegans embryogenesis. Our method can be generally used for protein expression and developmental studies at the embryonic level, but can also provide clues to understand the aging process and age-related diseases in particular.

[Applicability of non-invasive imaging methods in forensic medicine and forensic anthropology in particular].

PubMed

Marcinková, Mária; Straka, Ľubomír; Novomeský, František; Janík, Martin; Štuller, František; Krajčovič, Jozef

2018-01-01

Massive progress in developing even more precise imaging modalities influenced all medical branches including the forensic medicine. In forensic anthropology, an inevitable part of forensic medicine itself, the use of all imaging modalities becomes even more important. Despite of acquiring more accurate informations about the deceased, all of them can be used in the process of identification and/or age estimation. X - ray imaging is most commonly used in detecting foreign bodies or various pathological changes of the deceased. Computed tomography, on the other hand, can be very helpful in the process of identification, whereas outcomes of this examination can be used for virtual reconstruction of living objects. Magnetic resonance imaging offers new opportunities in detecting cardiovascular pathological processes or develompental anomalies. Ultrasonography provides promising results in age estimation of living subjects without excessive doses of radiation. Processing the latest information sources available, authors introduce the application examples of X - ray imaging, computed tomography, magnetic resonance imaging and ultrasonography in everyday forensic medicine routine, with particular focusing on forensic anthropology.

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells.

PubMed

Lajevardipour, Alireza; Chon, James W M; Chattopadhyay, Amitabha; Clayton, Andrew H A

2016-11-22

Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C 6 -NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

Correlative STED and Atomic Force Microscopy on Live Astrocytes Reveals Plasticity of Cytoskeletal Structure and Membrane Physical Properties during Polarized Migration

PubMed Central

Curry, Nathan; Ghézali, Grégory; Kaminski Schierle, Gabriele S.; Rouach, Nathalie; Kaminski, Clemens F.

2017-01-01

The plasticity of the cytoskeleton architecture and membrane properties is important for the establishment of cell polarity, adhesion and migration. Here, we present a method which combines stimulated emission depletion (STED) super-resolution imaging and atomic force microscopy (AFM) to correlate cytoskeletal structural information with membrane physical properties in live astrocytes. Using STED compatible dyes for live cell imaging of the cytoskeleton, and simultaneously mapping the cell surface topology with AFM, we obtain unprecedented detail of highly organized networks of actin and microtubules in astrocytes. Combining mechanical data from AFM with optical imaging of actin and tubulin further reveals links between cytoskeleton organization and membrane properties. Using this methodology we illustrate that scratch-induced migration induces cytoskeleton remodeling. The latter is caused by a polarization of actin and microtubule elements within astroglial cell processes, which correlates strongly with changes in cell stiffness. The method opens new avenues for the dynamic probing of the membrane structural and functional plasticity of living brain cells. It is a powerful tool for providing new insights into mechanisms of cell structural remodeling during physiological or pathological processes, such as brain development or tumorigenesis. PMID:28469559

Small-Animal Imaging Using Diffuse Fluorescence Tomography.

PubMed

Davis, Scott C; Tichauer, Kenneth M

2016-01-01

Diffuse fluorescence tomography (DFT) has been developed to image the spatial distribution of fluorescence-tagged tracers in living tissue. This capability facilitates the recovery of any number of functional parameters, including enzymatic activity, receptor density, blood flow, and gene expression. However, deploying DFT effectively is complex and often requires years of know-how, especially for newer mutlimodal systems that combine DFT with conventional imaging systems. In this chapter, we step through the process of using MRI-DFT imaging of a receptor-targeted tracer in small animals.

Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity

PubMed Central

Shen, Yi; Chen, Yingche; Wu, Jiahui; Shaner, Nathan C.; Campbell, Robert E.

2017-01-01

MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λex = 460 nm and λem = 610 nm) and a red-shifted (RDS)-mCherry variant (λex = 600 nm and λem = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry’s fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging. PMID:28241009

Design of a dynamic biofilm imaging cell for white-light interferometric microscopy

DOE PAGES

Larimer, Curtis; Brann, Michelle; Suter, Jonathan D.; ...

2017-05-10

In microbiology research there is a strong need for next generation imaging and sensing instrumentation that will enable minimally invasive and label-free investigation of soft, hydrated structures such as in bacterial biofilms. White light interferometry (WLI) can provide high resolution images of surface topology without the use of fluorescent labels but is not typically used to image biofilms because there is insufficient refractive index contrast to induce reflection from the biofilm’s interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ, especially in a non-destructive manner. In this report, we build onmore » our prior description of static biofilm imaging and describe the design of a dynamic imaging flow cell that enables monitoring the thickness and topology of live biofilms over time using a WLI microscope. The microfluidic system is specifically designed to create a reflective interface on the surface of biofilms while minimizing disruption of fragile structures. The imaging cell was also designed to accommodate limitations imposed by the depth of focus of the microscope’s objective lens. Example images of live biofilm samples are shown in order to illustrate the ability of the flow cell and WLI instrument to 1) support bacterial growth and biofilm development, 2) image biofilm structure that reflects growth in flow conditions, and 3) monitor biofilm development over time non-destructively. In future work, the apparatus described here will enable surface metrology measurements (roughness, surface area, etc.) of biofilms and may be used to observe changes in biofilm structure in response to changes in environmental conditions (e.g., flow velocity, availability of nutrients, and presence of biocides). Furthermore, this development will open new opportunities for the use of WLI in bioimaging.« less

Design of a dynamic biofilm imaging cell for white-light interferometric microscopy

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

Larimer, Curtis; Brann, Michelle; Suter, Jonathan D.

In microbiology research there is a strong need for next generation imaging and sensing instrumentation that will enable minimally invasive and label-free investigation of soft, hydrated structures such as in bacterial biofilms. White light interferometry (WLI) can provide high resolution images of surface topology without the use of fluorescent labels but is not typically used to image biofilms because there is insufficient refractive index contrast to induce reflection from the biofilm’s interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ, especially in a non-destructive manner. In this report, we build onmore » our prior description of static biofilm imaging and describe the design of a dynamic imaging flow cell that enables monitoring the thickness and topology of live biofilms over time using a WLI microscope. The microfluidic system is specifically designed to create a reflective interface on the surface of biofilms while minimizing disruption of fragile structures. The imaging cell was also designed to accommodate limitations imposed by the depth of focus of the microscope’s objective lens. Example images of live biofilm samples are shown in order to illustrate the ability of the flow cell and WLI instrument to 1) support bacterial growth and biofilm development, 2) image biofilm structure that reflects growth in flow conditions, and 3) monitor biofilm development over time non-destructively. In future work, the apparatus described here will enable surface metrology measurements (roughness, surface area, etc.) of biofilms and may be used to observe changes in biofilm structure in response to changes in environmental conditions (e.g., flow velocity, availability of nutrients, and presence of biocides). Furthermore, this development will open new opportunities for the use of WLI in bioimaging.« less

Long-term C. elegans immobilization enables high resolution developmental studies in vivo.

PubMed

Berger, Simon; Lattmann, Evelyn; Aegerter-Wilmsen, Tinri; Hengartner, Michael; Hajnal, Alex; deMello, Andrew; Casadevall I Solvas, Xavier

2018-05-01

Live-imaging of C. elegans is essential for the study of conserved cellular pathways (e.g. EGFR/Wnt signaling) and morphogenesis in vivo. However, the usefulness of live imaging as a research tool has been severely limited by the need to immobilize worms prior to and during imaging. Conventionally, immobilization is achieved by employing both physical and chemical interventions. These are known to significantly affect many physiological processes, and thus limit our understanding of dynamic developmental processes. Herein we present a novel, easy-to-use microfluidic platform for the long-term immobilization of viable, normally developing C. elegans, compatible with image acquisition at high resolution, thereby overcoming the limitations associated with conventional worm immobilization. The capabilities of the platform are demonstrated through the continuous assessment of anchor cell (AC) invasion and distal tip cell (DTC) migration in larval C. elegans and germ cell apoptosis in adult C. elegans in vivo for the first time.

CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging.

PubMed

Held, Michael; Schmitz, Michael H A; Fischer, Bernd; Walter, Thomas; Neumann, Beate; Olma, Michael H; Peter, Matthias; Ellenberg, Jan; Gerlich, Daniel W

2010-09-01

Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging-based screening with assays that directly score cellular dynamics.

Optical coherence tomography for embryonic imaging: a review

PubMed Central

Raghunathan, Raksha; Singh, Manmohan; Dickinson, Mary E.; Larin, Kirill V.

2016-01-01

Abstract. Embryogenesis is a highly complex and dynamic process, and its visualization is crucial for understanding basic physiological processes during development and for identifying and assessing possible defects, malformations, and diseases. While traditional imaging modalities, such as ultrasound biomicroscopy, micro-magnetic resonance imaging, and micro-computed tomography, have long been adapted for embryonic imaging, these techniques generally have limitations in their speed, spatial resolution, and contrast to capture processes such as cardiodynamics during embryogenesis. Optical coherence tomography (OCT) is a noninvasive imaging modality with micrometer-scale spatial resolution and imaging depth up to a few millimeters in tissue. OCT has bridged the gap between ultrahigh resolution imaging techniques with limited imaging depth like confocal microscopy and modalities, such as ultrasound sonography, which have deeper penetration but poorer spatial resolution. Moreover, the noninvasive nature of OCT has enabled live imaging of embryos without any external contrast agents. We review how OCT has been utilized to study developing embryos and also discuss advances in techniques used in conjunction with OCT to understand embryonic development. PMID:27228503

Constant-Distance Mode Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging of Biological Samples with Complex Topography

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

Nguyen, Son N.; Liyu, Andrey V.; Chu, Rosalie K.

A new approach for constant distance mode mass spectrometry imaging of biological samples using nanospray desorption electrospray ionization (nano-DESI MSI) was developed by integrating a shear-force probe with nano-DESI probe. The technical concept and basic instrumental setup as well as general operation of the system are described. Mechanical dampening of resonant oscillations due to the presence of shear forces between the probe and the sample surface enables constant-distance imaging mode via a computer controlled closed feedback loop. The capability of simultaneous chemical and topographic imaging of complex biological samples is demonstrated using living Bacillus Subtilis ATCC 49760 colonies on agarmore » plates. The constant-distance mode nano-DESI MSI enabled imaging of many metabolites including non-ribosomal peptides (surfactin, plipastatin and iturin) and iron-bound heme on the surface of living bacterial colonies ranging in diameter from 10 mm to 13 mm with height variations of up to 0.8 mm above the agar plate. Co-registration of ion images to topographic images provided higher-contrast images. Constant-mode nano-DESI MSI is ideally suited for imaging biological samples of complex topography in their native state.« less

Pushing the physical limits of spectroscopic imaging for new biology and better medicine (Conference Presentation)

NASA Astrophysics Data System (ADS)

Cheng, Ji-Xin

2017-02-01

In vivo molecular spectroscopic imaging is not a simple addition of a spectrometer to a microscope. Innovations are needed to break the physical limits in sensitivity, depth, speed and resolution perspectives. I will present our most recent advances in modality development, biological application, and clinical translation. My talk will focus on the development of mid-infrared photothermal microscope for depth-resolved vibrational imaging of living cells (Science Advances, in press), the discovery of a metabolic signature in cancer stem cells by hyperspectral stimulated Raman scattering imaging (Cell Stem Cell, in press), and the development of an intravascular vibrational photoacoustic catheter for label-free sensing of lipid laden plaques (Scientific Report 2016, 6:25236).

[A follow-up study on the degree of satisfaction regarding environment, life style and the coming Olympic events in the inhabitants living in the typical communities of Beijing].

PubMed

Zhang, Heng; Ma, Jun; Song, Yi; Li, Yan; Zong, Shu-ting; Xiao, Feng; Chen, Bo-wen

2008-08-01

To measure the degree of satisfaction on various environmental and health components and to discuss the impact of Olympic Games among the residents so as to make relative policy suggestions. In 2006, permanent residents over 15 years old lived in the Asian Games Village Community (where the 29th Olympic Games to be held) were selected to conduct a household's survey, while 1610 valid questionnaires were collected. The questionnaire included demographic information, degrees of satisfaction on various health-related environmental components, living condition and on Olympic events. The top 4 aspects with the highest satisfaction rates were "overall rates of satisfaction on current life" "green space", "housing conditions" and "water quality", which were 50.43%, 48.59%, 38.95%, 37.08%, respectively. Residents' satisfaction on "impact of hosting the Olympic Games on China's international image", "China's economic development level", "living conditions" and "personal life" were 65.53%, 56.09%, 47.27%, 46.40%, respectively. Data from partial correlation analysis showed that the total scores of satisfaction on environment and life had positive correlation with the total scores of Olympic satisfaction (P < 0.05). The satisfaction degree on Olympic event through factor analysis showed that 10 entries of the Olympic impact could be reflected by two factors--the influence of image to the nation and impact on personal income. Logistic regression showed that the impact of Olympic Games on personal income, the impact of Olympic Games on the image of the nation and standard of living, gender, education level were independent influencing factors of the total scores of environment and life satisfaction (P < 0.05). Other than "green space", most of health-related environment components of Beijing had low degree of satisfaction among inhabitants from the 'typical' communities in Beijing. However, residents had a higher degree of satisfaction on the impact of the Olympic Games to the country's image, the country's economic development level, the environment and personal standard of living.

Monitoring bacterial biofilms with a microfluidic flow chip designed for imaging with white-light interferometry

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

Brann, Michelle; Suter, Jonathan D.; Addleman, R. Shane

There is a need for imaging and sensing instrumentation that can monitor transitions in biofilm structure in order to better understand biofilm development and emergent properties such as anti-microbial resistance. Herein, we expanded on our previously reported technique for measuring and monitoring the thickness and topology of live biofilms using white-light interferometry (WLI). A flow cell designed for WLI enabled the use of this non-disruptive imaging method for the capture of high resolution three-dimensional profile images of biofilm growth over time. The fine axial resolution (3 nm) and wide field of view (>1 mm by 1 mm) enabled detection ofmore » biofilm formation as early as three hours after inoculation of the flow cell with a live bacterial culture (Pseudomonas fluorescens). WLI imaging facilitated monitoring the early stages of biofilm development and subtle variations in the structure of mature biofilms. Minimally-invasive imaging enabled monitoring of biofilm structure with surface metrology metrics (e.g., surface roughness). The system was used to observe a transition in biofilm structure that occurred in response to expsoure to a common antiseptic. In the future, WLI and the biofilm imaging cell described herein may be used to test the effectiveness of biofilm-specific therapies to combat common diseases associated with biofilm formation such as cystic fibrosis and periodontitis.« less

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

PubMed Central

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

Coherent anti-Stokes Raman scattering microscope with a high-signal-to-noise ratio, high stability, and high-speed imaging for live cell observation

NASA Astrophysics Data System (ADS)

Hayashi, Shinichi; Takimoto, Shinichi; Hashimoto, Takeshi

2007-02-01

Coherent anti-Stokes Raman scattering (CARS) microscopy, which can produce images of specific molecules without staining, has attracted the attention of researchers, as it matches the need for molecular imaging and pathway analysis of live cells. In particular, there have been an increasing number of CARS experimental results regarding lipids in live cells, which cannot be fluorescently tagged while keeping the cells alive. One of the important applications of lipid research is for the metabolic syndrome. Since the metabolic syndrome is said to be related to the lipids in lipocytes, blood, arterial vessels, and so on, the CARS technique is expected to find application in this field. However, CARS microscopy requires a pair of picosecond laser pulses, which overlap both temporally and spatially. This makes the optical adjustments of a CARS microscope challenging. The authors developed a CARS unit that includes optics for easy and stable adjustment of the overlap of these laser pulses. Adding the CARS unit to a laser scanning microscope provides CARS images of a high signal-to-noise ratio, with an acquisition rate as high as 2 microseconds per pixel. Thus, images of fast-moving lipid droplets in Hela cells were obtained.

A 3-D mixed-reality system for stereoscopic visualization of medical dataset.

PubMed

Ferrari, Vincenzo; Megali, Giuseppe; Troia, Elena; Pietrabissa, Andrea; Mosca, Franco

2009-11-01

We developed a simple, light, and cheap 3-D visualization device based on mixed reality that can be used by physicians to see preoperative radiological exams in a natural way. The system allows the user to see stereoscopic "augmented images," which are created by mixing 3-D virtual models of anatomies obtained by processing preoperative volumetric radiological images (computed tomography or MRI) with real patient live images, grabbed by means of cameras. The interface of the system consists of a head-mounted display equipped with two high-definition cameras. Cameras are mounted in correspondence of the user's eyes and allow one to grab live images of the patient with the same point of view of the user. The system does not use any external tracker to detect movements of the user or the patient. The movements of the user's head and the alignment of virtual patient with the real one are done using machine vision methods applied on pairs of live images. Experimental results, concerning frame rate and alignment precision between virtual and real patient, demonstrate that machine vision methods used for localization are appropriate for the specific application and that systems based on stereoscopic mixed reality are feasible and can be proficiently adopted in clinical practice.

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.

Vision-based overlay of a virtual object into real scene for designing room interior

NASA Astrophysics Data System (ADS)

Harasaki, Shunsuke; Saito, Hideo

2001-10-01

In this paper, we introduce a geometric registration method for augmented reality (AR) and an application system, interior simulator, in which a virtual (CG) object can be overlaid into a real world space. Interior simulator is developed as an example of an AR application of the proposed method. Using interior simulator, users can visually simulate the location of virtual furniture and articles in the living room so that they can easily design the living room interior without placing real furniture and articles, by viewing from many different locations and orientations in real-time. In our system, two base images of a real world space are captured from two different views for defining a projective coordinate of object 3D space. Then each projective view of a virtual object in the base images are registered interactively. After such coordinate determination, an image sequence of a real world space is captured by hand-held camera with tracking non-metric measured feature points for overlaying a virtual object. Virtual objects can be overlaid onto the image sequence by taking each relationship between the images. With the proposed system, 3D position tracking device, such as magnetic trackers, are not required for the overlay of virtual objects. Experimental results demonstrate that 3D virtual furniture can be overlaid into an image sequence of the scene of a living room nearly at video rate (20 frames per second).

Monitoring tumor metastases and osteolytic lesions with bioluminescence and micro CT imaging.

PubMed

Lim, Ed; Modi, Kshitij; Christensen, Anna; Meganck, Jeff; Oldfield, Stephen; Zhang, Ning

2011-04-14

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location. Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

Clinical evaluation of a confocal microendoscope system for imaging the ovary

NASA Astrophysics Data System (ADS)

Tanbakuchi, Anthony A.; Rouse, Andrew R.; Hatch, Kenneth D.; Sampliner, Richard E.; Udovich, Josh A.; Gmitro, Arthur F.

2008-02-01

We have developed a mobile confocal microendoscope system that provides live cellular imaging during surgery to aid in diagnosing microscopic abnormalities including cancer. We present initial clinical trial results using the device to image ovaries in-vivo using fluorescein and ex-vivo results using acridine orange. The imaging catheter has improved depth control and localized dye delivery mechanisms than previously presented. A manual control now provides a simple way for the surgeon to adjust and optimize imaging depth during the procedure while a tiny piezo valve in the imaging catheter controls the dye delivery.

Live-cell confocal microscopy and quantitative 4D image analysis of anchor cell invasion through the basement membrane in C. elegans

PubMed Central

Kelley, Laura C.; Wang, Zheng; Hagedorn, Elliott J.; Wang, Lin; Shen, Wanqing; Lei, Shijun; Johnson, Sam A.; Sherwood, David R.

2018-01-01

Cell invasion through basement membrane (BM) barriers is crucial during development, leukocyte trafficking, and for the spread of cancer. Despite its importance in normal and diseased states, the mechanisms that direct invasion are poorly understood, in large part because of the inability to visualize dynamic cell-basement membrane interactions in vivo. This protocol describes multi-channel time-lapse confocal imaging of anchor cell invasion in live C. elegans. Methods presented include outline slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min), and quantitative analysis (variable timing). Images acquired enable direct measurement of invasive dynamics including invadopodia formation, cell membrane protrusions, and BM removal. This protocol can be combined with genetic analysis, molecular activity probes, and optogenetic approaches to uncover molecular mechanisms underlying cell invasion. These methods can also be readily adapted for real-time analysis of cell migration, basement membrane turnover, and cell membrane dynamics by any worm laboratory. PMID:28880279

Fixed-Cell Imaging of Schizosaccharomyces pombe.

PubMed

Hagan, Iain M; Bagley, Steven

2016-07-01

The acknowledged genetic malleability of fission yeast has been matched by impressive cytology to drive major advances in our understanding of basic molecular cell biological processes. In many of the more recent studies, traditional approaches of fixation followed by processing to accommodate classical staining procedures have been superseded by live-cell imaging approaches that monitor the distribution of fusion proteins between a molecule of interest and a fluorescent protein. Although such live-cell imaging is uniquely informative for many questions, fixed-cell imaging remains the better option for others and is an important-sometimes critical-complement to the analysis of fluorescent fusion proteins by live-cell imaging. Here, we discuss the merits of fixed- and live-cell imaging as well as specific issues for fluorescence microscopy imaging of fission yeast. © 2016 Cold Spring Harbor Laboratory Press.

Live-Cell Imaging of Early Steps of Single HIV-1 Infection.

PubMed

Francis, Ashwanth C; Melikyan, Gregory B

2018-05-19

Live-cell imaging of single HIV-1 entry offers a unique opportunity to delineate the spatio-temporal regulation of infection. Novel virus labeling and imaging approaches enable the visualization of key steps of HIV-1 entry leading to nuclear import, integration into the host genome, and viral protein expression. Here, we discuss single virus imaging strategies, focusing on live-cell imaging of single virus fusion and productive uncoating that culminates in HIV-1 infection.

Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

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

Gambhir, Sanjiv; Pritha, Ray

Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

DOEpatents

Gambhir, Sanjiv; Pritha, Ray

2015-07-14

Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

Convolution Comparison Pattern: An Efficient Local Image Descriptor for Fingerprint Liveness Detection

PubMed Central

Gottschlich, Carsten

2016-01-01

We present a new type of local image descriptor which yields binary patterns from small image patches. For the application to fingerprint liveness detection, we achieve rotation invariant image patches by taking the fingerprint segmentation and orientation field into account. We compute the discrete cosine transform (DCT) for these rotation invariant patches and attain binary patterns by comparing pairs of two DCT coefficients. These patterns are summarized into one or more histograms per image. Each histogram comprises the relative frequencies of pattern occurrences. Multiple histograms are concatenated and the resulting feature vector is used for image classification. We name this novel type of descriptor convolution comparison pattern (CCP). Experimental results show the usefulness of the proposed CCP descriptor for fingerprint liveness detection. CCP outperforms other local image descriptors such as LBP, LPQ and WLD on the LivDet 2013 benchmark. The CCP descriptor is a general type of local image descriptor which we expect to prove useful in areas beyond fingerprint liveness detection such as biological and medical image processing, texture recognition, face recognition and iris recognition, liveness detection for face and iris images, and machine vision for surface inspection and material classification. PMID:26844544

Dynamic phase differences based on quantitative phase imaging for the objective evaluation of cell behavior.

PubMed

Krizova, Aneta; Collakova, Jana; Dostal, Zbynek; Kvasnica, Lukas; Uhlirova, Hana; Zikmund, Tomas; Vesely, Pavel; Chmelik, Radim

2015-01-01

Quantitative phase imaging (QPI) brought innovation to noninvasive observation of live cell dynamics seen as cell behavior. Unlike the Zernike phase contrast or differential interference contrast, QPI provides quantitative information about cell dry mass distribution. We used such data for objective evaluation of live cell behavioral dynamics by the advanced method of dynamic phase differences (DPDs). The DPDs method is considered a rational instrument offered by QPI. By subtracting the antecedent from the subsequent image in a time-lapse series, only the changes in mass distribution in the cell are detected. The result is either visualized as a two dimensional color-coded projection of these two states of the cell or as a time dependence of changes quantified in picograms. Then in a series of time-lapse recordings, the chain of cell mass distribution changes that would otherwise escape attention is revealed. Consequently, new salient features of live cell behavior should emerge. Construction of the DPDs method and results exhibiting the approach are presented. Advantage of the DPDs application is demonstrated on cells exposed to an osmotic challenge. For time-lapse acquisition of quantitative phase images, the recently developed coherence-controlled holographic microscope was employed.

Dynamic phase differences based on quantitative phase imaging for the objective evaluation of cell behavior

NASA Astrophysics Data System (ADS)

Krizova, Aneta; Collakova, Jana; Dostal, Zbynek; Kvasnica, Lukas; Uhlirova, Hana; Zikmund, Tomas; Vesely, Pavel; Chmelik, Radim

2015-11-01

Quantitative phase imaging (QPI) brought innovation to noninvasive observation of live cell dynamics seen as cell behavior. Unlike the Zernike phase contrast or differential interference contrast, QPI provides quantitative information about cell dry mass distribution. We used such data for objective evaluation of live cell behavioral dynamics by the advanced method of dynamic phase differences (DPDs). The DPDs method is considered a rational instrument offered by QPI. By subtracting the antecedent from the subsequent image in a time-lapse series, only the changes in mass distribution in the cell are detected. The result is either visualized as a two-dimensional color-coded projection of these two states of the cell or as a time dependence of changes quantified in picograms. Then in a series of time-lapse recordings, the chain of cell mass distribution changes that would otherwise escape attention is revealed. Consequently, new salient features of live cell behavior should emerge. Construction of the DPDs method and results exhibiting the approach are presented. Advantage of the DPDs application is demonstrated on cells exposed to an osmotic challenge. For time-lapse acquisition of quantitative phase images, the recently developed coherence-controlled holographic microscope was employed.

Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors.

PubMed

Danhier, Pierre; Krishnamachary, Balaji; Bharti, Santosh; Kakkad, Samata; Mironchik, Yelena; Bhujwalla, Zaver M

2015-12-01

Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF) activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc) and a variant of the enhanced green fluorescent protein (EGFP). Both ODD-luciferase and EGFP were under the promotion of a poly-hypoxia-response element sequence (5xHRE). The cells constitutively expressed tdTomato red fluorescent protein. For validating the imaging strategy, cells were incubated under hypoxia (1% O2) for 48 hours and then reoxygenated. The luciferase activity of PC3-HRE-EGFP/HRE-ODD-luc/tdtomato cells detected by bioluminescent imaging rapidly decreased after reoxygenation, whereas EGFP levels in these cells remained stable for several hours. After in vitro validation, PC3-HRE-EGFP/HRE-ODD-luc/tdtomato tumors were implanted subcutaneously and orthotopically in nude male mice and imaged in vivo and ex vivo using optical imaging in proof-of-principle studies to demonstrate differences in optical patterns between EGFP expression and bioluminescence. This novel "timer" imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP can provide a time frame of HRE activation in PC3 prostate cancer cells and will be useful to understand the temporal changes in hypoxia and HIF activity during cancer progression and following treatments including HIF targeting strategies. Copyright © 2015 Nencki Institute of Experimental Biology, Polish Academy of Sciences,. Published by Elsevier Inc. All rights reserved.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments.

PubMed

Giarmarco, Michelle M; Cleghorn, Whitney M; Hurley, James B; Brockerhoff, Susan E

2018-05-09

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method's versatility. This protocol was developed for imaging Ca 2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca 2+ or metabolites in Müller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca 2+ , and energy homeostasis.

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.

Development of Automatic Live Linux Rebuilding System with Flexibility in Science and Engineering Education and Applying to Information Processing Education

NASA Astrophysics Data System (ADS)

Sonoda, Jun; Yamaki, Kota

We develop an automatic Live Linux rebuilding system for science and engineering education, such as information processing education, numerical analysis and so on. Our system is enable to easily and automatically rebuild a customized Live Linux from a ISO image of Ubuntu, which is one of the Linux distribution. Also, it is easily possible to install/uninstall packages and to enable/disable init daemons. When we rebuild a Live Linux CD using our system, we show number of the operations is 8, and the rebuilding time is about 33 minutes on CD version and about 50 minutes on DVD version. Moreover, we have applied the rebuilded Live Linux CD in a class of information processing education in our college. As the results of a questionnaires survey from our 43 students who used the Live Linux CD, we obtain that the our Live Linux is useful for about 80 percents of students. From these results, we conclude that our system is able to easily and automatically rebuild a useful Live Linux in short time.

EXPLORER: Changing the molecular imaging paradigm with total-body PET/CT (Conference Presentation)

NASA Astrophysics Data System (ADS)

Cherry, Simon R.; Badawi, Ramsey D.; Jones, Terry

2016-04-01

Positron emission tomography (PET) is the highest sensitivity technique for human whole-body imaging studies. However, current clinical PET scanners do not make full use of the available signal, as they only permit imaging of a 15-25 cm segment of the body at one time. Given the limited sensitive region, whole-body imaging with clinical PET scanners requires relatively long scan times and subjects the patient to higher than necessary radiation doses. The EXPLORER initiative aims to build a 2-meter axial length PET scanner to allow imaging the entire subject at once, capturing nearly the entire available PET signal. EXPLORER will acquire data with ~40-fold greater sensitivity leading to a six-fold increase in reconstructed signal-to-noise ratio for imaging the total body. Alternatively, total-body images with the EXPLORER scanner will be able to be acquired in ~30 seconds or with ~0.15 mSv injected dose, while maintaining current PET image quality. The superior sensitivity will open many new avenues for biomedical research. Specifically for cancer applications, high sensitivity PET will enable detection of smaller lesions. Additionally, greater sensitivity will allow imaging out to 10 half-lives of positron emitting radiotracers. This will enable 1) metabolic ultra-staging with FDG by extending the uptake and clearance time to 3-5 hours to significantly improve contrast and 2) improved kinetic imaging with short-lived radioisotopes such as C-11, crucial for drug development studies. Frequent imaging studies of the same subject to study disease progression or to track response to therapy will be possible with the low dose capabilities of the EXPLORER scanner. The low dose capabilities will also open up new imaging possibilities in pediatrics and adolescents to better study developmental disorders. This talk will review the basis for developing total-body PET, potential applications, and review progress to date in developing EXPLORER, the first total-body PET scanner.

Live-Cell MicroRNA Imaging through MnO2 Nanosheet-Mediated DD-A Hybridization Chain Reaction.

PubMed

Ou, Min; Huang, Jin; Yang, Xiaohai; He, Xiaoxiao; Quan, Ke; Yang, Yanjing; Xie, Nuli; Li, Jing; Wang, Kemin

2018-01-18

Innovative techniques to visualize native microRNAs (miRNAs) in live cells can dramatically impact current research on the roles of miRNA in biology and medicine. Here, we report a novel approach for live-cell miRNA imaging using a biodegradable MnO 2 nanosheet-mediated DD-A FRET hybridization chain reaction (HCR). The MnO 2 nanosheets can adsorb DNA hairpin probes and deliver them into live cells. After entering cells, the MnO 2 nanosheets are degraded by cellular GSH. Then, the target miR-21 triggers cascaded assembly of the liberated hairpin probes into long dsDNA polymers, which brings each two FAMs (donor) and one TAMRA (acceptor) into close proximity to generate significantly enhanced DD-A FRET signals, which was discovered and proven by our previous report. We think the developed approach can serve as an excellent intracellular miRNAs detection tool, which promises the potential for biological and disease studies. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamic measurements of flowing cells labeled by gold nanoparticles using full-field photothermal interferometric imaging

NASA Astrophysics Data System (ADS)

Turko, Nir A.; Roitshtain, Darina; Blum, Omry; Kemper, Björn; Shaked, Natan T.

2017-06-01

We present highly dynamic photothermal interferometric phase microscopy for quantitative, selective contrast imaging of live cells during flow. Gold nanoparticles can be biofunctionalized to bind to specific cells, and stimulated for local temperature increase due to plasmon resonance, causing a rapid change of the optical phase. These phase changes can be recorded by interferometric phase microscopy and analyzed to form an image of the binding sites of the nanoparticles in the cells, gaining molecular specificity. Since the nanoparticle excitation frequency might overlap with the sample dynamics frequencies, photothermal phase imaging was performed on stationary or slowly dynamic samples. Furthermore, the computational analysis of the photothermal signals is time consuming. This makes photothermal imaging unsuitable for applications requiring dynamic imaging or real-time analysis, such as analyzing and sorting cells during fast flow. To overcome these drawbacks, we utilized an external interferometric module and developed new algorithms, based on discrete Fourier transform variants, enabling fast analysis of photothermal signals in highly dynamic live cells. Due to the self-interference module, the cells are imaged with and without excitation in video-rate, effectively increasing signal-to-noise ratio. Our approach holds potential for using photothermal cell imaging and depletion in flow cytometry.

A novel peptide-based fluorescence chemosensor for selective imaging of hydrogen sulfide both in living cells and zebrafish.

PubMed

Wang, Peng; Wu, Jiang; Di, Cuixia; Zhou, Rong; Zhang, Hong; Su, Pingru; Xu, Cong; Zhou, Panpan; Ge, Yushu; Liu, Dan; Liu, Weisheng; Tang, Yu

2017-06-15

Hydrogen sulfide (H 2 S) plays an important role as a signaling compound (gasotransmitter) in living systems. However, the development of an efficient imaging chemosensor of H 2 S in live animals is a challenging field for chemists. Herein, a novel peptide-based fluorescence chemosensor L-Cu was designed and synthesized on the basis of the copper chelating with the peptide ligand (FITC-Ahx-Ser-Pro-Gly-His-NH 2 , L), and its H 2 S sensing ability has been evaluated both in living cells and zebrafish. The peptide backbone and Cu 2+ -removal sensing mechanism are used to deliver rapid response time, high sensitivity, and good biocompatibility. After a fast fluorescence quench by Cu 2+ coordinated with L, the fluorescence of L is recovered by adding S 2- to form insoluble copper sulfide in aqueous solution with a detection limit for hydrogen sulfide measured to be 31nM. Furthermore, the fluorescence chemosensor L-Cu showed excellent cell permeation and low biotoxicity to realize the intracellular biosensing, L-Cu has also been applied to image hydrogen sulfide in live zebrafish larvae. We expect that this peptide-based fluorescence chemosensor L-Cu can be used to study H 2 S-related chemical biology in physiological and pathological events. Copyright © 2016 Elsevier B.V. All rights reserved.

iSBatch: a batch-processing platform for data analysis and exploration of live-cell single-molecule microscopy images and other hierarchical datasets.

PubMed

Caldas, Victor E A; Punter, Christiaan M; Ghodke, Harshad; Robinson, Andrew; van Oijen, Antoine M

2015-10-01

Recent technical advances have made it possible to visualize single molecules inside live cells. Microscopes with single-molecule sensitivity enable the imaging of low-abundance proteins, allowing for a quantitative characterization of molecular properties. Such data sets contain information on a wide spectrum of important molecular properties, with different aspects highlighted in different imaging strategies. The time-lapsed acquisition of images provides information on protein dynamics over long time scales, giving insight into expression dynamics and localization properties. Rapid burst imaging reveals properties of individual molecules in real-time, informing on their diffusion characteristics, binding dynamics and stoichiometries within complexes. This richness of information, however, adds significant complexity to analysis protocols. In general, large datasets of images must be collected and processed in order to produce statistically robust results and identify rare events. More importantly, as live-cell single-molecule measurements remain on the cutting edge of imaging, few protocols for analysis have been established and thus analysis strategies often need to be explored for each individual scenario. Existing analysis packages are geared towards either single-cell imaging data or in vitro single-molecule data and typically operate with highly specific algorithms developed for particular situations. Our tool, iSBatch, instead allows users to exploit the inherent flexibility of the popular open-source package ImageJ, providing a hierarchical framework in which existing plugins or custom macros may be executed over entire datasets or portions thereof. This strategy affords users freedom to explore new analysis protocols within large imaging datasets, while maintaining hierarchical relationships between experiments, samples, fields of view, cells, and individual molecules.

Spheroid imaging of phase-diversity homodyne OCT

NASA Astrophysics Data System (ADS)

Senda, Naoko; Osawa, Kentaro

2017-02-01

Non-invasive 3D imaging technique is essential for regenerative tissues evaluation. Optical coherence tomography (OCT) is one of 3D imaging tools with no staining and is used extensively for fundus examination. We have developed Phase-Diversity Homodyne OCT which enables cell imaging because of high resolution, whereas conventional OCT was not used for cell imaging because of low resolution. We demonstrated non-invasive imaging inside living spheroids with Phase-Diversity Homodyne OCT. Spheroids are spheroidal cell aggregates and used as regenerative tissues. Cartilage cells were cultured in low-adhesion 96-well plates and spheroids were manufactured. Cell membrane and cytoplasm of spheroid were imaged with OCT.

Imaging windows for long-term intravital imaging

PubMed Central

Alieva, Maria; Ritsma, Laila; Giedt, Randy J; Weissleder, Ralph; van Rheenen, Jacco

2014-01-01

Intravital microscopy is increasingly used to visualize and quantitate dynamic biological processes at the (sub)cellular level in live animals. By visualizing tissues through imaging windows, individual cells (e.g., cancer, host, or stem cells) can be tracked and studied over a time-span of days to months. Several imaging windows have been developed to access tissues including the brain, superficial fascia, mammary glands, liver, kidney, pancreas, and small intestine among others. Here, we review the development of imaging windows and compare the most commonly used long-term imaging windows for cancer biology: the cranial imaging window, the dorsal skin fold chamber, the mammary imaging window, and the abdominal imaging window. Moreover, we provide technical details, considerations, and trouble-shooting tips on the surgical procedures and microscopy setups for each imaging window and explain different strategies to assure imaging of the same area over multiple imaging sessions. This review aims to be a useful resource for establishing the long-term intravital imaging procedure. PMID:28243510

Imaging windows for long-term intravital imaging: General overview and technical insights.

PubMed

Alieva, Maria; Ritsma, Laila; Giedt, Randy J; Weissleder, Ralph; van Rheenen, Jacco

2014-01-01

Intravital microscopy is increasingly used to visualize and quantitate dynamic biological processes at the (sub)cellular level in live animals. By visualizing tissues through imaging windows, individual cells (e.g., cancer, host, or stem cells) can be tracked and studied over a time-span of days to months. Several imaging windows have been developed to access tissues including the brain, superficial fascia, mammary glands, liver, kidney, pancreas, and small intestine among others. Here, we review the development of imaging windows and compare the most commonly used long-term imaging windows for cancer biology: the cranial imaging window, the dorsal skin fold chamber, the mammary imaging window, and the abdominal imaging window. Moreover, we provide technical details, considerations, and trouble-shooting tips on the surgical procedures and microscopy setups for each imaging window and explain different strategies to assure imaging of the same area over multiple imaging sessions. This review aims to be a useful resource for establishing the long-term intravital imaging procedure.

Live-cell imaging of G-actin dynamics using sequential FDAP

PubMed Central

Kiuchi, Tai; Nagai, Tomoaki; Ohashi, Kazumasa; Watanabe, Naoki; Mizuno, Kensaku

2011-01-01

Various microscopic techniques have been developed to understand the mechanisms that spatiotemporally control actin filament dynamics in live cells. Kinetic data on the processes of actin assembly and disassembly on F-actin have been accumulated. However, the kinetics of cytoplasmic G-actin, a key determinant for actin polymerization, has remained unclear because of a lack of appropriate methods to measure the G-actin concentration quantitatively. We have developed two new microscopic techniques based on the fluorescence decay after photoactivation (FDAP) time-lapse imaging of photoswitchable Dronpa-labeled actin. These techniques, sequential FDAP (s-FDAP) and multipoint FDAP, were used to measure the time-dependent changes in and spatial distribution of the G-actin concentration in live cells. Use of s-FDAP provided data on changes in the G-actin concentration with high temporal resolution; these data were useful for the model analysis of actin assembly processes in live cells. The s-FDAP analysis also provided evidence that the cytoplasmic G-actin concentration substantially decreases after cell stimulation and that the extent of stimulus-induced actin assembly and cell size extension are linearly correlated with the G-actin concentration before cell stimulation. The advantages of using s-FDAP and multipoint FDAP to measure spatiotemporal G-actin dynamics and the roles of G-actin concentration and ADF/cofilin in stimulus-induced actin assembly and lamellipodium extension in live cells are discussed. PMID:22754616

Design of a dynamic biofilm imaging cell for white-light interferometric microscopy

NASA Astrophysics Data System (ADS)

Larimer, Curtis; Brann, Michelle; Suter, Jonathan D.; Addleman, R. Shane

2017-11-01

In microbiology research, there is a strong need for next-generation imaging and sensing instrumentation that will enable minimally invasive and label-free investigation of soft, hydrated structures, such as in bacterial biofilms. White-light interferometry (WLI) can provide high-resolution images of surface topology without the use of fluorescent labels but is not typically used to image biofilms because there is insufficient refractive index contrast to induce reflection from the biofilm's interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ, especially in a nondestructive manner. We build on our prior description of static biofilm imaging and describe the design of a dynamic growth flow cell that enables monitoring of the thickness and topology of live biofilms over time using a WLI microscope. The microfluidic system is designed to grow biofilms in dynamic conditions and to create a reflective interface on the surface while minimizing disruption of fragile structures. The imaging cell was also designed to accommodate limitations imposed by the depth of focus of the microscope's objective lens. Example images of live biofilm samples are shown to illustrate the ability of the flow cell and WLI instrument to (1) support bacterial growth and biofilm development, (2) image biofilm structure that reflects growth in flow conditions, and (3) monitor biofilm development over time nondestructively. In future work, the apparatus described here will enable surface metrology measurements (roughness, surface area, etc.) of biofilms and may be used to observe changes in biofilm structure in response to changes in environmental conditions (e.g., flow velocity, availability of nutrients, and presence of biocides). This development will open opportunities for the use of WLI in bioimaging.

Stimulated penetrating keratoplasty using real-time virtual intraoperative surgical optical coherence tomography

PubMed Central

Lee, Changho; Kim, Kyungun; Han, Seunghoon; Kim, Sehui; Lee, Jun Hoon; Kim, Hong kyun; Kim, Chulhong; Jung, Woonggyu; Kim, Jeehyun

2014-01-01

Abstract. An intraoperative surgical microscope is an essential tool in a neuro- or ophthalmological surgical environment. Yet, it has an inherent limitation to classify subsurface information because it only provides the surface images. To compensate for and assist in this problem, combining the surgical microscope with optical coherence tomography (OCT) has been adapted. We developed a real-time virtual intraoperative surgical OCT (VISOCT) system by adapting a spectral-domain OCT scanner with a commercial surgical microscope. Thanks to our custom-made beam splitting and image display subsystems, the OCT images and microscopic images are simultaneously visualized through an ocular lens or the eyepiece of the microscope. This improvement helps surgeons to focus on the operation without distraction to view OCT images on another separate display. Moreover, displaying the OCT live images on the eyepiece helps surgeon’s depth perception during the surgeries. Finally, we successfully processed stimulated penetrating keratoplasty in live rabbits. We believe that these technical achievements are crucial to enhance the usability of the VISOCT system in a real surgical operating condition. PMID:24604471

Imaging and quantifying ganglion cells and other transparent neurons in the living human retina.

PubMed

Liu, Zhuolin; Kurokawa, Kazuhiro; Zhang, Furu; Lee, John J; Miller, Donald T

2017-11-28

Ganglion cells (GCs) are fundamental to retinal neural circuitry, processing photoreceptor signals for transmission to the brain via their axons. However, much remains unknown about their role in vision and their vulnerability to disease leading to blindness. A major bottleneck has been our inability to observe GCs and their degeneration in the living human eye. Despite two decades of development of optical technologies to image cells in the living human retina, GCs remain elusive due to their high optical translucency. Failure of conventional imaging-using predominately singly scattered light-to reveal GCs has led to a focus on multiply-scattered, fluorescence, two-photon, and phase imaging techniques to enhance GC contrast. Here, we show that singly scattered light actually carries substantial information that reveals GC somas, axons, and other retinal neurons and permits their quantitative analysis. We perform morphometry on GC layer somas, including projection of GCs onto photoreceptors and identification of the primary GC subtypes, even beneath nerve fibers. We obtained singly scattered images by: ( i ) marrying adaptive optics to optical coherence tomography to avoid optical blurring of the eye; ( ii ) performing 3D subcellular image registration to avoid motion blur; and ( iii ) using organelle motility inside somas as an intrinsic contrast agent. Moreover, through-focus imaging offers the potential to spatially map individual GCs to underlying amacrine, bipolar, horizontal, photoreceptor, and retinal pigment epithelium cells, thus exposing the anatomical substrate for neural processing of visual information. This imaging modality is also a tool for improving clinical diagnosis and assessing treatment of retinal disease. Copyright © 2017 the Author(s). Published by PNAS.

Non-interferometric quantitative phase imaging of yeast cells

NASA Astrophysics Data System (ADS)

Poola, Praveen K.; Pandiyan, Vimal Prabhu; John, Renu

2015-12-01

Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.

Two-dimensional and three-dimensional dynamic imaging of live biofilms in a microchannel by time-of-flight secondary ion mass spectrometry

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

Hua, Xin; Marshall, Matthew J.; Xiong, Yijia

2015-05-01

A vacuum compatible microfluidic reactor, SALVI (System for Analysis at the Liquid Vacuum Interface) was employed for in situ chemical imaging of live biofilms using time-of-flight secondary ion mass spectrometry (ToF-SIMS). Depth profiling by sputtering materials in sequential layers resulted in live biofilm spatial chemical mapping. 2D images were reconstructed to report the first 3D images of hydrated biofilm elucidating spatial and chemical heterogeneity. 2D image principal component analysis (PCA) was conducted among biofilms at different locations in the microchannel. Our approach directly visualized spatial and chemical heterogeneity within the living biofilm by dynamic liquid ToF-SIMS.

Fabrication and application of heterogeneous printed mouse phantoms for whole animal optical imaging

PubMed Central

Bentz, Brian Z.; Chavan, Anmol V.; Lin, Dergan; Tsai, Esther H. R.; Webb, Kevin J.

2017-01-01

This work demonstrates the usefulness of 3D printing for optical imaging applications. Progress in developing optical imaging for biomedical applications requires customizable and often complex objects for testing and evaluation. There is therefore high demand for what have become known as tissue-simulating “phantoms.” We present a new optical phantom fabricated using inexpensive 3D printing methods with multiple materials, allowing for the placement of complex inhomogeneities in complex or anatomically realistic geometries, as opposed to previous phantoms, which were limited to simple shapes formed by molds or machining. We use diffuse optical imaging to reconstruct optical parameters in 3D space within a printed mouse to show the applicability of the phantoms for developing whole animal optical imaging methods. This phantom fabrication approach is versatile, can be applied to optical imaging methods besides diffusive imaging, and can be used in the calibration of live animal imaging data. PMID:26835763

Live imaging using adaptive optics with fluorescent protein guide-stars

PubMed Central

Tao, Xiaodong; Crest, Justin; Kotadia, Shaila; Azucena, Oscar; Chen, Diana C.; Sullivan, William; Kubby, Joel

2012-01-01

Spatially and temporally dependent optical aberrations induced by the inhomogeneous refractive index of live samples limit the resolution of live dynamic imaging. We introduce an adaptive optical microscope with a direct wavefront sensing method using a Shack-Hartmann wavefront sensor and fluorescent protein guide-stars for live imaging. The results of imaging Drosophila embryos demonstrate its ability to correct aberrations and achieve near diffraction limited images of medial sections of large Drosophila embryos. GFP-polo labeled centrosomes can be observed clearly after correction but cannot be observed before correction. Four dimensional time lapse images are achieved with the correction of dynamic aberrations. These studies also demonstrate that the GFP-tagged centrosome proteins, Polo and Cnn, serve as excellent biological guide-stars for adaptive optics based microscopy. PMID:22772285

Live-cell imaging combined with immunofluorescence, RNA, or DNA FISH to study the nuclear dynamics and expression of the X-inactivation center.

PubMed

Pollex, Tim; Piolot, Tristan; Heard, Edith

2013-01-01

Differentiation of embryonic stem cells is accompanied by changes of gene expression and chromatin and chromosome dynamics. One of the most impressive examples for these changes is inactivation of one of the two X chromosomes occurring upon differentiation of mouse female embryonic stem cells. With a few exceptions, these events have been mainly studied in fixed cells. In order to better understand the dynamics, kinetics, and order of events during differentiation, one needs to employ live-cell imaging techniques. Here, we describe a combination of live-cell imaging with techniques that can be used in fixed cells (e.g., RNA FISH) to correlate locus dynamics or subnuclear localization with, e.g., gene expression. To study locus dynamics in female ES cells, we generated cell lines containing TetO arrays in the X-inactivation center, the locus on the X chromosome regulating X-inactivation, which can be visualized upon expression of TetR fused to fluorescent proteins. We will use this system to elaborate on how to generate ES cell lines for live-cell imaging of locus dynamics, how to culture ES cells prior to live-cell imaging, and to describe typical live-cell imaging conditions for ES cells using different microscopes. Furthermore, we will explain how RNA, DNA FISH, or immunofluorescence can be applied following live-cell imaging to correlate gene expression with locus dynamics.

Discovery of a New Cellular Motion and Its Relevance to Breast Cancer and Involution

DTIC Science & Technology

2014-02-01

motion (CAMo), live cell imaging , confocal microscopy Overall Project Summary: During this first year of funding we have concentrated our work to...cell types in 3D cultures and in vivo. Subtask 1.1a: Real time live cell imaging using confocal microscopy will be used to image cellular movement...exciting as they are important steps in understanding behavior of normal myoepithelial cells using live cell imaging in physiologically

The development of biomedical engineering as experienced by one biomedical engineer

PubMed Central

2012-01-01

This personal essay described the development of the field of Biomedical Engineering from its early days, from the perspective of one who lived through that development. It describes the making of a major invention using data that had been rejected by other scientists, the re-discovery of an obscure fact of physiology and its use in developing a major medical instrument, the development of a new medical imaging modality, and the near-death rescue of a research project. The essay concludes with comments about the development and present status of impedance imaging, and recent changes in the evolution of biomedical engineering as a field. PMID:23234267

The development of biomedical engineering as experienced by one biomedical engineer.

PubMed

Newell, Jonathan C

2012-12-12

This personal essay described the development of the field of Biomedical Engineering from its early days, from the perspective of one who lived through that development. It describes the making of a major invention using data that had been rejected by other scientists, the re-discovery of an obscure fact of physiology and its use in developing a major medical instrument, the development of a new medical imaging modality, and the near-death rescue of a research project. The essay concludes with comments about the development and present status of impedance imaging, and recent changes in the evolution of biomedical engineering as a field.

Magnetically engineered smart thin films: toward lab-on-chip ultra-sensitive molecular imaging.

PubMed

Hassan, Muhammad A; Saqib, Mudassara; Shaikh, Haseeb; Ahmad, Nasir M; Elaissari, Abdelhamid

2013-03-01

Magnetically responsive engineered smart thin films of nanoferrites as contrast agent are employed to develop surface based magnetic resonance imaging to acquire simple yet fast molecular imaging. The work presented here can be of significant potential for future lab-on-chip point-of-care diagnostics from the whole blood pool on almost any substrates to reduce or even prevent clinical studies involve a living organism to enhance the non-invasive imaging to advance the '3Rs' of work in animals-replacement, refinement and reduction.

Scanning Ion Conductance Microscopy of Live Keratinocytes

NASA Astrophysics Data System (ADS)

Hegde, V.; Mason, A.; Saliev, T.; Smith, F. J. D.; McLean, W. H. I.; Campbell, P. A.

2012-07-01

Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (

A novel approach to 32-channel peripheral nervous system myelin imaging in vivo, with single axon resolution.

PubMed

Grochmal, Joey; Teo, Wulin; Gambhir, Hardeep; Kumar, Ranjan; Stratton, Jo Anne; Dhaliwal, Raveena; Brideau, Craig; Biernaskie, Jeff; Stys, Peter K; Midha, Rajiv

2018-01-19

OBJECTIVE Intravital spectral imaging of the large, deeply situated nerves in the rat peripheral nervous system (PNS) has not been well described. Here, the authors have developed a highly stable platform for performing imaging of the tibial nerve in live rodents, thus allowing the capture of high-resolution, high-magnification spectral images requiring long acquisition times. By further exploiting the qualities of the topically applied myelin dye Nile red, this technique is capable of visualizing the detailed microenvironment of peripheral nerve demyelination injury and recovery, while allowing us to obtain images of exogenous Schwann cell myelination in a living animal. METHODS The authors caused doxorubicin-induced focal demyelination in the tibial nerves of 25 Thy-1 GFP rats, of which 2 subsets (n = 10 each) received either BFP-labeled SKP-SCs or SCs to the zone of injury. Prior to acquiring images of myelin recovery in these nerves, a tibial nerve window was constructed using a silicone hemitube, a fast drying silicone polymer, and a small coverslip. This construct was then affixed to a 3D-printed nerve stage, which in turn was affixed to an external fixation/microscope stage device. Myelin visualization was facilitated by the topical application of Nile red. RESULTS The authors reliably demonstrated intravital peripheral nerve myelin imaging with micron-level resolution and magnification, and minimal movement artifact. The detailed microenvironment of nerve remyelination can be vividly observed, while exogenously applied Schwann cells and skin-derived precursor Schwann cells can be seen myelinating axons. CONCLUSIONS Topically applied Nile red enables intravital study of myelin in the living rat PNS. Furthermore, the use of a tibial nerve window facilitates stable intravital peripheral nerve imaging, making possible high-definition spectral imaging with long acquisition times.

Breast imaging technology: Recent advances in imaging endogenous or transferred gene expression utilizing radionuclide technologies in living subjects - applications to breast cancer

PubMed Central

Berger, Frank; Sam Gambhir, Sanjiv

2001-01-01

A variety of imaging technologies is being investigated as tools for studying gene expression in living subjects. Two technologies that use radiolabeled isotopes are single photon emission computed tomography (SPECT) and positron emission tomography (PET). A relatively high sensitivity, a full quantitative tomographic capability, and the ability to extend small animal imaging assays directly into human applications characterize radionuclide approaches. Various radiolabeled probes (tracers) can be synthesized to target specific molecules present in breast cancer cells. These include antibodies or ligands to target cell surface receptors, substrates for intracellular enzymes, antisense oligodeoxynucleotide probes for targeting mRNA, probes for targeting intracellular receptors, and probes for genes transferred into the cell. We briefly discuss each of these imaging approaches and focus in detail on imaging reporter genes. In a PET reporter gene system for in vivo reporter gene imaging, the protein products of the reporter genes sequester positron emitting reporter probes. PET subsequently measures the PET reporter gene dependent sequestration of the PET reporter probe in living animals. We describe and review reporter gene approaches using the herpes simplex type 1 virus thymidine kinase and the dopamine type 2 receptor genes. Application of the reporter gene approach to animal models for breast cancer is discussed. Prospects for future applications of the transgene imaging technology in human gene therapy are also discussed. Both SPECT and PET provide unique opportunities to study animal models of breast cancer with direct application to human imaging. Continued development of new technology, probes and assays should help in the better understanding of basic breast cancer biology and in the improved management of breast cancer patients. PMID:11250742

Live Cell Imaging and Measurements of Molecular Dynamics

PubMed Central

Frigault, M.; Lacoste, J.; Swift, J.; Brown, C.

2010-01-01

w3-2 Live cell microscopy is becoming widespread across all fields of the life sciences, as well as, many areas of the physical sciences. In order to accurately obtain live cell microscopy data, the live specimens must be properly maintained on the imaging platform. In addition, the fluorescence light path must be optimized for efficient light transmission in order to reduce the intensity of excitation light impacting the living sample. With low incident light intensities the processes under study should not be altered due to phototoxic effects from the light allowing for the long term visualization of viable living samples. Aspects for maintaining a suitable environment for the living sample, minimizing incident light and maximizing detection efficiency will be presented for various fluorescence based live cell instruments. Raster Image Correlation Spectroscopy (RICS) is a technique that uses the intensity fluctuations within laser scanning confocal images, as well as the well characterized scanning dynamics of the laser beam, to extract the dynamics, concentrations and clustering of fluorescent molecules within the cell. In addition, two color cross-correlation RICS can be used to determine protein-protein interactions in living cells without the many technical difficulties encountered in FRET based measurements. RICS is an ideal live cell technique for measuring cellular dynamics because the potentially damaging high intensity laser bursts required for photobleaching recovery measurements are not required, rather low laser powers, suitable for imaging, can be used. The RICS theory will be presented along with examples of live cell applications.

Using video playbacks to study visual communication in a marine fish, Salaria pavo.

PubMed

Gonçalves; Oliveira; Körner; Poschadel; Schlupp

2000-09-01

Video playbacks have been successfully applied to the study of visual communication in several groups of animals. However, this technique is controversial as video monitors are designed with the human visual system in mind. Differences between the visual capabilities of humans and other animals will lead to perceptually different interpretations of video images. We simultaneously presented males and females of the peacock blenny, Salaria pavo, with a live conspecific male and an online video image of the same individual. Video images failed to elicit appropriate responses. Males were aggressive towards the live male but not towards video images of the same male. Similarly, females courted only the live male and spent more time near this stimulus. In contrast, females of the gynogenetic poecilid Poecilia formosa showed an equal preference for a live and video image of a P. mexicana male, suggesting a response to live animals as strong as to video images. We discuss differences between the species that may explain their opposite reaction to video images. Copyright 2000 The Association for the Study of Animal Behaviour.

New integrative modules for multicolor-protein labeling and live-cell imaging in Saccharomyces cerevisiae.

PubMed

Malcova, Ivana; Farkasovsky, Marian; Senohrabkova, Lenka; Vasicova, Pavla; Hasek, Jiri

2016-05-01

Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chloro-Functionalized Photo-crosslinking BODIPY for Glutathione Sensing and Subcellular Trafficking.

PubMed

Murale, Dhiraj P; Hong, Seong Cheol; Haque, Md Mamunul; Lee, Jun-Seok

2018-05-18

Glutathione (GSH) is one of major antioxidants inside cells that regulates oxidoreduction homeostasis. Recently, there have been extensive efforts to visualize GSH in live cells, but most of the probes available today are simple detection sensors and do not provide details of cellular localization. A new fluorescent probe (pcBD2-Cl), which is cell permeable and selectively reacts with GSH in situ, has been developed. The in situ GSH-labeled probe (pcBD2-GSH) exhibited quenches fluorescence, but subsequent binding to cellular abundant glutathione S-transferase (GST) recovers the fluorescence intensity, which makes it possible to image the GSH-GST complex in live cells. Interactions between probe and GST were confirmed by means of photo-crosslinking under intact live-cell conditions. Interestingly, isomers of chloro-functionalized 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) compounds behaved very distinctively inside the cells. Following co-staining imaging with MitoTracker and mitochondria fractionation upon lipopolysaccharide-mediated reactive oxygen species induction experiments showed that pcBD2-GSH accumulated in mitochondria. This is the first example of a live-cell imaging probe to visualize translocation of GSH from the cytosol to mitochondria. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

PubMed

Andrews, Natalie; Ramel, Marie-Christine; Kumar, Sunil; Alexandrov, Yuriy; Kelly, Douglas J; Warren, Sean C; Kerry, Louise; Lockwood, Nicola; Frolov, Antonina; Frankel, Paul; Bugeon, Laurence; McGinty, James; Dallman, Margaret J; French, Paul M W

2016-04-01

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region. © 2016 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Weight Measurements and Standards for Soldiers

DTIC Science & Technology

2009-10-01

Dr. Corby Martin has expanded on this technology and has developed the Remote Food Photography Method (RFPM) for use in free-living conditions...Allen HR, Champagne CM, Anton SD. A novel method to remotely measure food intake of free-living individuals in real time: the remote food ... photography method. Br J Nutr. 2009 Feb;101:446-56. 5. Martin CK, Kaya S, Gunturk BK. Quantification of food intake using food image analysis. Conference

Live imaging of developmental processes in a living meristem of Davidia involucrata (Nyssaceae)

PubMed Central

Jerominek, Markus; Bull-Hereñu, Kester; Arndt, Melanie; Claßen-Bockhoff, Regine

2014-01-01

Morphogenesis in plants is usually reconstructed by scanning electron microscopy and histology of meristematic structures. These techniques are destructive and require many samples to obtain a consecutive series of states. Unfortunately, using this methodology the absolute timing of growth and complete relative initiation of organs remain obscure. To overcome this limitation, an in vivo observational method based on Epi-Illumination Light Microscopy (ELM) was developed and tested with a male inflorescence meristem (floral unit) of the handkerchief tree Davidia involucrata Baill. (Nyssaceae). We asked whether the most basal flowers of this floral unit arise in a basipetal sequence or, alternatively, are delayed in their development. The growing meristem was observed for 30 days, the longest live observation of a meristem achieved to date. The sequence of primordium initiation indicates a later initiation of the most basal flowers and not earlier or simultaneously as SEM images could suggest. D. involucrata exemplarily shows that live-ELM gives new insights into developmental processes of plants. In addition to morphogenetic questions such as the transition from vegetative to reproductive meristems or the absolute timing of ontogenetic processes, this method may also help to quantify cellular growth processes in the context of molecular physiology and developmental genetics studies. PMID:25431576

Live imaging of developmental processes in a living meristem of Davidia involucrata (Nyssaceae).

PubMed

Jerominek, Markus; Bull-Hereñu, Kester; Arndt, Melanie; Claßen-Bockhoff, Regine

2014-01-01

Morphogenesis in plants is usually reconstructed by scanning electron microscopy and histology of meristematic structures. These techniques are destructive and require many samples to obtain a consecutive series of states. Unfortunately, using this methodology the absolute timing of growth and complete relative initiation of organs remain obscure. To overcome this limitation, an in vivo observational method based on Epi-Illumination Light Microscopy (ELM) was developed and tested with a male inflorescence meristem (floral unit) of the handkerchief tree Davidia involucrata Baill. (Nyssaceae). We asked whether the most basal flowers of this floral unit arise in a basipetal sequence or, alternatively, are delayed in their development. The growing meristem was observed for 30 days, the longest live observation of a meristem achieved to date. The sequence of primordium initiation indicates a later initiation of the most basal flowers and not earlier or simultaneously as SEM images could suggest. D. involucrata exemplarily shows that live-ELM gives new insights into developmental processes of plants. In addition to morphogenetic questions such as the transition from vegetative to reproductive meristems or the absolute timing of ontogenetic processes, this method may also help to quantify cellular growth processes in the context of molecular physiology and developmental genetics studies.

Expanded palette of Nano-lanterns for real-time multicolor luminescence imaging.

PubMed

Takai, Akira; Nakano, Masahiro; Saito, Kenta; Haruno, Remi; Watanabe, Tomonobu M; Ohyanagi, Tatsuya; Jin, Takashi; Okada, Yasushi; Nagai, Takeharu

2015-04-07

Fluorescence live imaging has become an essential methodology in modern cell biology. However, fluorescence requires excitation light, which can sometimes cause potential problems, such as autofluorescence, phototoxicity, and photobleaching. Furthermore, combined with recent optogenetic tools, the light illumination can trigger their unintended activation. Because luminescence imaging does not require excitation light, it is a good candidate as an alternative imaging modality to circumvent these problems. The application of luminescence imaging, however, has been limited by the two drawbacks of existing luminescent protein probes, such as luciferases: namely, low brightness and poor color variants. Here, we report the development of bright cyan and orange luminescent proteins by extending our previous development of the bright yellowish-green luminescent protein Nano-lantern. The color change and the enhancement of brightness were both achieved by bioluminescence resonance energy transfer (BRET) from enhanced Renilla luciferase to a fluorescent protein. The brightness of these cyan and orange Nano-lanterns was ∼20 times brighter than wild-type Renilla luciferase, which allowed us to perform multicolor live imaging of intracellular submicron structures. The rapid dynamics of endosomes and peroxisomes were visualized at around 1-s temporal resolution, and the slow dynamics of focal adhesions were continuously imaged for longer than a few hours without photobleaching or photodamage. In addition, we extended the application of these multicolor Nano-lanterns to simultaneous monitoring of multiple gene expression or Ca(2+) dynamics in different cellular compartments in a single cell.

Compact ultrafast semiconductor disk laser for nonlinear imaging in living organisms

NASA Astrophysics Data System (ADS)

Aviles-Espinosa, Rodrigo; Filippidis, G.; Hamilton, Craig; Malcolm, Graeme; Weingarten, Kurt J.; Südmeyer, Thomas; Barbarin, Yohan; Keller, Ursula; Artigas, David; Loza-Alvarez, Pablo

2011-03-01

Ultrashort pulsed laser systems (such as Ti:sapphire) have been used in nonlinear microscopy during the last years. However, its implementation is not straight forward as they are maintenance-intensive, bulky and expensive. These limitations have prevented their wide-spread use for nonlinear imaging, especially in "real-life" biomedical applications. In this work we present the suitability of a compact ultrafast semiconductor disk laser source, with a footprint of 140x240x70 mm, to be used for nonlinear microscopy. The modelocking mechanism of the laser is based on a quantumdot semiconductor saturable absorber mirror (SESAM). The laser delivers an average output power of 287 mW with 1.5 ps pulses at 500 MHz, corresponding to a peak power of 0.4 kW. Its center wavelength is 965 nm which is ideally suited for two-photon excitation of the widely used Green Fluorescent Protein (GFP) marker as it virtually matches its twophoton action cross section. We reveal that it is possible to obtain two photon excited fluorescence images of GFP labeled neurons and secondharmonic generation images of pharynx and body wall muscles in living C. elegans nematodes. Our results demonstrate that this compact laser is well suited for long-term time-lapse imaging of living samples as very low powers provide a bright signal. Importantly this non expensive, turn-key, compact laser system could be used as a platform to develop portable nonlinear bio-imaging devices, facilitating its wide-spread adoption in "real-life" applications.

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

PubMed

Kadam, Parnika; McAllister, Ryan; Urbach, Jeffrey S; Sandberg, Kathryn; Mueller, Susette C

2017-03-27

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Degron Protease Blockade Sensor to Image Epigenetic Histone Protein Methylation in Cells and Living Animals

PubMed Central

2015-01-01

Lysine methylation of histone H3 and H4 has been identified as a promising therapeutic target in treating various cellular diseases. The availability of an in vivo assay that enables rapid screening and preclinical evaluation of drugs that potentially target this cellular process will significantly expedite the pace of drug development. This study is the first to report the development of a real-time molecular imaging biosensor (a fusion protein, [FLuc2]-[Suv39h1]-[(G4S)3]-[H3-K9]-[cODC]) that can detect and monitor the methylation status of a specific histone lysine methylation mark (H3-K9) in live animals. The sensitivity of this sensor was assessed in various cell lines, in response to down-regulation of methyltransferase EHMT2 by specific siRNA, and in nude mice with lysine replacement mutants. In vivo imaging in response to a combination of methyltransferase inhibitors BIX01294 and Chaetocin in mice reveals the potential of this sensor for preclinical drug evaluation. This biosensor thus has demonstrated its utility in the detection of H3-K9 methylations in vivo and potential value in preclinical drug development. PMID:25489787

Renaissance of morphological studies: the examination of functional structures in living animal organs using the in vivo cryotechnique.

PubMed

Ohno, Shinichi; Saitoh, Yurika; Ohno, Nobuhiko; Terada, Nobuo

2017-01-01

Medical and biological scientists wish to understand the in vivo structures of the cells and tissues that make up living animal organs, as well as the locations of their molecular components. Recently, the live imaging of animal cells and tissues with fluorescence-labeled proteins produced via gene manipulation has become increasingly common. Therefore, it is important to ensure that findings derived from histological or immunohistochemical tissue sections of living animal organs are compatible with those obtained from live images of the same organs, which can be assessed using recently developed digital imaging techniques. Over the past two decades, we have performed immunohistochemical and morphological studies of the cells and tissues in living animal organs using a novel in vivo cryotechnique. The use of a specially designed liquid cryogen system with or without a cryoknife during this cryotechnique solved the technical problems that inevitably arise during the conventional preparation methods employed prior to light or electron microscopic examinations. Our in vivo cryotechnique has been found to be extremely useful for arresting transient physiological processes in cells and tissues and for maintaining their functional components-such as rapidly changing signaling molecules, membrane channels, or receptors-in situ. The purpose of the present review is to describe the basic mechanism underlying cryotechniques and the significance of our in vivo cryotechnique. In addition, it describes various morphological or immunohistochemical findings, observations made using quantum dots, and a Raman cryomicroscopy-based method for assessing oxygen saturation in the erythrocytes flowing through intestinal tissues.

A reversible fluorescent probe for real-time live-cell imaging and quantification of endogenous hydropolysulfides.

PubMed

Umezawa, Keitaro; Kamiya, Mako; Urano, Yasuteru

2018-05-23

The chemical biology of reactive sulfur species, including hydropolysulfides, has been a subject undergoing intense study in recent years, but further understanding of their 'intact' function in living cells has been limited due to a lack of appropriate analytical tools. In order to overcome this limitation, we developed a new type of fluorescent probe which reversibly and selectively reacts to hydropolysulfides. The probe enables live-cell visualization and quantification of endogenous hydropolysulfides without interference from intrinsic thiol species such as glutathione. Additionally, real-time reversible monitoring of oxidative-stress-induced fluctuation of intrinsic hydropolysulfides has been achieved with a temporal resolution in the order of seconds, a result which has not yet been realized using conventional methods. These results reveal the probe's versatility as a new fluorescence imaging tool to understand the function of intracellular hydropolysulfides. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multi-pulse pumping for far-field super-resolution imaging

NASA Astrophysics Data System (ADS)

Requena, Sebastian; Raut, Sangram; Doan, Hung; Kimball, Joe; Fudala, Rafal; Borejdo, Julian; Gryczynski, Ignacy; Strzhemechny, Yuri; Gryczynski, Zygmunt

2016-02-01

Recently, far-field optical imaging with a resolution significantly beyond diffraction limit has attracted tremendous attention allowing for high resolution imaging in living objects. Various methods have been proposed that are divided in to two basic approaches; deterministic super-resolution like STED or RESOLFT and stochastic super-resolution like PALM or STORM. We propose to achieve super-resolution in far-field fluorescence imaging by the use of controllable (on-demand) bursts of pulses that can change the fluorescence signal of long-lived component over one order of magnitude. We demonstrate that two beads, one labeled with a long-lived dye and another with a short-lived dye, separated by a distance lower than 100 nm can be easily resolved in a single experiment. The proposed method can be used to separate two biological structures in a cell by targeting them with two antibodies labeled with long-lived and short-lived fluorophores.

Living into the imagined body: how the diagnostic image confronts the lived body.

PubMed

Stahl, Devan

2013-06-01

In this paper I will show how the medical image, presented to the patient by the physician, participates in medicine's cold culture of abstraction, objectification and mandated normativity. I begin by giving a brief account of the use of anatomical imaging since the Renaissance to show how images have historically functioned in contrast to how they are currently used in medical practice. Next, I examine how contemporary medical imaging techniques participate in a kind of knowledge production that objectifies the human body. Finally, I elucidate how physicians ought to place the medical image within the context of the lived body so as to create a healing relationship with the patient. In all this I hope to show that the medical image, far from a piece of objective data, testifies to the interplay of particular beliefs, practices and doctrines contemporary medicine holds dear. To best treat her patient, the physician must appreciate the influence of these images and appropriately place them within the context of the patient's lived experience.

Near-infrared fluorescent nanoprobes for cancer molecular imaging: status and challenges

PubMed Central

He, Xiaoxiao; Gao, Jinhao; Gambhir, Sanjiv Sam; Cheng, Zhen

2010-01-01

Near-infrared fluorescence (NIRF) imaging promises to improve cancer imaging and management; advances in nanomaterials allow scientists to combine new nanoparticles with NIRF imaging techniques, thereby fulfilling this promise. Here, we present a synopsis of current developments in NIRF nanoprobes, their use in imaging small living subjects, their pharmacokinetics and toxicity and finally their integration into multimodal imaging strategies. We also discuss challenges impeding the clinical translation of NIRF nanoprobes for molecular imaging of cancer. Whereas utilization of most NIRF nanoprobes remains at a proof-of-principle stage, optimizing the impact of nanomedicine in cancer patient diagnosis and management will likely be realized through persistent interdisciplinary amalgamation of diverse research fields. PMID:20870460

Particle tracking and extended object imaging by interferometric super resolution microscopy

NASA Astrophysics Data System (ADS)

Gdor, Itay; Yoo, Seunghwan; Wang, Xiaolei; Daddysman, Matthew; Wilton, Rosemarie; Ferrier, Nicola; Hereld, Mark; Cossairt, Oliver (Ollie); Katsaggelos, Aggelos; Scherer, Norbert F.

2018-02-01

An interferometric fluorescent microscope and a novel theoretic image reconstruction approach were developed and used to obtain super-resolution images of live biological samples and to enable dynamic real time tracking. The tracking utilizes the information stored in the interference pattern of both the illuminating incoherent light and the emitted light. By periodically shifting the interferometer phase and a phase retrieval algorithm we obtain information that allow localization with sub-2 nm axial resolution at 5 Hz.

Accurate cell counts in live mouse embryos using optical quadrature and differential interference contrast microscopy

NASA Astrophysics Data System (ADS)

Warger, William C., II; Newmark, Judith A.; Zhao, Bing; Warner, Carol M.; DiMarzio, Charles A.

2006-02-01

Present imaging techniques used in in vitro fertilization (IVF) clinics are unable to produce accurate cell counts in developing embryos past the eight-cell stage. We have developed a method that has produced accurate cell counts in live mouse embryos ranging from 13-25 cells by combining Differential Interference Contrast (DIC) and Optical Quadrature Microscopy. Optical Quadrature Microscopy is an interferometric imaging modality that measures the amplitude and phase of the signal beam that travels through the embryo. The phase is transformed into an image of optical path length difference, which is used to determine the maximum optical path length deviation of a single cell. DIC microscopy gives distinct cell boundaries for cells within the focal plane when other cells do not lie in the path to the objective. Fitting an ellipse to the boundary of a single cell in the DIC image and combining it with the maximum optical path length deviation of a single cell creates an ellipsoidal model cell of optical path length deviation. Subtracting the model cell from the Optical Quadrature image will either show the optical path length deviation of the culture medium or reveal another cell underneath. Once all the boundaries are used in the DIC image, the subtracted Optical Quadrature image is analyzed to determine the cell boundaries of the remaining cells. The final cell count is produced when no more cells can be subtracted. We have produced exact cell counts on 5 samples, which have been validated by Epi-Fluorescence images of Hoechst stained nuclei.

A fully actuated robotic assistant for MRI-guided prostate biopsy and brachytherapy

NASA Astrophysics Data System (ADS)

Li, Gang; Su, Hao; Shang, Weijian; Tokuda, Junichi; Hata, Nobuhiko; Tempany, Clare M.; Fischer, Gregory S.

2013-03-01

Intra-operative medical imaging enables incorporation of human experience and intelligence in a controlled, closed-loop fashion. Magnetic resonance imaging (MRI) is an ideal modality for surgical guidance of diagnostic and therapeutic procedures, with its ability to perform high resolution, real-time, high soft tissue contrast imaging without ionizing radiation. However, for most current image-guided approaches only static pre-operative images are accessible for guidance, which are unable to provide updated information during a surgical procedure. The high magnetic field, electrical interference, and limited access of closed-bore MRI render great challenges to developing robotic systems that can perform inside a diagnostic high-field MRI while obtaining interactively updated MR images. To overcome these limitations, we are developing a piezoelectrically actuated robotic assistant for actuated percutaneous prostate interventions under real-time MRI guidance. Utilizing a modular design, the system enables coherent and straight forward workflow for various percutaneous interventions, including prostate biopsy sampling and brachytherapy seed placement, using various needle driver configurations. The unified workflow compromises: 1) system hardware and software initialization, 2) fiducial frame registration, 3) target selection and motion planning, 4) moving to the target and performing the intervention (e.g. taking a biopsy sample) under live imaging, and 5) visualization and verification. Phantom experiments of prostate biopsy and brachytherapy were executed under MRI-guidance to evaluate the feasibility of the workflow. The robot successfully performed fully actuated biopsy sampling and delivery of simulated brachytherapy seeds under live MR imaging, as well as precise delivery of a prostate brachytherapy seed distribution with an RMS accuracy of 0.98mm.

A loop resonator for slice-selective in vivo EPR imaging in rats

PubMed Central

Hirata, Hiroshi; He, Guanglong; Deng, Yuanmu; Salikhov, Ildar; Petryakov, Sergey; Zweier, Jay L.

2008-01-01

A loop resonator was developed for 300-MHz continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy and imaging in live rats. A single-turn loop (55 mm in diameter) was used to provide sufficient space for the rat body. Efficiency for generating a radiofrequency magnetic field of 38 µT/W1/2 was achieved at the center of the loop. For the resonator itself, an unloaded quality factor of 430 was obtained. When a 350 g rat was placed in the resonator at the level of the lower abdomen, the quality factor decreased to 18. The sensitive volume in the loop was visualized with a bottle filled with an aqueous solution of the nitroxide spin probe 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy (3-CP). The resonator was shown to enable EPR imaging in live rats. Imaging was performed for 3-CP that had been infused intravenously into the rat and its distribution was visualized within the lower abdomen. PMID:18006343

Development and testing of a CW-EPR apparatus for imaging of short-lifetime nitroxyl radicals in mouse head

NASA Astrophysics Data System (ADS)

Sato-Akaba, Hideo; Fujii, Hirotada; Hirata, Hiroshi

2008-08-01

This article describes a method for reducing the acquisition time in three-dimensional (3D) continuous-wave electron paramagnetic resonance (CW-EPR) imaging. To visualize nitroxyl spin probes, which have a short lifetime in living organisms, the acquisition time for a data set of spectral projections should be shorter than the lifetime of the spin probes. To decrease the total time required for data acquisition, the duration of magnetic field scanning was reduced to 0.5 s. Moreover, the number of projections was decreased by using the concept of a uniform distribution. To demonstrate this faster data acquisition, two kinds of nitroxyl radicals with different decay rates were measured in mice. 3D EPR imaging of 4-hydroxy-2,2,6,6-tetramethylpiperidine-d 17-1- 15N-1-oxyl in mouse head was successfully carried out. 3D EPR imaging of nitroxyl spin probes with a half-life of a few minutes was achieved for the first time in live animals.

Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

PubMed Central

Wang, Zhonghai; Qin, Wan; Shao, Yonghong; Ma, Siyu; Borg, Thomas K.; Gao, Bruce Z.

2015-01-01

Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture. PMID:25767692

Live imaging of fluorescent proteins in chordate embryos: from ascidians to mice.

PubMed

Passamaneck, Yale J; Di Gregorio, Anna; Papaioannou, Virginia E; Hadjantonakis, Anna-Katerina

2006-03-01

Although we have advanced in our understanding of the molecular mechanisms intrinsic to the morphogenesis of chordate embryos, the question of how individual developmental events are integrated to generate the final morphological form is still unresolved. Microscopic observation is a pivotal tool in developmental biology, both for determining the normal course of events and for contrasting this with the results of experimental and pathological perturbations. Since embryonic development takes place in three dimensions over time, to fully understand the events required to build an embryo we must investigate embryo morphogenesis in multiple dimensions in situ. Recent advances in the isolation of naturally fluorescent proteins, and the refinement of techniques for in vivo microscopy offer unprecedented opportunities to study the cellular and molecular events within living, intact embryos using optical imaging. These technologies allow direct visual access to complex events as they happen in their native environment, and thus provide greater insights into cell behaviors operating during embryonic development. Since most fluorescent protein probes and modes of data acquisition are common across species, we have chosen the mouse and the ascidian, two model organisms at opposite ends of the chordate clade, to review the use of some of the current genetically-encoded fluorescent proteins and their visualization in vivo in living embryos for the generation of high-resolution imaging data. Microsc. Res. Tech. 69:160-167, 2006. (c) 2006 Wiley-Liss, Inc.

Noninvasive Detection and Imaging of Molecular Markers in Live Cardiomyocytes Derived from Human Embryonic Stem Cells

PubMed Central

Pascut, Flavius C.; Goh, Huey T.; Welch, Nathan; Buttery, Lee D.; Denning, Chris; Notingher, Ioan

2011-01-01

Raman microspectroscopy (RMS) was used to detect and image molecular markers specific to cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs). This technique is noninvasive and thus can be used to discriminate individual live CMs within highly heterogeneous cell populations. Principal component analysis (PCA) of the Raman spectra was used to build a classification model for identification of individual CMs. Retrospective immunostaining imaging was used as the gold standard for phenotypic identification of each cell. We were able to discriminate CMs from other phenotypes with >97% specificity and >96% sensitivity, as calculated with the use of cross-validation algorithms (target 100% specificity). A comparison between Raman spectral images corresponding to selected Raman bands identified by the PCA model and immunostaining of the same cells allowed assignment of the Raman spectral markers. We conclude that glycogen is responsible for the discrimination of CMs, whereas myofibril proteins have a lesser contribution. This study demonstrates the potential of RMS for allowing the noninvasive phenotypic identification of hESC progeny. With further development, such label-free optical techniques may enable the separation of high-purity cell populations with mature phenotypes, and provide repeated measurements to monitor time-dependent molecular changes in live hESCs during differentiation in vitro. PMID:21190678

Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

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

Wang, Ying; Li, Zhaohui; Liu, Misha

Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeledmore » single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.« less

A dual-function fluorescent probe for monitoring the degrees of hypoxia in living cells via the imaging of nitroreductase and adenosine triphosphate.

PubMed

Fang, Yu; Shi, Wen; Hu, Yiming; Li, Xiaohua; Ma, Huimin

2018-05-24

A new dual-function fluorescent probe is developed for detecting nitroreductase (NTR) and adenosine triphosphate (ATP) with different responses. Imaging application of the probe reveals that intracellular NTR and ATP display an adverse changing trend during a hypoxic process and ATP can serve as a new sign for cell hypoxia.

In situ fluorescence activation of DNA-silver nanoclusters as a label-free and general strategy for cell nucleus imaging.

PubMed

Li, Duo; Qiao, Zhenzhen; Yu, Yanru; Tang, Jinlu; He, Xiaoxiao; Shi, Hui; Ye, Xiaosheng; Lei, Yanli; Wang, Kemin

2018-01-25

A facile, general and turn-on nucleus imaging strategy was first developed based on in situ fluorescence activation of C-rich dark silver nanoclusters by G-rich telomeres. After a simple incubation without washing, nanoclusters could selectively stain the nucleus with intense red luminescence, which was confirmed using fixed/living cells and several cell lines.

Live imaging of mouse secondary palate fusion

PubMed Central

Kim, Seungil; Prochazka, Jan; Bush, Jeffrey O.

2017-01-01

LONG ABSTRACT The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled. PMID:28784960

Genetically Encoded Catalytic Hairpin Assembly for Sensitive RNA Imaging in Live Cells.

PubMed

Mudiyanselage, Aruni P K K Karunanayake; Yu, Qikun; Leon-Duque, Mark A; Zhao, Bin; Wu, Rigumula; You, Mingxu

2018-06-26

DNA and RNA nanotechnology has been used for the development of dynamic molecular devices. In particular, programmable enzyme-free nucleic acid circuits, such as catalytic hairpin assembly, have been demonstrated as useful tools for bioanalysis and to scale up system complexity to an extent beyond current cellular genetic circuits. However, the intracellular functions of most synthetic nucleic acid circuits have been hindered by challenges in the biological delivery and degradation. On the other hand, genetically encoded and transcribed RNA circuits emerge as alternative powerful tools for long-term embedded cellular analysis and regulation. Herein, we reported a genetically encoded RNA-based catalytic hairpin assembly circuit for sensitive RNA imaging inside living cells. The split version of Broccoli, a fluorogenic RNA aptamer, was used as the reporter. One target RNA can catalytically trigger the fluorescence from tens-to-hundreds of Broccoli. As a result, target RNAs can be sensitively detected. We have further engineered our circuit to allow easy programming to image various target RNA sequences. This design principle opens the arena for developing a large variety of genetically encoded RNA circuits for cellular applications.

In vivo imaging of cardiac development and function in zebrafish using light sheet microscopy.

PubMed

Weber, Michael; Huisken, Jan

2015-01-01

Detailed studies of heart development and function are crucial for our understanding of cardiac failures and pave the way for better diagnostics and treatment. However, the constant motion and close incorporation into the cardiovascular system prevent in vivo studies of the living, unperturbed heart. The complementary strengths of the zebrafish model and light sheet microscopy provide a useful platform to fill this gap. High-resolution images of the embryonic vertebrate heart are now recorded from within the living animal: deep inside the unperturbed heart we can follow cardiac contractions and measure action potentials and calcium transients. Three-dimensional reconstructions of the entire beating heart with cellular resolution give new insights into its ever-changing morphology and facilitate studies into how individual cells form the complex cardiac network. In addition, cardiac dynamics and robustness are now examined with targeted optical manipulation. Overall, the combination of zebrafish and light sheet microscopy represents a promising addition for cardiac research and opens the door to a better understanding of heart function and development.

Regulation of branching dynamics by axon-intrinsic asymmetries in Tyrosine Kinase Receptor signaling

PubMed Central

Zschätzsch, Marlen; Oliva, Carlos; Langen, Marion; De Geest, Natalie; Özel, Mehmet Neset; Williamson, W Ryan; Lemon, William C; Soldano, Alessia; Munck, Sebastian; Hiesinger, P Robin; Sanchez-Soriano, Natalia; Hassan, Bassem A

2014-01-01

Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors. DOI: http://dx.doi.org/10.7554/eLife.01699.001 PMID:24755286

Simultaneous immersion Mirau interferometry.

PubMed

Lyulko, Oleksandra V; Randers-Pehrson, Gerhard; Brenner, David J

2013-05-01

A novel technique for label-free imaging of live biological cells in aqueous medium that is insensitive to ambient vibrations is presented. This technique is a spin-off from previously developed immersion Mirau interferometry. Both approaches utilize a modified Mirau interferometric attachment for a microscope objective that can be used both in air and in immersion mode, when the device is submerged in cell medium and has its internal space filled with liquid. While immersion Mirau interferometry involves first capturing a series of images, the resulting images are potentially distorted by ambient vibrations. Overcoming these serial-acquisition challenges, simultaneous immersion Mirau interferometry incorporates polarizing elements into the optics to allow simultaneous acquisition of two interferograms. The system design and production are described and images produced with the developed techniques are presented.

Translational research of optical molecular imaging for personalized medicine.

PubMed

Qin, C; Ma, X; Tian, J

2013-12-01

In the medical imaging field, molecular imaging is a rapidly developing discipline and forms many imaging modalities, providing us effective tools to visualize, characterize, and measure molecular and cellular mechanisms in complex biological processes of living organisms, which can deepen our understanding of biology and accelerate preclinical research including cancer study and medicine discovery. Among many molecular imaging modalities, although the penetration depth of optical imaging and the approved optical probes used for clinics are limited, it has evolved considerably and has seen spectacular advances in basic biomedical research and new drug development. With the completion of human genome sequencing and the emergence of personalized medicine, the specific drug should be matched to not only the right disease but also to the right person, and optical molecular imaging should serve as a strong adjunct to develop personalized medicine by finding the optimal drug based on an individual's proteome and genome. In this process, the computational methodology and imaging system as well as the biomedical application regarding optical molecular imaging will play a crucial role. This review will focus on recent typical translational studies of optical molecular imaging for personalized medicine followed by a concise introduction. Finally, the current challenges and the future development of optical molecular imaging are given according to the understanding of the authors, and the review is then concluded.

Multiplexed 3D FRET imaging in deep tissue of live embryos

PubMed Central

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

2015-01-01

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

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

Nationwide Eclipse Ballooning Project

NASA Astrophysics Data System (ADS)

Colman Des Jardins, Angela; Berk Knighton, W.; Larimer, Randal; Mayer-Gawlik, Shane; Fowler, Jennifer; Harmon, Christina; Koehler, Christopher; Guzik, Gregory; Flaten, James; Nolby, Caitlin; Granger, Douglas; Stewart, Michael

2016-05-01

The purpose of the Nationwide Eclipse Ballooning Project is to make the most of the 2017 rare eclipse event in four main areas: public engagement, workforce development, partnership development, and science. The Project is focused on two efforts, both student-led: online live video of the eclipse from the edge of space and the study of the atmospheric response to the eclipse. These efforts, however, involving more than 60 teams across the US, are challenging in many ways. Therefore, the Project is leveraging the NASA Space Grant and NOAA atmospheric science communities to make it a success. The first and primary topic of this poster is the NASA Space Grant supported online live video effort. College and high school students on 48 teams from 31 states will conduct high altitude balloon flights from 15-20 locations across the 8/21/2017 total eclipse path, sending live video and images from near space to a national website. Video and images of a total solar eclipse from near space are fascinating and rare. It’s never been done live and certainly not in a network of coverage across a continent. In addition to the live video to the web, these teams are engaged in several other science experiments as secondary payloads. We also briefly highlight the eclipse atmospheric science effort, where about a dozen teams will launch over one hundred radiosondes from across the 2017 path, recording an unprecedented atmospheric data sample. Collected data will include temperature, density, wind, humidity, and ozone measurements.

Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

NASA Astrophysics Data System (ADS)

Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, Yongkeun

2016-09-01

Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

Multispectral Live-Cell Imaging.

PubMed

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

2018-06-01

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

Special issue on high-resolution optical imaging

NASA Astrophysics Data System (ADS)

Smith, Peter J. S.; Davis, Ilan; Galbraith, Catherine G.; Stemmer, Andreas

2013-09-01

The pace of development in the field of advanced microscopy is truly breath-taking, and is leading to major breakthroughs in our understanding of molecular machines and cell function. This special issue of Journal of Optics draws attention to a number of interesting approaches, ranging from fluorescence and imaging of unlabelled cells, to computational methods, all of which are describing the ever increasing detail of the dynamic behaviour of molecules in the living cell. This is a field which traditionally, and currently, demonstrates a marvellous interplay between the disciplines of physics, chemistry and biology, where apparent boundaries to resolution dissolve and living cells are viewed in ever more clarity. It is fertile ground for those interested in optics and non-conventional imaging to contribute high-impact outputs in the fields of cell biology and biomedicine. The series of articles presented here has been selected to demonstrate this interdisciplinarity and to encourage all those with a background in the physical sciences to 'dip their toes' into the exciting and dynamic discoveries surrounding cell function. Although single molecule super-resolution microscopy is commercially available, specimen preparation and interpretation of single molecule data remain a major challenge for scientists wanting to adopt the techniques. The paper by Allen and Davidson [1] provides a much needed detailed introduction to the practical aspects of stochastic optical reconstruction microscopy, including sample preparation, image acquisition and image analysis, as well as a brief description of the different variants of single molecule localization microscopy. Since super-resolution microscopy is no longer restricted to three-dimensional imaging of fixed samples, the review by Fiolka [2] is a timely introduction to techniques that have been successfully applied to four-dimensional live cell super-resolution microscopy. The combination of multiple high-resolution techniques, such as the combination of light sheet and structured illumination microscopy (SIM), which efficiently utilize photon budget and avoid illuminating regions of the specimen not currently being imaged, hold the greatest promise for future biological applications. Therefore, the combined setup for SIM and single molecule localization microscopy (SMLM) described by Rossberger et al [3] will be very helpful and stimulating to advanced microscopists in further modifying their setups. The SIM image helps in identifying artefacts in SMLM reconstruction, e.g. when two active fluorophores are close together and get rejected as 'out-of-focus'. This combined setup is another way to facilitate imaging live samples. The article by Thomas et al [4] presents another advance for biological super-resolution imaging with a new approach to reconstruct optically sectioned images using structured illumination. The method produces images with higher spatial resolution and greater signal to noise compared to existing approaches. This algorithm demonstrates great promise for reconstructing biological images where the signal intensities are inherently lower. Shevchuk et al [5] present a non-optic near field approach to imaging with a review of scanning ion-conductance microscopy. This is a powerful alternative approach for examining the surface dynamics of living cells including exo and endocytosis, unlabelled, and at the level of the single event. Here they present the first data on combining this approach with fluorescence confocal microscopy—adding that extra dimension. Different approaches to label-free live cell imaging are presented in the papers by Patel et al [6], Mehta and Oldenbourg [7], as well as Rogers and Zheludev [8]. All three papers bring home the excitement of looking at live cell dynamics without reporters—Patel et al [6] review both the potential of coherent anti-Stokes Raman scattering and biological applications, where specific biomolecules are detected on the basis of their biophysical properties. Polarized light microscopy as presented by Mehta and Oldenbourg [7], describe a novel implementation of this technology to detect dichroism, and demonstrate beautifully its use in imaging unlabelled microtubules, mitochondria and lipid droplets. Sub-wavelength light focusing provides another avenue to super-resolution, and this is presented by Rogers and Zheludev [8]. Speculating on further improvements, these authors expect a resolution of 0.15λ. To date, the method has not been applied to low contrast, squishy and motile biotargets, but is included here for the clear potential to drive label-free imaging in new directions. A similar logic lies behind the inclusion of Parsons et al [9] where ultraviolet coherent diffractive imaging is further developed. These authors have demonstrated a shrink-wrap technique which reduces the integration time by a factor of 5, bringing closer the time when we have lab based imaging systems based on extreme ultraviolet and soft x-ray sources using sophisticated phase retrieval algorithms. Real biological specimens have spatially varying refractive indices that inevitably lead to aberrations and image distortions. Global refractive index matching of the embedding medium has been an historic solution, but unfortunately is not practical for live cell imaging. Adaptive optics appears an attractive solution and Simmonds and Booth [10] demonstrate the theoretical benefits of applying several adaptive optical elements, placed in different conjugate planes, to create a kind of 'inverse specimen' that unwarps phase distortions of the sample—but these have yet to be tested on real specimens. A difficulty in single molecule localization microscopy has been the determination of whether or not two molecules are colocalized. Kim et al [11] present a method for correcting bleed-through during multi-colour, single molecule localization microscopy. Such methods are welcome standards when trying to quantifiably interpret how close two molecules actually are. Rees et al [12] provide an invaluable overview of key image processing steps in localization microscopy. This paper is an excellent starting point for anyone implementing localization algorithms and the Matlab software provided will be invaluable; a strong paper on which to conclude our overview of the excellent articles brought together in this issue. One aspect brought home in several of these articles is the volume of data now being collected by high resolution live cell imaging. Data processing and image reconstruction will continue to be pressure points in the further development of instrumentation and analyses. We would hope that the series of papers presented here will motivate software engineers, optical physicists and biologists to contribute to the further development of this exciting field. References [1] Allen J R et al 2013 J. Opt. 15 094001 [2] Fiolka R et al 2013 J. Opt. 15 094002 [3] Rossberger S et al 2013 J. Opt. 15 094003 [4] Thomas B et al 2013 J. Opt. 15 094004 [5] Shevchuk A et al 2013 J. Opt. 15 094005 [6] Patel I et al 2013 J. Opt. 15 094006 [7] Mehta S B et al 2013 J. Opt. 15 094007 [8] Rogers E T F et al 2013 J. Opt. 15 094008 [9] Parsons A D et al 2013 J. Opt. 15 094009 [10] Simmonds R et al 2013 J. Opt. 15 094010 [11] Kim D et al 2013 J. Opt. 15 094011 [12] Rees E J et al 2013 J. Opt. 15 094012

Beam-on imaging of short-lived positron emitters during proton therapy

NASA Astrophysics Data System (ADS)

Buitenhuis, H. J. T.; Diblen, F.; Brzezinski, K. W.; Brandenburg, S.; Dendooven, P.

2017-06-01

In vivo dose delivery verification in proton therapy can be performed by positron emission tomography (PET) of the positron-emitting nuclei produced by the proton beam in the patient. A PET scanner installed in the treatment position of a proton therapy facility that takes data with the beam on will see very short-lived nuclides as well as longer-lived nuclides. The most important short-lived nuclide for proton therapy is 12N (Dendooven et al 2015 Phys. Med. Biol. 60 8923-47), which has a half-life of 11 ms. The results of a proof-of-principle experiment of beam-on PET imaging of short-lived 12N nuclei are presented. The Philips Digital Photon Counting Module TEK PET system was used, which is based on LYSO scintillators mounted on digital SiPM photosensors. A 90 MeV proton beam from the cyclotron at KVI-CART was used to investigate the energy and time spectra of PET coincidences during beam-on. Events coinciding with proton bunches, such as prompt gamma rays, were removed from the data via an anti-coincidence filter with the cyclotron RF. The resulting energy spectrum allowed good identification of the 511 keV PET counts during beam-on. A method was developed to subtract the long-lived background from the 12N image by introducing a beam-off period into the cyclotron beam time structure. We measured 2D images and 1D profiles of the 12N distribution. A range shift of 5 mm was measured as 6  ±  3 mm using the 12N profile. A larger, more efficient, PET system with a higher data throughput capability will allow beam-on 12N PET imaging of single spots in the distal layer of an irradiation with an increased signal-to-background ratio and thus better accuracy. A simulation shows that a large dual panel scanner, which images a single spot directly after it is delivered, can measure a 5 mm range shift with millimeter accuracy: 5.5  ±  1.1 mm for 1  ×  108 protons and 5.2  ±  0.5 mm for 5  ×  108 protons. This makes fast and accurate feedback on the dose delivery during treatment possible.

A device for real-time live-cell microscopy during dynamic dual-modal mechanostimulation

NASA Astrophysics Data System (ADS)

Lorusso, D.; Nikolov, H. N.; Chmiel, T.; Beach, R. J.; Sims, S. M.; Dixon, S. J.; Holdsworth, D. W.

2017-03-01

Mechanotransduction - the process by which cells sense and respond to mechanical stimuli - is essential for several physiological processes including skeletal homeostasis. Mammalian cells are thought to be sensitive to different modes of mechanical stimuli, including vibration and fluid shear. To better understand the mechanisms underlying the early stages of mechanotransduction, we describe the development of devices for mechanostimulation (by vibration and fluid shear) of live cells that can be integrated with real-time optical microscopy. The integrated system can deliver up to 3 Pa of fluid shear simultaneous with high-frequency sinusoidal vibrations up to 1 g. Stimuli can be applied simultaneously or independently to cells during real-time microscopic imaging. A custom microfluidic chamber was prepared from polydimethylsiloxane on a glass-bottom cell culture dish. Fluid flow was applied with a syringe pump to induce shear stress. This device is compatible with a custom-designed motion control vibration system. A voice coil actuates the system that is suspended on linear air bushings. Accelerations produced by the system were monitored with an on-board accelerometer. Displacement was validated optically using particle tracking digital high-speed imaging (1200 frames per second). During operation at nominally 45 Hz and 0.3 g, displacements were observed to be within 3.56% of the expected value. MC3T3-E1 osteoblast like cells were seeded into the microfluidic device and loaded with the calcium sensitive fluorescent probe fura-2, then mounted onto the dual-modal mechanostimulation platform. Cells were then imaged and monitored for fluorescence emission. In summary, we have developed a system to deliver physiologically relevant vibrations and fluid shear to live cells during real-time imaging and photometry. Monitoring the behavior of live cells loaded with appropriate fluorescent probes will enable characterization of the signals activated during the initial stages of mechanotransduction.

The preparation of Drosophila embryos for live-imaging using the hanging drop protocol.

PubMed

Reed, Bruce H; McMillan, Stephanie C; Chaudhary, Roopali

2009-03-13

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

High-speed adaptive optics for imaging of the living human eye

PubMed Central

Yu, Yongxin; Zhang, Tianjiao; Meadway, Alexander; Wang, Xiaolin; Zhang, Yuhua

2015-01-01

The discovery of high frequency temporal fluctuation of human ocular wave aberration dictates the necessity of high speed adaptive optics (AO) correction for high resolution retinal imaging. We present a high speed AO system for an experimental adaptive optics scanning laser ophthalmoscope (AOSLO). We developed a custom high speed Shack-Hartmann wavefront sensor and maximized the wavefront detection speed based upon a trade-off among the wavefront spatial sampling density, the dynamic range, and the measurement sensitivity. We examined the temporal dynamic property of the ocular wavefront under the AOSLO imaging condition and improved the dual-thread AO control strategy. The high speed AO can be operated with a closed-loop frequency up to 110 Hz. Experiment results demonstrated that the high speed AO system can provide improved compensation for the wave aberration up to 30 Hz in the living human eye. PMID:26368408

Live-cell imaging of neurofilament transport in cultured neurons.

PubMed

Uchida, Atsuko; Monsma, Paula C; Fenn, J Daniel; Brown, Anthony

2016-01-01

Neurofilaments, which are the intermediate filaments of nerve cells, are space-filling cytoskeletal polymers that contribute to the growth of axonal caliber. In addition to their structural role, neurofilaments are cargos of axonal transport that move along microtubule tracks in a rapid, intermittent, and bidirectional manner. Though they measure just 10nm in diameter, which is well below the diffraction limit of optical microscopes, these polymers can reach 100 μm or more in length and are often packed densely, just tens of nanometers apart. These properties of neurofilaments present unique challenges for studies on their movement. In this article, we describe several live-cell fluorescence imaging strategies that we have developed to image neurofilament transport in axons of cultured neurons on short and long timescales. Together, these methods form a powerful set of complementary tools with which to study the axonal transport of these unique intracellular cargos. Copyright © 2016 Elsevier Inc. All rights reserved.

Localized Chemical Remodeling for Live Cell Imaging of Protein-Specific Glycoform.

PubMed

Hui, Jingjing; Bao, Lei; Li, Siqiao; Zhang, Yi; Feng, Yimei; Ding, Lin; Ju, Huangxian

2017-07-03

Live cell imaging of protein-specific glycoforms is important for the elucidation of glycosylation mechanisms and identification of disease states. The currently used metabolic oligosaccharide engineering (MOE) technology permits routinely global chemical remodeling (GCM) for carbohydrate site of interest, but can exert unnecessary whole-cell scale perturbation and generate unpredictable metabolic efficiency issue. A localized chemical remodeling (LCM) strategy for efficient and reliable access to protein-specific glycoform information is reported. The proof-of-concept protocol developed for MUC1-specific terminal galactose/N-acetylgalactosamine (Gal/GalNAc) combines affinity binding, off-on switchable catalytic activity, and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging. Noteworthy assay features associated with LCM as compared with MOE include minimum target cell perturbation, short reaction timeframe, effectiveness as a molecular ruler, and quantitative analysis capability. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plastic fiber optics for micro-imaging of fluorescence signals in living cells

NASA Astrophysics Data System (ADS)

Sakurai, Takashi; Natsume, Mitsuo; Koida, Kowa

2015-03-01

The fiber-coupled microscope (FCM) enables in vivo imaging at deep sites in the tissues or organs that other optical techniques are unable to reach. To develop FCM-based intravital imaging, we employed a plastic optical fiber (POF) bundle that included more than 10,000-units of polystyrene core and polymethyl methacrylate cladding. Each POF had a diameter of less than 5 μm the tip of the bundle was less than 0.5 mm wide, and the flexible wire had a length of 1,000 mm. The optical performance of the plastic FCM was sufficient for detection of significant signal changes in an acinus of rat pancreas labeled with a calcium ion-sensitive fluorescent dye. In the future, the potential power of plastic FCM is expected to increase, enabling analysis of structure and organization of specific functions in live cells within vulnerable organs.

Proton-Fueled, Reversible DNA Hybridization Chain Assembly for pH Sensing and Imaging.

PubMed

Liu, Lan; Liu, Jin-Wen; Huang, Zhi-Mei; Wu, Han; Li, Na; Tang, Li-Juan; Jiang, Jian-Hui

2017-07-05

Design of DNA self-assembly with reversible responsiveness to external stimuli is of great interest for diverse applications. We for the first time develop a pH-responsive, fully reversible hybridization chain reaction (HCR) assembly that allows sensitive sensing and imaging of pH in living cells. Our design relies on the triplex forming sequences that form DNA triplex with toehold regions under acidic conditions and then induce a cascade of strand displacement and DNA assembly. The HCR assembly has shown dynamic responses in physiological pH ranges with excellent reversibility and demonstrated the potential for in vitro detection and live-cell imaging of pH. Moreover, this method affords HCR assemblies with highly localized fluorescence responses, offering advantages of improving sensitivity and better selectivity. The proton-fueled, reversible HCR assembly may provide a useful approach for pH-related cell biology study and disease diagnostics.

Luciferase Protein Complementation Assays for Bioluminescence Imaging of Cells and Mice

PubMed Central

Luker, Gary D.; Luker, Kathryn E.

2015-01-01

Summary Protein fragment complementation assays (PCAs) with luciferase reporters currently are the preferred method for detecting and quantifying protein-protein interactions in living animals. At the most basic level, PCAs involve fusion of two proteins of interest to enzymatically inactive fragments of luciferase. Upon association of the proteins of interest, the luciferase fragments are capable of reconstituting enzymatic activity to generate luminescence in vivo. In addition to bi-molecular luciferase PCAs, unimolecular biosensors for hormones, kinases, and proteases also have been developed using target peptides inserted between inactive luciferase fragments. Luciferase PCAs offer unprecedented opportunities to quantify dynamics of protein-protein interactions in intact cells and living animals, but successful use of luciferase PCAs in cells and mice involves careful consideration of many technical factors. This chapter discusses the design of luciferase PCAs appropriate for animal imaging, including construction of reporters, incorporation of reporters into cells and mice, imaging techniques, and data analysis. PMID:21153371

Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

PubMed

Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

2018-01-01

Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real-time measurements of the ratio in living, functioning liver cells, overcoming many limitations of previous methods for measuring this important redox parameter. The feasibility of using Peredox in liver slices is particularly attractive because slices allow preservation of hepatic microanatomy and metabolic zonation of hepatocytes.

Longitudinal in vivo two-photon fluorescence imaging

PubMed Central

Crowe, Sarah E.; Ellis-Davies, Graham C.R.

2014-01-01

Fluorescence microscopy is an essential technique for the basic sciences, especially biomedical research. Since the invention of laser scanning confocal microscopy in 1980s, that enabled imaging both fixed and living biological tissue with three-dimensional precision, high-resolution fluorescence imaging has revolutionized biological research. Confocal microscopy, by its very nature, has one fundamental limitation. Due to the confocal pinhole, deep tissue fluorescence imaging is not practical. In contrast (no pun intended), two-photon fluorescence microscopy allows, in principle, the collection of all emitted photons from fluorophores in the imaged voxel, dramatically extending our ability to see deep into living tissue. Since the development of transgenic mice with genetically encoded fluorescent protein in neocortical cells in 2000, two-photon imaging has enabled the dynamics of individual synapses to be followed for up to two years. Since the initial landmark contributions to this field in 2002, the technique has been used to understand how neuronal structure are changed by experience, learning and memory and various diseases. Here we provide a basic summary of the crucial elements that are required for such studies, and discuss many applications of longitudinal two-photon fluorescence microscopy that have appeared since 2002. PMID:24214350

The Association Between Body Image and Smoking Cessation Among Individuals Living with HIV/AIDS

PubMed Central

Fingeret, Michelle Cororve; Vidrine, Damon J.; Arduino, Roberto C.; Gritz, Ellen R.

2007-01-01

Lower smoking cessation rates are associated with body image concerns in the general population. This relationship is particularly important to study in individuals living with HIV/AIDS due to alarmingly high smoking rates and considerable bodily changes experienced with HIV disease progression and treatment. The association between body image and smoking cessation rates was examined among individuals living with HIV/AIDS participating in a smoking cessation intervention. Body image concerns were significantly associated with depression, anxiety, stress, and social support, all variables known to affect cessation rates. However, reduced quit rates were found among individuals reporting elevated and low levels of body image concerns at the end of treatment. These findings suggest a unique relationship between smoking and body image among individuals living with HIV/AIDS. Further research is needed to examine these effects and whether moderate levels of body image concerns in this population reflect realistic body perceptions associated with positive mental health. PMID:18089265

Computational method for multi-modal microscopy based on transport of intensity equation

NASA Astrophysics Data System (ADS)

Li, Jiaji; Chen, Qian; Sun, Jiasong; Zhang, Jialin; Zuo, Chao

2017-02-01

In this paper, we develop the requisite theory to describe a hybrid virtual-physical multi-modal imaging system which yields quantitative phase, Zernike phase contrast, differential interference contrast (DIC), and light field moment imaging simultaneously based on transport of intensity equation(TIE). We then give the experimental demonstration of these ideas by time-lapse imaging of live HeLa cell mitosis. Experimental results verify that a tunable lens based TIE system, combined with the appropriate post-processing algorithm, can achieve a variety of promising imaging modalities in parallel with the quantitative phase images for the dynamic study of cellular processes.

Development and evaluation of a novel, real time mobile telesonography system in management of patients with abdominal trauma: study protocol.

PubMed

Ogedegbe, Chinwe; Morchel, Herman; Hazelwood, Vikki; Chaplin, William F; Feldman, Joseph

2012-12-18

Despite the use of e-FAST in management of patients with abdominal trauma, its utility in prehospital setting is not widely adopted. The goal of this study is to develop a novel portable telesonography (TS) system and evaluate the comparability of the quality of images obtained via this system among healthy volunteers who undergo e-FAST abdominal examination in a moving ambulance and at the ED. We hypothesize that: (1) real-time ultrasound images of acute trauma patients in the pre-hospital setting can be obtained and transmitted to the ED via the novel TS system; and (2) Ultrasound images transmitted to the hospital from the real-time TS system will be comparable in quality to those obtained in the ED. Study participants are three healthy volunteers (one each with normal, overweight and obese BMI category). The ultrasound images will be obtained by two ultrasound-trained physicians The TS is a portable sonogram (by Sonosite) interfaced with a portable broadcast unit (by Live-U). Two UTPs will conduct e-FAST examinations on healthy volunteers in moving ambulances and transmit the images via cellular network to the hospital server, where they are stored. Upon arrival in the ED, the same UTPs will obtain another set of images from the volunteers, which are then compared to those obtained in the moving ambulances by another set of blinded UTPs (evaluators) using a validated image quality scale, the Questionnaire for User Interaction Satisfaction (QUIS). Findings from this study will provide needed data on the validity of the novel TS in transmitting live images from moving ambulances to images obtained in the ED thus providing opportunity to facilitate medical care of a patient located in a remote or austere setting.

An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology

PubMed Central

Littlejohn, George R.; Mansfield, Jessica C.; Christmas, Jacqueline T.; Witterick, Eleanor; Fricker, Mark D.; Grant, Murray R.; Smirnoff, Nicholas; Everson, Richard M.; Moger, Julian; Love, John

2014-01-01

Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the “negative space” within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells. PMID:24795734

A turn-on fluorescent probe for endogenous formaldehyde in the endoplasmic reticulum of living cells

NASA Astrophysics Data System (ADS)

Tang, Yonghe; Ma, Yanyan; Xu, An; Xu, Gaoping; Lin, Weiying

2017-06-01

As the simplest aldehyde compounds, formaldehyde (FA) is implicated in nervous system diseases and cancer. Endoplasmic reticulum is an organelle that plays important functions in living cells. Accordingly, the development of efficient methods for FA detection in the endoplasmic reticulum (ER) is of great biomedical importance. In this work, we developed the first ER-targeted fluorescent FA probe Na-FA-ER. The detection is based on the condensation reaction of the hydrazine group and FA to suppress the photo-induced electron transfer (PET) pathway, resulting in a fluorescence increase. The novel Na-FA-ER showed high sensitivity to FA. In addition, the Na-FA-ER enabled the bio-imaging of exogenous and endogenous FA in living HeLa cells. Most significantly, the new Na-FA-ER was employed to visualize the endogenous FA in the ER in living cells for the first time.

Real-Time In Vivo Imaging of Butterfly Wing Development: Revealing the Cellular Dynamics of the Pupal Wing Tissue

PubMed Central

Iwata, Masaki; Ohno, Yoshikazu; Otaki, Joji M.

2014-01-01

Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the cellular dynamics of living wing tissues in butterflies. PMID:24586829

Real-time in vivo imaging of butterfly wing development: revealing the cellular dynamics of the pupal wing tissue.

PubMed

Iwata, Masaki; Ohno, Yoshikazu; Otaki, Joji M

2014-01-01

Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the cellular dynamics of living wing tissues in butterflies.

Line-Scanning Particle Image Velocimetry: An Optical Approach for Quantifying a Wide Range of Blood Flow Speeds in Live Animals

PubMed Central

Kim, Tyson N.; Goodwill, Patrick W.; Chen, Yeni; Conolly, Steven M.; Schaffer, Chris B.; Liepmann, Dorian; Wang, Rong A.

2012-01-01

Background The ability to measure blood velocities is critical for studying vascular development, physiology, and pathology. A key challenge is to quantify a wide range of blood velocities in vessels deep within living specimens with concurrent diffraction-limited resolution imaging of vascular cells. Two-photon laser scanning microscopy (TPLSM) has shown tremendous promise in analyzing blood velocities hundreds of micrometers deep in animals with cellular resolution. However, current analysis of TPLSM-based data is limited to the lower range of blood velocities and is not adequate to study faster velocities in many normal or disease conditions. Methodology/Principal Findings We developed line-scanning particle image velocimetry (LS-PIV), which used TPLSM data to quantify peak blood velocities up to 84 mm/s in live mice harboring brain arteriovenous malformation, a disease characterized by high flow. With this method, we were able to accurately detect the elevated blood velocities and exaggerated pulsatility along the abnormal vascular network in these animals. LS-PIV robustly analyzed noisy data from vessels as deep as 850 µm below the brain surface. In addition to analyzing in vivo data, we validated the accuracy of LS-PIV up to 800 mm/s using simulations with known velocity and noise parameters. Conclusions/Significance To our knowledge, these blood velocity measurements are the fastest recorded with TPLSM. Partnered with transgenic mice carrying cell-specific fluorescent reporters, LS-PIV will also enable the direct in vivo correlation of cellular, biochemical, and hemodynamic parameters in high flow vascular development and diseases such as atherogenesis, arteriogenesis, and vascular anomalies. PMID:22761686

Development of fluorescence based handheld imaging devices for food safety inspection

NASA Astrophysics Data System (ADS)

Lee, Hoyoung; Kim, Moon S.; Chao, Kuanglin; Lefcourt, Alan M.; Chan, Diane E.

2013-05-01

For sanitation inspection in food processing environment, fluorescence imaging can be a very useful method because many organic materials reveal unique fluorescence emissions when excited by UV or violet radiation. Although some fluorescence-based automated inspection instrumentation has been developed for food products, there remains a need for devices that can assist on-site inspectors performing visual sanitation inspection of the surfaces of food processing/handling equipment. This paper reports the development of an inexpensive handheld imaging device designed to visualize fluorescence emissions and intended to help detect the presence of fecal contaminants, organic residues, and bacterial biofilms at multispectral fluorescence emission bands. The device consists of a miniature camera, multispectral (interference) filters, and high power LED illumination. With WiFi communication, live inspection images from the device can be displayed on smartphone or tablet devices. This imaging device could be a useful tool for assessing the effectiveness of sanitation procedures and for helping processors to minimize food safety risks or determine potential problem areas. This paper presents the design and development including evaluation and optimization of the hardware components of the imaging devices.

SU-E-I-91: Quantitative Assessment of Early Hepatocellular Carcinoma and Cavernous Hemangioma of Live Using In-Line Phase-Contrast X-Ray Imaging

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

Duan, J

Purpose: To investigate the potential utility of in-line phase-contrast imaging (ILPCI) technique with synchrotron radiation in detecting early hepatocellular carcinoma and cavernous hemangioma of live using in vitro model system. Methods: Without contrast agents, three typical early hepatocellular carcinoma specimens and three typical cavernous hemangioma of live specimens were imaged using ILPCI. To quantitatively discriminate early hepatocellular carcinoma tissues and cavernous hemangioma tissues, the projection images texture feature based on gray level co-occurrence matrix (GLCM) were extracted. The texture parameters of energy, inertia, entropy, correlation, sum average, sum entropy, difference average, difference entropy and inverse difference moment, were obtained respectively.more » Results: In the ILPCI planar images of early hepatocellular carcinoma specimens, vessel trees were clearly visualized on the micrometer scale. Obvious distortion deformation was presented, and the vessel mostly appeared as a ‘dry stick’. Liver textures appeared not regularly. In the ILPCI planar images of cavernous hemangioma of live specimens, typical vessels had not been found compared with the early hepatocellular carcinoma planar images. The planar images of cavernous hemangioma of live specimens clearly displayed the dilated hepatic sinusoids with the diameter of less than 100 microns, but all of them were overlapped with each other. The texture parameters of energy, inertia, entropy, correlation, sum average, sum entropy, and difference average, showed a statistically significant between the two types specimens image (P<0.01), except the texture parameters of difference entropy and inverse difference moment(P>0.01). Conclusion: The results indicate that there are obvious changes in morphological levels including vessel structures and liver textures. The study proves that this imaging technique has a potential value in evaluating early hepatocellular carcinoma and cavernous hemangioma of live.« less

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles.

PubMed

Seynhaeve, Ann L B; Ten Hagen, Timo L M

2017-11-01

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Label-free optical imaging of nonfluorescent molecules by stimulated radiation.

PubMed

Min, Wei

2011-12-01

Imaging contrasts other than fluorescence are highly desirable for label-free detection and interrogation of nonfluorescent molecular species inside live cells, tissues, and organisms. The recently developed stimulated Raman scattering (SRS) and stimulated emission microscopy techniques provide sensitive and specific contrast mechanisms for nonfluorescent species, by employing the light amplification aspect of stimulated radiation. Compared to their spontaneous counterparts, stimulated radiation can enhance the imaging performance significantly, making the previously 'dark' molecules observable. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques. Copyright © 2011 Elsevier Ltd. All rights reserved.

Demonstration of in-vivo Multi-Probe Tracker Based on a Si/CdTe Semiconductor Compton Camera

NASA Astrophysics Data System (ADS)

Takeda, Shin'ichiro; Odaka, Hirokazu; Ishikawa, Shin-nosuke; Watanabe, Shin; Aono, Hiroyuki; Takahashi, Tadayuki; Kanayama, Yousuke; Hiromura, Makoto; Enomoto, Shuichi

2012-02-01

By using a prototype Compton camera consisting of silicon (Si) and cadmium telluride (CdTe) semiconductor detectors, originally developed for the ASTRO-H satellite mission, an experiment involving imaging multiple radiopharmaceuticals injected into a living mouse was conducted to study its feasibility for medical imaging. The accumulation of both iodinated (131I) methylnorcholestenol and 85Sr into the mouse's organs was simultaneously imaged by the prototype. This result implies that the Compton camera is expected to become a multi-probe tracker available in nuclear medicine and small animal imaging.

Photon-Counting H33D Detector for Biological Fluorescence Imaging

PubMed Central

Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Millaud, J.E.; Weiss, S.

2010-01-01

We have developed a photon-counting High-temporal and High-spatial resolution, High-throughput 3-Dimensional detector (H33D) for biological imaging of fluorescent samples. The design is based on a 25 mm diameter S20 photocathode followed by a 3-microchannel plate stack, and a cross delay line anode. We describe the bench performance of the H33D detector, as well as preliminary imaging results obtained with fluorescent beads, quantum dots and live cells and discuss applications of future generation detectors for single-molecule imaging and high-throughput study of biomolecular interactions. PMID:20151021

Live-cell imaging.

PubMed

Cole, Richard

2014-01-01

It would be hard to argue that live-cell imaging has not changed our view of biology. The past 10 years have seen an explosion of interest in imaging cellular processes, down to the molecular level. There are now many advanced techniques being applied to live cell imaging. However, cellular health is often under appreciated. For many researchers, if the cell at the end of the experiment has not gone into apoptosis or is blebbed beyond recognition, than all is well. This is simply incorrect. There are many factors that need to be considered when performing live-cell imaging in order to maintain cellular health such as: imaging modality, media, temperature, humidity, PH, osmolality, and photon dose. The wavelength of illuminating light, and the total photon dose that the cells are exposed to, comprise two of the most important and controllable parameters of live-cell imaging. The lowest photon dose that achieves a measureable metric for the experimental question should be used, not the dose that produces cover photo quality images. This is paramount to ensure that the cellular processes being investigated are in their in vitro state and not shifted to an alternate pathway due to environmental stress. The timing of the mitosis is an ideal canary in the gold mine, in that any stress induced from the imaging will result in the increased length of mitosis, thus providing a control model for the current imagining conditions.

Live-cell imaging

PubMed Central

Cole, Richard

2014-01-01

It would be hard to argue that live-cell imaging has not changed our view of biology. The past 10 years have seen an explosion of interest in imaging cellular processes, down to the molecular level. There are now many advanced techniques being applied to live cell imaging. However, cellular health is often under appreciated. For many researchers, if the cell at the end of the experiment has not gone into apoptosis or is blebbed beyond recognition, than all is well. This is simply incorrect. There are many factors that need to be considered when performing live-cell imaging in order to maintain cellular health such as: imaging modality, media, temperature, humidity, PH, osmolality, and photon dose. The wavelength of illuminating light, and the total photon dose that the cells are exposed to, comprise two of the most important and controllable parameters of live-cell imaging. The lowest photon dose that achieves a measureable metric for the experimental question should be used, not the dose that produces cover photo quality images. This is paramount to ensure that the cellular processes being investigated are in their in vitro state and not shifted to an alternate pathway due to environmental stress. The timing of the mitosis is an ideal canary in the gold mine, in that any stress induced from the imaging will result in the increased length of mitosis, thus providing a control model for the current imagining conditions. PMID:25482523

Imaging modes of atomic force microscopy for application in molecular and cell biology.

PubMed

Dufrêne, Yves F; Ando, Toshio; Garcia, Ricardo; Alsteens, David; Martinez-Martin, David; Engel, Andreas; Gerber, Christoph; Müller, Daniel J

2017-04-06

Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

PubMed

Peng, Tao; Hang, Howard C

2016-11-02

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Three-Dimensional Unstained Live-Cell Imaging Using Stimulated Parametric Emission Microscopy

NASA Astrophysics Data System (ADS)

Dang, Hieu M.; Kawasumi, Takehito; Omura, Gen; Umano, Toshiyuki; Kajiyama, Shin'ichiro; Ozeki, Yasuyuki; Itoh, Kazuyoshi; Fukui, Kiichi

2009-09-01

The ability to perform high-resolution unstained live imaging is very important to in vivo study of cell structures and functions. Stimulated parametric emission (SPE) microscopy is a nonlinear-optical microscopy based on ultra-fast electronic nonlinear-optical responses. For the first time, we have successfully applied this technique to archive three-dimensional (3D) images of unstained sub-cellular structures, such as, microtubules, nuclei, nucleoli, etc. in live cells. Observation of a complete cell division confirms the ability of SPE microscopy for long time-scale imaging.

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

PubMed

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

2015-01-01

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

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

PubMed Central

Zhang, Ming; Chakraborty, Subhasish K.; Sampath, Padma; Rojas, Juan J.; Hou, Weizhou; Saurabh, Saumya; Thorne, Steve H.; Bruchez, Marcel P.; Waggoner, Alan S.

2015-01-01

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule–based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter–tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene. PMID:26348895

PET radiopharmaceuticals for imaging of tumor hypoxia: a review of the evidence

PubMed Central

Lopci, Egesta; Grassi, Ilaria; Chiti, Arturo; Nanni, Cristina; Cicoria, Gianfranco; Toschi, Luca; Fonti, Cristina; Lodi, Filippo; Mattioli, Sandro; Fanti, Stefano

2014-01-01

Hypoxia is a pathological condition arising in living tissues when oxygen supply does not adequately cover the cellular metabolic demand. Detection of this phenomenon in tumors is of the utmost clinical relevance because tumor aggressiveness, metastatic spread, failure to achieve tumor control, increased rate of recurrence, and ultimate poor outcome are all associated with hypoxia. Consequently, in recent decades there has been increasing interest in developing methods for measurement of oxygen levels in tumors. Among the image-based modalities for hypoxia assessment, positron emission tomography (PET) is one of the most extensively investigated based on the various advantages it offers, i.e., broad range of radiopharmaceuticals, good intrinsic resolution, three-dimensional tumor representation, possibility of semiquantification/quantification of the amount of hypoxic tumor burden, overall patient friendliness, and ease of repetition. Compared with the other non-invasive techniques, the biggest advantage of PET imaging is that it offers the highest specificity for detection of hypoxic tissue. Starting with the 2-nitroimidazole family of compounds in the early 1980s, a great number of PET tracers have been developed for the identification of hypoxia in living tissue and solid tumors. This paper provides an overview of the principal PET tracers applied in cancer imaging of hypoxia and discusses in detail their advantages and pitfalls. PMID:24982822

Image-guided tissue engineering

PubMed Central

Ballyns, Jeffrey J; Bonassar, Lawrence J

2009-01-01

Replication of anatomic shape is a significant challenge in developing implants for regenerative medicine. This has lead to significant interest in using medical imaging techniques such as magnetic resonance imaging and computed tomography to design tissue engineered constructs. Implementation of medical imaging and computer aided design in combination with technologies for rapid prototyping of living implants enables the generation of highly reproducible constructs with spatial resolution up to 25 μm. In this paper, we review the medical imaging modalities available and a paradigm for choosing a particular imaging technique. We also present fabrication techniques and methodologies for producing cellular engineered constructs. Finally, we comment on future challenges involved with image guided tissue engineering and efforts to generate engineered constructs ready for implantation. PMID:19583811

EYE DEVELOPMENT

PubMed Central

Baker, Nicholas E.; Li, Ke; Quiquand, Manon; Ruggiero, Robert; Wang, Lan-Hsin

2014-01-01

The eye has been one of the most intensively studied organs in Drosophila. The wealth of knowledge about its development, as well as the reagents that have been developed, and the fact that the eye is dispensable for survival, also make the eye suitable for genetic interaction studies and genetic screens. This chapter provides a brief overview of the methods developed to image and probe eye development at multiple developmental stages, including live imaging, immunostaining of fixed tissues, in situ hybridizations, and scanning electron microscopy and color photography of adult eyes. Also summarized are genetic approaches that can be performed in the eye, including mosaic analysis and conditional mutation, gene misexpression and knockdown, and forward genetic and modifier screens. PMID:24784530

Scanless nonlinear optical microscope for image reconstruction and space-time correlation analysis

NASA Astrophysics Data System (ADS)

Ceffa, N. G.; Radaelli, F.; Pozzi, P.; Collini, M.; Sironi, L.; D'alfonso, L.; Chirico, G.

2017-06-01

Optical Microscopy has been applied to life science from its birth and reached widespread application due to its major advantages: limited perturbation of the biological tissue and the easy accessibility of the light sources. However, as the spatial and time resolution requirements and the time stability of the microscopes increase, researchers are struggling against some of its limitations: limited transparency and the refractivity of the living tissue to light and the field perturbations induced by the path in the tissue. We have developed a compact stand-alone, completely scan-less, optical setup that allows to acquire non-linear excitation images and to measure the sample dynamics simultaneously on an ensemble of arbitrary chosen regions of interests. The image is obtained by shining a square array of spots on the sample obtained by a spatial light modulator and by shifting it (10 ms refresh time) on the sample. The final image is computed from the superposition of (100-1000) images. Filtering procedures can be applied to the raw images of the excitation array before building the image. We discuss results that show how this setup can be used for the correction of wave front aberrations induced by turbid samples (such as living tissues) and for the computation of space-time cross-correlations in complex networks.

Single-cell resolution fluorescence imaging of circadian rhythms detected with a Nipkow spinning disk confocal system.

PubMed

Enoki, Ryosuke; Ono, Daisuke; Hasan, Mazahir T; Honma, Sato; Honma, Ken-Ichi

2012-05-30

Single-point laser scanning confocal imaging produces signals with high spatial resolution in living organisms. However, photo-induced toxicity, bleaching, and focus drift remain challenges, especially when recording over several days for monitoring circadian rhythms. Bioluminescence imaging is a tool widely used for this purpose, and does not cause photo-induced difficulties. However, bioluminescence signals are dimmer than fluorescence signals, and are potentially affected by levels of cofactors, including ATP, O(2), and the substrate, luciferin. Here we describe a novel time-lapse confocal imaging technique to monitor circadian rhythms in living tissues. The imaging system comprises a multipoint scanning Nipkow spinning disk confocal unit and a high-sensitivity EM-CCD camera mounted on an inverted microscope with auto-focusing function. Brain slices of the suprachiasmatic nucleus (SCN), the central circadian clock, were prepared from transgenic mice expressing a clock gene, Period 1 (Per1), and fluorescence reporter protein (Per1::d2EGFP). The SCN slices were cut out together with membrane, flipped over, and transferred to the collagen-coated glass dishes to obtain signals with a high signal-to-noise ratio and to minimize focus drift. The imaging technique and improved culture method enabled us to monitor the circadian rhythm of Per1::d2EGFP from optically confirmed single SCN neurons without noticeable photo-induced effects or focus drift. Using recombinant adeno-associated virus carrying a genetically encoded calcium indicator, we also monitored calcium circadian rhythms at a single-cell level in a large population of SCN neurons. Thus, the Nipkow spinning disk confocal imaging system developed here facilitates long-term visualization of circadian rhythms in living cells. Copyright © 2012 Elsevier B.V. All rights reserved.

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells.

PubMed

Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W; Gautier, Virginie W

2015-01-01

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip.

MATtrack: A MATLAB-Based Quantitative Image Analysis Platform for Investigating Real-Time Photo-Converted Fluorescent Signals in Live Cells

PubMed Central

Courtney, Jane; Woods, Elena; Scholz, Dimitri; Hall, William W.; Gautier, Virginie W.

2015-01-01

We introduce here MATtrack, an open source MATLAB-based computational platform developed to process multi-Tiff files produced by a photo-conversion time lapse protocol for live cell fluorescent microscopy. MATtrack automatically performs a series of steps required for image processing, including extraction and import of numerical values from Multi-Tiff files, red/green image classification using gating parameters, noise filtering, background extraction, contrast stretching and temporal smoothing. MATtrack also integrates a series of algorithms for quantitative image analysis enabling the construction of mean and standard deviation images, clustering and classification of subcellular regions and injection point approximation. In addition, MATtrack features a simple user interface, which enables monitoring of Fluorescent Signal Intensity in multiple Regions of Interest, over time. The latter encapsulates a region growing method to automatically delineate the contours of Regions of Interest selected by the user, and performs background and regional Average Fluorescence Tracking, and automatic plotting. Finally, MATtrack computes convenient visualization and exploration tools including a migration map, which provides an overview of the protein intracellular trajectories and accumulation areas. In conclusion, MATtrack is an open source MATLAB-based software package tailored to facilitate the analysis and visualization of large data files derived from real-time live cell fluorescent microscopy using photoconvertible proteins. It is flexible, user friendly, compatible with Windows, Mac, and Linux, and a wide range of data acquisition software. MATtrack is freely available for download at eleceng.dit.ie/courtney/MATtrack.zip. PMID:26485569

On measuring cell confluence in phase contrast microscopy

NASA Astrophysics Data System (ADS)

Dempsey, K. P.; Richardson, J. B.; Lam, K. P.

2014-03-01

A principal focus highlighting recent advances in cell based therapies concerns the development of effective treatments for osteoarthritis. Earlier clinicaltrials have shown that 80% of patients receiving mesenchymal stem cell(MSC) based treatment have improved their quality of life by alleviating pain whilst extending the life of their natural joints. The current challenge facing researchers is to identify the biological differences between the treatments that have worked and those which have shown little improvement. One possible candidate for the difference in treatment prognosis is an examination of the proliferation of the ( type) cells as they grow. To further understanding of the proliferation and differentiation of MSC, non-invasive live cell imaging techniques have been developed which capture important cell events and dynamics in cell divisions over an extended period of time. An automated image analysis procedure capable of tracking cell confluence over time has also been implemented, providing an objective and realistic estimation of cell growth within continuous live cell cultures. The proposed algorithm accounts for the halo artefacts that occur in phase microscopy. In addition to a favourable run-time performance, the method was also validated using continuous live MSC cultures, with consistent and meaningful results.

Spinning Disk Confocal Imaging of Neutrophil Migration in Zebrafish

PubMed Central

Lam, Pui-ying; Fischer, Robert S; Shin, William D.; Waterman, Clare M; Huttenlocher, Anna

2014-01-01

Live-cell imaging techniques have been substantially improved due to advances in confocal microscopy instrumentation coupled with ultrasensitive detectors. The spinning disk confocal system is capable of generating images of fluorescent live samples with broad dynamic range and high temporal and spatial resolution. The ability to acquire fluorescent images of living cells in vivo on a millisecond timescale allows the dissection of biological processes that have not previously been visualized in a physiologically relevant context. In vivo imaging of rapidly moving cells such as neutrophils can be technically challenging. In this chapter, we describe the practical aspects of imaging neutrophils in zebrafish embryos using spinning disk confocal microscopy. Similar setups can also be applied to image other motile cell types and signaling processes in translucent animals or tissues. PMID:24504955

Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice.

PubMed

Bhaumik, S; Lewis, X Z; Gambhir, S S

2004-01-01

We have recently demonstrated that Renilla luciferase (Rluc) is a promising bioluminescence reporter gene that can be used for noninvasive optical imaging of reporter gene expression in living mice, with the aid of a cooled charged couple device (CCD) camera. In the current study, we explore the expression of a novel synthetic Renilla luciferase reporter gene (hRluc) in living mice, which has previously been reported to be a more sensitive reporter than native Rluc in mammalian cells. We explore the strategies of simultaneous imaging of both Renilla luciferase enzyme (RL) and synthetic Renilla luciferase enzyme (hRL):coelenterazine (substrate for RL/hRL) in the same living mouse. We also demonstrate that hRL:coelenterazine can yield a higher signal when compared to Firefly luciferase enzyme (FL): D-Luciferin, both in cell culture studies and when imaged from cells at the surface and from lungs of living mice. These studies demonstrate that hRluc should be a useful primary reporter gene with high sensitivity when used alone or in conjunction with other bioluminescence reporter genes for imaging in living rodents. (c) 2004 Society of Photo-Optical Instrumentation Engineers.

Nanoscale live cell imaging using hopping probe ion conductance microscopy

PubMed Central

Novak, Pavel; Li, Chao; Shevchuk, Andrew I.; Stepanyan, Ruben; Caldwell, Matthew; Hughes, Simon; Smart, Trevor G.; Gorelik, Julia; Ostanin, Victor P.; Lab, Max J.; Moss, Guy W. J.; Frolenkov, Gregory I.; Klenerman, David; Korchev, Yuri E.

2009-01-01

We describe a major advance in scanning ion conductance microscopy: a new hopping mode that allows non-contact imaging of the complex surfaces of live cells with resolution better than 20 nm. The effectiveness of this novel technique was demonstrated by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allows studying nanoscale phenomena on the surface of live cells under physiological conditions. PMID:19252505

Anatomically Realistic Three-Dimensional Meshes of the Pelvic Floor & Anal Canal for Finite Element Analysis

PubMed Central

Noakes, Kimberley F.; Bissett, Ian P.; Pullan, Andrew J.; Cheng, Leo K.

2014-01-01

Three anatomically realistic meshes, suitable for finite element analysis, of the pelvic floor and anal canal regions have been developed to provide a framework with which to examine the mechanics, via finite element analysis of normal function within the pelvic floor. Two cadaver-based meshes were produced using the Visible Human Project (male and female) cryosection data sets, and a third mesh was produced based on MR image data from a live subject. The Visible Man (VM) mesh included 10 different pelvic structures while the Visible Woman and MRI meshes contained 14 and 13 structures respectively. Each image set was digitized and then finite element meshes were created using an iterative fitting procedure with smoothing constraints calculated from ‘L’-curves. These weights produced accurate geometric meshes of each pelvic structure with average Root Mean Square (RMS) fitting errors of less than 1.15 mm. The Visible Human cadaveric data provided high resolution images, however, the cadaveric meshes lacked the normal dynamic form of living tissue and suffered from artifacts related to postmortem changes. The lower resolution MRI mesh was able to accurately portray structure of the living subject and paves the way for dynamic, functional modeling. PMID:18317929

Optical Coherence Tomography for Retinal Surgery: Perioperative Analysis to Real-Time Four-Dimensional Image-Guided Surgery.

PubMed

Carrasco-Zevallos, Oscar M; Keller, Brenton; Viehland, Christian; Shen, Liangbo; Seider, Michael I; Izatt, Joseph A; Toth, Cynthia A

2016-07-01

Magnification of the surgical field using the operating microscope facilitated profound innovations in retinal surgery in the 1970s, such as pars plana vitrectomy. Although surgical instrumentation and illumination techniques are continually developing, the operating microscope for vitreoretinal procedures has remained essentially unchanged and currently limits the surgeon's depth perception and assessment of subtle microanatomy. Optical coherence tomography (OCT) has revolutionized clinical management of retinal pathology, and its introduction into the operating suite may have a similar impact on surgical visualization and treatment. In this article, we review the evolution of OCT for retinal surgery, from perioperative analysis to live volumetric (four-dimensional, 4D) image-guided surgery. We begin by briefly addressing the benefits and limitations of the operating microscope, the progression of OCT technology, and OCT applications in clinical/perioperative retinal imaging. Next, we review intraoperative OCT (iOCT) applications using handheld probes during surgical pauses, two-dimensional (2D) microscope-integrated OCT (MIOCT) of live surgery, and volumetric MIOCT of live surgery. The iOCT discussion focuses on technological advancements, applications during human retinal surgery, translational difficulties and limitations, and future directions.

Defining the Subcellular Interface of Nanoparticles by Live-Cell Imaging

PubMed Central

Hemmerich, Peter H.; von Mikecz, Anna H.

2013-01-01

Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes. PMID:23637951

Optical Coherence Tomography for Retinal Surgery: Perioperative Analysis to Real-Time Four-Dimensional Image-Guided Surgery

PubMed Central

Carrasco-Zevallos, Oscar M.; Keller, Brenton; Viehland, Christian; Shen, Liangbo; Seider, Michael I.; Izatt, Joseph A.; Toth, Cynthia A.

2016-01-01

Magnification of the surgical field using the operating microscope facilitated profound innovations in retinal surgery in the 1970s, such as pars plana vitrectomy. Although surgical instrumentation and illumination techniques are continually developing, the operating microscope for vitreoretinal procedures has remained essentially unchanged and currently limits the surgeon's depth perception and assessment of subtle microanatomy. Optical coherence tomography (OCT) has revolutionized clinical management of retinal pathology, and its introduction into the operating suite may have a similar impact on surgical visualization and treatment. In this article, we review the evolution of OCT for retinal surgery, from perioperative analysis to live volumetric (four-dimensional, 4D) image-guided surgery. We begin by briefly addressing the benefits and limitations of the operating microscope, the progression of OCT technology, and OCT applications in clinical/perioperative retinal imaging. Next, we review intraoperative OCT (iOCT) applications using handheld probes during surgical pauses, two-dimensional (2D) microscope-integrated OCT (MIOCT) of live surgery, and volumetric MIOCT of live surgery. The iOCT discussion focuses on technological advancements, applications during human retinal surgery, translational difficulties and limitations, and future directions. PMID:27409495

An interactive method based on the live wire for segmentation of the breast in mammography images.

PubMed

Zewei, Zhang; Tianyue, Wang; Li, Guo; Tingting, Wang; Lu, Xu

2014-01-01

In order to improve accuracy of computer-aided diagnosis of breast lumps, the authors introduce an improved interactive segmentation method based on Live Wire. This paper presents the Gabor filters and FCM clustering algorithm is introduced to the Live Wire cost function definition. According to the image FCM analysis for image edge enhancement, we eliminate the interference of weak edge and access external features clear segmentation results of breast lumps through improving Live Wire on two cases of breast segmentation data. Compared with the traditional method of image segmentation, experimental results show that the method achieves more accurate segmentation of breast lumps and provides more accurate objective basis on quantitative and qualitative analysis of breast lumps.

Fourier transform infrared spectroscopy imaging of live epithelial cancer cells under non-aqueous media.

PubMed

Soh, JunYi; Chueng, Adeline; Adio, Aminat; Cooper, Alan J; Birch, Brian R; Lwaleed, Bashir A

2013-04-01

Fourier transform infrared (FT-IR) imaging is increasingly being applied to biomedical specimens, but strong IR absorption by water complicates live cell imaging. This study investigates the viability of adherent epithelial cells maintained for short periods under mineral oils in order to facilitate live cell spectroscopy using FT-IR with subsequent imaging. The MGH-U1 urothelial or CaCo2 colorectal cancer cell lines were grown on plastic surfaces or mid-range infrared transparent windows. Medium in established cultures was replaced with paraffin mineral oil, or Fluorolube, for up to 2 h, and viability assessed by supravital staining. Drug handling characteristics were also assessed. Imaging of preparations was attempted by reflectance and transmission using a Varian FT-IR microscope. Cells covered by mineral oil remained viable for 2 h, with recovery into normal medium possible. MTT ((3-(4,5-dimethylthlazol-2-yl)-2,5-diphenyl tetrazolium) conversion to crystalline formazan and differential patterns of drug uptake were maintained. The combination of a calcium fluoride substrate, Fluorolube oil, and transmission optics proved best for spectroscopy. Spectral features were used to obtain images of live cells. The viability of cells overlaid with IR transparent oils was assessed as part of a technique to optimise conditions for FT-IR imaging. Images of untreated cells were obtained using both reflectance and transmission. This represents an effective means of imaging live cells by IR spectroscopy, and also means that imaging is not necessarily a terminal event. It also increases options for producing images based on real-time biochemistry in a range of in vitro experimental and 'optical biopsy' contexts.

Periorbital dirofilariasis—Clinical and imaging findings: Live worm on ultrasound

PubMed Central

Gopinath, Thandre N; Lakshmi, K P; Shaji, P C; Rajalakshmi, P C

2013-01-01

Ocular dirofilariasis is a zoonotic filariasis caused by nematode worm,Dirofilaria. We present a case of dirofilariasis affecting the upper eyelid in a 2-year-old child presenting as an acutely inflammed cyst, from southern Indian state of Kerala. Live adult worm was surgically removed and confirmed to be Dirofilaria repens. Live worm showing continuous movement was seen on the pre-operative high-resolution ultrasound. Ultrasound can be helpful in pre-operative identification of live worm. Imaging findings reported in literature are very few. We describe the clinical, ultrasound, and magnetic resonance imaging (MRI) findings. PMID:23803483

Spectro-microscopy of living plant cells.

PubMed

Harter, Klaus; Meixner, Alfred J; Schleifenbaum, Frank

2012-01-01

Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techniques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent developments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassinosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime (FLT). Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy (FIDSAM), has been developed. FIDSAM is capable of imaging low-expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of Förster resonance energy transfer (FRET) for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quantitative analysis of the photosystem I and II (PSI/PSII) ratio in the chloroplasts of living Arabidopsis plants at room temperature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiological and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.

Subcellular real-time in vivo imaging of intralymphatic and intravascular cancer-cell trafficking

NASA Astrophysics Data System (ADS)

McElroy, M.; Hayashi, K.; Kaushal, S.; Bouvet, M.; Hoffman, Robert M.

2008-02-01

With the use of fluorescent cells labeled with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and a highly sensitive small animal imaging system with both macro-optics and micro-optics, we have developed subcellular real-time imaging of cancer cell trafficking in live mice. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 small animal imaging system with a sensitive CCD camera and four objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. We have also developed real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with GFP and/or RFP were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked through lymphatic vessels where they were imaged via a skin flap in real-time at the cellular level until they entered the axillary lymph node. The bright dual-color fluorescence of the cancer cells and the real-time microscopic imaging capability of the Olympus OV100 enabled imaging the trafficking cancer cells in both blood vessels and lymphatics. With the dual-color cancer cells and the highly sensitive imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time.

Simultaneous immersion Mirau interferometry

PubMed Central

Lyulko, Oleksandra V.; Randers-Pehrson, Gerhard; Brenner, David J.

2013-01-01

A novel technique for label-free imaging of live biological cells in aqueous medium that is insensitive to ambient vibrations is presented. This technique is a spin-off from previously developed immersion Mirau interferometry. Both approaches utilize a modified Mirau interferometric attachment for a microscope objective that can be used both in air and in immersion mode, when the device is submerged in cell medium and has its internal space filled with liquid. While immersion Mirau interferometry involves first capturing a series of images, the resulting images are potentially distorted by ambient vibrations. Overcoming these serial-acquisition challenges, simultaneous immersion Mirau interferometry incorporates polarizing elements into the optics to allow simultaneous acquisition of two interferograms. The system design and production are described and images produced with the developed techniques are presented. PMID:23742552

Molecular imaging promotes progress in orthopedic research.

PubMed

Mayer-Kuckuk, Philipp; Boskey, Adele L

2006-11-01

Modern orthopedic research is directed towards the understanding of molecular mechanisms that determine development, maintenance and health of musculoskeletal tissues. In recent years, many genetic and proteomic discoveries have been made which necessitate investigation under physiological conditions in intact, living tissues. Molecular imaging can meet this demand and is, in fact, the only strategy currently available for noninvasive, quantitative, real-time biology studies in living subjects. In this review, techniques of molecular imaging are summarized, and applications to bone and joint biology are presented. The imaging modality most frequently used in the past was optical imaging, particularly bioluminescence and near-infrared fluorescence imaging. Alternate technologies including nuclear and magnetic resonance imaging were also employed. Orthopedic researchers have applied molecular imaging to murine models including transgenic mice to monitor gene expression, protein degradation, cell migration and cell death. Within the bone compartment, osteoblasts and their stem cells have been investigated, and the organic and mineral bone phases have been assessed. These studies addressed malignancy and injury as well as repair, including fracture healing and cell/gene therapy for skeletal defects. In the joints, molecular imaging has focused on the inflammatory and tissue destructive processes that cause arthritis. As described in this review, the feasibility of applying molecular imaging to numerous areas of orthopedic research has been demonstrated and will likely result in an increase in research dedicated to this powerful strategy. Molecular imaging holds great promise in the future for preclinical orthopedic research as well as next-generation clinical musculoskeletal diagnostics.

In vivo metabolic fingerprinting of neutral lipids with hyperspectral stimulated Raman scattering microscopy.

PubMed

Fu, Dan; Yu, Yong; Folick, Andrew; Currie, Erin; Farese, Robert V; Tsai, Tsung-Huang; Xie, Xiaoliang Sunney; Wang, Meng C

2014-06-18

Metabolic fingerprinting provides valuable information on the physiopathological states of cells and tissues. Traditional imaging mass spectrometry and magnetic resonance imaging are unable to probe the spatial-temporal dynamics of metabolites at the subcellular level due to either lack of spatial resolution or inability to perform live cell imaging. Here we report a complementary metabolic imaging technique that is based on hyperspectral stimulated Raman scattering (hsSRS). We demonstrated the use of hsSRS imaging in quantifying two major neutral lipids: cholesteryl ester and triacylglycerol in cells and tissues. Our imaging results revealed previously unknown changes of lipid composition associated with obesity and steatohepatitis. We further used stable-isotope labeling to trace the metabolic dynamics of fatty acids in live cells and live Caenorhabditis elegans with hsSRS imaging. We found that unsaturated fatty acid has preferential uptake into lipid storage while saturated fatty acid exhibits toxicity in hepatic cells. Simultaneous metabolic fingerprinting of deuterium-labeled saturated and unsaturated fatty acids in living C. elegans revealed that there is a lack of interaction between the two, unlike previously hypothesized. Our findings provide new approaches for metabolic tracing of neutral lipids and their precursors in living cells and organisms, and could potentially serve as a general approach for metabolic fingerprinting of other metabolites.

Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging

NASA Astrophysics Data System (ADS)

Chen, Wei-Wen; Yi, Yung-Hsiang; Chien, Cheng-Hao; Hsiung, Kuei-Ching; Ma, Tian-Hsiang; Lin, Yi-Chun; Lo, Szecheng J.; Chang, Ta-Chau

2016-08-01

Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm-1) and lipid (~2845 cm-1) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans.

Super-resolution optical imaging and magnetometry using NV centers in diamond

NASA Astrophysics Data System (ADS)

Jaskula, Jean-Christophe; Trifonov, Alexei; Glenn, David; Bar-Gill, Nir; Walsworth, Ronald

2013-05-01

We report progress done on the development and application of depletion-based techniques for super-resolution (nanoscale) optical imaging and magnetometry using NV centers in diamond. In particulare we are integrating stimulated emission depletion (STED) and ground state depletion (GSD) imaging techniques with advanced pulsed sequences for AC magnetometry. NV centers in diamond do not bleach under optical excitation, have long-lived electronic spin coherence and spin-state-dependent fluorescence, and are not biotoxic. Thus NV-diamond has great potential in quantum science and as a nanoscale magnetic biosensor.

Quantitative depth resolved microcirculation imaging with optical coherence tomography angiography (Part ΙΙ): Microvascular network imaging.

PubMed

Gao, Wanrong

2017-04-17

In this work, we review the main phenomena that have been explored in OCT angiography to image the vessels of the microcirculation within living tissues with the emphasis on how the different processing algorithms were derived to circumvent specific limitations. Parameters are then discussed that can quantitatively describe the depth-resolved microvascular network for possible clinic diagnosis applications. Finally,future directions in continuing OCT development are discussed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Advances in bioluminescence imaging: new probes from old recipes.

PubMed

Yao, Zi; Zhang, Brendan S; Prescher, Jennifer A

2018-06-04

Bioluminescent probes are powerful tools for visualizing biology in live tissues and whole animals. Recent years have seen a surge in the number of new luciferases, luciferins, and related tools available for bioluminescence imaging. Many were crafted using classic methods of optical probe design and engineering. Here we highlight recent advances in bioluminescent tool discovery and development, along with applications of the probes in cells, tissues, and organisms. Collectively, these tools are improving in vivo imaging capabilities and bolstering new research directions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

USDA-ARS?s Scientific Manuscript database

Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

Autofluorescence-Free Live-Cell Imaging Using Terbium Nanoparticles.

PubMed

Cardoso Dos Santos, M; Goetz, J; Bartenlian, H; Wong, K-L; Charbonnière, L J; Hildebrandt, N

2018-04-18

Fluorescent nanoparticles (NPs) have become irreplaceable tools for advanced cellular and subcellular imaging. While very bright NPs require excitation with UV or visible light, which can create strong autofluorescence of biological components, NIR-excitable NPs without autofluorescence issues exhibit much lower brightness. Here, we show the application of a new type of surface-photosensitized terbium NPs (Tb-NPs) for autofluorescence-free intracellular imaging in live HeLa cells. The combination of exceptionally high brightness, high photostability, and long photoluminecence (PL) lifetimes for highly efficient suppression of the short-lived autofluorescence allowed for time-gated PL imaging of intracellular vesicles over 72 h without toxicity and at extremely low Tb-NP concentrations down to 12 pM. Detection of highly resolved long-lifetime (ms) PL decay curves from small (∼10 μm 2 ) areas within single cells within a few seconds emphasized the unprecedented photophysical properties of Tb-NPs for live-cell imaging that extend well beyond currently available nanometric imaging agents.

Evaluation of living liver transplant donors: method for precise anatomic definition by using a dedicated contrast-enhanced MR imaging protocol.

PubMed

Sahani, Dushyant; D'souza, Roy; Kadavigere, Rajagopal; Hertl, Martin; McGowan, Jennifer; Saini, Sanjay; Mueller, Peter R

2004-01-01

Liver transplantation from a living donor involves removal of part of the donor liver in a fashion that does not endanger its vascular supply or metabolic function. The radiologist plays an important role in evaluation of the living donor to define the conditions under which graft donation is contraindicated and to identify anatomic variations that may alter the surgical approach. In the past, diagnostic work-up of the donor involved costly and invasive tests. Currently, dynamic contrast material-enhanced computed tomography and magnetic resonance (MR) imaging are the imaging tests performed, each of which has advantages and limitations. MR imaging performed with liver-specific and extravascular contrast agents may be used as a single imaging test for comprehensive noninvasive evaluation of living liver transplant donors. MR imaging provides valuable information about variations in the vascular and biliary anatomy and allows evaluation of the hepatic parenchyma for diffuse or focal abnormalities. Copyright RSNA, 2004

Live-cell imaging of endogenous mRNAs with a small molecule.

PubMed

Sato, Shin-ichi; Watanabe, Mizuki; Katsuda, Yousuke; Murata, Asako; Wang, Dan Ohtan; Uesugi, Motonari

2015-02-02

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non-engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene-specific RNA aptamer, combined with a cell-permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin-2, cortactin, and cytoplasmic FMR1-interacting protein 2. The RNA-imaging technology and its further optimization might permit live-cell imaging of any RNA molecules. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Influence of the environment and phototoxicity of the live cell imaging system at IMP microbeam facility

NASA Astrophysics Data System (ADS)

Liu, Wenjing; Du, Guanghua; Guo, Jinlong; Wu, Ruqun; Wei, Junzhe; Chen, Hao; Li, Yaning; Zhao, Jing; Li, Xiaoyue

2017-08-01

To investigate the spatiotemporal dynamics of DNA damage and repair after the ion irradiation, an online live cell imaging system has been established based on the microbeam facility at Institute of Modern Physics (IMP). The system could provide a sterile and physiological environment by making use of heating plate and live cell imaging solution. The phototoxicity was investigated through the evaluation of DNA repair protein XRCC1 foci formed in HT1080-RFP cells during the imaging exposure. The intensity of the foci induced by phototoxicity was much lower compared with that of the foci induced by heavy ion hits. The results showed that although spontaneous foci were formed due to RFP exposure during live cell imaging, they had little impact on the analysis of the recruitment kinetics of XRCC1 in the foci induced by the ion irradiation.

Creating a histology-embryology free digital image database using high-end microscopy and computer techniques for on-line biomedical education.

PubMed

Silva-Lopes, Victor W; Monteiro-Leal, Luiz H

2003-07-01

The development of new technology and the possibility of fast information delivery by either Internet or Intranet connections are changing education. Microanatomy education depends basically on the correct interpretation of microscopy images by students. Modern microscopes coupled to computers enable the presentation of these images in a digital form by creating image databases. However, the access to this new technology is restricted entirely to those living in cities and towns with an Information Technology (IT) infrastructure. This study describes the creation of a free Internet histology database composed by high-quality images and also presents an inexpensive way to supply it to a greater number of students through Internet/Intranet connections. By using state-of-the-art scientific instruments, we developed a Web page (http://www2.uerj.br/~micron/atlas/atlasenglish/index.htm) that, in association with a multimedia microscopy laboratory, intends to help in the reduction of the IT educational gap between developed and underdeveloped regions. Copyright 2003 Wiley-Liss, Inc.

Simple synthesis of carbon-11 labeled styryl dyes as new potential PET RNA-specific, living cell imaging probes.

PubMed

Wang, Min; Gao, Mingzhang; Miller, Kathy D; Sledge, George W; Hutchins, Gary D; Zheng, Qi-Huang

2009-05-01

A new type of styryl dyes have been developed as RNA-specific, live cell imaging probes for fluorescent microscopy technology to study nuclear structure and function. This study was designed to develop carbon-11 labeled styryl dyes as new probes for biomedical imaging technique positron emission tomography (PET) imaging of RNA in living cells. Precursors (E)-2-(2-(1-(triisopropylsilyl)-1H-indol-3-yl)vinyl)quinoline (2), (E)-2-(2,4,6-trimethoxystyryl)quinoline (3) and (E)-4-(2-(6-methoxyquinolin-2-yl)vinyl)-N,N-diemthylaniline (4), and standards styryl dyes E36 (6), E144 (7) and F22 (9) were synthesized in multiple steps with moderate to high chemical yields. Precursor 2 was labeled by [(11)C]CH(3)OTf, trapped on a cation-exchange CM Sep-Pak cartridge following a quick deprotecting reaction by addition of (n-Bu)(4)NF in THF, and isolated by solid-phase extraction (SPE) purification to provide target tracer [(11)C]E36 ([(11)C]6) in 40-50% radiochemical yields, decay corrected to end of bombardment (EOB), based on [(11)C]CO(2). The target tracers [(11)C]E144 ([(11)C]7) and [(11)C]F22 ([(11)C]9) were prepared by N-[(11)C]methylation of the precursors 3 and 4, respectively, using [(11)C]CH(3)OTf and isolated by SPE method in 50-70% radiochemical yields at EOB. The specific activity of the target tracers [(11)C]6, [(11)C]7 and [(11)C]9 was in a range of 74-111GBq/mumol at the end of synthesis (EOS).

Orai1 as New Therapeutic Target for Inhibiting Breast Tumor Metastasis

DTIC Science & Technology

2009-09-01

includes focal adhesion assembly (formation of focal complex) and focal adhesion disassembly, we used live - cell imaging to quantify the rates of assembly...A and B) Live cell imaging of paxillin-GFP transfected MEF cells in the absence (A) or presence (B) of SKF96365. Scale bar: 10 µm. (C and D...includes focal adhesion assembly (formation of focal complexes) and focal adhesion disassembly, we used live - cell imaging to quantify the rates of focal

Investigating the Functional Role of Prostate-Specific Membrane Antigen and its Enzymatic Activity in Prostate Cancer Metastasis

DTIC Science & Technology

2008-02-01

fluorescent probes for live cell imaging . PSMA distribution of cells grown on different extracellular matrices will be characterized to provide guidance...PCa migration, using in vitro cell model systems and live - cell imaging methods, we characterized the role of PSMA in cell motility and adhesion. Using...Generated fluorescently conjugated anti-PSMA antibodies for live cell imaging . 2. Optimized the siRNA-PSMA transfection and achieved an approximately

Live-cell imaging of rice cytological changes reveals the importance of host vacuole maintenance for biotrophic invasion by blast fungus, Magnaporthe oryzae.

PubMed

Mochizuki, Susumu; Minami, Eiichi; Nishizawa, Yoko

2015-12-01

The rice blast fungus Magnaporthe oryzae grows inside living host cells. Cytological analyses by live-cell imaging have revealed characteristics of the biotrophic invasion, particularly the extrainvasive hyphal membrane (EIHM) originating from the host plasma membrane and a host membrane-rich structure, biotrophic interfacial complex (BIC). Here, we observed rice subcellular changes associated with invasive hyphal growth using various transformants expressing specifically localized fluorescent proteins. The invasive hyphae did not penetrate across but were surrounded by the host vacuolar membrane together with EIHM even after branching. High-resolution imaging of BICs revealed that the host cytosol was accumulated at BIC with aggregated EIHM and a symplastic effector, Pwl2, in a punctate form. The vacuolar membrane did not aggregate in but closely surrounded the BIC. A good correlation was observed between the early collapse of vacuoles and damage of invasive hyphae in the first-invaded cell. Furthermore, a newly developed, long-term imaging method has revealed that the central vacuole gradually shrank until collapse, which was caused by the hyphal invasion occurring earlier in the neighboring cells than in the first-invaded cells. These data suggest that M. oryzae may suppress host vacuole collapse during early infection stages for successful infection. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

N-doped carbon dots derived from bovine serum albumin and formic acid with one- and two-photon fluorescence for live cell nuclear imaging.

PubMed

Tan, Mingqian; Li, Xintong; Wu, Hao; Wang, Beibei; Wu, Jing

2015-12-01

Carbon dots with both one- and two-photon fluorescence have drawn great attention for biomedical imaging. Herein, nitrogen-doped carbon dots were facilely developed by one-pot hydrothermal method using bovine serum albumin and formic acid as carbon sources. They are highly water-soluble with strong fluorescence when excited with ultraviolet or near infrared light. The carbon dots have a diameter of ~8.32 nm and can emit strong two-photon induced fluorescence upon excitation at 750 nm with a femtosecond laser. X-ray photoelectron spectrometer analysis revealed that the carbon dots contained three components, C, N and O, corresponding to the peak at 285, 398 and 532 eV, respectively. The Fourier-transform infrared spectroscopy analysis revealed that there are carboxyl and carboxylic groups on the surface, which allowed further linking of functional molecules. pH stability study demonstrated that the carbon dots are able to be used in a wide range of pH values. The fluorescence mechanism is also discussed in this study. Importantly, these carbon dots are biocompatible and highly photostable, which can be directly applied for both one- and two-photon living cell imaging. After proper surface functionalization with TAT peptide, they can be used as fluorescent probes for live cell nuclear-targeted imaging. Copyright © 2015 Elsevier B.V. All rights reserved.

Assessing resolution in live cell structured illumination microscopy

NASA Astrophysics Data System (ADS)

Pospíšil, Jakub; Fliegel, Karel; Klíma, Miloš

2017-12-01

Structured Illumination Microscopy (SIM) is a powerful super-resolution technique, which is able to enhance the resolution of optical microscope beyond the Abbe diffraction limit. In the last decade, numerous SIM methods that achieve the resolution of 100 nm in the lateral dimension have been developed. The SIM setups with new high-speed cameras and illumination pattern generators allow rapid acquisition of the live specimen. Therefore, SIM is widely used for investigation of the live structures in molecular and live cell biology. Quantitative evaluation of resolution enhancement in a real sample is essential to describe the efficiency of super-resolution microscopy technique. However, measuring the resolution of a live cell sample is a challenging task. Based on our experimental findings, the widely used Fourier ring correlation (FRC) method does not seem to be well suited for measuring the resolution of SIM live cell video sequences. Therefore, the resolution assessing methods based on Fourier spectrum analysis are often used. We introduce a measure based on circular average power spectral density (PSDca) estimated from a single SIM image (one video frame). PSDca describes the distribution of the power of a signal with respect to its spatial frequency. Spatial resolution corresponds to the cut-off frequency in Fourier space. In order to estimate the cut-off frequency from a noisy signal, we use a spectral subtraction method for noise suppression. In the future, this resolution assessment approach might prove useful also for single-molecule localization microscopy (SMLM) live cell imaging.

A group intervention to improve body image satisfaction and dietary habits in gay and bisexual men living with HIV/AIDS.

PubMed

Feldman, Matthew B; Torino, Jenny A; Swift, Margaret

2011-01-01

A healthy diet is essential to maintaining a strong immune system for people living with HIV and AIDS. Prior studies have shown that HIV-positive gay and bisexual men are more susceptible to poor body image, which can negatively impact dietary habits. Interventions that simultaneously address body image and nutrition are therefore critical for this population. This paper describes the curriculum for a 14-week group designed to improve body image satisfaction and dietary habits in gay and bisexual men living with HIV/AIDS.

Single-Molecule and Superresolution Imaging in Live Bacteria Cells

PubMed Central

Biteen, Julie S.; Moerner, W.E.

2010-01-01

Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells. PMID:20300204

In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles.

PubMed

Gu, Luo; Hall, David J; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J; Howell, Stephen B; Sailor, Michael J

2013-01-01

Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (<10 ns) emission signals from organic chromophores or tissue autofluorescence. Here using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by >50-fold in vitro and by >20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.

In situ synthesis of alkenyl tetrazines for highly fluorogenic bioorthogonal live-cell imaging probes.

PubMed

Wu, Haoxing; Yang, Jun; Šečkutė, Jolita; Devaraj, Neal K

2014-06-02

In spite of the wide application potential of 1,2,4,5-tetrazines, particularly in live-cell and in vivo imaging, a major limitation has been the lack of practical synthetic methods. Here we report the in situ synthesis of (E)-3-substituted 6-alkenyl-1,2,4,5-tetrazine derivatives through an elimination-Heck cascade reaction. By using this strategy, we provide 24 examples of π-conjugated tetrazine derivatives that can be conveniently prepared from tetrazine building blocks and related halides. These include tetrazine analogs of biological small molecules, highly conjugated buta-1,3-diene-substituted tetrazines, and a diverse array of fluorescent probes suitable for live-cell imaging. These highly conjugated probes show very strong fluorescence turn-on (up to 400-fold) when reacted with dienophiles such as cyclopropenes and trans-cyclooctenes, and we demonstrate their application for live-cell imaging. This work provides an efficient and practical synthetic methodology for tetrazine derivatives and will facilitate the application of conjugated tetrazines, particularly as fluorogenic probes for live-cell imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tilted light sheet microscopy with 3D point spread functions for single-molecule super-resolution imaging in mammalian cells

NASA Astrophysics Data System (ADS)

Gustavsson, Anna-Karin; Petrov, Petar N.; Lee, Maurice Y.; Shechtman, Yoav; Moerner, W. E.

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D superresolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Biophotonics for imaging and cell manipulation: quo vadis?

NASA Astrophysics Data System (ADS)

Serafetinides, Alexandros A.; Makropoulou, Mirsini; Kotsifaki, Domna G.; Tsigaridas, Giorgos

2016-01-01

As one of the major health problems for mankind is cancer, any development for the early detection and effective treatment of cancer is crucial to saving lives. Worldwide, the dream for the anti-cancer procedure of attack is the development of a safe and efficient early diagnosis technique, the so called "optical biopsy". As early diagnosis of cancer is associated with improved prognosis, several laser based optical diagnostic methods were developed to enable earlier, non-invasive detection of human cancer, as Laser Induced Fluorescence spectroscopy (LIFs), Diffuse Reflectance spectroscopy (DRs), confocal microscopy, and Optical Coherence Tomography (OCT). Among them, Optical Coherence Tomography (OCT) imaging is considered to be a useful tool to differentiate healthy from malignant (e.g. basal cell carcinoma, squamous cell carcinoma) skin tissue. If the demand is to perform imaging in sub-tissular or even sub-cellular level, optical tweezers and atomic force microscopy have enabled the visualization of molecular events underlying cellular processes in live cells, as well as the manipulation and characterization of microscale or even nanoscale biostructures. In this work, we will present the latest advances in the field of laser imaging and manipulation techniques, discussing some representative experimental data focusing on the 21th century biophotonics roadmap of novel diagnostic and therapeutical approaches. As an example of a recently discussed health and environmental problem, we studied both experimentally and theoretically the optical trapping forces exerted on yeast cells and modified with estrogen-like acting compounds yeast cells, suspended in various buffer media.

A Highly Specific Gold Nanoprobe for Live-Cell Single-Molecule Imaging

NASA Astrophysics Data System (ADS)

Leduc, Cécile; Si, Satyabrata; Gautier, Jérémie; Soto-Ribeiro, Martinho; Wehrle-Haller, Bernhard; Gautreau, Alexis; Giannone, Grégory; Cognet, Laurent; Lounis, Brahim

2013-04-01

Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Due to these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5-nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used GFPs with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.

In vivo evidence of significant levator ani muscle stretch on MR images of a live childbirth.

PubMed

Sindhwani, Nikhil; Bamberg, Christian; Famaey, Nele; Callewaert, Geertje; Dudenhausen, Joachim W; Teichgräber, Ulf; Deprest, Jan

2017-08-01

Vaginal childbirth is believed to be a significant risk factor for the development of pelvic floor dysfunction later in life. Previous studies have explored the use of medical imaging and simulations of childbirth to determine the stretch in the levator ani muscle. A report in 2012 has recorded magnetic resonance images of a live childbirth of a 24 year old woman giving birth vaginally for the second time, using a 1.0 Tesla open, high-field scanner. Our objective was to determine the stretch ratios in the levator muscle using these magnetic resonance images of live childbirth. Three-dimensional magnetic resonance image sequences were obtained to visualize coronal and axial planes before and after the childbirth. These images were obtained before the expulsion phase without pushing and were used to reconstruct the levator muscle and the fetal head in 3 dimensions. The fetal head was approximated to be an ellipsoid, and it is assumed that its middle section is visible in dynamic magnetic resonance images. Assuming incompressibility, the full deformation field of the fetal head is then calculated. Real-time cine magnetic resonance images were acquired for the during the expulsion phase, occurring over 2 contractions in the midsagittal plane. The levator muscle stretch is estimated using a custom program. The program calculates points of contact between the fetal head ellipsoid and the levator ani muscle model as the head descends down the birth canal and moves them orthogonal to its surface. Circumferential stretch was calculated to represent the extension needed to allow the passage of the fetal head. Starting from a position in the preexpulsion phase, the levator muscle experiences a maximum circumferential stretch of 248% on the posterior-medial portion of the levator ani muscle, as shown in previously published finite element simulations. However, the maximal stretch was notably less than that predicted by finite element models. This is because our baseline 3-dimensional model of the levator muscle is created from images taken shortly before expulsion and thus is already in a stretched state. Furthermore, the finite element models are created from images of a healthy nulliparous woman, while this study uses images from a para 2 woman. This study is the first attempt to estimate the stretch in levator ani muscle using magnetic resonance images of a live childbirth. The stretch was significant and the locations corroborate with previous findings of finite element models. Copyright © 2017 Elsevier Inc. All rights reserved.

Depletion-based techniques for super-resolution imaging of NV-diamond

NASA Astrophysics Data System (ADS)

Jaskula, Jean-Christophe; Trifonov, Alexei; Glenn, David; Walsworth, Ronald

2012-06-01

We discuss the development and application of depletion-based techniques for super-resolution imaging of NV centers in diamond: stimulated emission depletion (STED), metastable ground state depletion (GSD), and dark state depletion (DSD). NV centers in diamond do not bleach under optical excitation, are not biotoxic, and have long-lived electronic spin coherence and spin-state-dependent fluorescence. Thus NV-diamond has great potential as a fluorescent biomarker and as a magnetic biosensor.

Light microscopy applications in systems biology: opportunities and challenges

PubMed Central

2013-01-01

Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various biological scales in living cells. The combination of molecular biology and imaging provides a bottom-up tool for direct insight into how molecular processes work on a cellular scale. However, imaging can also be used as a top-down approach to study the behavior of a system without detailed prior knowledge about its underlying molecular mechanisms. In this review, we highlight the recent developments on microscopy-based systems analyses and discuss the complementary opportunities and different challenges with high-content screening and high-throughput imaging. Furthermore, we provide a comprehensive overview of the available platforms that can be used for image analysis, which enable community-driven efforts in the development of image-based systems biology. PMID:23578051

Imaging light responses of foveal ganglion cells in the living macaque eye.

PubMed

Yin, Lu; Masella, Benjamin; Dalkara, Deniz; Zhang, Jie; Flannery, John G; Schaffer, David V; Williams, David R; Merigan, William H

2014-05-07

The fovea dominates primate vision, and its anatomy and perceptual abilities are well studied, but its physiology has been little explored because of limitations of current physiological methods. In this study, we adapted a novel in vivo imaging method, originally developed in mouse retina, to explore foveal physiology in the macaque, which permits the repeated imaging of the functional response of many retinal ganglion cells (RGCs) simultaneously. A genetically encoded calcium indicator, G-CaMP5, was inserted into foveal RGCs, followed by calcium imaging of the displacement of foveal RGCs from their receptive fields, and their intensity-response functions. The spatial offset of foveal RGCs from their cone inputs makes this method especially appropriate for fovea by permitting imaging of RGC responses without excessive light adaptation of cones. This new method will permit the tracking of visual development, progression of retinal disease, or therapeutic interventions, such as insertion of visual prostheses.

Carbon-11 and fluorine-18 chemistry devoted to molecular probes for imaging the brain with positron emission tomography.

PubMed

Dollé, Frédéric

2013-01-01

Exploration of the living human brain in real-time and in a noninvasive way was for centuries only a dream, made, however, possible today with the remarkable development during the four last decades of powerful molecular imaging techniques, and especially positron emission tomography (PET). Molecular PET imaging relies, from a chemical point of view, on the use and preparation of a positron-emitting radiolabelled probe or radiotracer, notably compounds incorporating one of two short-lived radionuclides fluorine-18 (T1/2 : 109.8 min) and carbon-11 (T1/2 : 20.38 min). The growing availability and interest for the radiohalogen fluorine-18 in radiopharmaceutical chemistry undoubtedly results from its convenient half-life and the successful use in clinical oncology of 2-[(18) F]fluoro-2-deoxy-d-glucose ([(18) F]FDG). The special interest of carbon-11 is not only that carbon is present in virtually all biomolecules and drugs allowing therefore for isotopic labelling of their chemical structures but also that a given molecule could be radiolabelled at different functions or sites, permitting to explore (or to take advantage of) in vivo metabolic pathways. PET chemistry includes production of these short-lived radioactive isotopes via nuclear transmutation reactions using a cyclotron, and is directed towards the development of rapid synthetic methods, at the trace level, for the introduction of these nuclides into a molecule, as well as the use of fast purification, analysis and formulation techniques. PET chemistry is the driving force in molecular PET imaging, and this special issue of the Journal of Labelled Compounds and Radiopharmaceuticals, which is strongly chemistry and radiochemistry-oriented, aims at illustrating, be it in part only, the state-of-the-art arsenal of reactions currently available and its potential for the research and development of specific molecular probes labelled with the positron emitters carbon-11 and fluorine-18, with optimal imaging properties for PET exploration of the brain. Copyright © 2013 John Wiley & Sons, Ltd.

Alterations of the cytoskeleton in human cells in space proved by life-cell imaging.

PubMed

Corydon, Thomas J; Kopp, Sascha; Wehland, Markus; Braun, Markus; Schütte, Andreas; Mayer, Tobias; Hülsing, Thomas; Oltmann, Hergen; Schmitz, Burkhard; Hemmersbach, Ruth; Grimm, Daniela

2016-01-28

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24(th) DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31(st) parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization.

Alterations of the cytoskeleton in human cells in space proved by life-cell imaging

PubMed Central

Corydon, Thomas J.; Kopp, Sascha; Wehland, Markus; Braun, Markus; Schütte, Andreas; Mayer, Tobias; Hülsing, Thomas; Oltmann, Hergen; Schmitz, Burkhard; Hemmersbach, Ruth; Grimm, Daniela

2016-01-01

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24th DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31st parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization. PMID:26818711

Improving high resolution retinal image quality using speckle illumination HiLo imaging

PubMed Central

Zhou, Xiaolin; Bedggood, Phillip; Metha, Andrew

2014-01-01

Retinal image quality from flood illumination adaptive optics (AO) ophthalmoscopes is adversely affected by out-of-focus light scatter due to the lack of confocality. This effect is more pronounced in small eyes, such as that of rodents, because the requisite high optical power confers a large dioptric thickness to the retina. A recently-developed structured illumination microscopy (SIM) technique called HiLo imaging has been shown to reduce the effect of out-of-focus light scatter in flood illumination microscopes and produce pseudo-confocal images with significantly improved image quality. In this work, we adopted the HiLo technique to a flood AO ophthalmoscope and performed AO imaging in both (physical) model and live rat eyes. The improvement in image quality from HiLo imaging is shown both qualitatively and quantitatively by using spatial spectral analysis. PMID:25136486

Improving high resolution retinal image quality using speckle illumination HiLo imaging.

PubMed

Zhou, Xiaolin; Bedggood, Phillip; Metha, Andrew

2014-08-01

Retinal image quality from flood illumination adaptive optics (AO) ophthalmoscopes is adversely affected by out-of-focus light scatter due to the lack of confocality. This effect is more pronounced in small eyes, such as that of rodents, because the requisite high optical power confers a large dioptric thickness to the retina. A recently-developed structured illumination microscopy (SIM) technique called HiLo imaging has been shown to reduce the effect of out-of-focus light scatter in flood illumination microscopes and produce pseudo-confocal images with significantly improved image quality. In this work, we adopted the HiLo technique to a flood AO ophthalmoscope and performed AO imaging in both (physical) model and live rat eyes. The improvement in image quality from HiLo imaging is shown both qualitatively and quantitatively by using spatial spectral analysis.

A Microplate Reader-Based System for Visualizing Transcriptional Activity During in vivo Microbial Interactions in Space and Time.

PubMed

Hennessy, Rosanna C; Stougaard, Peter; Olsson, Stefan

2017-03-21

Here, we report the development of a microplate reader-based system for visualizing gene expression dynamics in living bacterial cells in response to a fungus in space and real-time. A bacterium expressing the red fluorescent protein mCherry fused to the promoter region of a regulator gene nunF indicating activation of an antifungal secondary metabolite gene cluster was used as a reporter system. Time-lapse image recordings of the reporter red signal and a green signal from fluorescent metabolites combined with microbial growth measurements showed that nunF-regulated gene transcription is switched on when the bacterium enters the deceleration growth phase and upon physical encounter with fungal hyphae. This novel technique enables real-time live imaging of samples by time-series multi-channel automatic recordings using a microplate reader as both an incubator and image recorder of general use to researchers. The technique can aid in deciding when to destructively sample for other methods e.g. transcriptomics and mass spectrometry imaging to study gene expression and metabolites exchanged during the interaction.

Advances in single-cell experimental design made possible by automated imaging platforms with feedback through segmentation.

PubMed

Crick, Alex J; Cammarota, Eugenia; Moulang, Katie; Kotar, Jurij; Cicuta, Pietro

2015-01-01

Live optical microscopy has become an essential tool for studying the dynamical behaviors and variability of single cells, and cell-cell interactions. However, experiments and data analysis in this area are often extremely labor intensive, and it has often not been achievable or practical to perform properly standardized experiments on a statistically viable scale. We have addressed this challenge by developing automated live imaging platforms, to help standardize experiments, increasing throughput, and unlocking previously impossible ones. Our real-time cell tracking programs communicate in feedback with microscope and camera control software, and they are highly customizable, flexible, and efficient. As examples of our current research which utilize these automated platforms, we describe two quite different applications: egress-invasion interactions of malaria parasites and red blood cells, and imaging of immune cells which possess high motility and internal dynamics. The automated imaging platforms are able to track a large number of motile cells simultaneously, over hours or even days at a time, greatly increasing data throughput and opening up new experimental possibilities. Copyright © 2015 Elsevier Inc. All rights reserved.

Live Cell Imaging of Viscosity in 3D Tumour Cell Models.

PubMed

Shirmanova, Marina V; Shimolina, Lubov' E; Lukina, Maria M; Zagaynova, Elena V; Kuimova, Marina K

2017-01-01

Abnormal levels of viscosity in tissues and cells are known to be associated with disease and malfunction. While methods to measure bulk macroscopic viscosity of bio-tissues are well developed, imaging viscosity at the microscopic scale remains a challenge, especially in vivo. Molecular rotors are small synthetic viscosity-sensitive fluorophores in which fluorescence parameters are strongly correlated to the microviscosity of their immediate environment. Hence, molecular rotors represent a promising instrument for mapping of viscosity in living cells and tissues at the microscopic level. Quantitative measurements of viscosity can be achieved by recording time-resolved fluorescence decays of molecular rotor using fluorescence lifetime imaging microscopy (FLIM), which is also suitable for dynamic viscosity mapping, both in cellulo and in vivo. Among tools of experimental oncology, 3D tumour cultures, or spheroids, are considered a more adequate in vitro model compared to a cellular monolayer, and represent a less labour-intensive and more unified approach compared to animal tumour models. This chapter describes a methodology for microviscosity imaging in tumour spheroids using BODIPY-based molecular rotors and two photon-excited FLIM.

Applications of nanopipettes in bionanotechnology.

PubMed

Ying, Liming

2009-08-01

At present, technical hurdles remain in probing biochemical processes in living cells and organisms at nanometre spatial resolution, millisecond time resolution and with high specificity and single-molecule sensitivity. Owing to its unique shape, size and electrical properties, the nanopipette has been used to obtain high-resolution topographic images of live cells under physiological conditions, and to create nanoscale features by controlled delivery of biomolecules. In the present paper, I discuss recent progress in the development of a family of new methods for nanosensing and nanomanipulation using nanopipettes.

High sensitivity contrast enhanced optical coherence tomography for functional in vivo imaging

NASA Astrophysics Data System (ADS)

Liba, Orly; SoRelle, Elliott D.; Sen, Debasish; de la Zerda, Adam

2017-02-01

In this study, we developed and applied highly-scattering large gold nanorods (LGNRs) and custom spectral detection algorithms for high sensitivity contrast-enhanced optical coherence tomography (OCT). We were able to detect LGNRs at a concentration as low as 50 pM in blood. We used this approach for noninvasive 3D imaging of blood vessels deep in solid tumors in living mice. Additionally, we demonstrated multiplexed imaging of spectrally-distinct LGNRs that enabled observations of functional drainage in lymphatic networks. This method, which we call MOZART, provides a platform for molecular imaging and characterization of tissue noninvasively at cellular resolution.

A simple microfluidic device for live cell imaging of Arabidopsis cotyledons, leaves, and seedlings.

PubMed

Vang, Shia; Seitz, Kati; Krysan, Patrick J

2018-06-01

One of the challenges of performing live-cell imaging in plants is establishing a system for securing the sample during imaging that allows for the rapid addition of treatments. Here we report how a commercially available device called a HybriWell ™ can be repurposed to create an imaging chamber suitable for Arabidopsis seedlings, cotyledons and leaves. Liquid in the imaging chamber can be rapidly exchanged to introduce chemical treatments via microfluidic passive pumping. When used in conjunction with fluorescent biosensors, this system can facilitate live-cell imaging studies of signal transduction pathways triggered by different treatments. As a demonstration, we show how the HybriWell can be used to monitor flg22-induced calcium transients using the R-GECO1 calcium indicator in detached Arabidopsis leaves.

In vivo imaging of protease activity by Probody therapeutic activation

PubMed Central

Wong, Kenneth R.; Menendez, Elizabeth; Craik, Charles S.; Kavanaugh, W. Michael; Vasiljeva, Olga

2017-01-01

Probody™ therapeutics are recombinant, proteolytically-activated antibody prodrugs, engineered to remain inert until activated locally by tumor-associated proteases. Probody therapeutics exploit the fundamental dysregulation of extracellular protease activity that exists in tumors relative to healthy tissue. Leveraging the ability of a Probody therapeutic to bind its target at the site of disease after proteolytic cleavage, we developed a novel method for profiling protease activity in living animals. Using NIR optical imaging, we demonstrated that a non-labeled anti-EGFR Probody therapeutic can become activated and compete for binding to tumor cells in vivo with a labeled anti-EGFR monoclonal antibody. Furthermore, by inhibiting matriptase activity in vivo with a blocking-matriptase antibody, we show that the ability of the Probody therapeutic to bind EGFR in vivo was dependent on protease activity. These results demonstrate that in vivo imaging of Probody therapeutic activation can be used for screening and characterization of protease activity in living animals, and provide a method that avoids some of the limitations of prior methods. This approach can improve our understanding of the activity of proteases in disease models and help to develop efficient strategies for cancer diagnosis and treatment. PMID:26546838

The changing image of the Kurds in Turkish cities: middle-class perceptions of Kurdish migrants in Izmir.

PubMed

Saraçoğlu, Cenk

2010-01-01

Saraçoğlu deals with the ways in which the Kurdish migrants living in the western cities of Turkey have been identified in middle-class discourse by certain pejorative labels and stereotypes. He argues that this new Kurdish image demonstrates the ethnicization of longstanding anti-migrant sentiments in Turkey. He develops and substantiates the argument by means of qualitative data gathered in a field study in zmir between June 2006 and July 2007. The study involved ninety in-depth interviews with middle-class individuals living in the city and explored their anti-Kurdish attitudes. Through a close analysis of two of the common stereotypes that these interviewees deployed in the interviews-namely, that the Kurds were 'benefit scroungers' and that they 'disrupt urban life'- Saraçoğlu explores the formation of the urban social context in which such perceptions have emerged. Close examination of the narratives of the middle-class respondents indicates that the development of a new image of the Kurds has occurred in an urban context shaped by the neoliberal transformation of Turkish cities, on the one hand, and the internal displacement of Kurdish migrants, on the other.

Are those bugs reflective? Non-destructive biofilm imaging with white light interferometry

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

Larimer, Curtis J.; Brann, Michelle R.; Suter, Jonathan D.

White light interferometry (WLI) is not typically used to image bacterial biofilms that are immersed in water because there is insufficient refractive index contrast to induce reflection from the biofilm’s interface. The soft structure and water-like bulk properties of hydrated biofilms make them difficult to characterize in situ by any means, especially in a non-destructive manner. Here we describe a new method for measuring and monitoring the thickness and topology of live biofilms using a WLI microscope. A microfluidic system was used to create a reflective interface on the surface of biofilms. Live biofilm samples were monitored non-destructively over time.more » The method enables surface metrology measurements (roughness, surface area) and a novel approach to measuring thickness of the thin hydrated biofilms. Increase in surface roughness preceded observable increase in biofilm thickness, indicating that this measure may be used to predict future development of biofilms. We have also developed a flow cell that enables WLI biofilm imaging in a dynamic environment. We have used this flow cell to observe changes in biofilm structure in response to changes in environmental conditions - flow velocity, availability of nutrients, and presence of biocides.« less

Live imaging of muscle histolysis in Drosophila metamorphosis.

PubMed

Kuleesha, Yadav; Puah, Wee Choo; Wasser, Martin

2016-05-04

The contribution of programmed cell death (PCD) to muscle wasting disorders remains a matter of debate. Drosophila melanogaster metamorphosis offers the opportunity to study muscle cell death in the context of development. Using live cell imaging of the abdomen, two groups of larval muscles can be observed, doomed muscles that undergo histolysis and persistent muscles that are remodelled and survive into adulthood. To identify and characterize genes that control the decision between survival and cell death of muscles, we developed a method comprising in vivo imaging, targeted gene perturbation and time-lapse image analysis. Our approach enabled us to study the cytological and temporal aspects of abnormal cell death phenotypes. In a previous genetic screen for genes controlling muscle size and cell death in metamorphosis, we identified gene perturbations that induced cell death of persistent or inhibit histolysis of doomed larval muscles. RNA interference (RNAi) of the genes encoding the helicase Rm62 and the lysosomal Cathepsin-L homolog Cysteine proteinase 1 (Cp1) caused premature cell death of persistent muscle in early and mid-pupation, respectively. Silencing of the transcriptional co-repressor Atrophin inhibited histolysis of doomed muscles. Overexpression of dominant-negative Target of Rapamycin (TOR) delayed the histolysis of a subset of doomed and induced ablation of all persistent muscles. RNAi of AMPKα, which encodes a subunit of the AMPK protein complex that senses AMP and promotes ATP formation, led to loss of attachment and a spherical morphology. None of the perturbations affected the survival of newly formed adult muscles, suggesting that the method is useful to find genes that are crucial for the survival of metabolically challenged muscles, like those undergoing atrophy. The ablation of persistent muscles did not affect eclosion of adult flies. Live imaging is a versatile approach to uncover gene functions that are required for the survival of muscle undergoing remodelling, yet are dispensable for other adult muscles. Our approach promises to identify molecular mechanisms that can explain the resilience of muscles to PCD.

Perylene-diimide-based nanoparticles as highly efficient photoacoustic agents for deep brain tumor imaging in living mice

DOE PAGES

Fan, Quli; Cheng, Kai; Yang, Zhen; ...

2014-11-06

In order to promote preclinical and clinical applications of photoacoustic imaging, novel photoacoustic contrast agents are highly desired for molecular imaging of diseases, especially for deep tumor imaging. In this paper, perylene-3,4,9,10-tetracarboxylic diiimide-based near-infrared-absorptive organic nanoparticles are reported as an efficient agent for photoacoustic imaging of deep brain tumors in living mice with enhanced permeability and retention effect

Imaging of mesoscopic-scale organisms using selective-plane optoacoustic tomography.

PubMed

Razansky, Daniel; Vinegoni, Claudio; Ntziachristos, Vasilis

2009-05-07

Mesoscopic-scale living organisms (i.e. 1 mm to 1 cm sized) remain largely inaccessible by current optical imaging methods due to intensive light scattering in tissues. Therefore, imaging of many important model organisms, such as insects, fishes, worms and similarly sized biological specimens, is currently limited to embryonic or other transparent stages of development. This makes it difficult to relate embryonic cellular and molecular mechanisms to consequences in organ function and animal behavior in more advanced stages and adults. Herein, we have developed a selective-plane illumination optoacoustic tomography technique for in vivo imaging of optically diffusive organisms and tissues. The method is capable of whole-body imaging at depths from the sub-millimeter up to centimeter range with a scalable spatial resolution in the order of magnitude of a few tenths of microns. In contrast to pure optical methods, the spatial resolution here is not determined nor limited by light diffusion; therefore, such performance cannot be achieved by any other optical imaging technology developed so far. The utility of the method is demonstrated on several whole-body models and small-animal extremities.

Comparison of optical projection tomography and optical coherence tomography for assessment of murine embryonic development

NASA Astrophysics Data System (ADS)

Singh, Manmohan; Nair, Achuth; Vadakkan, Tegy; Piazza, Victor; Udan, Ryan; Frazier, Michael V.; Janecek, Trevor; Dickinson, Mary E.; Larin, Kirill V.

2015-03-01

The murine model is a common model for studying developmental diseases. In this study, we compare the performance of the relatively new method of Optical Projection Tomography (OPT) to the well-established technique of Optical Coherence Tomography (OCT) to assess murine embryonic development at three stages, 9.5, 11.5, and 13.5 days post conception. While both methods can provide spatial resolution at the micrometer scale, OPT can provide superior imaging depth compared to OCT. However, OPT requires samples to be fixed, placed in an immobilization media such as agar, and cleared before imaging. Because OCT does not require fixing, it can be used to image embryos in vivo and in utero. In this study, we compare the efficacy of OPT and OCT for imaging murine embryonic development. The data demonstrate the superior capability of OPT for imaging fine structures with high resolution in optically-cleared embryos while only OCT can provide structural and functional imaging of live embryos ex vivo and in utero with micrometer scale resolution.

Development of a General Aza-Cope Reaction Trigger Applied to Fluorescence Imaging of Formaldehyde in Living Cells.

PubMed

Bruemmer, Kevin J; Walvoord, Ryan R; Brewer, Thomas F; Burgos-Barragan, Guillermo; Wit, Niek; Pontel, Lucas B; Patel, Ketan J; Chang, Christopher J

2017-04-19

Formaldehyde (FA) is a reactive signaling molecule that is continuously produced through a number of central biological pathways spanning epigenetics to one-carbon metabolism. On the other hand, aberrant, elevated levels of FA are implicated in disease states ranging from asthma to neurodegenerative disorders. In this context, fluorescence-based probes for FA imaging are emerging as potentially powerful chemical tools to help disentangle the complexities of FA homeostasis and its physiological and pathological contributions. Currently available FA indicators require direct modification of the fluorophore backbone through complex synthetic considerations to enable FA detection, often limiting the generalization of designs to other fluorophore classes. To address this challenge, we now present the rational, iterative development of a general reaction-based trigger utilizing 2-aza-Cope reactivity for selective and sensitive detection of FA in living systems. Specifically, we developed a homoallylamine functionality that can undergo a subsequent self-immolative β-elimination, creating a FA-responsive trigger that is capable of masking a phenol on a fluorophore or any other potential chemical scaffold for related imaging and/or therapeutic applications. We demonstrate the utility of this trigger by creating a series of fluorescent probes for FA with excitation and emission wavelengths that span the UV to visible spectral regions through caging of a variety of dye units. In particular, Formaldehyde Probe 573 (FAP573), based on a resorufin scaffold, is the most red-shifted and FA sensitive in this series in terms of signal-to-noise responses and enables identification of alcohol dehydrogenase 5 (ADH5) as an enzyme that regulates FA metabolism in living cells. The results provide a starting point for the broader use of 2-aza-Cope reactivity for probing and manipulating FA biology.

Detection and tracking of dual-labeled HIV particles using wide-field live cell imaging to follow viral core integrity

PubMed Central

Mamede, Joao I.; Hope, Thomas J.

2016-01-01

Summary Live cell imaging is a valuable technique that allows the characterization of the dynamic processes of the HIV-1 life-cycle. Here, we present a method of production and imaging of dual-labeled HIV viral particles that allows the visualization of two events. Varying release of the intravirion fluid phase marker reveals virion fusion and the loss of the integrity of HIV viral cores with the use of live wide-field fluorescent microscopy. PMID:26714704

In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy.

PubMed

Uddin, Md Imam; Evans, Stephanie M; Craft, Jason R; Capozzi, Megan E; McCollum, Gary W; Yang, Rong; Marnett, Lawrence J; Uddin, Md Jashim; Jayagopal, Ashwath; Penn, John S

2016-08-05

Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs.

In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy

PubMed Central

Uddin, Md. Imam; Evans, Stephanie M.; Craft, Jason R.; Capozzi, Megan E.; McCollum, Gary W.; Yang, Rong; Marnett, Lawrence J.; Uddin, Md. Jashim; Jayagopal, Ashwath; Penn, John S.

2016-01-01

Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs. PMID:27491345

Dances with Membranes: Breakthroughs from Super-resolution Imaging

PubMed Central

Curthoys, Nikki M.; Parent, Matthew; Mlodzianoski, Michael; Nelson, Andrew J.; Lilieholm, Jennifer; Butler, Michael B.; Valles, Matthew; Hess, Samuel T.

2017-01-01

Biological membrane organization mediates numerous cellular functions and has also been connected with an immense number of human diseases. However, until recently, experimental methodologies have been unable to directly visualize the nanoscale details of biological membranes, particularly in intact living cells. Numerous models explaining membrane organization have been proposed, but testing those models has required indirect methods; the desire to directly image proteins and lipids in living cell membranes is a strong motivation for the advancement of technology. The development of super-resolution microscopy has provided powerful tools for quantification of membrane organization at the level of individual proteins and lipids, and many of these tools are compatible with living cells. Previously inaccessible questions are now being addressed, and the field of membrane biology is developing rapidly. This chapter discusses how the development of super-resolution microscopy has led to fundamental advances in the field of biological membrane organization. We summarize the history and some models explaining how proteins are organized in cell membranes, and give an overview of various super-resolution techniques and methods of quantifying super-resolution data. We discuss the application of super-resolution techniques to membrane biology in general, and also with specific reference to the fields of actin and actin-binding proteins, virus infection, mitochondria, immune cell biology, and phosphoinositide signaling. Finally, we present our hopes and expectations for the future of super-resolution microscopy in the field of membrane biology. PMID:26015281

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

NASA Astrophysics Data System (ADS)

Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

2015-03-01

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy.

PubMed

Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

2015-03-09

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

Imaging C. elegans embryos using an epifluorescent microscope and open source software.

PubMed

Verbrugghe, Koen J C; Chan, Raymond C

2011-03-24

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples(1,2). Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage(3), thus providing an ideal experiment model for studying questions in cell biology(4,5)and development(6-9). C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis(10,11)) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis(12-15)). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters(16,17). These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo(18-21). In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Contrast imaging in mouse embryos using high-frequency ultrasound.

PubMed

Denbeigh, Janet M; Nixon, Brian A; Puri, Mira C; Foster, F Stuart

2015-03-04

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvβ3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Aberration-free FTIR spectroscopic imaging of live cells in microfluidic devices.

PubMed

Chan, K L Andrew; Kazarian, Sergei G

2013-07-21

The label-free, non-destructive chemical analysis offered by FTIR spectroscopic imaging is a very attractive and potentially powerful tool for studies of live biological cells. FTIR imaging of live cells is a challenging task, due to the fact that cells are cultured in an aqueous environment. While the synchrotron facility has proven to be a valuable tool for FTIR microspectroscopic studies of single live cells, we have demonstrated that high quality infrared spectra of single live cells using an ordinary Globar source can also be obtained by adding a pair of lenses to a common transmission liquid cell. The lenses, when placed on the transmission cell window, form pseudo hemispheres which removes the refraction of light and hence improve the imaging and spectral quality of the obtained data. This study demonstrates that infrared spectra of single live cells can be obtained without the focus shifting effect at different wavenumbers, caused by the chromatic aberration. Spectra of the single cells have confirmed that the measured spectral region remains in focus across the whole range, while spectra of the single cells measured without the lenses have shown some erroneous features as a result of the shift of focus. It has also been demonstrated that the addition of lenses can be applied to the imaging of cells in microfabricated devices. We have shown that it was not possible to obtain a focused image of an isolated cell in a droplet of DPBS in oil unless the lenses are applied. The use of the approach described herein allows for well focused images of single cells in DPBS droplets to be obtained.

Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolavirus-Infected Cells.

PubMed

Schudt, Gordian; Dolnik, Olga; Kolesnikova, Larissa; Biedenkopf, Nadine; Herwig, Astrid; Becker, Stephan

2015-10-01

Transport of ebolavirus (EBOV) nucleocapsids from perinuclear viral inclusions, where they are formed, to the site of budding at the plasma membrane represents an obligatory step of virus assembly. Until now, no live-cell studies on EBOV nucleocapsid transport have been performed, and participation of host cellular factors in this process, as well as the trajectories and speed of nucleocapsid transport, remain unknown. Live-cell imaging of EBOV-infected cells treated with different inhibitors of cellular cytoskeleton was used for the identification of cellular proteins involved in the nucleocapsid transport. EBOV nucleocapsids were visualized by expression of green fluorescent protein (GFP)-labeled nucleocapsid viral protein 30 (VP30) in EBOV-infected cells. Incorporation of the fusion protein VP30-GFP into EBOV nucleocapsids was confirmed by Western blot and indirect immunofluorescence analyses. Importantly, VP30-GFP fluorescence was readily detectable in the densely packed nucleocapsids inside perinuclear viral inclusions and in the dispersed rod-like nucleocapsids located outside of viral inclusions. Live-cell imaging of EBOV-infected cells revealed exit of single nucleocapsids from the viral inclusions and their intricate transport within the cytoplasm before budding at the plasma membrane. Nucleocapsid transport was arrested upon depolymerization of actin filaments (F-actin) and inhibition of the actin-nucleating Arp2/3 complex, and it was not altered upon depolymerization of microtubules or inhibition of N-WASP. Actin comet tails were often detected at the rear end of nucleocapsids. Marginally located nucleocapsids entered filopodia, moved inside, and budded from the tip of these thin cellular protrusions. Live-cell imaging of EBOV-infected cells revealed actin-dependent long-distance transport of EBOV nucleocapsids before budding at the cell surface. These findings provide useful insights into EBOV assembly and have potential application in the development of antivirals. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A Fluorogenic TMP-tag for High Signal-to-Background Intracellular Live Cell Imaging

PubMed Central

Jing, Chaoran

2013-01-01

Developed to compliment the use of fluorescent proteins in live cell imaging, chemical tags enjoy the benefit of modular incorporation of organic fluorophores, opening the possibility of high photon output and special photophysical properties. However, the theoretical challenge in using chemical tags as opposed to fluorescent proteins for high-resolution imaging is background noise from unbound and/or non-specifically bound ligand-fluorophore. We envisioned we could overcome this limit by engineering fluorogenic trimethoprim-based chemical tags (TMP-tags) in which the fluorophore is quenched until binding with E. coli dihydrofolate reductase (eDHFR) tagged protein displaces the quencher. Thus, we began by building a non-fluorogenic, covalent TMP-tag based on a proximity-induced reaction known to achieve rapid and specific labeling both in vitro and inside of living cells. Here we take the final step and render the covalent TMP-tag fluorogenic. In brief, we designed a trimeric TMP-fluorophore-quencher molecule (TMP-Q-Atto520) with the quencher attached to a leaving group that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue (Cys) installed just outside the binding pocket of eDHFR. We present the in vitro experiments showing that the eDHFR:L28C nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting in covalent labeling and remarkable fluorescence enhancement. Most significantly, while only our initial design, TMP-Q-Atto520 achieved the demanding goal of not only labeling highly abundant, localized intracellular proteins, but also less abundant, more dynamic cytoplasmic proteins. These results suggest that fluorogenic TMP-tag can significantly impact highresolution live cell imaging and further establish the potential of proximity-induced reactivity and organic chemistry more broadly as part of the growing toolbox for synthetic biology and cell engineering. PMID:23745575

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development.

PubMed

Hirsinger, Estelle; Steventon, Ben

2017-01-26

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

The application of computer image analysis in life sciences and environmental engineering

NASA Astrophysics Data System (ADS)

Mazur, R.; Lewicki, A.; Przybył, K.; Zaborowicz, M.; Koszela, K.; Boniecki, P.; Mueller, W.; Raba, B.

2014-04-01

The main aim of the article was to present research on the application of computer image analysis in Life Science and Environmental Engineering. The authors used different methods of computer image analysis in developing of an innovative biotest in modern biomonitoring of water quality. Created tools were based on live organisms such as bioindicators Lemna minor L. and Hydra vulgaris Pallas as well as computer image analysis method in the assessment of negatives reactions during the exposition of the organisms to selected water toxicants. All of these methods belong to acute toxicity tests and are particularly essential in ecotoxicological assessment of water pollutants. Developed bioassays can be used not only in scientific research but are also applicable in environmental engineering and agriculture in the study of adverse effects on water quality of various compounds used in agriculture and industry.

In vivo bioluminescence and reflectance imaging of multiple organs in bioluminescence reporter mice by bundled-fiber-coupled microscopy

PubMed Central

Ando, Yoriko; Sakurai, Takashi; Koida, Kowa; Tei, Hajime; Hida, Akiko; Nakao, Kazuki; Natsume, Mistuo; Numano, Rika

2016-01-01

Bioluminescence imaging (BLI) is used in biomedical research to monitor biological processes within living organisms. Recently, fiber bundles with high transmittance and density have been developed to detect low light with high resolution. Therefore, we have developed a bundled-fiber-coupled microscope with a highly sensitive cooled-CCD camera that enables the BLI of organs within the mouse body. This is the first report of in vivo BLI of the brain and multiple organs in luciferase-reporter mice using bundled-fiber optics. With reflectance imaging, the structures of blood vessels and organs can be seen clearly with light illumination, and it allowed identification of the structural details of bioluminescence images. This technique can also be applied to clinical diagnostics in a low invasive manner. PMID:27231601

Using Digital Microscopy

ERIC Educational Resources Information Center

Travaille, Madelaine; Adams, Sandra D.

2006-01-01

Studying "Caenorhabditis elegans" ("C. elegans") live cultures provides excellent opportunities for authentic inquiry in a high school anatomy and physiology or other biology lab course. Using a digital dissection microscope, a student can photograph the organism during various stages of development and study and analyze the images. In this…

Sparking Interest

ERIC Educational Resources Information Center

Guth, Douglas J.

2018-01-01

Community colleges are attempting to bridge America's widening blue-collar skills gap through workforce development programs promising living-wage jobs that don't require four years of college. While trade, construction and manufacturing companies are starving for talented workers, these fields also suffer from an image problem, one fueled by…

Luminescence and fluorescence of essential oils. Fluorescence imaging in vivo of wild chamomile oil.

PubMed

Boschi, F; Fontanella, M; Calderan, L; Sbarbati, A

2011-06-16

Essential oils are currently of great importance to pharmaceutical companies, cosmetics producers and manufacturers of veterinary products. They are found in perfumes, creams, bath products, and household cleaning substances, and are used for flavouring food and drinks. It is well known that some of them act on the respiratory apparatus. The increasing interest in optical imaging techniques and the development of related technologies have made possible the investigation of the optical properties of several compounds. Luminescent properties of essential oils have not been extensively investigated. We evaluated the luminescent and fluorescent emissions of several essential oils, in order to detect them in living organisms by exploiting their optical properties. Some fluorescent emission data were high enough to be detected in dermal treatments. Consequently, we demonstrated how the fluorescent signal can be monitored for at least three hours on the skin of living mice treated with wild chamomile oil. The results encourage development of this technique to investigate the properties of drugs and cosmetics containing essential oils.

Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development

PubMed Central

Mora-Bermúdez, Felipe; Badsha, Farhath; Kanton, Sabina; Camp, J Gray; Vernot, Benjamin; Köhler, Kathrin; Voigt, Birger; Okita, Keisuke; Maricic, Tomislav; He, Zhisong; Lachmann, Robert; Pääbo, Svante; Treutlein, Barbara; Huttner, Wieland B

2016-01-01

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution. DOI: http://dx.doi.org/10.7554/eLife.18683.001 PMID:27669147

Nanohybrids with Magnetic and Persistent Luminescence Properties for Cell Labeling, Tracking, In Vivo Real-Time Imaging, and Magnetic Vectorization.

PubMed

Teston, Eliott; Maldiney, Thomas; Marangon, Iris; Volatron, Jeanne; Lalatonne, Yoann; Motte, Laurence; Boisson-Vidal, Catherine; Autret, Gwennhael; Clément, Olivier; Scherman, Daniel; Gazeau, Florence; Richard, Cyrille

2018-04-01

Once injected into a living organism, cells diffuse or migrate around the initial injection point and become impossible to be visualized and tracked in vivo. The present work concerns the development of a new technique for therapeutic cell labeling and subsequent in vivo visualization and magnetic retention. It is hypothesized and subsequently demonstrated that nanohybrids made of persistent luminescence nanoparticles and ultrasmall superparamagnetic iron oxide nanoparticles incorporated into a silica matrix can be used as an effective nanoplatform to label therapeutic cells in a nontoxic way in order to dynamically track them in real-time in vitro and in living mice. As a proof-of-concept, it is shown that once injected, these labeled cells can be visualized and attracted in vivo using a magnet. This first step suggests that these nanohybrids represent efficient multifunctional nanoprobes for further imaging guided cell therapies development. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Luminescence and fluorescence of essential oils. Fluorescence imaging in vivo of wild chamomile oil

PubMed Central

Boschi, F.; Fontanella, M.; Calderan, L.; Sbarbati, A.

2011-01-01

Essential oils are currently of great importance to pharmaceutical companies, cosmetics producers and manufacturers of veterinary products. They are found in perfumes, creams, bath products, and household cleaning substances, and are used for flavouring food and drinks. It is well known that some of them act on the respiratory apparatus. The increasing interest in optical imaging techniques and the development of related technologies have made possible the investigation of the optical properties of several compounds. Luminescent properties of essential oils have not been extensively investigated. We evaluated the luminescent and fluorescent emissions of several essential oils, in order to detect them in living organisms by exploiting their optical properties. Some fluorescent emission data were high enough to be detected in dermal treatments. Consequently, we demonstrated how the fluorescent signal can be monitored for at least three hours on the skin of living mice treated with wild chamomile oil. The results encourage development of this technique to investigate the properties of drugs and cosmetics containing essential oils. PMID:22193298

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

PubMed

Higuchi-Sanabria, Ryo; Garcia, Enrique J; Tomoiaga, Delia; Munteanu, Emilia L; Feinstein, Paul; Pon, Liza A

2016-01-01

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

Bioorthogonal Chemical Imaging for Biomedicine

NASA Astrophysics Data System (ADS)

Min, Wei

2017-06-01

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because relatively bulky fluorescent labels could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, we have developed a bioorthogonal chemical imaging platform. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes, nitriles and stable isotopes including 2H and 13C), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, multiplicity and biocompatibility for imaging small biomolecules in live systems including tissues and organisms. Exciting biomedical applications such as imaging fatty acid metabolism related to lipotoxicity, glucose uptake and metabolism, drug trafficking, protein synthesis, DNA replication, protein degradation, RNA synthesis and tumor metabolism will be presented. This bioorthogonal chemical imaging platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, further chemical and spectroscopic strategies allow for multicolor bioorthogonal chemical imaging, a valuable technique in the era of "omics". We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species, bringing small molecules under the illumination of modern light microscopy.

Labiopexy and labioplasty for labium majus rejuvenation in light of a newly discovered anatomic structure.

PubMed

Ostrzenski, Adam

2014-06-01

Currently, removal of excessive cutis with subdermal adipose tissues is done for labium majus rejuvenation. The objectives of this study were to determine the accurate labium majus anatomy, to develop new labiopexy and labioplasty techniques, to attest to the applicability of these two new operations, and to determine the impact of the operations on self-perceived body image and sensation of nerve endings. A prospective case series study was conducted. Eleven fresh human female cadavers and three living subjects were studied. Living subjects were healthy women and presented with labium majus deformities. The study was conducted in three phases: phase I, the labium majus anatomy of the cadavers was studied; phase II, anatomic findings from phase I were used to develop and standardize new stepwise surgical interventions; and phase III, newly developed operations were implemented on living subjects. Labial sensation tests of nerve endings were performed preoperatively and at 6 months postoperatively. The primary outcome measured the labial anatomy and applicability of the new operations. The secondary outcome measured the self-perceived body image and sensation of nerve endings before and after surgery. A new anatomic feature, the labium majus adipose sac, was discovered and was present in each subject. Labium majus labiopexy and labioplasty were executed without technical difficulties or complications. Postoperatively, the self-perceived body image improved and sensation of nerve endings was intact. The labium majus adipose sac is present in each woman. Intraoperatively, newly developed labium majus labiopexy and/or labioplasty do not create technical problems. Self-perceived body image improves and sensation of the nerve endings is intact after labiopexy or labioplasty. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266.

Quantitative label-free sperm imaging by means of transport of intensity

NASA Astrophysics Data System (ADS)

Poola, Praveen Kumar; Pandiyan, Vimal Prabhu; Jayaraman, Varshini; John, Renu

2016-03-01

Most living cells are optically transparent which makes it difficult to visualize them under bright field microscopy. Use of contrast agents or markers and staining procedures are often followed to observe these cells. However, most of these staining agents are toxic and not applicable for live cell imaging. In the last decade, quantitative phase imaging has become an indispensable tool for morphological characterization of the phase objects without any markers. In this paper, we report noninterferometric quantitative phase imaging of live sperm cells by solving transport of intensity equations with recorded intensity measurements along optical axis on a commercial bright field microscope.

Optical system for monitoring the internal image of foods and the human body

NASA Astrophysics Data System (ADS)

Aisha, Nur; Fugang, Lee; Genta, Tsuneaki; Yamaguchi, Kenzo; Fukuda, Mitsuo

2011-10-01

We present a technique for monitoring alien substances in foods and blood vessels in the human body. A prototype of the system using near-infrared rays is developed, and its applicability to food is analyzed in detail. The system developed is basically composed of an optical source and a CMOS sensor. Some optical components adjusted at 850-nm band are also set in the system. The system can monitor organic alien substances intentionally added to foods and blood vessels. The clarity of the image increased with decreasing water content and homogeneous material density. The resolving power of the images was confirmed to be about 100 μm. This technique will be useful for our safety and health in our daily lives.

Animals In Synchrotrons: Overcoming Challenges For High-Resolution, Live, Small-Animal Imaging

NASA Astrophysics Data System (ADS)

Donnelley, Martin; Parsons, David; Morgan, Kaye; Siu, Karen

2010-07-01

Physiological studies in small animals can be complicated, but the complexity is increased dramatically when performing live-animal synchrotron X-ray imaging studies. Our group has extensive experience in high-resolution live-animal imaging at the Japanese SPring-8 synchrotron, primarily examining airways in two-dimensions. These experiments normally image an area of 1.8 mm×1.2 mm at a pixel resolution of 0.45 μm and are performed with live, intact, anaesthetized mice. There are unique challenges in this experimental setting. Importantly, experiments must be performed in an isolated imaging hutch not specifically designed for small-animal imaging. This requires equipment adapted to remotely monitor animals, maintain their anesthesia, and deliver test substances while collecting images. The horizontal synchrotron X-ray beam has a fixed location and orientation that limits experimental flexibility. The extremely high resolution makes locating anatomical regions-of-interest slow and can result in a high radiation dose, and at this level of magnification small animal movements produce motion-artifacts that can render acquired images unusable. Here we describe our experimental techniques and how we have overcome several challenges involved in performing live mouse synchrotron imaging. Experiments have tested different mouse strains, with hairless strains minimizing overlying skin and hair artifacts. Different anesthetics have also be trialed due to the limited choices available at SPring-8. Tracheal-intubation methods have been refined and controlled-ventilation is now possible using a specialized small-animal ventilator. With appropriate animal restraint and respiratory-gating, motion-artifacts have been minimized. The animal orientation (supine vs. head-high) also appears to affect animal physiology, and can alter image quality. Our techniques and image quality at SPring-8 have dramatically improved and in the near future we plan to translate this experience to the Imaging and Medical Beamline at the Australian Synchrotron. Overcoming these challenges has permitted increasingly sophisticated imaging of animals with synchrotron X-rays, and we expect a bright future for these techniques.

Animals In Synchrotrons: Overcoming Challenges For High-Resolution, Live, Small-Animal Imaging

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

Donnelley, Martin; Parsons, David; Women's and Children's Health Research Institute, Adelaide, South Australia

Physiological studies in small animals can be complicated, but the complexity is increased dramatically when performing live-animal synchrotron X-ray imaging studies. Our group has extensive experience in high-resolution live-animal imaging at the Japanese SPring-8 synchrotron, primarily examining airways in two-dimensions. These experiments normally image an area of 1.8 mmx1.2 mm at a pixel resolution of 0.45 {mu}m and are performed with live, intact, anaesthetized mice.There are unique challenges in this experimental setting. Importantly, experiments must be performed in an isolated imaging hutch not specifically designed for small-animal imaging. This requires equipment adapted to remotely monitor animals, maintain their anesthesia, andmore » deliver test substances while collecting images. The horizontal synchrotron X-ray beam has a fixed location and orientation that limits experimental flexibility. The extremely high resolution makes locating anatomical regions-of-interest slow and can result in a high radiation dose, and at this level of magnification small animal movements produce motion-artifacts that can render acquired images unusable. Here we describe our experimental techniques and how we have overcome several challenges involved in performing live mouse synchrotron imaging.Experiments have tested different mouse strains, with hairless strains minimizing overlying skin and hair artifacts. Different anesthetics have also be trialed due to the limited choices available at SPring-8. Tracheal-intubation methods have been refined and controlled-ventilation is now possible using a specialized small-animal ventilator. With appropriate animal restraint and respiratory-gating, motion-artifacts have been minimized. The animal orientation (supine vs. head-high) also appears to affect animal physiology, and can alter image quality. Our techniques and image quality at SPring-8 have dramatically improved and in the near future we plan to translate this experience to the Imaging and Medical Beamline at the Australian Synchrotron.Overcoming these challenges has permitted increasingly sophisticated imaging of animals with synchrotron X-rays, and we expect a bright future for these techniques.« less

Living cell dry mass measurement using quantitative phase imaging with quadriwave lateral shearing interferometry: an accuracy and sensitivity discussion.

PubMed

Aknoun, Sherazade; Savatier, Julien; Bon, Pierre; Galland, Frédéric; Abdeladim, Lamiae; Wattellier, Benoit; Monneret, Serge

2015-01-01

Single-cell dry mass measurement is used in biology to follow cell cycle, to address effects of drugs, or to investigate cell metabolism. Quantitative phase imaging technique with quadriwave lateral shearing interferometry (QWLSI) allows measuring cell dry mass. The technique is very simple to set up, as it is integrated in a camera-like instrument. It simply plugs onto a standard microscope and uses a white light illumination source. Its working principle is first explained, from image acquisition to automated segmentation algorithm and dry mass quantification. Metrology of the whole process, including its sensitivity, repeatability, reliability, sources of error, over different kinds of samples and under different experimental conditions, is developed. We show that there is no influence of magnification or spatial light coherence on dry mass measurement; effect of defocus is more critical but can be calibrated. As a consequence, QWLSI is a well-suited technique for fast, simple, and reliable cell dry mass study, especially for live cells.

Imaging and modeling of collagen architecture in living tissue with polarized light transfer (Conference Presentation)

NASA Astrophysics Data System (ADS)

Ramella-Roman, Jessica C.; Stoff, Susan; Chue-Sang, Joseph; Bai, Yuqiang

2016-03-01

The extra-cellular space in connective tissue of animals and humans alike is comprised in large part of collagen. Monitoring of collagen arrangement and cross-linking has been utilized to diagnose a variety of medical conditions and guide surgical intervention. For example, collagen monitoring is useful in the assessment and treatment of cervical cancer, skin cancer, myocardial infarction, and non-arteritic anterior ischemic optic neuropathy. We have developed a suite of tools and models based on polarized light transfer for the assessment of collagen presence, cross-linking, and orientation in living tissue. Here we will present some example of such approach applied to the human cervix. We will illustrate a novel Mueller Matrix (MM) imaging system for the study of cervical tissue; furthermore we will show how our model of polarized light transfer through cervical tissue compares to the experimental findings. Finally we will show validation of the methodology through histological results and Second Harmonic imaging microscopy.

Non-invasive airway health assessment: synchrotron imaging reveals effects of rehydrating treatments on mucociliary transit in-vivo.

PubMed

Donnelley, Martin; Morgan, Kaye S; Siu, Karen K W; Farrow, Nigel R; Stahr, Charlene S; Boucher, Richard C; Fouras, Andreas; Parsons, David W

2014-01-14

To determine the efficacy of potential cystic fibrosis (CF) therapies we have developed a novel mucociliary transit (MCT) measurement that uses synchrotron phase contrast X-ray imaging (PCXI) to non-invasively measure the transit rate of individual micron-sized particles deposited into the airways of live mice. The aim of this study was to image changes in MCT produced by a rehydrating treatment based on hypertonic saline (HS), a current CF clinical treatment. Live mice received HS containing a long acting epithelial sodium channel blocker (P308); isotonic saline; or no treatment, using a nebuliser integrated within a small-animal ventilator circuit. Marker particle motion was tracked for 20 minutes using PCXI. There were statistically significant increases in MCT in the isotonic and HS-P308 groups. The ability to quantify in vivo changes in MCT may have utility in pre-clinical research studies designed to bring new genetic and pharmaceutical treatments for respiratory diseases into clinical trials.

Radiosyntheses using Fluorine-18: the Art and Science of Late Stage Fluorination

PubMed Central

Cole, Erin L.; Stewart, Megan N.; Littich, Ryan; Hoareau, Raphael; Scott, Peter J. H.

2014-01-01

Positron (β+) emission tomography (PE) is a powerful, noninvasive tool for the in vivo, three-dimensional imaging of physiological structures and biochemical pathways. The continued growth of PET imaging relies on a corresponding increase in access to radiopharmaceuticals (biologically active molecules labeled with short-lived radionuclides such as fluorine-18). This unique need to incorporate the short-lived fluorine-18 atom (t1/2 = 109.77 min) as late in the synthetic pathway as possible has made development of methodologies that enable rapid and efficient late stage fluorination an area of research within its own right. In this review we describe strategies for radiolabeling with fluorine-18, including classical fluorine-18 radiochemistry and emerging techniques for late stage fluorination reactions, as well as labeling technologies such as microfluidics and solid-phase radiochemistry. The utility of fluorine-18 labeled radiopharmaceuticals is showcased through recent applications of PET imaging in the healthcare, personalized medicine and drug discovery settings. PMID:24484425

Non-invasive airway health assessment: Synchrotron imaging reveals effects of rehydrating treatments on mucociliary transit in-vivo

NASA Astrophysics Data System (ADS)

Donnelley, Martin; Morgan, Kaye S.; Siu, Karen K. W.; Farrow, Nigel R.; Stahr, Charlene S.; Boucher, Richard C.; Fouras, Andreas; Parsons, David W.

2014-01-01

To determine the efficacy of potential cystic fibrosis (CF) therapies we have developed a novel mucociliary transit (MCT) measurement that uses synchrotron phase contrast X-ray imaging (PCXI) to non-invasively measure the transit rate of individual micron-sized particles deposited into the airways of live mice. The aim of this study was to image changes in MCT produced by a rehydrating treatment based on hypertonic saline (HS), a current CF clinical treatment. Live mice received HS containing a long acting epithelial sodium channel blocker (P308); isotonic saline; or no treatment, using a nebuliser integrated within a small-animal ventilator circuit. Marker particle motion was tracked for 20 minutes using PCXI. There were statistically significant increases in MCT in the isotonic and HS-P308 groups. The ability to quantify in vivo changes in MCT may have utility in pre-clinical research studies designed to bring new genetic and pharmaceutical treatments for respiratory diseases into clinical trials.

Mapping impervious surface expansion using medium resolution satellite image time series: A case study in Yangtze river delta, China

USDA-ARS?s Scientific Manuscript database

Due to the rapid growth of population and economic development in the developing countries, more people are now living in the cities than in the rural areas in the world for the first time in human history. As a result, cities are sprawling rapidly into their surroundings. A characteristic change as...

Image Processing for Bioluminescence Resonance Energy Transfer Measurement-BRET-Analyzer.

PubMed

Chastagnier, Yan; Moutin, Enora; Hemonnot, Anne-Laure; Perroy, Julie

2017-01-01

A growing number of tools now allow live recordings of various signaling pathways and protein-protein interaction dynamics in time and space by ratiometric measurements, such as Bioluminescence Resonance Energy Transfer (BRET) Imaging. Accurate and reproducible analysis of ratiometric measurements has thus become mandatory to interpret quantitative imaging. In order to fulfill this necessity, we have developed an open source toolset for Fiji- BRET-Analyzer -allowing a systematic analysis, from image processing to ratio quantification. We share this open source solution and a step-by-step tutorial at https://github.com/ychastagnier/BRET-Analyzer. This toolset proposes (1) image background subtraction, (2) image alignment over time, (3) a composite thresholding method of the image used as the denominator of the ratio to refine the precise limits of the sample, (4) pixel by pixel division of the images and efficient distribution of the ratio intensity on a pseudocolor scale, and (5) quantification of the ratio mean intensity and standard variation among pixels in chosen areas. In addition to systematize the analysis process, we show that the BRET-Analyzer allows proper reconstitution and quantification of the ratiometric image in time and space, even from heterogeneous subcellular volumes. Indeed, analyzing twice the same images, we demonstrate that compared to standard analysis BRET-Analyzer precisely define the luminescent specimen limits, enlightening proficient strengths from small and big ensembles over time. For example, we followed and quantified, in live, scaffold proteins interaction dynamics in neuronal sub-cellular compartments including dendritic spines, for half an hour. In conclusion, BRET-Analyzer provides a complete, versatile and efficient toolset for automated reproducible and meaningful image ratio analysis.

SPLASSH: Open source software for camera-based high-speed, multispectral in-vivo optical image acquisition

PubMed Central

Sun, Ryan; Bouchard, Matthew B.; Hillman, Elizabeth M. C.

2010-01-01

Camera-based in-vivo optical imaging can provide detailed images of living tissue that reveal structure, function, and disease. High-speed, high resolution imaging can reveal dynamic events such as changes in blood flow and responses to stimulation. Despite these benefits, commercially available scientific cameras rarely include software that is suitable for in-vivo imaging applications, making this highly versatile form of optical imaging challenging and time-consuming to implement. To address this issue, we have developed a novel, open-source software package to control high-speed, multispectral optical imaging systems. The software integrates a number of modular functions through a custom graphical user interface (GUI) and provides extensive control over a wide range of inexpensive IEEE 1394 Firewire cameras. Multispectral illumination can be incorporated through the use of off-the-shelf light emitting diodes which the software synchronizes to image acquisition via a programmed microcontroller, allowing arbitrary high-speed illumination sequences. The complete software suite is available for free download. Here we describe the software’s framework and provide details to guide users with development of this and similar software. PMID:21258475

Computational high-resolution optical imaging of the living human retina

NASA Astrophysics Data System (ADS)

Shemonski, Nathan D.; South, Fredrick A.; Liu, Yuan-Zhi; Adie, Steven G.; Scott Carney, P.; Boppart, Stephen A.

2015-07-01

High-resolution in vivo imaging is of great importance for the fields of biology and medicine. The introduction of hardware-based adaptive optics (HAO) has pushed the limits of optical imaging, enabling high-resolution near diffraction-limited imaging of previously unresolvable structures. In ophthalmology, when combined with optical coherence tomography, HAO has enabled a detailed three-dimensional visualization of photoreceptor distributions and individual nerve fibre bundles in the living human retina. However, the introduction of HAO hardware and supporting software adds considerable complexity and cost to an imaging system, limiting the number of researchers and medical professionals who could benefit from the technology. Here we demonstrate a fully automated computational approach that enables high-resolution in vivo ophthalmic imaging without the need for HAO. The results demonstrate that computational methods in coherent microscopy are applicable in highly dynamic living systems.

Aerospace Technology Innovation. Volume 10

NASA Technical Reports Server (NTRS)

Turner, Janelle (Editor); Cousins, Liz (Editor); Bennett, Evonne (Editor); Vendette, Joel (Editor); West, Kenyon (Editor)

2002-01-01

Whether finding new applications for existing NASA technologies or developing unique marketing strategies to demonstrate them, NASA's offices are committed to identifying unique partnering opportunities. Through their efforts NASA leverages resources through joint research and development, and gains new insight into the core areas relevant to all NASA field centers. One of the most satisfying aspects of my job comes when I learn of a mission-driven technology that can be spun-off to touch the lives of everyday people. NASA's New Partnerships in Medical Diagnostic Imaging is one such initiative. Not only does it promise to provide greater dividends for the country's investment in aerospace research, but also to enhance the American quality of life. This issue of Innovation highlights the new NASA-sponsored initiative in medical imaging. Early in 2001, NASA announced the launch of the New Partnerships in Medical Diagnostic Imaging initiative to promote the partnership and commercialization of NASA technologies in the medical imaging industry. NASA and the medical imaging industry share a number of crosscutting technologies in areas such as high-performance detectors and image-processing tools. Many of the opportunities for joint development and technology transfer to the medical imaging market also hold the promise for future spin back to NASA.

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

PubMed Central

Gustafsson, Nils; Culley, Siân; Ashdown, George; Owen, Dylan M.; Pereira, Pedro Matos; Henriques, Ricardo

2016-01-01

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours. PMID:27514992

In Vivo Optical Imaging for Targeted Drug Kinetics and Localization for Oral Surgery and Super-Resolution, Facilitated by Printed Phantoms

NASA Astrophysics Data System (ADS)

Bentz, Brian Z.

Many human cancer cell types over-express folate receptors, and this provides an opportunity to develop targeted anti-cancer drugs. For these drugs to be effective, their kinetics must be well understood in vivo and in deep tissue where tumors occur. We demonstrate a method for imaging these parameters by incorporating a kinetic compartment model and fluorescence into optical diffusion tomography (ODT). The kinetics were imaged in a live mouse, and found to be in agreement with previous in vitro studies, demonstrating the validity of the method and its feasibility as an effective tool in preclinical drug development studies. Progress in developing optical imaging for biomedical applications requires customizable and often complex objects known as "phantoms" for testing and evaluation. We present new optical phantoms fabricated using inexpensive 3D printing methods with multiple materials, allowing for the placement of complex inhomogeneities in heterogeneous or anatomically realistic geometries, as opposed to previous phantoms which were limited to simple shapes formed by molds or machining. Furthermore, we show that Mie theory can be used to design the optical properties to match a target tissue. The phantom fabrication methods are versatile, can be applied to optical imaging methods besides diffusive imaging, and can be used in the calibration of live animal imaging data. Applications of diffuse optical imaging in the operating theater have been limited in part due to computational burden. We present an approach for the fast localization of arteries in the roof of the mouth that has the potential to reduce complications. Furthermore, we use the extracted position information to fabricate a custom surgical guide using 3D printing that could protect the arteries during surgery. The resolution of ODT is severely limited by the attenuation of high spatial frequencies. We present a super-resolution method achieved through the point localization of fluorescent inhomogeneities in a tissue-like scattering medium, and examine the localization uncertainty numerically and experimentally. Furthermore, we show numerical results for the localization of multiple fluorescent inhomogeneities by distinguishing them based on temporal characteristics. Potential applications include imaging neuron activation in the brain.

A philosophy for CNS radiotracer design.

PubMed

Van de Bittner, Genevieve C; Ricq, Emily L; Hooker, Jacob M

2014-10-21

Decades after its discovery, positron emission tomography (PET) remains the premier tool for imaging neurochemistry in living humans. Technological improvements in radiolabeling methods, camera design, and image analysis have kept PET in the forefront. In addition, the use of PET imaging has expanded because researchers have developed new radiotracers that visualize receptors, transporters, enzymes, and other molecular targets within the human brain. However, of the thousands of proteins in the central nervous system (CNS), researchers have successfully imaged fewer than 40 human proteins. To address the critical need for new radiotracers, this Account expounds on the decisions, strategies, and pitfalls of CNS radiotracer development based on our current experience in this area. We discuss the five key components of radiotracer development for human imaging: choosing a biomedical question, selection of a biological target, design of the radiotracer chemical structure, evaluation of candidate radiotracers, and analysis of preclinical imaging. It is particularly important to analyze the market of scientists or companies who might use a new radiotracer and carefully select a relevant biomedical question(s) for that audience. In the selection of a specific biological target, we emphasize how target localization and identity can constrain this process and discuss the optimal target density and affinity ratios needed for binding-based radiotracers. In addition, we discuss various PET test-retest variability requirements for monitoring changes in density, occupancy, or functionality for new radiotracers. In the synthesis of new radiotracer structures, high-throughput, modular syntheses have proved valuable, and these processes provide compounds with sites for late-stage radioisotope installation. As a result, researchers can manage the time constraints associated with the limited half-lives of isotopes. In order to evaluate brain uptake, a number of methods are available to predict bioavailability, blood-brain barrier (BBB) permeability, and the associated issues of nonspecific binding and metabolic stability. To evaluate the synthesized chemical library, researchers need to consider high-throughput affinity assays, the analysis of specific binding, and the importance of fast binding kinetics. Finally, we describe how we initially assess preclinical radiotracer imaging, using brain uptake, specific binding, and preliminary kinetic analysis to identify promising radiotracers that may be useful for human brain imaging. Although we discuss these five design components separately and linearly in this Account, in practice we develop new PET-based radiotracers using these design components nonlinearly and iteratively to develop new compounds in the most efficient way possible.

Modeling in vivo fluorescence of small animals using TracePro software

NASA Astrophysics Data System (ADS)

Leavesley, Silas; Rajwa, Bartek; Freniere, Edward R.; Smith, Linda; Hassler, Richard; Robinson, J. Paul

2007-02-01

The theoretical modeling of fluorescence excitation, emission, and propagation within living tissue has been a limiting factor in the development and calibration of in vivo small animal fluorescence imagers. To date, no definitive calibration standard, or phantom, has been developed for use with small animal fluorescence imagers. Our work in the theoretical modeling of fluorescence in small animals using solid modeling software is useful in optimizing the design of small animal imaging systems, and in predicting their response to a theoretical model. In this respect, it is also valuable in the design of a fluorescence phantom for use in in vivo small animal imaging. The use of phantoms is a critical step in the testing and calibration of most diagnostic medical imaging systems. Despite this, a realistic, reproducible, and informative phantom has yet to be produced for use in small animal fluorescence imaging. By modeling the theoretical response of various types of phantoms, it is possible to determine which parameters are necessary for accurately modeling fluorescence within inhomogenous scattering media such as tissue. Here, we present the model that has been developed, the challenges and limitations associated with developing such a model, and the applicability of this model to experimental results obtained in a commercial small animal fluorescence imager.

Correlation of live-cell imaging with volume scanning electron microscopy.

PubMed

Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

2017-01-01

Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

Equally sloped X-ray microtomography of living insects with low radiation dose and improved resolution capability

NASA Astrophysics Data System (ADS)

Yao, Shengkun; Fan, Jiadong; Zong, Yunbing; He, You; Zhou, Guangzhao; Sun, Zhibin; Zhang, Jianhua; Huang, Qingjie; Xiao, Tiqiao; Jiang, Huaidong

2016-03-01

Three-dimensional X-ray imaging of living specimens is challenging due to the limited resolution of conventional absorption contrast X-ray imaging and potential irradiation damage of biological specimens. In this letter, we present microtomography of a living specimen combining phase-contrast imaging and a Fourier-based iterative algorithm termed equally sloped tomography. Non-destructive 3D imaging of an anesthetized living yellow mealworm Tenebrio molitor was demonstrated with a relatively low dose using synchrotron generated X-rays. Based on the high-quality 3D images, branching tracheoles and different tissues of the insect in a natural state were identified and analyzed, demonstrating a significant advantage of the technique over conventional X-ray radiography or histotomy. Additionally, the insect survived without problem after a 1.92-s X-ray exposure and subsequent absorbed radiation dose of ˜1.2 Gy. No notable physiological effects were observed after reviving the insect from anesthesia. The improved static tomographic method demonstrated in this letter shows advantage in the non-destructive structural investigation of living insects in three dimensions due to the low radiation dose and high resolution capability, and offers many potential applications in biological science.

Benzofurazan Sulfides for Thiol Imaging and Quantification in Live Cells through Fluorescence Microscopy

PubMed Central

Li, Yinghong; Yang, Yang; Guan, Xiangming

2012-01-01

Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a–e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH2, -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 nm and 520 nm as the excitation and emission wavelengths respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an HPLC/UV total thiol assay method. The reagents and method will be of a great value to thiol redox-related research. PMID:22794193

Equally sloped X-ray microtomography of living insects with low radiation dose and improved resolution capability

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

Yao, Shengkun; Fan, Jiadong; Zong, Yunbing

Three-dimensional X-ray imaging of living specimens is challenging due to the limited resolution of conventional absorption contrast X-ray imaging and potential irradiation damage of biological specimens. In this letter, we present microtomography of a living specimen combining phase-contrast imaging and a Fourier-based iterative algorithm termed equally sloped tomography. Non-destructive 3D imaging of an anesthetized living yellow mealworm Tenebrio molitor was demonstrated with a relatively low dose using synchrotron generated X-rays. Based on the high-quality 3D images, branching tracheoles and different tissues of the insect in a natural state were identified and analyzed, demonstrating a significant advantage of the technique overmore » conventional X-ray radiography or histotomy. Additionally, the insect survived without problem after a 1.92-s X-ray exposure and subsequent absorbed radiation dose of ∼1.2 Gy. No notable physiological effects were observed after reviving the insect from anesthesia. The improved static tomographic method demonstrated in this letter shows advantage in the non-destructive structural investigation of living insects in three dimensions due to the low radiation dose and high resolution capability, and offers many potential applications in biological science.« less

Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy

PubMed Central

Vélez-Ortega, A. Catalina; Frolenkov, Gregory I.

2016-01-01

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3 to 4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette –which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier– is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface. Here we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations. PMID:27259929

Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy.

PubMed

Vélez-Ortega, A Catalina; Frolenkov, Gregory I

2016-01-01

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3-4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette-which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier-is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface.Here, we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations.

Design and implementation of a PC-based image-guided surgical system.

PubMed

Stefansic, James D; Bass, W Andrew; Hartmann, Steven L; Beasley, Ryan A; Sinha, Tuhin K; Cash, David M; Herline, Alan J; Galloway, Robert L

2002-11-01

In interactive, image-guided surgery, current physical space position in the operating room is displayed on various sets of medical images used for surgical navigation. We have developed a PC-based surgical guidance system (ORION) which synchronously displays surgical position on up to four image sets and updates them in real time. There are three essential components which must be developed for this system: (1) accurately tracked instruments; (2) accurate registration techniques to map physical space to image space; and (3) methods to display and update the image sets on a computer monitor. For each of these components, we have developed a set of dynamic link libraries in MS Visual C++ 6.0 supporting various hardware tools and software techniques. Surgical instruments are tracked in physical space using an active optical tracking system. Several of the different registration algorithms were developed with a library of robust math kernel functions, and the accuracy of all registration techniques was thoroughly investigated. Our display was developed using the Win32 API for windows management and tomographic visualization, a frame grabber for live video capture, and OpenGL for visualization of surface renderings. We have begun to use this current implementation of our system for several surgical procedures, including open and minimally invasive liver surgery.

Mosaicing of single plane illumination microscopy images using groupwise registration and fast content-based image fusion

NASA Astrophysics Data System (ADS)

Preibisch, Stephan; Rohlfing, Torsten; Hasak, Michael P.; Tomancak, Pavel

2008-03-01

Single Plane Illumination Microscopy (SPIM; Huisken et al., Nature 305(5686):1007-1009, 2004) is an emerging microscopic technique that enables live imaging of large biological specimens in their entirety. By imaging the living biological sample from multiple angles SPIM has the potential to achieve isotropic resolution throughout even relatively large biological specimens. For every angle, however, only a relatively shallow section of the specimen is imaged with high resolution, whereas deeper regions appear increasingly blurred. In order to produce a single, uniformly high resolution image, we propose here an image mosaicing algorithm that combines state of the art groupwise image registration for alignment with content-based image fusion to prevent degrading of the fused image due to regional blurring of the input images. For the registration stage, we introduce an application-specific groupwise transformation model that incorporates per-image as well as groupwise transformation parameters. We also propose a new fusion algorithm based on Gaussian filters, which is substantially faster than fusion based on local image entropy. We demonstrate the performance of our mosaicing method on data acquired from living embryos of the fruit fly, Drosophila, using four and eight angle acquisitions.

WE-FG-BRA-04: A Portable Confocal Microscope to Image Live Cell Damage Response Induced by Therapeutic Radiation

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

McFadden, C; Flint, D; Grosshans, D

Purpose: To construct a custom and portable fluorescence confocal laser-scanning microscope (FCLSM) that can be placed in the path of therapeutic radiation beams to study real-time radiation-induced damage response in live cells. Methods: We designed and constructed a portable FCLSM with three laser diodes for excitation (405, 488, and 635 nm). An objective lens focuses the excitation light and collects fluorescence from the sample. A pair of galvanometer mirrors scans/collects the laser beam/fluorescence along the focal plane (x/y-directions). A stepper motor stage scans in the axial direction and positions the x/y of the image field. Barrier filters and dichroic mirrorsmore » are used to route the spectral emission bands to the appropriate photodetector. An avalanche photodiode collects near-infrared fluorescence; a photodiode collects back-reflected 635 nm light; and a photomultiplier tube collects green fluorescence in the range of eGFP/eYFP. A 200-µm diameter pinhole was used to implement the confocal geometry for near-infrared and red channels and a 150-µm diameter pinhole for the green channel. Data acquisition and system control were achieved using a high-throughput data acquisition card. In-house software developed in LabVIEW was used to control the hardware, collect data from the photodetectors and reconstruct the confocal images. Results: 6 frames/s can be acquired for a 25 µm{sup 2} (128×128 pixels) field of view, visualizing the entire volume of the cell nucleus (∼10 µm depth) in <10 s. To demonstrate the usefulness of our FCLSM, we imaged gold nanoshells in live cells, radiation-induced damage in fibrosarcoma cells expressing eGFP tagged to a DNA repair protein, and neurons expressing eGFP. The system can also image particle tracks in fluorescent nuclear track detectors. Conclusion: We developed a versatile and portable FCLSM that allows radiobiology studies in live cells exposed to therapeutic radiation. The FCLSM can be placed in any vertical beam line for top-to-bottom exposures. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.« less

Parallel-multiplexed excitation light-sheet microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Zhou, Weibin; Peng, Leilei

2017-02-01

Laser scanning light-sheet imaging allows fast 3D image of live samples with minimal bleach and photo-toxicity. Existing light-sheet techniques have very limited capability in multi-label imaging. Hyper-spectral imaging is needed to unmix commonly used fluorescent proteins with large spectral overlaps. However, the challenge is how to perform hyper-spectral imaging without sacrificing the image speed, so that dynamic and complex events can be captured live. We report wavelength-encoded structured illumination light sheet imaging (λ-SIM light-sheet), a novel light-sheet technique that is capable of parallel multiplexing in multiple excitation-emission spectral channels. λ-SIM light-sheet captures images of all possible excitation-emission channels in true parallel. It does not require compromising the imaging speed and is capable of distinguish labels by both excitation and emission spectral properties, which facilitates unmixing fluorescent labels with overlapping spectral peaks and will allow more labels being used together. We build a hyper-spectral light-sheet microscope that combined λ-SIM with an extended field of view through Bessel beam illumination. The system has a 250-micron-wide field of view and confocal level resolution. The microscope, equipped with multiple laser lines and an unlimited number of spectral channels, can potentially image up to 6 commonly used fluorescent proteins from blue to red. Results from in vivo imaging of live zebrafish embryos expressing various genetic markers and sensors will be shown. Hyper-spectral images from λ-SIM light-sheet will allow multiplexed and dynamic functional imaging in live tissue and animals.

In Vivo Time-gated Fluorescence Imaging with Biodegradable Luminescent Porous Silicon Nanoparticles

PubMed Central

Gu, Luo; Hall, David J.; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J.; Howell, Stephen B.; Sailor, Michael J.

2014-01-01

Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (< 10 ns) emission signals from organic chromophores or tissue autofluorescence.Here, using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by > 50-fold in vitro and by > 20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed. PMID:23933660

Technical Advance: Live-imaging analysis of human dendritic cell migrating behavior under the influence of immune-stimulating reagents in an organotypic model of lung

PubMed Central

Nguyen Hoang, Anh Thu; Chen, Puran; Björnfot, Sofia; Högstrand, Kari; Lock, John G.; Grandien, Alf; Coles, Mark; Svensson, Mattias

2014-01-01

This manuscript describes technical advances allowing manipulation and quantitative analyses of human DC migratory behavior in lung epithelial tissue. DCs are hematopoietic cells essential for the maintenance of tissue homeostasis and the induction of tissue-specific immune responses. Important functions include cytokine production and migration in response to infection for the induction of proper immune responses. To design appropriate strategies to exploit human DC functional properties in lung tissue for the purpose of clinical evaluation, e.g., candidate vaccination and immunotherapy strategies, we have developed a live-imaging assay based on our previously described organotypic model of the human lung. This assay allows provocations and subsequent quantitative investigations of DC functional properties under conditions mimicking morphological and functional features of the in vivo parental tissue. We present protocols to set up and prepare tissue models for 4D (x, y, z, time) fluorescence-imaging analysis that allow spatial and temporal studies of human DCs in live epithelial tissue, followed by flow cytometry analysis of DCs retrieved from digested tissue models. This model system can be useful for elucidating incompletely defined pathways controlling DC functional responses to infection and inflammation in lung epithelial tissue, as well as the efficacy of locally administered candidate interventions. PMID:24899587

Bioorthogonal chemical imaging of metabolic activities in live mammalian hippocampal tissues with stimulated Raman scattering

NASA Astrophysics Data System (ADS)

Hu, Fanghao; Lamprecht, Michael R.; Wei, Lu; Morrison, Barclay; Min, Wei

2016-12-01

Brain is an immensely complex system displaying dynamic and heterogeneous metabolic activities. Visualizing cellular metabolism of nucleic acids, proteins, and lipids in brain with chemical specificity has been a long-standing challenge. Recent development in metabolic labeling of small biomolecules allows the study of these metabolisms at the global level. However, these techniques generally require nonphysiological sample preparation for either destructive mass spectrometry imaging or secondary labeling with relatively bulky fluorescent labels. In this study, we have demonstrated bioorthogonal chemical imaging of DNA, RNA, protein and lipid metabolism in live rat brain hippocampal tissues by coupling stimulated Raman scattering microscopy with integrated deuterium and alkyne labeling. Heterogeneous metabolic incorporations for different molecular species and neurogenesis with newly-incorporated DNA were observed in the dentate gyrus of hippocampus at the single cell level. We further applied this platform to study metabolic responses to traumatic brain injury in hippocampal slice cultures, and observed marked upregulation of protein and lipid metabolism particularly in the hilus region of the hippocampus within days of mechanical injury. Thus, our method paves the way for the study of complex metabolic profiles in live brain tissue under both physiological and pathological conditions with single-cell resolution and minimal perturbation.