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.

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

Long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular

NASA Astrophysics Data System (ADS)

Kimura, Hiroaki; Momiyama, Masashi; Tomita, Katsuro; Tsuchiya, Hiroyuki; Hoffman, Robert M.

2010-11-01

We demonstrate the development of a long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular. To observe cytoplasmic and nuclear dynamics of cancer cells in the living mouse, 143B human osteosarcoma cells are labeled with green fluorescent protein in the nucleus and red fluorescent protein in the cytoplasm. These dual-color cells are injected by a vascular route in an abdominal skin flap in nude mice. The mice are then imaged with the Olympus MVX10 macroview fluorescence microscope. With the MVX10, the nuclear and cytoplasmic behavior of cancer cells trafficking in blood vessels of live mice is observed. We also image lung metastases in live mice from the macro- to the subcellular level by opening the chest wall and imaging the exposed lung in live mice. Injected splenocytes, expressing cyan fluorescent protein, could also be imaged on the lung of live mice. We demonstrate that the MVX10 microscope offers the possibility of full-range in vivo fluorescence imaging from macro- to subcellular and should enable widespread use of powerful imaging technologies enabled by genetic reporters and other fluorophores.

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.

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.

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.

Green light for quantitative live-cell imaging in plants.

PubMed

Grossmann, Guido; Krebs, Melanie; Maizel, Alexis; Stahl, Yvonne; Vermeer, Joop E M; Ott, Thomas

2018-01-29

Plants exhibit an intriguing morphological and physiological plasticity that enables them to thrive in a wide range of environments. To understand the cell biological basis of this unparalleled competence, a number of methodologies have been adapted or developed over the last decades that allow minimal or non-invasive live-cell imaging in the context of tissues. Combined with the ease to generate transgenic reporter lines in specific genetic backgrounds or accessions, we are witnessing a blooming in plant cell biology. However, the imaging of plant cells entails a number of specific challenges, such as high levels of autofluorescence, light scattering that is caused by cell walls and their sensitivity to environmental conditions. Quantitative live-cell imaging in plants therefore requires adapting or developing imaging techniques, as well as mounting and incubation systems, such as micro-fluidics. Here, we discuss some of these obstacles, and review a number of selected state-of-the-art techniques, such as two-photon imaging, light sheet microscopy and variable angle epifluorescence microscopy that allow high performance and minimal invasive live-cell imaging in plants. © 2018. Published by The Company of Biologists Ltd.

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.

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.

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

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.

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.

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

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

GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells.

PubMed

Wang, Sheng; Chen, Xuanze; Chang, Lei; Ding, Miao; Xue, Ruiying; Duan, Haifeng; Sun, Yujie

2018-06-05

Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

Live-cell imaging of mammalian RNAs with Spinach2.

PubMed

Strack, Rita L; Jaffrey, Samie R

2015-01-01

The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of "RNA mimics of GFP" or Spinach technology for tagging mammalian RNAs and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag. © 2015 Elsevier Inc. All rights reserved.

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.

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.

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 (

ExTzBox: A Glowing Cyclophane for Live-Cell Imaging.

PubMed

Roy, Indranil; Bobbala, Sharan; Zhou, Jiawang; Nguyen, Minh T; Nalluri, Siva Krishna Mohan; Wu, Yilei; Ferris, Daniel P; Scott, Evan Alexander; Wasielewski, Michael R; Stoddart, J Fraser

2018-06-13

The ideal fluorescent probe for live-cell imaging is bright and non-cytotoxic and can be delivered easily into the living cells in an efficient manner. The design of synthetic fluorophores having all three of these properties, however, has proved to be challenging. Here, we introduce a simple, yet effective, strategy based on well-established chemistry for designing a new class of fluorescent probes for live-cell imaging. A box-like hybrid cyclophane, namely ExTzBox·4X (6·4X, X = PF 6 - , Cl - ), has been synthesized by connecting an extended viologen (ExBIPY) and a dipyridyl thiazolothiazole (TzBIPY) unit in an end-to-end fashion with two p-xylylene linkers. Photophysical studies show that 6·4Cl has a quantum yield Φ F = 1.00. Furthermore, unlike its ExBIPY 2+ and TzBIPY 2+ building units, 6·4Cl is non-cytotoxic to RAW 264.7 macrophages, even with a loading concentration as high as 100 μM, presumably on account of its rigid box-like structure which prevents its intercalation into DNA and may inhibit other interactions with it. After gaining an understanding of the toxicity profile of 6·4Cl, we employed it in live-cell imaging. Confocal microscopy has demonstrated that 6 4+ is taken up by the RAW 264.7 macrophages, allowing the cells to glow brightly with blue laser excitation, without any hint of photobleaching or disruption of normal cell behavior under the imaging conditions. By contrast, the acyclic reference compound Me 2 TzBIPY·2Cl (4·2Cl) shows very little fluorescence inside the cells, which is quenched completely under the same imaging conditions. In vitro cell investigations underscore the significance of using highly fluorescent box-like rigid cyclophanes for live-cell imaging.

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.

Live-Cell Imaging of Filoviruses.

PubMed

Schudt, Gordian; Dolnik, Olga; Becker, Stephan

2017-01-01

Observation of molecular processes inside living cells is fundamental to a deeper understanding of virus-host interactions in filoviral-infected cells. These observations can provide spatiotemporal insights into protein synthesis, protein-protein interaction dynamics, and transport processes of these highly pathogenic viruses. Thus, live-cell imaging provides the possibility for antiviral screening in real time and gives mechanistic insights into understanding filovirus assembly steps that are dependent on cellular factors, which then represent potential targets against this highly fatal disease. Here we describe analysis of living filovirus-infected cells under maximum biosafety (i.e., BSL4) conditions using plasmid-driven expression of fluorescently labeled viral and cellular proteins and/or viral genome-encoded expression of fluorescently labeled proteins. Such multiple-color and multidimensional time-lapse live-cell imaging analyses are a powerful method to gain a better understanding of the filovirus infection cycle.

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.

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.

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

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.

Imaging single cells in a beam of live cyanobacteria with an X-ray laser (CXIDB ID 26)

DOE Data Explorer

Schot, Gijs, vander

2015-02-10

This entry contains ten diffraction patterns, and reconstructions images, of individual living Cyanobium gracile cells, imaged using 517 eV X-rays from the LCLS XFEL. The Hawk software package was used for phasing. The Uppsala aerosol injector was used for sample injection, assuring very low noise levels. The cells come from various stages of the cell cycle, and were imaged in random orientations.

Photoelectrocyclization as an activation mechanism for organelle-specific live-cell imaging probes.

PubMed

Tran, Mai N; Chenoweth, David M

2015-05-26

Photoactivatable fluorophores are useful tools in live-cell imaging owing to their potential for precise spatial and temporal control. In this report, a new photoactivatable organelle-specific live-cell imaging probe based on a 6π electrocyclization/oxidation mechanism is described. It is shown that this new probe is water-soluble, non-cytotoxic, cell-permeable, and useful for mitochondrial imaging. The probe displays large Stokes shifts in both pre-activated and activated forms, allowing simultaneous use with common dyes and fluorescent proteins. Sequential single-cell activation experiments in dense cellular environments demonstrate high spatial precision and utility in single- or multi-cell labeling experiments. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nanofiber Nerve Guide for Peripheral Nerve Repair and Regeneration

DTIC Science & Technology

2014-01-01

observing cell migration using live - cell imaging microscopy, and analyzing cell migration with our MATLAB-based programs. Our studies...are then pipetted into the chamber and their path of migration is observed using a live - cell imaging microscope (Fig. 6d). Utilizing this migration

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

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

PubMed Central

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

2013-01-01

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

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.

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.

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

Label-free chemical imaging of live Euglena gracilis by high-speed SRS spectral microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wakisaka, Yoshifumi; Suzuki, Yuta; Tokunaga, Kyoya; Hirose, Misa; Domon, Ryota; Akaho, Rina; Kuroshima, Mai; Tsumura, Norimichi; Shimobaba, Tomoyoshi; Iwata, Osamu; Suzuki, Kengo; Nakashima, Ayaka; Goda, Keisuke; Ozeki, Yasuyuki

2016-03-01

Microbes, especially microalgae, have recently been of great interest for developing novel biofuels, drugs, and biomaterials. Imaging-based screening of live cells can provide high selectivity and is attractive for efficient bio-production from microalgae. Although conventional cellular screening techniques use cell labeling, labeling of microbes is still under development and can interfere with their cellular functions. Furthermore, since live microbes move and change their shapes rapidly, a high-speed imaging technique is required to suppress motion artifacts. Stimulated Raman scattering (SRS) microscopy allows for label-free and high-speed spectral imaging, which helps us visualize chemical components inside biological cells and tissues. Here we demonstrate high-speed SRS imaging, with temporal resolution of 0.14 seconds, of intracellular distributions of lipid, polysaccharide, and chlorophyll concentrations in rapidly moving Euglena gracilis, a unicellular phytoflagellate. Furthermore, we show that our method allows us to analyze the amount of chemical components inside each living cell. Our results indicate that SRS imaging may be applied to label-free screening of living microbes based on chemical information.

Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

PubMed

Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A; Bathe, Mark

2016-01-01

Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments.

PubMed

Van Valen, David A; Kudo, Takamasa; Lane, Keara M; Macklin, Derek N; Quach, Nicolas T; DeFelice, Mialy M; Maayan, Inbal; Tanouchi, Yu; Ashley, Euan A; Covert, Markus W

2016-11-01

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

DOE PAGES

Van Valen, David A.; Kudo, Takamasa; Lane, Keara M.; ...

2016-11-04

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domainsmore » of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.« less

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

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

Van Valen, David A.; Kudo, Takamasa; Lane, Keara M.

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domainsmore » of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.« less

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

PubMed Central

Van Valen, David A.; Lane, Keara M.; Quach, Nicolas T.; Maayan, Inbal

2016-01-01

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems. PMID:27814364

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

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.

High Resolution Live Cell Raman Imaging Using Subcellular Organelle-Targeting SERS-Sensitive Gold Nanoparticles with Highly Narrow Intra-Nanogap

PubMed Central

Kang, Jeon Woong; So, Peter T. C.; Dasari, Ramachandra R.; Lim, Dong-Kwon

2015-01-01

We report a method to achieve high speed and high resolution live cell Raman images using small spherical gold nanoparticles with highly narrow intra-nanogap structures responding to NIR excitation (785 nm) and high-speed confocal Raman microscopy. The three different Raman-active molecules placed in the narrow intra-nanogap showed a strong and uniform Raman intensity in solution even under transient exposure time (10 ms) and low input power of incident laser (200 μW), which lead to obtain high-resolution single cell image within 30 s without inducing significant cell damage. The high resolution Raman image showed the distributions of gold nanoparticles for their targeted sites such as cytoplasm, mitochondria, or nucleus. The high speed Raman-based live cell imaging allowed us to monitor rapidly changing cell morphologies during cell death induced by the addition of highly toxic KCN solution to cells. These results strongly suggest that the use of SERS-active nanoparticle can greatly improve the current temporal resolution and image quality of Raman-based cell images enough to obtain the detailed cell dynamics and/or the responses of cells to potential drug molecules. PMID:25646716

Regulation of Cell Migration in Breast Cancer

DTIC Science & Technology

2011-04-01

the wound healing, assay by scarring and Oris plate migration assay, transwell migration assay and live - cell imaging studies. Cell migration capacity...evaluated by the use of techniques that include the wound healing assay by scarring and Oris plate migration assay, transwell migration assay and live - cell imaging studies

Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

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

Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak

2014-04-20

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from themore » GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.« less

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

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.

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.

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.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

PubMed Central

Maynard, Carrie; Plaks, Vicki

2016-01-01

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment. PMID:26862704

Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

PubMed

Wöllert, Torsten; Langford, George M

2016-01-01

Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live cell imaging systems are provided.

A system and methodology for high-content visual screening of individual intact living cells in suspension

NASA Astrophysics Data System (ADS)

Renaud, Olivier; Heintzmann, Rainer; Sáez-Cirión, Asier; Schnelle, Thomas; Mueller, Torsten; Shorte, Spencer

2007-02-01

Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

PubMed

Fujimoto, Tomokazu; Inoue, Toshihiro; Inoue-Mochita, Miyuki; Tanihara, Hidenobu

2016-04-01

The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 μM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Atomic force microscopy as a tool for the investigation of living cells.

PubMed

Morkvėnaitė-Vilkončienė, Inga; Ramanavičienė, Almira; Ramanavičius, Arūnas

2013-01-01

Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low scanning speed, and (iii) usage of "soft," low resolution cantilevers. The tapping mode allows a cell to behave like a very solid material, and destructive shear forces are minimized, but imaging in liquid is difficult. The force spectroscopy mode is used for measuring the mechanical properties of cells; however, obtained results strongly depend on the cell fixation method. In this paper, the application of 3 atomic force microscopy modes including (i) contact, (ii) tapping, and (iii) force spectroscopy for the investigation of cells is described. The possibilities of cell preparation for the measurements, imaging, and determination of mechanical properties of cells are provided. The applicability of atomic force microscopy to diagnostics and other biomedical purposes is discussed.

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.

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.

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.

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

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.

Fluorescence Live Cell Imaging

PubMed Central

Ettinger, Andreas

2014-01-01

Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

4Pi-confocal microscopy of live cells

NASA Astrophysics Data System (ADS)

Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

2002-06-01

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

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.

Live-Cell Imaging of the Adult Drosophila Ovary Using Confocal Microscopy.

PubMed

Shalaby, Nevine A; Buszczak, Michael

2017-01-01

The Drosophila ovary represents a key in vivo model used to study germline stem cell (GSC) maintenance and stem cell daughter differentiation because these cells and their somatic cell neighbors can be identified at single-cell resolution within their native environment. Here we describe a fluorescent-based technique for the acquisition of 4D datasets of the Drosophila ovariole for periods that can exceed 12 consecutive hours. Live-cell imaging facilitates the investigation of molecular and cellular dynamics that were not previously possible using still images.

Synthetic aperture tomographic phase microscopy for 3D imaging of live cells in translational motion

PubMed Central

Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

2009-01-01

We present a technique for 3D imaging of live cells in translational motion without need of axial scanning of objective lens. A set of transmitted electric field images of cells at successive points of transverse translation is taken with a focused beam illumination. Based on Hyugens’ principle, angular plane waves are synthesized from E-field images of a focused beam. For a set of synthesized angular plane waves, we apply a filtered back-projection algorithm and obtain 3D maps of refractive index of live cells. This technique, which we refer to as synthetic aperture tomographic phase microscopy, can potentially be combined with flow cytometry or microfluidic devices, and will enable high throughput acquisition of quantitative refractive index data from large numbers of cells. PMID:18825263

Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

PubMed

MacLean, Lorna; Price, Helen; O'Toole, Peter

2016-01-01

Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye

PubMed Central

Guevara-Torres, A.; Joseph, A.; Schallek, J. B.

2016-01-01

Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level. PMID:27867728

Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye.

PubMed

Guevara-Torres, A; Joseph, A; Schallek, J B

2016-10-01

Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level.

Enhanced Fluorescence Imaging of Live Cells by Effective Cytosolic Delivery of Probes

PubMed Central

Massignani, Marzia; Canton, Irene; Sun, Tao; Hearnden, Vanessa; MacNeil, Sheila; Blanazs, Adam; Armes, Steven P.; Lewis, Andrew; Battaglia, Giuseppe

2010-01-01

Background Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. Principal Findings We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes) that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. Significance We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation. PMID:20454666

Systems Imaging of the Immune Synapse.

PubMed

Ambler, Rachel; Ruan, Xiangtao; Murphy, Robert F; Wülfing, Christoph

2017-01-01

Three-dimensional live cell imaging of the interaction of T cells with antigen-presenting cells (APCs) visualizes the subcellular distributions of signaling intermediates during T cell activation at thousands of resolved positions within a cell. These information-rich maps of local protein concentrations are a valuable resource in understanding T cell signaling. Here, we describe a protocol for the efficient acquisition of such imaging data and their computational processing to create four-dimensional maps of local concentrations. This protocol allows quantitative analysis of T cell signaling as it occurs inside live cells with resolution in time and space across thousands of cells.

Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

PubMed Central

Wei, Lu; Yu, Yong; Shen, Yihui; Wang, Meng C.; Min, Wei

2013-01-01

Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo. PMID:23798434

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

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

Ptychography: use of quantitative phase information for high-contrast label free time-lapse imaging of living cells

NASA Astrophysics Data System (ADS)

Suman, Rakesh; O'Toole, Peter

2014-03-01

Here we report a novel label free, high contrast and quantitative method for imaging live cells. The technique reconstructs an image from overlapping diffraction patterns using a ptychographical algorithm. The algorithm utilises both amplitude and phase data from the sample to report on quantitative changes related to the refractive index (RI) and thickness of the specimen. We report the ability of this technique to generate high contrast images, to visualise neurite elongation in neuronal cells, and to provide measure of cell proliferation.

Live-cell imaging for the assessment of the dynamics of autophagosome formation: focus on early steps.

PubMed

Karanasios, Eleftherios; Ktistakis, Nicholas T

2015-03-01

Autophagy is a cytosolic degradative pathway, which through a series of complicated membrane rearrangements leads to the formation of a unique double membrane vesicle, the autophagosome. The use of fluorescent proteins has allowed visualizing the autophagosome formation in live cells and in real time, almost 40 years after electron microscopy studies observed these structures for the first time. In the last decade, live-cell imaging has been extensively used to study the dynamics of autophagosome formation in cultured mammalian cells. Hereby we will discuss how the live-cell imaging studies have tried to settle the debate about the origin of the autophagosome membrane and how they have described the way different autophagy proteins coordinate in space and time in order to drive autophagosome formation. Copyright © 2014 Elsevier Inc. All rights reserved.

A Graphene-enhanced imaging of microRNA with enzyme-free signal amplification of catalyzed hairpin assembly in living cells.

PubMed

Liu, Haiyun; Tian, Tian; Ji, Dandan; Ren, Na; Ge, Shenguang; Yan, Mei; Yu, Jinghua

2016-11-15

In situ imaging of miRNA in living cells could help us to monitor the miRNA expression in real time and obtain accurate information for studying miRNA related bioprocesses and disease. Given the low-level expression of miRNA, amplification strategies for intracellular miRNA are imperative. Here, we propose an amplification strategy with a non-destructive enzyme-free manner in living cells using catalyzed hairpin assembly (CHA) based on graphene oxide (GO) for cellular miRNA imaging. The enzyme-free CHA exhibits stringent recognition and excellent signal amplification of miRNA in the living cells. GO is a good candidate as a fluorescence quencher and cellular carrier. Taking the advantages of the CHA and GO, we can monitor the miRNA at low level in living cells with a simple, sensitive and real-time manner. Finally, imaging of miRNAs in the different expression cells is realized. The novel method could supply an effective tool to visualize intracellular low-level miRNAs and help us to further understand the role of miRNAs in cellular processes. Copyright © 2016 Elsevier B.V. All rights reserved.

Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.

PubMed

Lopata, Anna; Hughes, Ruth; Tiede, Christian; Heissler, Sarah M; Sellers, James R; Knight, Peter J; Tomlinson, Darren; Peckham, Michelle

2018-04-26

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.

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.

Inter-chromosomal Contact Properties in Live-Cell Imaging and in Hi-C.

PubMed

Maass, Philipp G; Barutcu, A Rasim; Weiner, Catherine L; Rinn, John L

2018-03-15

Imaging (fluorescence in situ hybridization [FISH]) and genome-wide chromosome conformation capture (Hi-C) are two major approaches to the study of higher-order genome organization in the nucleus. Intra-chromosomal and inter-chromosomal interactions (referred to as non-homologous chromosomal contacts [NHCCs]) have been observed by several FISH-based studies, but locus-specific NHCCs have not been detected by Hi-C. Due to crosslinking, neither of these approaches assesses spatiotemporal properties. Toward resolving the discrepancies between imaging and Hi-C, we sought to understand the spatiotemporal properties of NHCCs in living cells by CRISPR/Cas9 live-cell imaging (CLING). In mammalian cells, we find that NHCCs are stable and occur as frequently as intra-chromosomal interactions, but NHCCs occur at farther spatial distance that could explain their lack of detection in Hi-C. By revealing the spatiotemporal properties in living cells, our study provides fundamental insights into the biology of NHCCs. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

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.

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

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.

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

Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

PubMed

Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

2014-01-01

Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

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.

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.

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.

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.

Generation of HIV-1 based bi-cistronic lentiviral vectors for stable gene expression and live cell imaging.

PubMed

Sehgal, Lalit; Budnar, Srikanth; Bhatt, Khyati; Sansare, Sneha; Mukhopadhaya, Amitabha; Kalraiya, Rajiv D; Dalal, Sorab N

2012-10-01

The study of protein-protein interactions, protein localization, protein organization into higher order structures and organelle dynamics in live cells, has greatly enhanced the understanding of various cellular processes. Live cell imaging experiments employ plasmid or viral vectors to express the protein/proteins of interest fused to a fluorescent protein. Unlike plasmid vectors, lentiviral vectors can be introduced into both dividing and non dividing cells, can be pseudotyped to infect a broad or narrow range of cells, and can be used to generate transgenic animals. However, the currently available lentiviral vectors are limited by the choice of fluorescent protein tag, choice of restriction enzyme sites in the Multiple Cloning Sites (MCS) and promoter choice for gene expression. In this report, HIV-1 based bi-cistronic lentiviral vectors have been generated that drive the expression of multiple fluorescent tags (EGFP, mCherry, ECFP, EYFP and dsRed), using two different promoters. The presence of a unique MCS with multiple restriction sites allows the generation of fusion proteins with the fluorescent tag of choice, allowing analysis of multiple fusion proteins in live cell imaging experiments. These novel lentiviral vectors are improved delivery vehicles for gene transfer applications and are important tools for live cell imaging in vivo.

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

Integrin Alpha-v and HER2 in Breast Cancer Brain Metastasis

DTIC Science & Technology

2015-10-01

ZOOM live cell imaging machine (ESSEN Bioscience; Figure 2). c. Interactions of αv integrin and HER2 in breast cancer brain metastases. We found...HCC1954 breast cancer cells. C) Real time live cell imaging of MM2BH cells treated with cilengitide (0, .3, 1, 3, and 10 µg/mL) using IncuCyte ZOOM

Molecular Basis of Genomic Instability in Breast Cancer: Regulation of the Centrosome Duplication Cycle

DTIC Science & Technology

2005-06-01

anaphase mitotic figures were found in these transfected cells, suggesting cells arrested at prometaphase (Fig. 3b). Live cell imaging over a 4-h time...Bubulya for help in live cell imaging , M. McCurrach for advice in BrdUrd labeling, A. Denli and A. Caudy for their critical reading of the manuscript and

A protocol for preparing, characterizing and using three RNA-specific, live cell imaging probes: E36, E144 and F22.

PubMed

Li, Qian; Chang, Young-Tae

2006-01-01

This protocol outlines a methodology for the preparation and characterization of three RNA-specific fluorescent probes (E36, E144 and F22) and their use in live cell imaging. It describes a detailed procedure for their chemical synthesis and purification; serial product characterization and quality control tests, including measurements of their fluorescence properties in solution, measurement of RNA specificity and analysis of cellular toxicity; and live cell staining and counterstaining with Hoechst or DAPI. Preparation and application of these RNA imaging probes takes 1 week.

A portable low-cost long-term live-cell imaging platform for biomedical research and education.

PubMed

Walzik, Maria P; Vollmar, Verena; Lachnit, Theresa; Dietz, Helmut; Haug, Susanne; Bachmann, Holger; Fath, Moritz; Aschenbrenner, Daniel; Abolpour Mofrad, Sepideh; Friedrich, Oliver; Gilbert, Daniel F

2015-02-15

Time-resolved visualization and analysis of slow dynamic processes in living cells has revolutionized many aspects of in vitro cellular studies. However, existing technology applied to time-resolved live-cell microscopy is often immobile, costly and requires a high level of skill to use and maintain. These factors limit its utility to field research and educational purposes. The recent availability of rapid prototyping technology makes it possible to quickly and easily engineer purpose-built alternatives to conventional research infrastructure which are low-cost and user-friendly. In this paper we describe the prototype of a fully automated low-cost, portable live-cell imaging system for time-resolved label-free visualization of dynamic processes in living cells. The device is light-weight (3.6 kg), small (22 × 22 × 22 cm) and extremely low-cost (<€1250). We demonstrate its potential for biomedical use by long-term imaging of recombinant HEK293 cells at varying culture conditions and validate its ability to generate time-resolved data of high quality allowing for analysis of time-dependent processes in living cells. While this work focuses on long-term imaging of mammalian cells, the presented technology could also be adapted for use with other biological specimen and provides a general example of rapidly prototyped low-cost biosensor technology for application in life sciences and education. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D-Printed Parts, a Smartphone, and Off-The-Shelf Electronics

PubMed Central

Schwan, Emil; Fatsis-Kavalopoulos, Nikos; Kreuger, Johan

2016-01-01

Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community. PMID:28002463

A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D-Printed Parts, a Smartphone, and Off-The-Shelf Electronics.

PubMed

Hernández Vera, Rodrigo; Schwan, Emil; Fatsis-Kavalopoulos, Nikos; Kreuger, Johan

2016-01-01

Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community.

Cell lineage and cell cycling analyses of the 4d micromere using live imaging in the marine annelid Platynereis dumerilii

PubMed Central

Handberg-Thorsager, Mette; Vervoort, Michel

2017-01-01

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos. PMID:29231816

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

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.

Confocal bioimaging the living cornea with autofluorescence and specific fluorescent probes

NASA Astrophysics Data System (ADS)

Masters, Barry R.; Paddock, Stephen W.

1990-08-01

Confocal bioimaging of the fine structure of the living rabbit cornea with both reflected light and fluorescent light has been demonstrated with a laser scanning confocal imaging system. Kalman averaging was used to reduce the noise in the images. Superficial epithelial, basal epithelial cells, stromal keratocytes, and endothelial cells were imaged. These cells and their subcellular structures were imaged in the two modes for comparison. The superficial epithelial cells were imaged by their autofluorescence (488/520 nm). This fluorescence signal may be due to the mitochondrial flavoproteins and can be used as a noninvasive indicator of cellular oxidative function. Thiazole orange was used to stain cell nuclei for fluorescence imaging. DiOC6 was used to stain the endoplasmic reticulum for fluorescence imaging. Fluorescein- conjugated phalloidin was used to stain actin for fluorescence imaging.

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

Label-free imaging of the dynamics of cell-to-cell string-like structure bridging in the free-space by low-coherent quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

2013-03-01

We succeeded in utilizing our low-coherent quantitative phase microscopy (LC-QPM) to achieve label-free and three-dimensional imaging of string-like structures bridging the free-space between live cells. In past studies, three dimensional morphology of the string-like structures between cells had been investigated by electron microscopies and fluorescence microscopies and these structures were called "membrane nanotubes" or "tunneling nanotubes." However, use of electron microscopy inevitably kills these cells and fluorescence microscopy is itself a potentially invasive method. To achieve noninvasive imaging of live cells, we applied our LC-QPM which is a reflection-type, phase resolved and full-field interference microscope employing a low-coherent light source. LC-QPM is able to visualize the three-dimensional morphology of live cells without labeling by means of low-coherence interferometry. The lateral (diffraction limit) and longitudinal (coherence-length) spatial resolution of LC-QPM were respectively 0.49 and 0.93 micrometers and the repeatability of the phase measurement was 0.02 radians (1.0 nm). We successfully obtained three-dimensional morphology of live cultured epithelial cells (cell type: HeLa, derived from cervix cancer) and were able to clearly observe the individual string-like structures interconnecting the cells. When we performed volumetric imaging, a 80 micrometer by 60 micrometer by 6.5 micrometer volume was scanned every 5.67 seconds and 70 frames of a three-dimensional movie were recorded for a duration of 397 seconds. Moreover, the optical phase images gave us detailed information about the three-dimensional morphology of the string-like structure at sub-wavelength resolution. We believe that our LC-QPM will be a useful tool for the study of three-dimensional morphology of live cells.

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae

PubMed Central

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. PMID:26727004

Live Cell Imaging of a Fluorescent Gentamicin Conjugate

PubMed Central

Escobedo, Jorge O.; Chu, Yu-Hsuan; Wang, Qi; Steyger, Peter S.; Strongin, Robert M.

2012-01-01

Understanding cellular mechanisms of ototoxic and nephrotoxic drug uptake, intracellular distribution, and molecular trafficking across cellular barrier systems aids the study of potential uptake blockers that preserve sensory and renal function during critical life-saving therapy. Herein we report the design, synthesis characterization and evaluation of a fluorescent conjugate of the aminoglycoside antibiotic gentamicin. Live cell imaging results show the potential utility of this new material. Related gentamicin conjugates studied to date quench in live kindney cells, and have been largely restricted to use in fixed (delipidated) cells. PMID:22545403

Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status.

PubMed

Zhang, Jing; Moradi, Emilia; Somekh, Michael G; Mather, Melissa L

2018-01-15

A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system's performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.

Noninvasive imaging of protein-protein interactions in living animals

NASA Astrophysics Data System (ADS)

Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

2002-05-01

Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

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.

Noncontact Viscoelastic Imaging of Living Cells Using a Long-Needle Atomic Force Microscope with Dual-Frequency Modulation

NASA Astrophysics Data System (ADS)

Guan, Dongshi; Charlaix, Elisabeth; Qi, Robert Z.; Tong, Penger

2017-10-01

Imaging of surface topography and elasticity of living cells can provide insight into the roles played by the cells' volumetric and mechanical properties and their response to external forces in regulating the essential cellular events and functions. Here, we report a unique technique of noncontact viscoelastic imaging of live cells using atomic force microscopy (AFM) with a long-needle glass probe. Because only the probe tip is placed in a liquid medium near the cell surface, the AFM cantilever in air functions well under dual-frequency modulation, retaining its high-quality resonant modes. The probe tip interacts with the cell surface through a minute hydrodynamic flow in the nanometer-thin gap region between them without physical contact. Quantitative measurements of the cell height, volume, and Young's modulus are conducted simultaneously. The experiment demonstrates that the long-needle AFM has a wide range of applications in the study of cell mechanics.

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.

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.

Imaging single cells in a beam of live cyanobacteria with an X-ray laser.

PubMed

van der Schot, Gijs; Svenda, Martin; Maia, Filipe R N C; Hantke, Max; DePonte, Daniel P; Seibert, M Marvin; Aquila, Andrew; Schulz, Joachim; Kirian, Richard; Liang, Mengning; Stellato, Francesco; Iwan, Bianca; Andreasson, Jakob; Timneanu, Nicusor; Westphal, Daniel; Almeida, F Nunes; Odic, Dusko; Hasse, Dirk; Carlsson, Gunilla H; Larsson, Daniel S D; Barty, Anton; Martin, Andrew V; Schorb, Sebastian; Bostedt, Christoph; Bozek, John D; Rolles, Daniel; Rudenko, Artem; Epp, Sascha; Foucar, Lutz; Rudek, Benedikt; Hartmann, Robert; Kimmel, Nils; Holl, Peter; Englert, Lars; Duane Loh, Ne-Te; Chapman, Henry N; Andersson, Inger; Hajdu, Janos; Ekeberg, Tomas

2015-02-11

There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential.

NanoLuc reporter for dual luciferase imaging in living animals.

PubMed

Stacer, Amanda C; Nyati, Shyam; Moudgil, Pranav; Iyengar, Rahul; Luker, Kathryn E; Rehemtulla, Alnawaz; Luker, Gary D

2013-10-01

Bioluminescence imaging is widely used for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on the high signal to background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events, as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, adenosine triphosphate–independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc [NL]) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although the detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in transforming growth factor β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as a new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development.

NanoLuc Reporter for Dual Luciferase Imaging in Living Animals

PubMed Central

Stacer, Amanda C.; Nyati, Shyam; Moudgil, Pranav; Iyengar, Rahul; Luker, Kathryn E.; Rehemtulla, Alnawaz; Luker, Gary D.

2014-01-01

Bioluminescence imaging is utilized widely for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on high signal-to-background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, ATP-independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc, NL) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in TGF-β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development. PMID:24371848

Dynamics of the DNA damage response: insights from live-cell imaging

PubMed Central

Karanam, Ketki; Loewer, Alexander

2013-01-01

All organisms have to safeguard the integrity of their genome to prevent malfunctioning and oncogenic transformation. Sophisticated DNA damage response mechanisms have evolved to detect and repair genomic lesions. With the emergence of live-cell microscopy of individual cells, we now begin to appreciate the complex spatiotemporal kinetics of the DNA damage response and can address the causes and consequences of the heterogeneity in the responses of genetically identical cells. Here, we highlight key discoveries where live-cell imaging has provided unprecedented insights into how cells respond to DNA double-strand breaks and discuss the main challenges and promises in using this technique. PMID:23292635

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

PubMed Central

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-01-01

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min−1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics. PMID:27991512

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

NASA Astrophysics Data System (ADS)

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-12-01

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions.

PubMed

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-12-19

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min -1 . The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear

PubMed Central

Drummond, Meghan C.; Barzik, Melanie; Bird, Jonathan E.; Zhang, Duan-Sun; Lechene, Claude P.; Corey, David P.; Cunningham, Lisa L.; Friedman, Thomas B.

2015-01-01

The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24–48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of 15N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips. PMID:25898120

Imaging cell biology in live animals: ready for prime time.

PubMed

Weigert, Roberto; Porat-Shliom, Natalie; Amornphimoltham, Panomwat

2013-06-24

Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.

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

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

Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging

PubMed Central

2015-01-01

Bioorthogonal reactions, including the strain-promoted azide–alkyne cycloaddition (SPAAC) and inverse electron demand Diels–Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines. PMID:26270632

Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging.

PubMed

Murrey, Heather E; Judkins, Joshua C; Am Ende, Christopher W; Ballard, T Eric; Fang, Yinzhi; Riccardi, Keith; Di, Li; Guilmette, Edward R; Schwartz, Joel W; Fox, Joseph M; Johnson, Douglas S

2015-09-09

Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.

Engineered core-shell magnetic nanoparticle for MR dual-modal tracking and safe magnetic manipulation of ependymal cells in live rodents

NASA Astrophysics Data System (ADS)

Peng, Yung-Kang; Lui, Cathy N. P.; Chen, Yu-Wei; Chou, Shang-Wei; Chou, Pi-Tai; Yung, Ken K. L.; Edman Tsang, S. C.

2018-01-01

Tagging recognition group(s) on superparamagnetic iron oxide is known to aid localisation (imaging), stimulation and separation of biological entities using magnetic resonance imaging (MRI) and magnetic agitation/separation (MAS) techniques. Despite the wide applicability of iron oxide nanoparticles in T 2-weighted MRI and MAS, the quality of the images and safe manipulation of the exceptionally delicate neural cells in a live brain are currently the key challenges. Here, we demonstrate the engineered manganese oxide clusters-iron oxide core-shell nanoparticle as an MR dual-modal contrast agent for neural stem cells (NSCs) imaging and magnetic manipulation in live rodents. As a result, using this engineered nanoparticle and associated technologies, identification, stimulation and transportation of labelled potentially multipotent NSCs from a specific location of a live brain to another by magnetic means for self-healing therapy can therefore be made possible.

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.

Micro patterned surfaces: an effective tool for long term digital holographic microscopy cell imaging

NASA Astrophysics Data System (ADS)

Mues, Sarah; Lilge, Inga; Schönherr, Holger; Kemper, Björn; Schnekenburger, Jürgen

2017-02-01

The major problem of Digital Holographic Microscopy (DHM) long term live cell imaging is that over time most of the tracked cells move out of the image area and other ones move in. Therefore, most of the cells are lost for the evaluation of individual cellular processes. Here, we present an effective solution for this crucial problem of long-term microscopic live cell analysis. We have generated functionalized slides containing areas of 250 μm per 200 μm. These micropatterned biointerfaces consist of passivating polyaclrylamide brushes (PAAm). Inner areas are backfilled with octadecanthiol (ODT), which allows cell attachment. The fouling properties of these surfaces are highly controllable and therefore the defined areas designed for the size our microscopic image areas were effective in keeping all cells inside the rectangles over the selected imaging period.

Live-cell imaging of cyanobacteria.

PubMed

Yokoo, Rayka; Hood, Rachel D; Savage, David F

2015-10-01

Cyanobacteria are a diverse bacterial phylum whose members possess a high degree of ultrastructural organization and unique gene regulatory mechanisms. Unraveling this complexity will require the use of live-cell fluorescence microscopy, but is impeded by the inherent fluorescent background associated with light-harvesting pigments and the need to feed photosynthetic cells light. Here, we outline a roadmap for overcoming these challenges. Specifically, we show that although basic cyanobacterial biology creates challenging experimental constraints, these restrictions can be mitigated by the careful choice of fluorophores and microscope instrumentation. Many of these choices are motivated by recent successful live-cell studies. We therefore also highlight how live-cell imaging has advanced our understanding of bacterial microcompartments, circadian rhythm, and the organization and segregation of the bacterial nucleoid.

Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer.

PubMed

De, Abhijit; Gambhir, Sanjiv Sam

2005-12-01

This study demonstrates a significant advancement of imaging of a distance-dependent physical process, known as the bioluminescent resonance energy transfer (BRET2) signal in living subjects, by using a cooled charge-coupled device (CCD) camera. A CCD camera-based spectral imaging strategy enables simultaneous visualization and quantitation of BRET signal from live cells and cells implanted in living mice. We used the BRET2 system, which utilizes Renilla luciferase (hRluc) protein and its substrate DeepBlueC (DBC) as an energy donor and a mutant green fluorescent protein (GFP2) as the acceptor. To accomplish this objective in this proof-of-principle study, the donor and acceptor proteins were fused to FKBP12 and FRB, respectively, which are known to interact only in the presence of the small molecule mediator rapamycin. Mammalian cells expressing these fusion constructs were imaged using a cooled-CCD camera either directly from culture dishes or by implanting them into mice. By comparing the emission photon yields in the presence and absence of rapamycin, the specific BRET signal was determined. The CCD imaging approach of BRET signal is particularly appealing due to its capacity to seamlessly bridge the gap between in vitro and in vivo studies. This work validates BRET as a powerful tool for interrogating and observing protein-protein interactions directly at limited depths in living mice.

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.

Simple and fast spectral domain algorithm for quantitative phase imaging of living cells with digital holographic microscopy

NASA Astrophysics Data System (ADS)

Min, Junwei; Yao, Baoli; Ketelhut, Steffi; Kemper, Björn

2017-02-01

The modular combination of optical microscopes with digital holographic microscopy (DHM) has been proven to be a powerful tool for quantitative live cell imaging. The introduction of condenser and different microscope objectives (MO) simplifies the usage of the technique and makes it easier to measure different kinds of specimens with different magnifications. However, the high flexibility of illumination and imaging also causes variable phase aberrations that need to be eliminated for high resolution quantitative phase imaging. The existent phase aberrations compensation methods either require add additional elements into the reference arm or need specimen free reference areas or separate reference holograms to build up suitable digital phase masks. These inherent requirements make them unpractical for usage with highly variable illumination and imaging systems and prevent on-line monitoring of living cells. In this paper, we present a simple numerical method for phase aberration compensation based on the analysis of holograms in spatial frequency domain with capabilities for on-line quantitative phase imaging. From a single shot off-axis hologram, the whole phase aberration can be eliminated automatically without numerical fitting or pre-knowledge of the setup. The capabilities and robustness for quantitative phase imaging of living cancer cells are demonstrated.

Fluorescent water-Soluble Probes Based on Ammonium Cation Peg Substituted Perylenepisimides: Synthesis, Photophysical Properties, and Live Cell Images

NASA Astrophysics Data System (ADS)

Yang, Wei; Cai, Jiaxuan; Zhang, Shuchen; Yi, Xuegang; Gao, Baoxiang

2018-01-01

To synthesize perylenbisimides (PBI) fluorescent probes that will improve the water-soluble ability and the cytocompatibility, the synthesis and properties of fluorescent water-soluble probes based on dendritic ammonium cation polyethylene glycol (PEG) substituted perylenebisimides(GPDIs) are presented. As we expected, with increased ammonium cation PEG, the aggregation of the PBI in an aqueous solution is completely suppressed by the hydrophilic ammonium cation PEG groups. And the fluorescence quantum yield increases from 25% for GPDI-1 to 62% for GPDI-2. When incubated with Hela cells for 48 h, the viabilities are 71% (for GPDI-1) and 76% (for GPDI-2). Live cell imaging shows that these probes are efficiently internalized by HeLa cells. The study of the photophysical properties indicated increasing the ammonium cation PEG generation can increase the fluorescence quantum yield. Live cell imaging shows that with the ammonium cation PEG chains of perylenebisimides has high biocompatibility. The exceptionally low cytotoxicity is ascribed to the ammonium cation PEG chains, which protect the dyes from nonspecifically interacting with the extracellular proteins. Live cell imaging shows that ammonium cations PEG chains can promote the internalization of these probes.

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

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.

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.

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.

Three-dimensional label-free imaging and quantification of lipid droplets in live hepatocytes

NASA Astrophysics Data System (ADS)

Kim, Kyoohyun; Lee, Seoeun; Yoon, Jonghee; Heo, Jihan; Choi, Chulhee; Park, Yongkeun

2016-11-01

Lipid droplets (LDs) are subcellular organelles with important roles in lipid storage and metabolism and involved in various diseases including cancer, obesity, and diabetes. Conventional methods, however, have limited ability to provide quantitative information on individual LDs and have limited capability for three-dimensional (3-D) imaging of LDs in live cells especially for fast acquisition of 3-D dynamics. Here, we present an optical method based on 3-D quantitative phase imaging to measure the 3-D structural distribution and biochemical parameters (concentration and dry mass) of individual LDs in live cells without using exogenous labelling agents. The biochemical change of LDs under oleic acid treatment was quantitatively investigated, and 4-D tracking of the fast dynamics of LDs revealed the intracellular transport of LDs in live cells.

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.

Optical magnetic imaging of living cells

PubMed Central

Le Sage, D.; Arai, K.; Glenn, D. R.; DeVience, S. J.; Pham, L. M.; Rahn-Lee, L.; Lukin, M. D.; Yacoby, A.; Komeili, A.; Walsworth, R. L.

2013-01-01

Magnetic imaging is a powerful tool for probing biological and physical systems. However, existing techniques either have poor spatial resolution compared to optical microscopy and are hence not generally applicable to imaging of sub-cellular structure (e.g., magnetic resonance imaging [MRI]1), or entail operating conditions that preclude application to living biological samples while providing sub-micron resolution (e.g., scanning superconducting quantum interference device [SQUID] microscopy2, electron holography3, and magnetic resonance force microscopy [MRFM]4). Here we demonstrate magnetic imaging of living cells (magnetotactic bacteria) under ambient laboratory conditions and with sub-cellular spatial resolution (400 nm), using an optically-detected magnetic field imaging array consisting of a nanoscale layer of nitrogen-vacancy (NV) colour centres implanted at the surface of a diamond chip. With the bacteria placed on the diamond surface, we optically probe the NV quantum spin states and rapidly reconstruct images of the vector components of the magnetic field created by chains of magnetic nanoparticles (magnetosomes) produced in the bacteria, and spatially correlate these magnetic field maps with optical images acquired in the same apparatus. Wide-field sCMOS acquisition allows parallel optical and magnetic imaging of multiple cells in a population with sub-micron resolution and >100 micron field-of-view. Scanning electron microscope (SEM) images of the bacteria confirm that the correlated optical and magnetic images can be used to locate and characterize the magnetosomes in each bacterium. The results provide a new capability for imaging bio-magnetic structures in living cells under ambient conditions with high spatial resolution, and will enable the mapping of a wide range of magnetic signals within cells and cellular networks5, 6. PMID:23619694

Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Nonprotein Thiol Imaging and Quantification in Mitochondria.

PubMed

Wang, Shenggang; Yin, Huihui; Huang, Yue; Guan, Xiangming

2018-06-11

Cellular thiols are divided into two major categories: nonprotein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7'-thiobis( N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence intensity. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λ ex = 550 nm, λ em = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two colocalization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols.

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).

A tunable refractive index matching medium for live imaging cells, tissues and model organisms

PubMed Central

Boothe, Tobias; Hilbert, Lennart; Heide, Michael; Berninger, Lea; Huttner, Wieland B; Zaburdaev, Vasily; Vastenhouw, Nadine L; Myers, Eugene W; Drechsel, David N; Rink, Jochen C

2017-01-01

In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids. DOI: http://dx.doi.org/10.7554/eLife.27240.001 PMID:28708059

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

Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance.

PubMed

Ghodke, Harshad; Ho, Han; van Oijen, Antoine M

2018-02-19

Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow individual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA-repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA-repair processes in prokaryotes. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

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.

Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

PubMed

Hayashi, Takahiro; Hamachi, Itaru

2012-09-18

Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional affinity labeling method and allows for real-time monitoring of protein activity. With the high target specificity and biocompatibility of this technique, we have achieved individual labeling and imaging of endogenously expressed proteins in samples of high biological complexity. We also highlight applications in which our current approach enabled the monitoring of important biological events, such as ligand binding, in living cells. These novel chemical labeling techniques are expected to provide a molecular toolbox for studying a wide variety of proteins and beyond in living cells.

High resolution quantitative phase imaging of live cells with constrained optimization approach

NASA Astrophysics Data System (ADS)

Pandiyan, Vimal Prabhu; Khare, Kedar; John, Renu

2016-03-01

Quantitative phase imaging (QPI) aims at studying weakly scattering and absorbing biological specimens with subwavelength accuracy without any external staining mechanisms. Use of a reference beam at an angle is one of the necessary criteria for recording of high resolution holograms in most of the interferometric methods used for quantitative phase imaging. The spatial separation of the dc and twin images is decided by the reference beam angle and Fourier-filtered reconstructed image will have a very poor resolution if hologram is recorded below a minimum reference angle condition. However, it is always inconvenient to have a large reference beam angle while performing high resolution microscopy of live cells and biological specimens with nanometric features. In this paper, we treat reconstruction of digital holographic microscopy images as a constrained optimization problem with smoothness constraint in order to recover only complex object field in hologram plane even with overlapping dc and twin image terms. We solve this optimization problem by gradient descent approach iteratively and the smoothness constraint is implemented by spatial averaging with appropriate size. This approach will give excellent high resolution image recovery compared to Fourier filtering while keeping a very small reference angle. We demonstrate this approach on digital holographic microscopy of live cells by recovering the quantitative phase of live cells from a hologram recorded with nearly zero reference angle.

Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes.

PubMed

Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus

2015-02-11

Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

Live-cell stimulated Raman scattering imaging of alkyne-tagged biomolecules.

PubMed

Hong, Senlian; Chen, Tao; Zhu, Yuntao; Li, Ang; Huang, Yanyi; Chen, Xing

2014-06-02

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman-silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label-free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state-of-the-art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non-invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live-cell imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

PubMed Central

Warren, Sean C.; Margineanu, Anca; Katan, Matilda; Dunsby, Chris; French, Paul M. W.

2015-01-01

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. PMID:26133241

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

Rotation of single live mammalian cells using dynamic holographic optical tweezers

NASA Astrophysics Data System (ADS)

Bin Cao; Kelbauskas, Laimonas; Chan, Samantha; Shetty, Rishabh M.; Smith, Dean; Meldrum, Deirdre R.

2017-05-01

We report on a method for rotating single mammalian cells about an axis perpendicular to the optical system axis through the imaging plane using dynamic holographic optical tweezers (HOTs). Two optical traps are created on the opposite edges of a mammalian cell and are continuously transitioned through the imaging plane along the circumference of the cell in opposite directions, thus providing the torque to rotate the cell in a controlled fashion. The method enables a complete 360° rotation of live single mammalian cells with spherical or near-to spherical shape in 3D space, and represents a useful tool suitable for the single cell analysis field, including tomographic imaging.

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.

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.

RNA imaging: tracking in real-time RNA transport in neurons using molecular beacons and confocal microscopy.

PubMed

Zepeda, Angélica; Arias, Clorinda; Flores-Jasso, Fabian; Vaca, Luis

2013-01-01

RNAs are present within eukaryotic cells and are involved in several biological processes. RNA transport within cell compartments is important for proper cell function. To understand in depth the cellular processes in which RNA is involved requires a method that reveals RNA localization in real time in a sub-cellular context in living cells. In this protocol we describe a method for imaging RNA in living cells and in particular in neuronal cultures based on cell microinjection of molecular beacons in conjunction with confocal microscopy. This methodology overcomes some of the main obstacles for imaging RNA in live cells since microinjection allows the delivery of the probe to a desired cellular compartment and MBs bind with high specificity to its target RNA without inhibiting its function. The proper design of the MBs is essential to obtain RNA-MB association at the temperature of the cell cytosol. MBs design with other purposes in mind (such as PCR experiments) have a design that facilitates association to its target at high temperatures, rendering them unsuitable for live cell imaging. Using the methodology described in this chapter allows the study of RNA transport to different regions of neurons and may be combined with the tagging of proteins of interest to measure co-transport of the protein and the RNA to different cellular regions. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

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.

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

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

PubMed

Lu, Yongbo; Kamel-El Sayed, Suzan A; Wang, Kun; Tiede-Lewis, LeAnn M; Grillo, Michael A; Veno, Patricia A; Dusevich, Vladimir; Phillips, Charlotte L; Bonewald, Lynda F; Dallas, Sarah L

2018-06-01

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Research.

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.

Imaging Single Cells in the Living Retina

PubMed Central

Williams, David R.

2011-01-01

A quarter century ago, we were limited to a macroscopic view of the retina inside the living eye. Since then, new imaging technologies, including confocal scanning laser ophthalmoscopy, optical coherence tomography, and adaptive optics fundus imaging, transformed the eye into a microscope in which individual cells can now be resolved noninvasively. These technologies have enabled a wide range of studies of the retina that were previously impossible. PMID:21596053

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.

Photothermal quantitative phase imaging of living cells with nanoparticles utilizing a cost-efficient setup

NASA Astrophysics Data System (ADS)

Turko, Nir A.; Isbach, Michael; Ketelhut, Steffi; Greve, Burkhard; Schnekenburger, Jürgen; Shaked, Natan T.; Kemper, Björn

2017-02-01

We explored photothermal quantitative phase imaging (PTQPI) of living cells with functionalized nanoparticles (NPs) utilizing a cost-efficient setup based on a cell culture microscope. The excitation light was modulated by a mechanical chopper wheel with low frequencies. Quantitative phase imaging (QPI) was performed with Michelson interferometer-based off-axis digital holographic microscopy and a standard industrial camera. We present results from PTQPI observations on breast cancer cells that were incubated with functionalized gold NPs binding to the epidermal growth factor receptor. Moreover, QPI was used to quantify the impact of the NPs and the low frequency light excitation on cell morphology and viability.

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.

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

Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging

PubMed Central

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

2017-01-01

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. PMID:28835375

Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

PubMed

Park, Jae Woo; Na, Sang Cheol; Nguyen, Thanh Qua; Paik, Sang-Min; Kang, Myeongwoo; Hong, Daewha; Choi, Insung S; Lee, Jae-Hyeok; Jeon, Noo Li

2015-03-01

This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. © 2014 Wiley Periodicals, Inc.

Imaging molecular dynamics in vivo--from cell biology to animal models.

PubMed

Timpson, Paul; McGhee, Ewan J; Anderson, Kurt I

2011-09-01

Advances in fluorescence microscopy have enabled the study of membrane diffusion, cell adhesion and signal transduction at the molecular level in living cells grown in culture. By contrast, imaging in living organisms has primarily been restricted to the localization and dynamics of cells in tissues. Now, imaging of molecular dynamics is on the cusp of progressing from cell culture to living tissue. This transition has been driven by the understanding that the microenvironment critically determines many developmental and pathological processes. Here, we review recent progress in fluorescent protein imaging in vivo by drawing primarily on cancer-related studies in mice. We emphasize the need for techniques that can be easily combined with genetic models and complement fluorescent protein imaging by providing contextual information about the cellular environment. In this Commentary we will consider differences between in vitro and in vivo experimental design and argue for an approach to in vivo imaging that is built upon the use of intermediate systems, such as 3-D and explant culture models, which offer flexibility and control that is not always available in vivo. Collectively, these methods present a paradigm shift towards the molecular-level investigation of disease and therapy in animal models of disease.

An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

NASA Astrophysics Data System (ADS)

Wollman, Adam J. M.; Miller, Helen; Foster, Simon; Leake, Mark C.

2016-10-01

Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphologically complex structures of fluorescently labelled proteins present in clusters of other types of cells.

Monitoring of live cell cultures during apoptosis by phase imaging and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Sharikova, Anna; Saide, George; Sfakis, Lauren; Park, Jun Yong; Desta, Habben; Maloney, Maxwell C.; Castracane, James; Mahajan, Supriya D.; Khmaladze, Alexander

2017-02-01

Non-invasive live cell measurements are an important tool in biomedical research. We present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy records an interference pattern between object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information about the sample. When the phase is mapped across the sample and converted into height information for each pixel, a three dimensional image is obtained. The measurement of live cell cultures by digital holographic microscopy yields information about cell shape and volume, changes to which are reflective of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopy, on the other hand, is sensitive to rotational and vibrational molecular transitions, as well as intermolecular vibrations. Therefore, Raman spectroscopy provides complementary information about cells, such as protein, lipid and nucleic acid content, and, particularly, the spectral signatures associated with structural changes in molecules. The cell cultures are kept in the temperature-controlled environmental chamber during the experiment, which allows monitoring over multiple cell cycles. The DHM system combines a visible (red) laser source with conventional microscope base, and LabVIEW-run data processing. We analyzed and compared cell culture information obtained by these two methods.

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

Open-dish incubator for live cell imaging with an inverted microscope.

PubMed

Heidemann, Steven R; Lamoureux, Phillip; Ngo, Kha; Reynolds, Matthew; Buxbaum, Robert E

2003-10-01

Here we describe the design and fabrication of an inexpensive cell culture incubator for the stage of an inverted light microscope for use in live cell imaging. This device maintains the temperature of the cell culture at 37 degrees C with great stability and, after reaching equilibrium, provides focal stability of an image for 20-25 min with oil-immersion lenses. We describe two versions of the incubator: one for use with standard 60-mm plastic culture dishes, and the other version for imaging of cells on glass coverslips. Either can be made for less than $400. Most components are widely available commercially, and it requires only simple wiring and 3 h to assemble. Although the device is generally useful for live cell imaging on an inverted microscope, it is particularly suitable for work in which instruments are introduced into the culture, such as electrophysiology or micromanipulation. The design is based on the principle that control performance is limited by the lag time between detection and response. The key element of the design is a heated, temperature-controlled aluminum ring serving as a mini-incubator surrounding the culture vessel. For this reason, we call our design a "ringcubator."

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

PubMed Central

Peckys, Diana B.; Mazur, Peter; Gould, Kathleen L.; de Jonge, Niels

2011-01-01

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. PMID:21575587

Fully hydrated yeast cells imaged with electron microscopy.

PubMed

Peckys, Diana B; Mazur, Peter; Gould, Kathleen L; de Jonge, Niels

2011-05-18

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The live cell DNA stain SiR-Hoechst induces DNA damage responses and impairs cell cycle progression.

PubMed

Sen, Onur; Saurin, Adrian T; Higgins, Jonathan M G

2018-05-21

SiR-Hoechst (SiR-DNA) is a far-red fluorescent DNA probe being used widely for time-lapse imaging of living cells that is reported to be minimally toxic at concentrations as high as 10-25 µM. However, measuring nuclear import of Cyclin B1, inhibition of mitotic entry, and the induction of γH2AX foci in cultured human cells reveals that SiR-Hoechst induces DNA damage responses and G2 arrest at concentrations well below 1 µM. SiR-Hoechst is useful for live cell imaging, but it should be used with caution and at the lowest practicable concentration.

Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution

PubMed Central

Zhang, Delong; Li, Chen; Zhang, Chi; Slipchenko, Mikhail N.; Eakins, Gregory; Cheng, Ji-Xin

2016-01-01

Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption–induced photothermal effect by a visible laser beam. Our mid-infrared photothermal (MIP) approach reached 10 μM detection sensitivity and submicrometer lateral spatial resolution. This performance has exceeded the diffraction limit of infrared microscopy and allowed label-free three-dimensional chemical imaging of live cells and organisms. Distributions of endogenous lipid and exogenous drug inside single cells were visualized. We further demonstrated in vivo MIP imaging of lipids and proteins in Caenorhabditis elegans. The reported MIP imaging technology promises broad applications from monitoring metabolic activities to high-resolution mapping of drug molecules in living systems, which are beyond the reach of current infrared microscopy. PMID:27704043

Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution.

PubMed

Zhang, Delong; Li, Chen; Zhang, Chi; Slipchenko, Mikhail N; Eakins, Gregory; Cheng, Ji-Xin

2016-09-01

Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption-induced photothermal effect by a visible laser beam. Our mid-infrared photothermal (MIP) approach reached 10 μM detection sensitivity and submicrometer lateral spatial resolution. This performance has exceeded the diffraction limit of infrared microscopy and allowed label-free three-dimensional chemical imaging of live cells and organisms. Distributions of endogenous lipid and exogenous drug inside single cells were visualized. We further demonstrated in vivo MIP imaging of lipids and proteins in Caenorhabditis elegans . The reported MIP imaging technology promises broad applications from monitoring metabolic activities to high-resolution mapping of drug molecules in living systems, which are beyond the reach of current infrared microscopy.

Long-term live-cell imaging reveals new roles for Salmonella effector proteins SseG and SteA.

PubMed

McQuate, Sarah E; Young, Alexandra M; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S; Palmer, Amy E

2017-01-01

Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single-cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here, we establish a pipeline for long-term (17 h) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyper-replication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. © 2016 John Wiley & Sons Ltd.

Long-Term Live Cell Imaging Reveals New Roles For Salmonella Effector Proteins SseG and SteA

PubMed Central

McQuate, Sarah E.; Young, Alexandra M.; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S.; Palmer, Amy E.

2016-01-01

Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. PMID:27376507

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.

Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens.

PubMed

Tran, Mai L; McCarthy, Thomas W; Sun, Hao; Wu, Shu-Zon; Norris, Joanna H; Bezanilla, Magdalena; Vidali, Luis; Anderson, Charles T; Roberts, Alison W

2018-01-15

Results from live cell imaging of fluorescently tagged Cellulose Synthase (CESA) proteins in Cellulose Synthesis Complexes (CSCs) have enhanced our understanding of cellulose biosynthesis, including the mechanisms of action of cellulose synthesis inhibitors. However, this method has been applied only in Arabidopsis thaliana and Brachypodium distachyon thus far. Results from freeze fracture electron microscopy of protonemal filaments of the moss Funaria hygrometrica indicate that a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), fragments CSCs and clears them from the plasma membrane. This differs from Arabidopsis, in which DCB causes CSC accumulation in the plasma membrane and a different cellulose synthesis inhibitor, isoxaben, clears CSCs from the plasma membrane. In this study, live cell imaging of the moss Physcomitrella patens indicated that DCB and isoxaben have little effect on protonemal growth rates, and that only DCB causes tip rupture. Live cell imaging of mEGFP-PpCESA5 and mEGFP-PpCESA8 showed that DCB and isoxaben substantially reduced CSC movement, but had no measureable effect on CSC density in the plasma membrane. These results suggest that DCB and isoxaben have similar effects on CSC movement in P. patens and Arabidopsis, but have different effects on CSC intracellular trafficking, cell growth and cell integrity in these divergent plant lineages.

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.

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.

A Scalable Perfusion Culture System with Miniature Peristaltic Pumps for Live-Cell Imaging Assays with Provision for Microfabricated Scaffolds

PubMed Central

Balakrishnan, Sreenath; Suma, M.S.; Raju, Shilpa R.; Bhargav, Santosh D.B.; Arunima, S.; Das, Saumitra

2015-01-01

Abstract We present a perfusion culture system with miniature bioreactors and peristaltic pumps. The bioreactors are designed for perfusion, live-cell imaging studies, easy incorporation of microfabricated scaffolds, and convenience of operation in standard cell culture techniques. By combining with miniature peristaltic pumps—one for each bioreactor to avoid cross-contamination and to maintain desired flow rate in each—we have made a culture system that facilitates perfusion culture inside standard incubators. This scalable system can support multiple parallel perfusion experiments. The major components are fabricated by three-dimensional printing using VeroWhite, which we show to be amenable to ex vivo cell culture. Furthermore, the components of the system can be reused, thus making it economical. We validate the system and illustrate its versatility by culturing primary rat hepatocytes, live imaging the growth of mouse fibroblasts (NIH 3T3) on microfabricated ring-scaffolds inserted into the bioreactor, performing perfusion culture of breast cancer cells (MCF7), and high-magnification imaging of hepatocarcinoma cells (HuH7). PMID:26309810

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

PubMed

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

2015-11-02

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

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

PubMed Central

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

2015-01-01

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

Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy.

PubMed

Fermie, Job; Liv, Nalan; Ten Brink, Corlinda; van Donselaar, Elly G; Müller, Wally H; Schieber, Nicole L; Schwab, Yannick; Gerritsen, Hans C; Klumperman, Judith

2018-05-01

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

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

Optical method for high magnification imaging and video recording of live cells at sub-micron resolution

NASA Astrophysics Data System (ADS)

Romo, Jaime E., Jr.

Optical microscopy, the most common technique for viewing living microorganisms, is limited in resolution by Abbe's criterion. Recent microscopy techniques focus on circumnavigating the light diffraction limit by using different methods to obtain the topography of the sample. Systems like the AFM and SEM provide images with fields of view in the nanometer range with high resolvable detail, however these techniques are expensive, and limited in their ability to document live cells. The Dino-Lite digital microscope coupled with the Zeiss Axiovert 25 CFL microscope delivers a cost-effective method for recording live cells. Fields of view ranging from 8 microns to 300 microns with fair resolution provide a reliable method for discovering native cell structures at the nanoscale. In this report, cultured HeLa cells are recorded using different optical configurations resulting in documentation of cell dynamics at high magnification and resolution.

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

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

Insights into nuclear dynamics using live-cell imaging approaches.

PubMed

Bigley, Rachel B; Payumo, Alexander Y; Alexander, Jeffrey M; Huang, Guo N

2017-03-01

The nucleus contains the genetic blueprint of the cell and myriad interactions within this subcellular structure are required for gene regulation. In the current scientific era, characterization of these gene regulatory networks through biochemical techniques coupled with systems-wide 'omic' approaches has become commonplace. However, these strategies are limited because they represent a mere snapshot of the cellular state. To obtain a holistic understanding of nuclear dynamics, relevant molecules must be studied in their native contexts in living systems. Live-cell imaging approaches are capable of providing quantitative assessment of the dynamics of gene regulatory interactions within the nucleus. We survey recent insights into what live-cell imaging approaches have provided the field of nuclear dynamics. In this review, we focus on interactions of DNA with other DNA loci, proteins, RNA, and the nuclear envelope. WIREs Syst Biol Med 2017, 9:e1372. doi: 10.1002/wsbm.1372 For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

Photoacoustic imaging of single circulating melanoma cells in vivo

NASA Astrophysics Data System (ADS)

Wang, Lidai; Yao, Junjie; Zhang, Ruiying; Xu, Song; Li, Guo; Zou, Jun; Wang, Lihong V.

2015-03-01

Melanoma, one of the most common types of skin cancer, has a high mortality rate, mainly due to a high propensity for tumor metastasis. The presence of circulating tumor cells (CTCs) is a potential predictor for metastasis. Label-free imaging of single circulating melanoma cells in vivo provides rich information on tumor progress. Here we present photoacoustic microscopy of single melanoma cells in living animals. We used a fast-scanning optical-resolution photoacoustic microscope to image the microvasculature in mouse ears. The imaging system has sub-cellular spatial resolution and works in reflection mode. A fast-scanning mirror allows the system to acquire fast volumetric images over a large field of view. A 500-kHz pulsed laser was used to image blood and CTCs. Single circulating melanoma cells were imaged in both capillaries and trunk vessels in living animals. These high-resolution images may be used in early detection of CTCs with potentially high sensitivity. In addition, this technique enables in vivo study of tumor cell extravasation from a primary tumor, which addresses an urgent pre-clinical need.

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.

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

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.

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.

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.

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.

A multiplexed method for kinetic measurements of apoptosis and proliferation using live-content imaging.

PubMed

Artymovich, Katherine; Appledorn, Daniel M

2015-01-01

In vitro cell proliferation and apoptosis assays are widely used to study cancer cell biology. Commonly used methodologies are however performed at a single, user-defined endpoint. We describe a kinetic multiplex assay incorporating the CellPlayer(TM) NucLight Red reagent to measure proliferation and the CellPlayer(TM) Caspase-3/7 reagent to measure apoptosis using the two-color, live-content imaging platform, IncuCyte(TM) ZOOM. High-definition phase-contrast images provide an additional qualitative validation of cell death based on morphological characteristics. The kinetic data generated using this strategy can be used to derive informed pharmacology measurements to screen potential cancer therapeutics.

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.

A bioaccumulative cyclometalated platinum(II) complex with two-photon-induced emission for live cell imaging.

PubMed

Koo, Chi-Kin; Wong, Ka-Leung; Man, Cornelia Wing-Yin; Lam, Yun-Wah; So, Leo King-Yan; Tam, Hoi-Lam; Tsao, Sai-Wah; Cheah, Kok-Wai; Lau, Kai-Chung; Yang, Yang-Yi; Chen, Jin-Can; Lam, Michael Hon-Wah

2009-02-02

The cyclometalated platinum(II) complex [Pt(L)Cl], where HL is a new cyclometalating ligand 2-phenyl-6-(1H-pyrazol-3-yl)pyridine containing C(phenyl), N(pyridyl), and N(pyrazolyl) donor moieties, was found to possess two-photon-induced luminescent properties. The two-photon-absorption cross section of the complex in N,N-dimethylformamide at room temperature was measured to be 20.8 GM. Upon two-photon excitation at 730 nm from a Ti:sapphire laser, bright-green emission was observed. Besides its two-photon-induced luminescent properties, [Pt(L)Cl] was able to be rapidly accumulated in live HeLa and NIH3T3 cells. The two-photon-induced luminescence of the complex was retained after live cell internalization and can be observed by two-photon confocal microscopy. Its bioaccumulation properties enabled time-lapse imaging of the internalization process of the dye into living cells. Cytotoxicity of [Pt(L)Cl] to both tested cell lines was low, according to MTT assays, even at loadings as high as 20 times the dose concentration for imaging for 6 h.

Imaging cell competition in Drosophila imaginal discs.

PubMed

Ohsawa, Shizue; Sugimura, Kaoru; Takino, Kyoko; Igaki, Tatsushi

2012-01-01

Cell competition is a process in which cells with higher fitness ("winners") survive and proliferate at the expense of less fit neighbors ("losers"). It has been suggested that cell competition is involved in a variety of biological processes such as organ size control, tissue homeostasis, cancer progression, and the maintenance of stem cell population. By advent of a genetic mosaic technique, which enables to generate fluorescently marked somatic clones in Drosophila imaginal discs, recent studies have presented some aspects of molecular mechanisms underlying cell competition. Now, with a live-imaging technique using ex vivo-cultured imaginal discs, we can dissect the spatiotemporal nature of competitive cell behaviors within multicellular communities. Here, we describe procedures and tips for live imaging of cell competition in Drosophila imaginal discs. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Fluorescent probes for nucleic Acid visualization in fixed and live cells.

PubMed

Boutorine, Alexandre S; Novopashina, Darya S; Krasheninina, Olga A; Nozeret, Karine; Venyaminova, Alya G

2013-12-11

This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i) sequence-specific peptides and proteins; (ii) triplex-forming oligonucleotides and (iii) polyamide oligo(N-methylpyrrole/N-methylimidazole) minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

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.

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.

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.

A CRISPR/molecular beacon hybrid system for live-cell genomic imaging.

PubMed

Wu, Xiaotian; Mao, Shiqi; Yang, Yantao; Rushdi, Muaz N; Krueger, Christopher J; Chen, Antony K

2018-04-30

The clustered regularly interspersed short palindromic repeat (CRISPR) gene-editing system has been repurposed for live-cell genomic imaging, but existing approaches rely on fluorescent protein reporters, making sensitive and continuous imaging difficult. Here, we present a fluorophore-based live-cell genomic imaging system that consists of a nuclease-deactivated mutant of the Cas9 protein (dCas9), a molecular beacon (MB), and an engineered single-guide RNA (sgRNA) harboring a unique MB target sequence (sgRNA-MTS), termed CRISPR/MB. Specifically, dCas9 and sgRNA-MTS are first co-expressed to target a specific locus in cells, followed by delivery of MBs that can then hybridize to MTS to illuminate the target locus. We demonstrated the feasibility of this approach for quantifying genomic loci, for monitoring chromatin dynamics, and for dual-color imaging when using two orthogonal MB/MTS pairs. With flexibility in selecting different combinations of fluorophore/quencher pairs and MB/MTS sequences, our CRISPR/MB hybrid system could be a promising platform for investigating chromatin activities.

Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

NASA Astrophysics Data System (ADS)

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Feletti, Lely; Lasser, Theo; Radenovic, Aleksandra

2017-02-01

Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM-SOFI concept as a quantitative imaging framework by investigating moving focal adhesions 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.

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

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.

Three dimensional live-cell STED microscopy at increased depth using a water immersion objective

NASA Astrophysics Data System (ADS)

Heine, Jörn; Wurm, Christian A.; Keller-Findeisen, Jan; Schönle, Andreas; Harke, Benjamin; Reuss, Matthias; Winter, Franziska R.; Donnert, Gerald

2018-05-01

Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope's resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5-180 μm.

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

Micro patterned surfaces allow long-term digital holographic microscopy live cell imaging

NASA Astrophysics Data System (ADS)

Mues, Sarah; Lilge, Inga; Schönherr, Holger; Kemper, Björn; Schnekenburger, Jürgen

2017-07-01

During long-term imaging, cells move out of the field of view. We have generated functionalized substrates containing rectangular areas, which were capable in keeping cells over the whole observation period.

Tracking the Dynamic Folding and Unfolding of RNA G-Quadruplexes in Live Cells.

PubMed

Chen, Xiu-Cai; Chen, Shuo-Bin; Dai, Jing; Yuan, Jia-Hao; Ou, Tian-Miao; Huang, Zhi-Shu; Tan, Jia-Heng

2018-04-16

Because of the absence of methods for tracking RNA G-quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G-quadruplexes is so far limited. Herein, we report a new red-emitting fluorescent probe, QUMA-1, for the selective, continuous, and real-time visualization of RNA G-quadruplexes in live cells. The applications of QUMA-1 in several previously intractable applications, including live-cell imaging of the dynamic folding, unfolding, and movement of RNA G-quadruplexes and the visualization of the unwinding of RNA G-quadruplexes by RNA helicase have been demonstrated. Notably, our real-time results revealed the complexity of the dynamics of RNA G-quadruplexes in live cells. We anticipate that the further application of QUMA-1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G-quadruplexes will uncover more information about the biological roles of RNA G-quadruplexes. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-speed AFM for scanning the architecture of living cells

NASA Astrophysics Data System (ADS)

Li, Jing; Deng, Zhifeng; Chen, Daixie; Ao, Zhuo; Sun, Quanmei; Feng, Jiantao; Yin, Bohua; Han, Li; Han, Dong

2013-08-01

We address the modelling of tip-cell membrane interactions under high speed atomic force microscopy. Using a home-made device with a scanning area of 100 × 100 μm2, in situ imaging of living cells is successfully performed under loading rates from 1 to 50 Hz, intending to enable detailed descriptions of physiological processes in living samples.We address the modelling of tip-cell membrane interactions under high speed atomic force microscopy. Using a home-made device with a scanning area of 100 × 100 μm2, in situ imaging of living cells is successfully performed under loading rates from 1 to 50 Hz, intending to enable detailed descriptions of physiological processes in living samples. Electronic supplementary information (ESI) available: Movie of the real-time change of inner surface within fresh blood vessel. The movie was captured at a speed of 30 Hz in the range of 80 μm × 80 μm. See DOI: 10.1039/c3nr01464a

Surface-enhanced Raman scattering (SERS) imaging of alkyne-tagged small molecule drug in live cells with endocytosed gold nanoparticles

NASA Astrophysics Data System (ADS)

Ando, Jun; Sekiya, Takumasa; Ka, Den; Yamakoshi, Hiroyuki; Dodo, Kosuke; Sodeoka, Mikiko; Kawata, Satoshi; Fujita, Katsumasa

2017-02-01

We propose the combination of alkyne-tag and surface-enhanced Raman scattering (SERS) spectroscopy to perform highly-sensitive and selective drug imaging in live cells. Gold nanoparticles are introduced in lysosomes through endocytosis as SERS agents, and the alkyne-tagged drugs are subsequently administered in cells. Raman microscopic observation reveals the arrival of drug in lysosome through enhanced Raman signal of alkyne. Since the peak of alkyne appears in Raman-silent region of biomolecules, selective detection of drugs is possible without background signal of endogenous molecules. From endocytosed gold nanoparticles in living HeLa cells, we observed distinct Raman signal from alkyne-tagged inhibitor of lysosomal enzyme.

Synthesis, biological evaluation, and live cell imaging of novel fluorescent duocarmycin analogs.

PubMed

Tietze, Lutz F; Behrendt, Frank; Pestel, Galina F; Schuberth, Ingrid; Mitkovski, Mišo

2012-11-01

For a better understanding of the mode of action of duocarmycin and its analogs, the novel fluorescent duocarmycin derivatives 13-15 and 17b-19b were synthesized, and their bioactivity as well as their cellular uptake investigated using confocal laser scanning microscopy (CLSM) in live-cell imaging experiments. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging.

PubMed

Nakamura, Akinobu; Takigawa, Kazumasa; Kurishita, Yasutaka; Kuwata, Keiko; Ishida, Manabu; Shimoda, Yasushi; Hamachi, Itaru; Tsukiji, Shinya

2014-06-11

We report a general strategy to create small-molecule fluorescent probes for the nucleus in living cells. Our strategy is based on the attachment of the DNA-binding Hoechst compound to a fluorophore of interest. Using this approach, simple fluorescein, BODIPY, and rhodamine dyes were readily converted to novel turn-on fluorescent nucleus-imaging probes.

Live cell imaging combined with high-energy single-ion microbeam

NASA Astrophysics Data System (ADS)

Guo, Na; Du, Guanghua; Liu, Wenjing; Guo, Jinlong; Wu, Ruqun; Chen, Hao; Wei, Junzhe

2016-03-01

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10-3 s-1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10-2 s-1.

[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.

Highly luminescent, biocompatible ytterbium(iii) complexes as near-infrared fluorophores for living cell imaging.

PubMed

Ning, Yingying; Tang, Juan; Liu, Yi-Wei; Jing, Jing; Sun, Yuansheng; Zhang, Jun-Long

2018-04-21

Herein, we report the design and synthesis of biocompatible Yb 3+ complexes for near-infrared (NIR) living cell imaging. Upon excitation at either the visible (Soret band) or red region (Q band), these β-fluorinated Yb 3+ complexes display high NIR luminescence (quantum yields up to 23% and 13% in dimethyl sulfoxide and water, respectively) and have higher stabilities and prolonged decay lifetimes (up to 249 μs) compared to the β-non-fluorinated counterparts. This renders the β-fluorinated Yb 3+ complexes as a new class of biological optical probes in both steady-state imaging and time-resolved fluorescence lifetime imaging (FLIM). NIR confocal fluorescence images showed strong and specific intracellular Yb 3+ luminescence signals when the biocompatible Yb 3+ complexes were uptaken into the living cells. Importantly, FLIM measurements showed an intracellular lifetime distribution between 100 and 200 μs, allowing an effective discrimination from cell autofluorescence, and afforded high signal-to-noise ratios as firstly demonstrated in the NIR region. These results demonstrated the prospects of NIR lanthanide complexes as biological probes for NIR steady-state fluorescence and time-resolved fluorescence lifetime imaging.

Detecting cells in time varying intensity images in confocal microscopy for gene expression studies in living cells

NASA Astrophysics Data System (ADS)

Mitra, Debasis; Boutchko, Rostyslav; Ray, Judhajeet; Nilsen-Hamilton, Marit

2015-03-01

In this work we present a time-lapsed confocal microscopy image analysis technique for an automated gene expression study of multiple single living cells. Fluorescence Resonance Energy Transfer (FRET) is a technology by which molecule-to-molecule interactions are visualized. We analyzed a dynamic series of ~102 images obtained using confocal microscopy of fluorescence in yeast cells containing RNA reporters that give a FRET signal when the gene promoter is activated. For each time frame, separate images are available for three spectral channels and the integrated intensity snapshot of the system. A large number of time-lapsed frames must be analyzed to identify each cell individually across time and space, as it is moving in and out of the focal plane of the microscope. This makes it a difficult image processing problem. We have proposed an algorithm here, based on scale-space technique, which solves the problem satisfactorily. The algorithm has multiple directions for even further improvement. The ability to rapidly measure changes in gene expression simultaneously in many cells in a population will open the opportunity for real-time studies of the heterogeneity of genetic response in a living cell population and the interactions between cells that occur in a mixed population, such as the ones found in the organs and tissues of multicellular organisms.

Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

NASA Astrophysics Data System (ADS)

Jünger, Felix; Olshausen, Philipp V.; Rohrbach, Alexander

2016-07-01

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

PubMed Central

Jünger, Felix; Olshausen, Philipp v.; Rohrbach, Alexander

2016-01-01

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes. PMID:27465033

Live cell refractometry using microfluidic devices.

PubMed

Lue, Niyom; Popescu, Gabriel; Ikeda, Takahiro; Dasari, Ramachandra R; Badizadegan, Kamran; Feld, Michael S

2006-09-15

Using Hilbert phase microscopy for extracting quantitative phase images, we measured the average refractive index associated with live cells in culture. To decouple the contributions to the phase signal from the cell refractive index and thickness, we confined the cells in microchannels. The results are confirmed by comparison with measurements of spherical cells in suspension.

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.

Multiphoton luminescent graphene quantum dots for in vivo tracking of human adipose-derived stem cells

NASA Astrophysics Data System (ADS)

Kim, Jin; Song, Sung Ho; Jin, Yoonhee; Park, Hyun-Ji; Yoon, Hyewon; Jeon, Seokwoo; Cho, Seung-Woo

2016-04-01

The applicability of graphene quantum dots (GQDs) for the in vitro and in vivo live imaging and tracking of different types of human stem cells is investigated. GQDs synthesized by the modified graphite intercalated compound method show efficient cellular uptake with improved biocompatibility and highly sensitive optical properties, indicating their feasibility as a bio-imaging probe for stem cell therapy.The applicability of graphene quantum dots (GQDs) for the in vitro and in vivo live imaging and tracking of different types of human stem cells is investigated. GQDs synthesized by the modified graphite intercalated compound method show efficient cellular uptake with improved biocompatibility and highly sensitive optical properties, indicating their feasibility as a bio-imaging probe for stem cell therapy. Electronic supplementary information (ESI) available: Additional results. See DOI: 10.1039/c6nr02143c

Sequential processing of quantitative phase images for the study of cell behaviour in real-time digital holographic microscopy.

PubMed

Zikmund, T; Kvasnica, L; Týč, M; Křížová, A; Colláková, J; Chmelík, R

2014-11-01

Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time-lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long-term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least-squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Swept Field Laser Confocal Microscopy for Enhanced Spatial and Temporal Resolution in Live-Cell Imaging

PubMed Central

Castellano-Muñoz, Manuel; Peng, Anthony Wei; Salles, Felipe T.; Ricci, Anthony J.

2013-01-01

Confocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality. PMID:22831554

Evaluating novel synthetic compounds active against Bacillus subtilis and Bacillus cereus spores using Live imaging with SporeTrackerX.

PubMed

Omardien, Soraya; Ter Beek, Alexander; Vischer, Norbert; Montijn, Roy; Schuren, Frank; Brul, Stanley

2018-06-14

An empirical approach was taken to screen a novel synthetic compound library designed to be active against Gram-positive bacteria. We obtained five compounds that were active against spores from the model organism Bacillus subtilis and the food-borne pathogen Bacillus cereus during our population based experiments. Using single cell live imaging we were able to observe effects of the compounds on spore germination and outgrowth. Difference in sensitivity to the compounds could be observed between B. subtilis and B. cereus using live imaging, with minor difference in the minimal inhibitory and bactericidal concentrations of the compounds against the spores. The compounds all delayed the bursting time of germinated spores and affected the generation time of vegetative cells at sub-inhibitory concentrations. At inhibitory concentrations spore outgrowth was prevented. One compound showed an unexpected potential for preventing spore germination at inhibitory concentrations, which merits further investigation. Our study shows the valuable role single cell live imaging can play in the final selection process of antimicrobial compounds.

The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

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

Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique

2013-04-01

Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and themore » ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.« less

Use of an optical trap for study of host-pathogen interactions for dynamic live cell imaging.

PubMed

Tam, Jenny M; Castro, Carlos E; Heath, Robert J W; Mansour, Michael K; Cardenas, Michael L; Xavier, Ramnik J; Lang, Matthew J; Vyas, Jatin M

2011-07-28

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system(1, 2); however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis(3) have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture. Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions(4). Radiation pressure was first observed and applied to optical tweezer systems in 1970(5, 6), and was first used to control biological specimens in 1987(7). Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena(8-13). We describe a method(14) that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals(15, 16) (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging

PubMed Central

Devaraj, Neal K.; Weissleder, Ralph; Hilderbrand, Scott A.

2009-01-01

Bioorthogonal tetrazine cycloadditions have been applied to live cell labeling. Tetrazines react irreversibly with the strained dienophile norbornene forming dihydropyrazine products and dinitrogen. The reaction is high yielding, selective, and fast in aqueous media. Her2/neu receptors on live human breast cancer cells were targeted with a monoclonal antibody modified with a norbornene. Tetrazines conjugated to a near-infrared fluorochrome selectively and rapidly label the pretargeted antibody in the presence of serum. These findings indicate that this chemistry is suitable for in vitro labeling experiments, and suggests that it may prove a useful strategy for in vivo pretargeted imaging under numerous modalities. PMID:19053305

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.

Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor.

PubMed

Panda, Koustubh; Chawla-Sarkar, Mamta; Santos, Cecile; Koeck, Thomas; Erzurum, Serpil C; Parkinson, John F; Stuehr, Dennis J

2005-07-19

The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression.

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.

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.

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.

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.

Two-photon imaging of formaldehyde in live cells and animals utilizing a lysosome-targetable and acidic pH-activatable fluorescent probe.

PubMed

Xie, Xilei; Tang, Fuyan; Shangguan, Xiaoyan; Che, Shiyi; Niu, Jinye; Xiao, Yongsheng; Wang, Xu; Tang, Bo

2017-06-13

Lyso-TPFP presents lysosomal targetability and an acidic pH-activatable response toward formaldehyde. Thus, it exclusively visualizes lysosomal formaldehyde and is immune against it in neutral cytosol and other organelles. In addition, two-photon fluorescence imaging endows Lyso-TPFP with the capability of in situ tracking formaldehyde in live cells and animals.

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.

New water soluble Hg2 + selective fluorescent calix[4]arenes: Synthesis and application in living cells imaging

NASA Astrophysics Data System (ADS)

Oguz, Mehmet; Bhatti, Asif Ali; Karakurt, Serdar; Aktas, Mehmet; Yilmaz, Mustafa

2017-01-01

The present study demonstrates the synthesis of water-soluble fluorescent calix[4]arenes (6 and 7) and its application in living cell imaging for Hg2 + detection at a low level. The synthesized fluorescent ligands 6 and 7 were characterized by 1H NMR technique. The fluorescent study showed both water soluble ligands were Hg2 + selective and follow photo-induced electron transfer (PET) process. From the fluorimeter titration experiment detection limit was calculated as 1.14 × 10- 5 and 3.42 × 10- 5 for ligand 6 and 7, respectively. From the Benesi-Hildebrand plot binding constant values were evaluated as 666.7 and 733.3 M- 1 for 6 and 7, respectively. The interactions between ligands 6 and 7 and Hg2 + were also demonstrated in living cells, SW-620, using Fluorescent Cell Imager. While ligands 6 and 7 alone show fluorescent properties, they loss their action with the presence of Hg2 + in SW-620 cells.

Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly.

PubMed

Baumgärtel, Viola; Müller, Barbara; Lamb, Don C

2012-05-01

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy.

PubMed

Traenkle, Bjoern; Rothbauer, Ulrich

2017-01-01

Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies) have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

Quantitative Live-Cell Imaging of Human Immunodeficiency Virus (HIV-1) Assembly

PubMed Central

Baumgärtel, Viola; Müller, Barbara; Lamb, Don C.

2012-01-01

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site. PMID:22754649

RNA Imaging with Dimeric Broccoli in Live Bacterial and Mammalian Cells

PubMed Central

Filonov, Grigory S.

2016-01-01

RNA spatial dynamics play a crucial role in cell physiology and thus the ability to monitor RNA localization in live cells can provide insight into important biological problems. This article focuses on imaging RNAs using an “RNA mimic of GFP”. This approach relies on a RNA aptamer, called dimeric Broccoli, which binds to and switches on the fluorescence of DFHBI, a small molecule mimicking the fluorophore in GFP. Dimeric Broccoli is tagged to heterologously expressed RNAs and upon DFHBI binding the fluorescent signal of dimeric Broccoli reports the transcript’s localization in cells. This protocol describes the process of validating the fluorescence of dimeric Broccoli-labeled transcripts in vitro and in cells, flow cytometry analysis to determine overall fluorescence levels in cells, and fluorescence imaging in bacterial and mammalian cells. Overall, the current protocol should be useful for researchers seeking to image high abundance RNAs, such as transcribed off the T7 promoter in bacteria or off Pol III-dependent promoters in mammalian cells. PMID:26995352

Remnant living cells that escape cell loss in late-stage tumors exhibit cancer stem cell-like characteristics

PubMed Central

Chen, Y-L; Wang, S-Y; Liu, R-S; Wang, H-E; Chen, J-C; Chiou, S-H; Chang, C A; Lin, L-T; Tan, D T W; Lee, Y-J

2012-01-01

A balance between cell proliferation and cell loss is essential for tumor progression. Although up to 90% of cells are lost in late-stage carcinomas, the progression and characteristics of remnant living cells in tumor mass are unclear. Here we used molecular imaging to track the progression of living cells in a syngeneic tumor model, and ex vivo investigated the properties of this population at late-stage tumor. The piggyBac transposon system was used to stably introduce the dual reporter genes, including monomeric red fluorescent protein (mRFP) and herpes simplex virus type-1 thymidine kinase (HSV1-tk) genes for fluorescence-based and radionuclide-based imaging of tumor growth in small animals, respectively. Iodine-123-labeled 5-iodo-2′-fluoro-1-beta-𝒟-arabinofuranosyluracil was used as a radiotracer for HSV1-tk gene expression in tumors. The fluorescence- and radionuclide-based imaging using the single-photon emission computed tomography/computed tomography revealed that the number of living cells reached the maximum at 1 week after implantation of 4T1 tumors, and gradually decreased and clustered near the side of the body until 4 weeks accompanied by enlargement of tumor mass. The remnant living cells at late-stage tumor were isolated and investigated ex vivo. The results showed that these living cells could form mammospheres and express cancer stem cell (CSC)-related biomarkers, including octamer-binding transcription factor 4, SRY (sex-determining region Y)-box 2, and CD133 genes compared with those cultured in vitro. Furthermore, this HSV1-tk-expressing CSC-like population was sensitive to ganciclovir applied for the suicide therapy. Taken together, the current data suggested that cells escaping from cell loss in late-stage tumors exhibit CSC-like characteristics, and HSV1-tk may be considered a theranostic agent for targeting this population in vivo. PMID:23034334

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.

Label-free and live cell imaging by interferometric scattering microscopy.

PubMed

Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

2018-03-14

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

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

Coherent imaging with incoherent light in digital holographic microscopy

NASA Astrophysics Data System (ADS)

Chmelik, Radim

2012-01-01

Digital holographic microscope (DHM) allows for imaging with a quantitative phase contrast. In this way it becomes an important instrument, a completely non-invasive tool for a contrast intravital observation of living cells and a cell drymass density distribution measurement. A serious drawback of current DHMs is highly coherent illumination which makes the lateral resolution worse and impairs the image quality by a coherence noise and a parasitic interference. An uncompromising solution to this problem can be found in the Leith concept of incoherent holography. An off-axis hologram can be formed with arbitrary degree of light coherence in systems equipped with an achromatic interferometer and thus the resolution and the image quality typical for an incoherent-light wide-field microscopy can be achieved. In addition, advanced imaging modes based on limited coherence can be utilized. The typical example is a coherence-gating effect which provides a finite axial resolution and makes DHM image similar to that of a confocal microscope. These possibilities were described theoretically using the formalism of three-dimensional coherent transfer functions and proved experimentally by the coherence-controlled holographic microscope which is DHM based on the Leith achromatic interferometer. Quantitative-phase-contrast imaging is demonstrated with incoherent light by the living cancer cells observation and their motility evaluation. The coherence-gating effect was proved by imaging of model samples through a scattering layer and living cells inside an opalescent medium.

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.

Intracellular imaging of docosanol in living cells by coherent anti-Stokes Raman scattering microscopy

NASA Astrophysics Data System (ADS)

You, Sixian; Liu, Yuan; Arp, Zane; Zhao, Youbo; Chaney, Eric J.; Marjanovic, Marina; Boppart, Stephen A.

2017-07-01

Docosanol is an over-the-counter topical agent that has proved to be one of the most effective therapies for treating herpes simplex labialis. However, the mechanism by which docosanol suppresses lesion formation remains poorly understood. To elucidate its mechanism of action, we investigated the uptake of docosanol in living cells using coherent anti-Stokes Raman scattering microscopy. Based on direct visualization of the deuterated docosanol, we observed highly concentrated docosanol inside living cells 24 h after drug treatment. In addition, different spatial patterns of drug accumulation were observed in different cell lines. In keratinocytes, which are the targeted cells of docosanol, the drug molecules appeared to be docking at the periphery of the cell membrane. In contrast, the drug molecules in fibroblasts appeared to accumulate in densely packed punctate regions throughout the cytoplasm. These results suggest that this molecular imaging approach is suitable for the longitudinal tracking of drug molecules in living cells to identify cell-specific trafficking and may also have implications for elucidating the mechanism by which docosanol suppresses lesion formation.

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 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

Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging.

PubMed

Mendive-Tapia, Lorena; Subiros-Funosas, Ramon; Zhao, Can; Albericio, Fernando; Read, Nick D; Lavilla, Rodolfo; Vendrell, Marc

2017-08-01

Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences. Fmoc-Trp(C 2 -BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C-C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH (3-4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6-7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging).

Live-Cell Imaging of Mitochondria and the Actin Cytoskeleton in Budding Yeast.

PubMed

Higuchi-Sanabria, Ryo; Swayne, Theresa C; Boldogh, Istvan R; Pon, Liza A

2016-01-01

Maintenance and regulation of proper mitochondrial dynamics and functions are necessary for cellular homeostasis. Numerous diseases, including neurodegeneration and muscle myopathies, and overall cellular aging are marked by declining mitochondrial function and subsequent loss of multiple other cellular functions. For these reasons, optimized protocols are needed for visualization and quantification of mitochondria and their function and fitness. In budding yeast, mitochondria are intimately associated with the actin cytoskeleton and utilize actin for their movement and inheritance. This chapter describes optimal approaches for labeling mitochondria and the actin cytoskeleton in living budding yeast cells, for imaging the labeled cells, and for analyzing the resulting images.

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.

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.

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.

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.

Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes.

PubMed

Rakers, Lena; Grill, David; Matos, Anna L L; Wulff, Stephanie; Wang, Da; Börgel, Jonas; Körsgen, Martin; Arlinghaus, Heinrich F; Galla, Hans-Joachim; Gerke, Volker; Glorius, Frank

2018-05-01

Cholesterol is an essential component of most biological membranes and serves important functions in controlling membrane integrity, organization, and signaling. However, probes to follow the dynamic distribution of cholesterol in live cells are scarce and so far show only limited applicability. Herein, we addressed this problem by synthesizing and characterizing a class of versatile and clickable cholesterol-based imidazolium salts. We show that these cholesterol analogs faithfully mimic the biophysical properties of natural cholesterol in phospholipid mono- and bilayers, and that they integrate into the plasma membrane of cultured and primary human cells. The membrane-incorporated cholesterol analogs can be specifically labeled by click chemistry and visualized in live-cell imaging experiments that show a distribution and behavior comparable with that of endogenous membrane cholesterol. These results indicate that the cholesterol analogs can be used to reveal the dynamic distribution of cholesterol in live cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Live cell imaging combined with high-energy single-ion microbeam

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

Guo, Na; Du, Guanghua, E-mail: gh-du@impcas.ac.cn; Liu, Wenjing

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in themore » cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10{sup −3} s{sup −1} and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10{sup −2} s{sup −1}.« less

Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

PubMed

Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

2016-10-01

The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing.

PubMed

Moo, Eng Kuan; Abusara, Ziad; Abu Osman, Noor Azuan; Pingguan-Murphy, Belinda; Herzog, Walter

2013-08-09

Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Prospects and challenges of quantitative phase imaging in tumor cell biology

NASA Astrophysics Data System (ADS)

Kemper, Björn; Götte, Martin; Greve, Burkhard; Ketelhut, Steffi

2016-03-01

Quantitative phase imaging (QPI) techniques provide high resolution label-free quantitative live cell imaging. Here, prospects and challenges of QPI in tumor cell biology are presented, using the example of digital holographic microscopy (DHM). It is shown that the evaluation of quantitative DHM phase images allows the retrieval of different parameter sets for quantification of cellular motion changes in migration and motility assays that are caused by genetic modifications. Furthermore, we demonstrate simultaneously label-free imaging of cell growth and morphology properties.

Live-cell imaging of Salmonella Typhimurium interaction with zebrafish larvae after injection and immersion delivery methods.

PubMed

Varas, Macarena; Fariña, Alonso; Díaz-Pascual, Francisco; Ortíz-Severín, Javiera; Marcoleta, Andrés E; Allende, Miguel L; Santiviago, Carlos A; Chávez, Francisco P

2017-04-01

The zebrafish model has been used to determine the role of vertebrate innate immunity during bacterial infections. Here, we compare the in vivo immune response induced by GFP-tagged Salmonella Typhimurium inoculated by immersion and microinjection in transgenic zebrafish larvae. Our novel infection protocols in zebrafish allow live-cell imaging of Salmonella colonization. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

Gold nanorods for cell imaging with confocal reflectance microscopy and two-photon fluorescence microscopy

NASA Astrophysics Data System (ADS)

Chen, Ji-Yao; Wang, Pei-Nan

2010-02-01

Gold nanorods have unique optical properties as their two photon absorption cross sections are very high and their spectral positions of extinction bands can be controlled by their aspect ratio only, so that gold nanorods have been considered as agents for cell imaging. Two-photon photoluminescence imaging could be used to detect the cellular gold nanorods with the high power femto-second (fs) infrared laser, but may cause the photothermal effect melting the rods. The 3-D distribution of gold nanorods in living cells also can be measured by confocal reflectance microscopy with a very low laser power, and thus the cell damaging can be avoided. In this work, these two methods were comparatively studied in living rat basophilic leukemia (RBL-2H3) cells.

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.

Microemulsion-Based Soft Bacteria-Driven Microswimmers for Active Cargo Delivery.

PubMed

Singh, Ajay Vikram; Hosseinidoust, Zeinab; Park, Byung-Wook; Yasa, Oncay; Sitti, Metin

2017-10-24

Biohybrid cell-driven microsystems offer unparalleled possibilities for realization of soft microrobots at the micron scale. Here, we introduce a bacteria-driven microswimmer that combines the active locomotion and sensing capabilities of bacteria with the desirable encapsulation and viscoelastic properties of a soft double-micelle microemulsion for active transport and delivery of cargo (e.g., imaging agents, genes, and drugs) to living cells. Quasi-monodisperse double emulsions were synthesized with an aqueous core that encapsulated the fluorescence imaging agents, as a proof-of-concept cargo in this study, and an outer oil shell that was functionalized with streptavidin for specific and stable attachment of biotin-conjugated Escherichia coli. Motile bacteria effectively propelled the soft microswimmers across a Transwell membrane, actively delivering imaging agents (i.e., dyes) encapsulated inside of the micelles to a monolayer of cultured MCF7 breast cancer and J744A.1 macrophage cells, which enabled real-time, live-cell imaging of cell organelles, namely mitochondria, endoplasmic reticulum, and Golgi body. This in vitro model demonstrates the proof-of-concept feasibility of the proposed soft microswimmers and offers promise for potential biomedical applications in active and/or targeted transport and delivery of imaging agents, drugs, stem cells, siRNA, and therapeutic genes to live tissue in in vitro disease models (e.g., organ-on-a-chip devices) and stagnant or low-flow-velocity fluidic regions of the human body.

Col-F, a fluorescent probe for ex vivo confocal imaging of collagen and elastin in animal tissues.

PubMed

Biela, Ewa; Galas, Jerzy; Lee, Brian; Johnson, Gary L; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

2013-06-01

A new low-molecular-weight fluorescent probe, Col-F, that exhibits affinity to collagen and elastin, was used successfully in imaging of extracellular matrix in freshly excised animal tissues. Col-F readily penetrates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Fibers of collagen and elastin have been stained in a variety of tissues, including tendon, skeletal muscle, connective tissue, and arteries. Cells migrating in a Col-F-stained collagenous biomaterial were also imaged. No phototoxic effects were detected when live keratocytes were imaged in the in vitro culture in the presence of Col-F. In conclusion, Col-F provides a simple and convenient tool for fluorescence three-dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen-containing biomaterials. Copyright © 2013 International Society for Advancement of Cytometry.

Motility contrast imaging of live porcine cumulus-oocyte complexes

NASA Astrophysics Data System (ADS)

An, Ran; Turek, John; Machaty, Zoltan; Nolte, David

2013-02-01

Freshly-harvested porcine oocytes are invested with cumulus granulosa cells in cumulus-oocyte complexes (COCs). The cumulus cell layer is usually too thick to image the living oocyte under a conventional microscope. Therefore, it is difficult to assess the oocyte viability. The low success rate of implantation is the main problem for in vitro fertilization. In this paper, we demonstrate our dynamic imaging technique called motility contrast imaging (MCI) that provides a non-invasive way to monitor the COCs before and after maturation. MCI shows a change of intracellular activity during oocyte maturation, and a measures dynamic contrast between the cumulus granulosa shell and the oocytes. MCI also shows difference in the spectral response between oocytes that were graded into quality classes. MCI is based on shortcoherence digital holography. It uses intracellular motility as the endogenous imaging contrast of living tissue. MCI presents a new approach for cumulus-oocyte complex assessment.

[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.

Two-colour live-cell nanoscale imaging of intracellular targets

NASA Astrophysics Data System (ADS)

Bottanelli, Francesca; Kromann, Emil B.; Allgeyer, Edward S.; Erdmann, Roman S.; Wood Baguley, Stephanie; Sirinakis, George; Schepartz, Alanna; Baddeley, David; Toomre, Derek K.; Rothman, James E.; Bewersdorf, Joerg

2016-03-01

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution.

Cd(II)-terpyridine-based complex as a ratiometric fluorescent probe for pyrophosphate detection in solution and as an imaging agent in living cells.

PubMed

Jiao, Shu-Yan; Li, Kun; Zhang, Wei; Liu, Yan-Hong; Huang, Zeng; Yu, Xiao-Qi

2015-01-21

The terpyridine anthracene ligand was synthesized and characterized. is a ratiometric fluorescent probe for Cd(2+) with a recognition mechanism based on intramolecular charge transfer (ICT). An complex was isolated, and its structure was established using single-crystal XRD. The complex was able to serve as a novel reversible chemosensing ensemble to allow ratiometric response to pyrophosphate (PPi) in aqueous media. Moreover, the fluorescence imaging in living cells from these two emission channels suggested that was a ratiometric probe for Cd(2+), and the in situ generated complex was also a ratiometric ensemble for PPi detection in living cells.

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

Reaction-based small-molecule fluorescent probes for dynamic detection of ROS and transient redox changes in living cells and small animals.

PubMed

Lü, Rui

2017-09-01

Dynamic detection of transient redox changes in living cells and animals has broad implications for human health and disease diagnosis, because intracellular redox homeostasis regulated by reactive oxygen species (ROS) plays important role in cell functions, normal physiological functions and some serious human diseases (e.g., cancer, Alzheimer's disease, diabetes, etc.) usually have close relationship with the intracellular redox status. Small-molecule ROS-responsive fluorescent probes can act as powerful tools for dynamic detection of ROS and redox changes in living cells and animals through fluorescence imaging techniques; and great advances have been achieved recently in the design and synthesis of small-molecule ROS-responsive fluorescent probes. This article highlights up-to-date achievements in designing and using the reaction-based small-molecule fluorescent probes (with high sensitivity and selectivity to ROS and redox cycles) in the dynamic detection of ROS and transient redox changes in living cells and animals through fluorescence imaging. Copyright © 2017. Published by Elsevier Ltd.

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.

Spatially coordinated dynamic gene transcription in living pituitary tissue

PubMed Central

Featherstone, Karen; Hey, Kirsty; Momiji, Hiroshi; McNamara, Anne V; Patist, Amanda L; Woodburn, Joanna; Spiller, David G; Christian, Helen C; McNeilly, Alan S; Mullins, John J; Finkenstädt, Bärbel F; Rand, David A; White, Michael RH; Davis, Julian RE

2016-01-01

Transcription at individual genes in single cells is often pulsatile and stochastic. A key question emerges regarding how this behaviour contributes to tissue phenotype, but it has been a challenge to quantitatively analyse this in living cells over time, as opposed to studying snap-shots of gene expression state. We have used imaging of reporter gene expression to track transcription in living pituitary tissue. We integrated live-cell imaging data with statistical modelling for quantitative real-time estimation of the timing of switching between transcriptional states across a whole tissue. Multiple levels of transcription rate were identified, indicating that gene expression is not a simple binary ‘on-off’ process. Immature tissue displayed shorter durations of high-expressing states than the adult. In adult pituitary tissue, direct cell contacts involving gap junctions allowed local spatial coordination of prolactin gene expression. Our findings identify how heterogeneous transcriptional dynamics of single cells may contribute to overall tissue behaviour. DOI: http://dx.doi.org/10.7554/eLife.08494.001 PMID:26828110

Optimization of cell morphology measurement via single-molecule tracking PALM.

PubMed

Frost, Nicholas A; Lu, Hsiangmin E; Blanpied, Thomas A

2012-01-01

In neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution of molecular positions mapped via live-cell Photoactivated Localization Microscopy (PALM) is a powerful approach, but molecular motion complicates this analysis and can degrade overall resolution of the morphological reconstruction. Nevertheless, the motion is of additional interest because tracking single molecules provides diffusion coefficients, bound fraction, and other key functional parameters. We used Monte Carlo simulations to examine features of single-molecule tracking of practical utility for the simultaneous determination of cell morphology. We find that the accuracy of determining both distance and angle of motion depend heavily on the precision with which molecules are localized. Strikingly, diffusion within a bounded region resulted in an inward bias of localizations away from the edges, inaccurately reflecting the region structure. This inward bias additionally resulted in a counterintuitive reduction of measured diffusion coefficient for fast-moving molecules; this effect was accentuated by the long camera exposures typically used in single-molecule tracking. Thus, accurate determination of cell morphology from rapidly moving molecules requires the use of short integration times within each image to minimize artifacts caused by motion during image acquisition. Sequential imaging of neuronal processes using excitation pulses of either 2 ms or 10 ms within imaging frames confirmed this: processes appeared erroneously thinner when imaged using the longer excitation pulse. Using this pulsed excitation approach, we show that PALM can be used to image spine and spine neck morphology in living neurons. These results clarify a number of issues involved in interpretation of single-molecule data in living cells and provide a method to minimize artifacts in single-molecule experiments.

Off-on fluorescence monitoring of intracellular Ag+ in single living cells using an Ag+-responsive probe

NASA Astrophysics Data System (ADS)

Lv, Jian; Zhao, Li-Jun; Qian, Ruo-Can; Long, Yi-Tao

2017-12-01

Detection of silver ions (Ag+) in living cells has becoming more and more attractive due to the important biological impact of Ag+ on cellular functions. Here, we put forward a new approach to realize the in situ fluorescence imaging and detection of Ag+ in single cells via an ultrasensitive Ag+-responsive probe, 3‧,6‧-bis (diethylamino)-2-(2-iodoethyl) spiro[isoindoline-1,9‧-xanthen]-3-one (BDISIX). In the presence of Ag+, the fluorescence of the probe can be turned ‘on’, generating strong red fluorescence. Using breast cancer cells (MCF-7) as the example, we successfully realize the imaging of intracellular Ag+ through one-step incubation of the probe, which is especially convenient and fast for the in situ intact detection of Ag+ in living cells.

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.

Does Increased Expression of the Plasma Membrane Calcium-ATPase Isoform 2 Confer Resistance to Apoptosis on Breast Cancer Cells?

DTIC Science & Technology

2008-09-01

T47D control cells with the highest sustained levels of intracellular calcium in the  live   cell   imaging  experiments (Figure 9). Membrane blebbing is a...classic hallmark, and early indicator of apoptosis. No  membrane blebbing was observed in T47D/PMCA2 cells during the  live   cell   imaging  studies.     10

Improved two-photon imaging of living neurons in brain tissue through temporal gating

PubMed Central

Gautam, Vini; Drury, Jack; Choy, Julian M. C.; Stricker, Christian; Bachor, Hans-A.; Daria, Vincent R.

2015-01-01

We optimize two-photon imaging of living neurons in brain tissue by temporally gating an incident laser to reduce the photon flux while optimizing the maximum fluorescence signal from the acquired images. Temporal gating produces a bunch of ~10 femtosecond pulses and the fluorescence signal is improved by increasing the bunch-pulse energy. Gating is achieved using an acousto-optic modulator with a variable gating frequency determined as integral multiples of the imaging sampling frequency. We hypothesize that reducing the photon flux minimizes the photo-damage to the cells. Our results, however, show that despite producing a high fluorescence signal, cell viability is compromised when the gating and sampling frequencies are equal (or effectively one bunch-pulse per pixel). We found an optimum gating frequency range that maintains the viability of the cells while preserving a pre-set fluorescence signal of the acquired two-photon images. The neurons are imaged while under whole-cell patch, and the cell viability is monitored as a change in the membrane’s input resistance. PMID:26504651

Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells.

PubMed

Hebert, Benedict; Costantino, Santiago; Wiseman, Paul W

2005-05-01

We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of alpha-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mum/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mum/min) and diffusion of interacting alpha5 integrin/enhanced cyan fluorescent protein and alpha-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.

Fluorescence Lifetime Imaging Microscopy (FLIM) of quantum dots in living cells

NASA Astrophysics Data System (ADS)

Nadeau, Jay; Carlini, Lina

2013-02-01

Fluorescence lifetime imaging microscopy (FLIM) is an emerging imaging technique that can indicate environmental factors such as pH and redox potential by the effect of these factors on the fluorescence lifetimes of fluorophores. Semiconductor quantum dots (QDs) are highly sensitive to environment and so are ideal for use in FLIM, although certain experimental parameters must be carefully considered for QD imaging to account for their long lifetimes and two-photon behavior. We image the uptake of three types of QDs in cultured fibroblasts and show some preliminary results on the effects of endosomes and lysosomes on QD lifetimes. These results indicate the feasibility of FLIM for studies using QDs in live cells.

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

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.

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

Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging.

PubMed

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

2017-10-15

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. © 2017 Schvartz et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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

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.

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.

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.

A dual pH and temperature responsive polymeric fluorescent sensor and its imaging application in living cells.

PubMed

Yin, Liyan; He, Chunsheng; Huang, Chusen; Zhu, Weiping; Wang, Xin; Xu, Yufang; Qian, Xuhong

2012-05-11

A polymeric fluorescent sensor PNME, consisting of A4 and N-isopropylacrylamide (NIPAM) units, was synthesized. PNME exhibited dual responses to pH and temperature, and could be used as an intracellular pH sensor for lysosomes imaging. Moreover, it also could sense different temperature change in living cells at 25 and 37 °C, respectively. This journal is © The Royal Society of Chemistry 2012

Hydrothermal synthesis of nitrogen-doped carbon dots with real-time live-cell imaging and blood-brain barrier penetration capabilities.

PubMed

Lu, Shousi; Guo, Shanshan; Xu, Pingxiang; Li, Xiaorong; Zhao, Yuming; Gu, Wei; Xue, Ming

Nitrogen-doped carbon dots (N-CDs) were synthesized using a one-pot hydrothermal treatment with citric acid in the presence of polyethylenimine. Transmission electron microscopy analysis revealed that the N-CDs were monodispersed and quasi-spherical with an average size of ~2.6 nm. Under ultraviolet irradiation the N-CDs emitted a strong blue luminescence with a quantum yield as high as 51%. Moreover, the N-CDs exhibited a negligible cytotoxicity and could be applied as efficient nanoprobes for real-time imaging of live cells. In addition, the ability of the N-CDs to cross the blood-brain barrier (BBB) in a concentration-dependent manner was demonstrated using an in vitro BBB model. Therefore, these PEI-passivated N-CDs with real-time live-cell imaging and BBB-penetration capabilities hold promise for traceable drug delivery to the brain.

Hydrothermal synthesis of nitrogen-doped carbon dots with real-time live-cell imaging and blood–brain barrier penetration capabilities

PubMed Central

Lu, Shousi; Guo, Shanshan; Xu, Pingxiang; Li, Xiaorong; Zhao, Yuming; Gu, Wei; Xue, Ming

2016-01-01

Nitrogen-doped carbon dots (N-CDs) were synthesized using a one-pot hydrothermal treatment with citric acid in the presence of polyethylenimine. Transmission electron microscopy analysis revealed that the N-CDs were monodispersed and quasi-spherical with an average size of ~2.6 nm. Under ultraviolet irradiation the N-CDs emitted a strong blue luminescence with a quantum yield as high as 51%. Moreover, the N-CDs exhibited a negligible cytotoxicity and could be applied as efficient nanoprobes for real-time imaging of live cells. In addition, the ability of the N-CDs to cross the blood–brain barrier (BBB) in a concentration-dependent manner was demonstrated using an in vitro BBB model. Therefore, these PEI-passivated N-CDs with real-time live-cell imaging and BBB-penetration capabilities hold promise for traceable drug delivery to the brain. PMID:27932880

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

NASA Astrophysics Data System (ADS)

Kano, Hideaki; Hamaguchi, Hiro-O.

2006-04-01

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

A single frame: imaging live cells twenty-five years ago.

PubMed

Fink, Rachel

2011-07-01

In the mid-1980s live-cell imaging was changed by the introduction of video techniques, allowing new ways to collect and store data. The increased resolution obtained by manipulating video signals, the ability to use time-lapse videocassette recorders to study events that happen over long time intervals, and the introduction of fluorescent probes and sensitive video cameras opened research avenues previously unavailable. The author gives a personal account of this evolution, focusing on cell migration studies at the Marine Biological Laboratory 25 years ago. Copyright © 2011 Wiley-Liss, Inc.

Deciphering the internal complexity of living cells with quantitative phase microscopy: a multiscale approach

NASA Astrophysics Data System (ADS)

Martinez-Torres, Cristina; Laperrousaz, Bastien; Berguiga, Lotfi; Boyer-Provera, Elise; Elezgaray, Juan; Nicolini, Franck E.; Maguer-Satta, Veronique; Arneodo, Alain; Argoul, Françoise

2015-09-01

The distribution of refractive indices (RIs) of a living cell contributes in a nonintuitive manner to its optical phase image and quite rarely can be inverted to recover its internal structure. The interpretation of the quantitative phase images of living cells remains a difficult task because (1) we still have very little knowledge on the impact of its internal macromolecular complexes on the local RI and (2) phase changes produced by light propagation through the sample are mixed with diffraction effects by the internal cell bodies. We propose to implement a two-dimensional wavelet-based contour chain detection method to distinguish internal boundaries based on their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are the morphological indicators suited for comparing cells of different origins and/or to follow their transformation in pathologic situations. We use this method to compare nonadherent blood cells from primary and laboratory culture origins and to assess the internal transformation of hematopoietic stem cells by the transduction of the BCR-ABL oncogene responsible for the chronic myelogenous leukemia.

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

Designed Proteins as Novel Imaging Reagents in Living Escherichia coli.

PubMed

Pratt, Susan E; Speltz, Elizabeth B; Mochrie, Simon G J; Regan, Lynne

2016-09-02

Fluorescence imaging is a powerful tool to study protein function in living cells. Here, we introduce a novel imaging strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the protein of interest (POI). Our method was based on a set of designed tetratricopeptide repeat affinity proteins (TRAPs) that specifically and reversibly interact with a short, extended peptide tag. We co-expressed the TRAPs fused to fluorescent proteins (FPs) and the peptide tags fused to the POIs. We illustrated the method using the Escherichia coli protein FtsZ and showed that our system could track distinct FtsZ structures under both low and high expression conditions in live cells. We anticipate that our imaging strategy will be a useful tool for imaging the subcellular localization of many proteins, especially those recalcitrant to imaging by direct tagging with FPs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Photoluminescent graphene quantum dots for in vivo imaging of apoptotic cells

NASA Astrophysics Data System (ADS)

Roy, Prathik; Periasamy, Arun Prakash; Lin, Chiu-Ya; Her, Guor-Mour; Chiu, Wei-Jane; Li, Chi-Lin; Shu, Chia-Lun; Huang, Chih-Ching; Liang, Chi-Te; Chang, Huan-Tsung

2015-01-01

Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL-1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications.Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL-1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications. Electronic supplementary information (ESI) available: Experimental discussion on synthesis, characterization, cellular imaging, cytotoxicity of GQDs in addition to its effect on zebrafish embryos, preparation of annexin V (A5)-modified GQDs (AbA5-GQDs), staining procedures and imaging are given. Figures for XRD, UV-vis absorption, photoluminescence of GQDs, mortality of zebrafish, time course recording of morphology of zebrafish embryos and morphology of adult zebrafish exposed to GQDs are illustrated. See DOI: 10.1039/c4nr07005d

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

PubMed Central

Lee, Aih Cheun; Decourt, Boris; Suter, Daniel

2008-01-01

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones. PMID:19066568

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.

Rapid 3D Refractive‐Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation

PubMed Central

Habaza, Mor; Kirschbaum, Michael; Guernth‐Marschner, Christian; Dardikman, Gili; Barnea, Itay; Korenstein, Rafi; Duschl, Claus

2016-01-01

A major challenge in the field of optical imaging of live cells is achieving rapid, 3D, and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved. A new label‐free tomographic interferometry approach is presented. This approach provides rapid capturing of the 3D refractive‐index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped, and then rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3D refractive‐index map. Uniquely, this approach provides full (360°) coverage of the rotation angular range around any axis, and knowledge on the viewing angle. The experimental demonstrations presented include 3D, label‐free imaging of cancer cells and three types of white blood cells. This approach is expected to be useful for label‐free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of specific cell types in blood or other body fluids. PMID:28251046

Quantitative phase microscopy for cellular dynamics based on transport of intensity equation.

PubMed

Li, Ying; Di, Jianglei; Ma, Chaojie; Zhang, Jiwei; Zhong, Jinzhan; Wang, Kaiqiang; Xi, Teli; Zhao, Jianlin

2018-01-08

We demonstrate a simple method for quantitative phase imaging of tiny transparent objects such as living cells based on the transport of intensity equation. The experiments are performed using an inverted bright field microscope upgraded with a flipping imaging module, which enables to simultaneously create two laterally separated images with unequal defocus distances. This add-on module does not include any lenses or gratings and is cost-effective and easy-to-alignment. The validity of this method is confirmed by the measurement of microlens array and human osteoblastic cells in culture, indicating its potential in the applications of dynamically measuring living cells and other transparent specimens in a quantitative, non-invasive and label-free manner.

Imaging in living cells using νB-H Raman spectroscopy: monitoring COSAN uptake.

PubMed

Tarrés, Màrius; Canetta, Elisabetta; Viñas, Clara; Teixidor, Francesc; Harwood, Adrian J

2014-03-28

The boron-rich cobaltabisdicarbollide (COSAN) and its 8,8'-I2 derivative (I2-COSAN), both of purely inorganic nature, are shown to accumulate within living cells, where they can be detected using νB-H Raman microspectroscopy. This demonstrates an alternative method for cell labelling and detection.

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.

A Checklist for Successful Quantitative Live Cell Imaging in Systems Biology

PubMed Central

Sung, Myong-Hee

2013-01-01

Mathematical modeling of signaling and gene regulatory networks has provided unique insights about systems behaviors for many cell biological problems of medical importance. Quantitative single cell monitoring has a crucial role in advancing systems modeling of molecular networks. However, due to the multidisciplinary techniques that are necessary for adaptation of such systems biology approaches, dissemination to a wide research community has been relatively slow. In this essay, I focus on some technical aspects that are often under-appreciated, yet critical in harnessing live cell imaging methods to achieve single-cell-level understanding and quantitative modeling of molecular networks. The importance of these technical considerations will be elaborated with examples of successes and shortcomings. Future efforts will benefit by avoiding some pitfalls and by utilizing the lessons collectively learned from recent applications of imaging in systems biology. PMID:24709701

Imaging transplanted stem cells in real time using an MRI dual-contrast method

PubMed Central

Ngen, Ethel J.; Wang, Lee; Kato, Yoshinori; Krishnamachary, Balaji; Zhu, Wenlian; Gandhi, Nishant; Smith, Barbara; Armour, Michael; Wong, John; Gabrielson, Kathleen; Artemov, Dmitri

2015-01-01

Stem cell therapies are currently being investigated for the repair of brain injuries. Although exogenous stem cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) prior to transplantation provides a means to noninvasively monitor stem cell transplantation by magnetic resonance imaging (MRI), monitoring cell death is still a challenge. Here, we investigate the feasibility of using an MRI dual-contrast technique to detect cell delivery, cell migration and cell death after stem cell transplantation. Human mesenchymal stem cells were dual labelled with SPIONs and gadolinium-based chelates (GdDTPA). The viability, proliferation rate, and differentiation potential of the labelled cells were then evaluated. The feasibility of this MRI technique to distinguish between live and dead cells was next evaluated using MRI phantoms, and in vivo using both immune-competent and immune-deficient mice, following the induction of brain injury in the mice. All results were validated with bioluminescence imaging. In live cells, a negative (T2/T2*) MRI contrast predominates, and is used to track cell delivery and cell migration. Upon cell death, a diffused positive (T1) MRI contrast is generated in the vicinity of the dead cells, and serves as an imaging marker for cell death. Ultimately, this technique could be used to manage stem cell therapies. PMID:26330231

Imaging transplanted stem cells in real time using an MRI dual-contrast method.

PubMed

Ngen, Ethel J; Wang, Lee; Kato, Yoshinori; Krishnamachary, Balaji; Zhu, Wenlian; Gandhi, Nishant; Smith, Barbara; Armour, Michael; Wong, John; Gabrielson, Kathleen; Artemov, Dmitri

2015-09-02

Stem cell therapies are currently being investigated for the repair of brain injuries. Although exogenous stem cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) prior to transplantation provides a means to noninvasively monitor stem cell transplantation by magnetic resonance imaging (MRI), monitoring cell death is still a challenge. Here, we investigate the feasibility of using an MRI dual-contrast technique to detect cell delivery, cell migration and cell death after stem cell transplantation. Human mesenchymal stem cells were dual labelled with SPIONs and gadolinium-based chelates (GdDTPA). The viability, proliferation rate, and differentiation potential of the labelled cells were then evaluated. The feasibility of this MRI technique to distinguish between live and dead cells was next evaluated using MRI phantoms, and in vivo using both immune-competent and immune-deficient mice, following the induction of brain injury in the mice. All results were validated with bioluminescence imaging. In live cells, a negative (T2/T2*) MRI contrast predominates, and is used to track cell delivery and cell migration. Upon cell death, a diffused positive (T1) MRI contrast is generated in the vicinity of the dead cells, and serves as an imaging marker for cell death. Ultimately, this technique could be used to manage stem cell therapies.

Hyaluronan functionalizing QDs as turn-on fluorescent probe for targeted recognition CD44 receptor

NASA Astrophysics Data System (ADS)

Zhou, Shang; Huo, Danqun; Hou, Changjun; Yang, Mei; Fa, Huanbao

2017-09-01

The recognition of tumor markers in living cancer cells has attracted increasing interest. In the present study, the turn-on fluorescence probe was designed based on the fluorescence of thiolated chitosan-coated CdTe QDs (CdTe/TCS QDs) quenched by hyaluronan, which could provide the low background signal for sensitive cellular imaging. This system is expected to offer specific recognition of CD44 receptor over other substances owing to the specific affinity of hyaluronan and CD44 receptor ( 8-9 kcal/mol). The probe is stable in aqueous and has little toxicity to living cells; thus, it can be utilized for targeted cancer cell imaging. The living lung cancer cell imaging experiments further demonstrate its value in recognizing cell-surface CD44 receptor with turn-on mode. In addition, the probe can be used to recognize and differentiate the subtypes of lung cancer cells based on the difference of CD44 expression on the surface of lung cancer cells. And, the western blot test further confirmed that the expression level of the CD44 receptor in lung cancer cells is different. Therefore, this probe may be potentially applied in recognizing lung cancer cells with higher contrast and sensitivity and provide new tools for cancer prognosis and therapy. [Figure not available: see fulltext.

Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein

NASA Astrophysics Data System (ADS)

Biteen, Julie S.; Thompson, Michael A.; Tselentis, Nicole K.; Shapiro, Lucy; Moerner, W. E.

2009-02-01

Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

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.

Advanced Image Acquisition and Analytical Techniques for Studies of Living Cells and Tissue Sections.

PubMed

Franek, Michal; Suchánková, Jana; Sehnalová, Petra; Krejčí, Jana; Legartová, Soňa; Kozubek, Stanislav; Večeřa, Josef; Sorokin, Dmitry V; Bártová, Eva

2016-04-01

Studies on fixed samples or genome-wide analyses of nuclear processes are useful for generating snapshots of a cell population at a particular time point. However, these experimental approaches do not provide information at the single-cell level. Genome-wide studies cannot assess variability between individual cells that are cultured in vitro or originate from different pathological stages. Immunohistochemistry and immunofluorescence are fundamental experimental approaches in clinical laboratories and are also widely used in basic research. However, the fixation procedure may generate artifacts and prevents monitoring of the dynamics of nuclear processes. Therefore, live-cell imaging is critical for studying the kinetics of basic nuclear events, such as DNA replication, transcription, splicing, and DNA repair. This review is focused on the advanced microscopy analyses of the cells, with a particular focus on live cells. We note some methodological innovations and new options for microscope systems that can also be used to study tissue sections. Cornerstone methods for the biophysical research of living cells, such as fluorescence recovery after photobleaching and fluorescence resonance energy transfer, are also discussed, as are studies on the effects of radiation at the individual cellular level.

Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy

PubMed Central

Yan, Yuling; Petchprayoon, Chutima; Mao, Shu; Marriott, Gerard

2013-01-01

Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens. PMID:23267183

Culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy

PubMed Central

Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

2017-01-01

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina i.e. they migrate between the basal inner nuclear layer (INL) and the outer nuclear layer (ONL), respectively, in a process described as interkinetic nuclear migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM. PMID:28287581

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy.

PubMed

Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

2017-02-24

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP] mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Phase contrast microscopy of living cells within the whole lens: spatial correlations and morphological dynamics

PubMed Central

Kong, Zhiying; Zhu, Xiangjia; Zhang, Shenghai; Wu, Jihong

2012-01-01

Purpose Images from cultured lens cells do not convey enough spatial information, and imaging of fixed lens specimens cannot reveal dynamic changes in the cells. As such, a real-time, convenient approach for monitoring label-free imaging of dynamic processes of living cells within the whole lens is urgently needed. Methods Female Wistar rat lenses were kept in organ culture. Insulin-like growth factor-I was added to the culture medium to induce cell mitosis. A novel method of ultraviolet (UV) irradiation was used to induce cell apoptosis and fiber damage. The cellular morphological dynamics within the whole lens were monitored by inverted phase contrast microscopy. Apoptosis was assessed using a commercial kit with Hoechst 33342/YO-PRO®-1/propidium iodide (PI). Results The intrinsic transparency and low-light scattering property of the rat lens permitted direct imaging of the lens epithelial cells (LECs) and the superficial fiber cells. We visualized the processes of mitosis and apoptosis of the LECs, and we obtained dynamic images of posterior fiber cells following UVA irradiation. Conclusions This method opens a new window for observing lens cells in their physiologic location, and it can be readily applied in studies on lens physiology and pathology. PMID:22879736

Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters.

PubMed

Maryu, Gembu; Miura, Haruko; Uda, Youichi; Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

2018-04-25

Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions. Several genetically encoded kinase activity reporters, which are based on the modes of action of kinase activation and phosphorylation, are currently available. These reporters are classified into single-fluorophore kinase activity reporters and Förster (or fluorescence) resonance energy transfer (FRET)-based kinase activity reporters. Here, we introduce the principles of genetically encoded kinase activity reporters, and discuss the advantages and disadvantages of these reporters.Key words: kinase, FRET, phosphorylation, KTR.

Measuring spatial and temporal Ca2+ signals in Arabidopsis plants.

PubMed

Zhu, Xiaohong; Taylor, Aaron; Zhang, Shenyu; Zhang, Dayong; Feng, Ying; Liang, Gaimei; Zhu, Jian-Kang

2014-09-02

Developmental and environmental cues induce Ca(2+) fluctuations in plant cells. Stimulus-specific spatial-temporal Ca(2+) patterns are sensed by cellular Ca(2+) binding proteins that initiate Ca(2+) signaling cascades. However, we still know little about how stimulus specific Ca(2+) signals are generated. The specificity of a Ca(2+) signal may be attributed to the sophisticated regulation of the activities of Ca(2+) channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca(2+) signals at both the tissue and cellular levels. Genetically encoded Ca(2+) indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca(2+) signals. Here we describe instructions for the use of two Ca(2+) detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca(2+) imaging and case12 based live cell confocal fluorescence Ca(2+) imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca(2+) signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca(2+) signals at a high resolution.

An authentic imaging probe to track cell fate from beginning to end.

PubMed

Lee, Seung Koo; Mortensen, Luke J; Lin, Charles P; Tung, Ching-Hsuan

2014-10-17

Accurate tracing of cell viability is critical for optimizing delivery methods and evaluating the efficacy and safety of cell therapeutics. A nanoparticle-based cell tracker is developed to image cell fate from live to dead. The particle is fabricated from two types of optically quenched polyelectrolytes, a life indicator and a death indicator, through electrostatic interactions. On incubation with cells, the fabricated bifunctional nanoprobes are taken up efficiently and the first colour is produced by normal intracellular proteolysis, reflecting the healthy status of the cells. Depending on the number of coated layers, the signal can persist for several replication cycles. However, as the cells begin dying, the second colour appears quickly to reflect the new cell status. Using this chameleon-like cell tracker, live cells can be distinguished from apoptotic and necrotic cells instantly and definitively.

Halo-free Phase Contrast Microscopy

NASA Astrophysics Data System (ADS)

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

2017-03-01

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. Acquiring 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.

Live imaging reveals the progenitors and cell dynamics of limb regeneration

PubMed Central

Alwes, Frederike; Enjolras, Camille; Averof, Michalis

2016-01-01

Regeneration is a complex and dynamic process, mobilizing diverse cell types and remodelling tissues over long time periods. Tracking cell fate and behaviour during regeneration in active adult animals is especially challenging. Here, we establish continuous live imaging of leg regeneration at single-cell resolution in the crustacean Parhyale hawaiensis. By live recordings encompassing the first 4-5 days after amputation, we capture the cellular events that contribute to wound closure and morphogenesis of regenerating legs with unprecedented resolution and temporal detail. Using these recordings we are able to track cell lineages, to generate fate maps of the blastema and to identify the progenitors of regenerated epidermis. We find that there are no specialized stem cells for the epidermis. Most epidermal cells in the distal part of the leg stump proliferate, acquire new positional values and contribute to new segments in the regenerating leg. DOI: http://dx.doi.org/10.7554/eLife.19766.001 PMID:27776632

Imaging the Effects of Prostaglandin Analogues on Cultured Trabecular Meshwork Cells by Coherent Anti-Stokes Raman Scattering

PubMed Central

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

2013-01-01

Purpose. The aim of this study was to nondestructively monitor morphological changes to the lipid membranes of primary cultures of living human trabecular meshwork cells (hTMC) without the application of exogenous label. Methods. Live hTMC were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). The hTMC were treated with a commercial formulation of latanoprost (0.5 μg/mL) for 24 hours before imaging. Untreated cells and cells treated with vehicle containing the preservative benzalkonium chloride (BAK; 2 μg/mL) were imaged as controls. After CARS/TPAF imaging, hTMC were fixed, stained with the fluorescent lipid dye Nile Red, and imaged by conventional confocal microscopy to verify lipid membrane structures. Results. Analysis of CARS/TPAF images of hTMC treated with latanoprost revealed multiple intracellular lipid membranes absent from untreated or BAK-treated hTMC. Treatment of hTMC with sodium fluoride or ouabain, agents shown to cause morphological changes to hTMC, also did not induce formation of intracellular lipid membranes. Conclusions. CARS microscopy detected changes in living hTMC morphology that were validated by subsequent histological stain. Prostaglandin-induced changes to hTMC involved rearrangement of lipid membranes within these cells. These in vitro results identify a novel biological response to a class of antiglaucoma drugs, and further experiments are needed to establish how this effect is involved in the hypotensive action of prostaglandin analogues in vivo. PMID:23900606

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.

Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.

PubMed

Mahen, Robert; Koch, Birgit; Wachsmuth, Malte; Politi, Antonio Z; Perez-Gonzalez, Alexis; Mergenthaler, Julia; Cai, Yin; Ellenberg, Jan

2014-11-05

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells. © 2014 Mahen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Long term imaging of living brain cancer cells

NASA Astrophysics Data System (ADS)

Farias, Patricia M. A.; Galembeck, André; Milani, Raquel; Andrade, Arnaldo C. D. S.; Stingl, Andreas

2018-02-01

QDs synthesized in aqueous medium and functionalized with polyethylene glycol were used as fluorescent probes. They label and monitor living healthy and cancer brain glial cells in culture. Physical-chemical characterization was performed. Toxicological studies were performed by in vivo short and long-term inhalation in animal models. Healthy and cancer glial living cells were incubated in culture media with highly controlled QDs. Specific features of glial cancer cells were enhanced by QD labelling. Cytoplasmic labelling pattern was clearly distinct for healthy and cancer cells. Labelled cells kept their normal activity for same period as non-labelled control samples.

Real-time monitoring of endogenous cysteine levels in living cells using a CD-based ratiometric fluorescent nanoprobe.

PubMed

Wang, Hong; Zhang, Peisheng; Tian, Yong; Zhang, Yuan; Yang, Heping; Chen, Shu; Zeng, Rongjin; Long, Yunfei; Chen, Jian

2018-04-30

A simple and readily available fluorescent probe is needed for the real-time monitoring of endogenous cysteine (Cys) levels in living cells, as such a probe could be used to study the role of Cys in related diseases. Herein, we report the first fluorescent probe based on carbon dots (CDs-FITA) for the selective and ratiometric imaging of endogenous Cys in live cells. In this ratiometric fluorescent probe, a fluorescein derivative (FITA) that recognizes Cys is covalently linked to the surfaces of carbon dots (CDs); employing CDs greatly improves the water solubility of the probe. Acrylate on FITA is selectively cleaved by Cys in aqueous solution under mild conditions, leading to a dramatic increase in the fluorescence from fluorescein. The probe therefore allows the highly selective ratiometric fluorescent detection of Cys even in the presence of various interferents. The as-prepared CDs-FITA showed excellent performance when applied to detect Cys in blood serum. In addition, due to its negligible cytotoxicity, the CDs-FITA can also be utilized for the real-time monitoring of endogenous cysteine (Cys) levels in living cells. Graphical abstract Illustration of the CD-based probe for Cys imaging in living cells.

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.

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

Influence of sample preparation and identification of subcelluar structures in quantitative holographic phase contrast microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Schmidt, Lisa; Przibilla, Sabine; Rommel, Christina; Vollmer, Angelika; Ketelhut, Steffi; Schnekenburger, Jürgen; von Bally, Gert

2010-04-01

Digital holographic microscopy (DHM) provides label-free quantitative phase contrast with low demands on sample preparation. Nevertheless, for DHM measurements on fixed cells the mounting medium has to be considered while the phase contrast of living cells may be influenced by the used buffer solution. To quantify these effects, the maximum cell caused phase contrast and the visibility of the nucleoli were analyzed. A second aim of the study was to identify subcellular components in DHM phase contrast images. Therefore, comparative investigations using bright field imaging, DHM and fluorescence microscopy with 4',6- Diamidino-2-phenylindol (DAPI) staining were performed. DAPI-staining visualizes cell components containing DNA. The obtained results demonstrate exemplarily for two tumor cell lines that from DHM phase contrast images of fixed cells in phosphate buffer saline (PBS) cell thickness values are obtained which are comparable to living cells. Furthermore, it is shown that in many cases nucleus components can be identified only by DHM phase contrast.

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.

A precise pointing nanopipette for single-cell imaging via electroosmotic injection.

PubMed

Lv, Jian; Qian, Ruo-Can; Hu, Yong-Xu; Liu, Shao-Chuang; Cao, Yue; Zheng, Yong-Jie; Long, Yi-Tao

2016-11-24

The precise transportation of fluorescent probes to the designated location in living cells is still a challenge. Here, we present a new addition to nanopipettes as a powerful tool to deliver fluorescent molecules to a given place in a single cell by electroosmotic flow, indicating favorable potential for further application in single-cell imaging.

Mathematical imaging methods for mitosis analysis in live-cell phase contrast microscopy.

PubMed

Grah, Joana Sarah; Harrington, Jennifer Alison; Koh, Siang Boon; Pike, Jeremy Andrew; Schreiner, Alexander; Burger, Martin; Schönlieb, Carola-Bibiane; Reichelt, Stefanie

2017-02-15

In this paper we propose a workflow to detect and track mitotic cells in time-lapse microscopy image sequences. In order to avoid the requirement for cell lines expressing fluorescent markers and the associated phototoxicity, phase contrast microscopy is often preferred over fluorescence microscopy in live-cell imaging. However, common specific image characteristics complicate image processing and impede use of standard methods. Nevertheless, automated analysis is desirable due to manual analysis being subjective, biased and extremely time-consuming for large data sets. Here, we present the following workflow based on mathematical imaging methods. In the first step, mitosis detection is performed by means of the circular Hough transform. The obtained circular contour subsequently serves as an initialisation for the tracking algorithm based on variational methods. It is sub-divided into two parts: in order to determine the beginning of the whole mitosis cycle, a backwards tracking procedure is performed. After that, the cell is tracked forwards in time until the end of mitosis. As a result, the average of mitosis duration and ratios of different cell fates (cell death, no division, division into two or more daughter cells) can be measured and statistics on cell morphologies can be obtained. All of the tools are featured in the user-friendly MATLAB®Graphical User Interface MitosisAnalyser. Copyright © 2017. Published by Elsevier Inc.

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

Quantitative phase imaging of living cells with a swept laser source

NASA Astrophysics Data System (ADS)

Chen, Shichao; Zhu, Yizheng

2016-03-01

Digital holographic phase microscopy is a well-established quantitative phase imaging technique. However, interference artifacts from inside the system, typically induced by elements whose optical thickness are within the source coherence length, limit the imaging quality as well as sensitivity. In this paper, a swept laser source based technique is presented. Spectra acquired at a number of wavelengths, after Fourier Transform, can be used to identify the sources of the interference artifacts. With proper tuning of the optical pathlength difference between sample and reference arms, it is possible to avoid these artifacts and achieve sensitivity below 0.3nm. Performance of the proposed technique is examined in live cell imaging.

Cytosolic delivery of materials with endosome-disrupting colloids

DOEpatents

Helms, Brett A.; Bayles, Andrea R.

2016-03-15

A facile procedure to deliver nanocrystals to the cytosol of live cells that is both rapid and general. The technique employs a unique cationic core-shell polymer colloid that directs nanocrystals to the cytosol of living cells within a few hours of incubation. The present methods and compositions enable a host of advanced applications arising from efficient cytosolic delivery of nanocrystal imaging probes: from single particle tracking experiments to monitoring protein-protein interactions in live cells for extended periods.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.

PubMed

Gómez-Villafuertes, Rosa; Paniagua-Herranz, Lucía; Gascon, Sergio; de Agustín-Durán, David; Ferreras, María de la O; Gil-Redondo, Juan Carlos; Queipo, María José; Menendez-Mendez, Aida; Pérez-Sen, Ráquel; Delicado, Esmerilda G; Gualix, Javier; Costa, Marcos R; Schroeder, Timm; Miras-Portugal, María Teresa; Ortega, Felipe

2017-12-16

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

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

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.

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

Practical three color live cell imaging by widefield microscopy

PubMed Central

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

2006-01-01

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

Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2011-04-13

The intracellular uptake of 30 nm diameter gold nanoparticles (Au-NPs) was studied at the nanoscale in pristine eukaryotic cells. Live COS-7 cells were maintained in a microfluidic chamber and imaged using scanning transmission electron microscopy. A quantitative image analysis showed that Au-NPs bound to the membranes of vesicles, possibly lysosomes, and occupied 67% of the available surface area. The vesicles accumulated to form a micrometer-sized cluster after 24 h of incubation. Two clusters were analyzed and found to consist of 117 ± 9 and 164 ± 4 NP-filled vesicles.

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.

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.

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.

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).

Techniques for 3D tracking of single molecules with nanometer accuracy in living cells

NASA Astrophysics Data System (ADS)

Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S.

2013-06-01

We describe a microscopy technique that, combining wide-field single molecule microscopy, bifocal imaging and Highly Inclined and Laminated Optical sheet (HILO) microscopy, allows a 3D tracking with nanometer accuracy of single fluorescent molecules in vitro and in living cells.

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.

cGMP Signaling in the Cardiovascular System—The Role of Compartmentation and Its Live Cell Imaging

PubMed Central

Bork, Nadja I.; Nikolaev, Viacheslav O.

2018-01-01

The ubiquitous second messenger 3′,5′-cyclic guanosine monophosphate (cGMP) regulates multiple physiologic processes in the cardiovascular system. Its intracellular effects are mediated by stringently controlled subcellular microdomains. In this review, we will illustrate the current techniques available for real-time cGMP measurements with a specific focus on live cell imaging methods. We will also discuss currently accepted and emerging mechanisms of cGMP compartmentation in the cardiovascular system. PMID:29534460

Steady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane

PubMed Central

Devauges, Viviane; Matthews, Daniel R.; Aluko, Justin; Nedbal, Jakub; Levitt, James A.; Poland, Simon P.; Coban, Oana; Weitsman, Gregory; Monypenny, James; Ng, Tony; Ameer-Beg, Simon M.

2014-01-01

We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor. PMID:25360776

Live Imaging of LysoTracker-Labelled Phagolysosomes Tracks Diurnal Phagocytosis of Photoreceptor Outer Segment Fragments in Rat RPE Tissue Ex Vivo.

PubMed

Mao, Yingyu; Finnemann, Silvia C

2016-01-01

Renewal of rod photoreceptor outer segments in the mammalian eye involves synchronized diurnal shedding after light onset of spent distal outer segment fragments (POS) linked to swift clearance of shed POS from the subretinal space by the adjacent retinal pigment epithelium (RPE). Engulfed POS phagosomes in RPE cells mature to acidified phagolysosomes, which accomplish enzymatic degradation of POS macromolecules. Here, we used an acidophilic fluorophore LysoTracker to label acidic organelles in freshly dissected, live rat RPE tissue flat mounts. We observed that all RPE cells imaged contained numerous acidified POS phagolysosomes whose abundance per cell was dramatically increased 2 h after light onset as compared to either 1 h before or 4 h after light onset. Lack of organelles of similar diameter (of 1-2 μm) in phagocytosis-defective mutant RCS rat RPE confirmed that LysoTracker live imaging detected POS phagolysosomes. Lack of increase in lysosomal membrane protein LAMP-1 in RPE/choroid during the diurnal phagocytic burst suggests that formation of POS phagolysosomes in RPE in situ may not involve extra lysosome membrane biogenesis. Taken together, we report a new imaging approach that directly detects POS phagosome acidification and allows rapid tracking and quantification of POS phagocytosis by live RPE -tissue ex situ.

Subcellular analysis of interaction between breast cancer cells and drug by digital holography

NASA Astrophysics Data System (ADS)

Zhao, Jie; Lin, Qiaowen; Wang, Dayong; Wang, Yunxin; Ouyang, Liting; Guo, Sha; Yao, Qian

2017-10-01

Digital holographic microscopy is a promising quantitative phase-contrast imaging technique, which exhibits the advantages of non-destruction, full field of view, quasi-real time, and don't need dye and external marker to the living biological sample. In this paper, the inverted off-axis image-plane digital holography with pre-magnification is built up to study the living MDA-MB-231 breast cancer cells. The lateral resolution of the proposed experimental setup is 0.87μm, which is verified by the standard USAF test target. Then the system is used to visualize the interaction between living breast cancer cells and drug. The blebbing is observed after the cells are treated by paclitaxel drug, and the distribution of the paclitaxel inside the cells is detected, which is near the cytomembrane, or in other words the end of the microtubules. It will stop the mitosis and cause the death of the cells. It is helpful to reveal the anticancer mechanism of paclitaxel in the subcellular scale.

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 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.

Versatile quantitative phase imaging system applied to high-speed, low noise and multimodal imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Federici, Antoine; Aknoun, Sherazade; Savatier, Julien; Wattellier, Benoit F.

2017-02-01

Quadriwave lateral shearing interferometry (QWLSI) is a well-established quantitative phase imaging (QPI) technique based on the analysis of interference patterns of four diffraction orders by an optical grating set in front of an array detector [1]. As a QPI modality, this is a non-invasive imaging technique which allow to measure the optical path difference (OPD) of semi-transparent samples. We present a system enabling QWLSI with high-performance sCMOS cameras [2] and apply it to perform high-speed imaging, low noise as well as multimodal imaging. This modified QWLSI system contains a versatile optomechanical device which images the optical grating near the detector plane. Such a device is coupled with any kind of camera by varying its magnification. In this paper, we study the use of a sCMOS Zyla5.5 camera from Andor along with our modified QWLSI system. We will present high-speed live cell imaging, up to 200Hz frame rate, in order to follow intracellular fast motions while measuring the quantitative phase information. The structural and density information extracted from the OPD signal is complementary to the specific and localized fluorescence signal [2]. In addition, QPI detects cells even when the fluorophore is not expressed. This is very useful to follow a protein expression with time. The 10 µm spatial pixel resolution of our modified QWLSI associated to the high sensitivity of the Zyla5.5 enabling to perform high quality fluorescence imaging, we have carried out multimodal imaging revealing fine structures cells, like actin filaments, merged with the morphological information of the phase. References [1]. P. Bon, G. Maucort, B. Wattellier, and S. Monneret, "Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells," Opt. Express, vol. 17, pp. 13080-13094, 2009. [2] P. Bon, S. Lécart, E. Fort and S. Lévêque-Fort, "Fast label-free cytoskeletal network imaging in living mammalian cells," Biophysical journal, 106(8), pp. 1588-1595, 2014

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.

Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis

PubMed Central

Maes, Margaret E.; Schlamp, Cassandra L.

2017-01-01

The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies. PMID:28880942

Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis.

PubMed

Maes, Margaret E; Schlamp, Cassandra L; Nickells, Robert W

2017-01-01

The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells.

PubMed

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2013-01-01

Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50-60 nm on a time scale of 2.3 s. Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells

PubMed Central

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2016-01-01

Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level. PMID:27795878

Application of novel low-intensity nonscanning fluorescence lifetime imaging microscopy for monitoring excited state dynamics in individual chloroplasts and living cells of photosynthetic organisms

NASA Astrophysics Data System (ADS)

Eckert, Hann-Jörg; Petrášek, Zdeněk; Kemnitz, Klaus

2006-10-01

Picosecond fluorescence lifetime imaging microscopy (FLIM) provides a most valuable tool to analyze the primary processes of photosynthesis in individual cells and chloroplasts of living cells. In order to obtain correct lifetimes of the excited states, the peak intensity of the exciting laser pulses as well as the average intensity has to be sufficiently low to avoid distortions of the kinetics by processes such as singlet-singlet annihilation, closing of the reaction centers or photoinhibition. In the present study this requirement is achieved by non-scanning wide-field FLIM based on time- and space-correlated single-photon counting (TSCSPC) using a novel microchannel plate photomultiplier with quadrant anode (QA-MCP) that allows parallel acquisition of time-resolved images under minimally invasive low-excitation conditions. The potential of the wide-field TCSPC method is demonstrated by presenting results obtained from measurements of the fluorescence dynamics in individual chloroplasts of moss leaves and living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.

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

Live cell imaging reveals marked variability in myoblast proliferation and fate

PubMed Central

2013-01-01

Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

Super-Resolution Imaging of the Golgi in Live Cells with a Bio-orthogonal Ceramide Probe**

PubMed Central

Erdmann, Roman S.; Takakura, Hideo; Thompson, Alexander D.; Rivera-Molina, Felix; Allgeyer, Edward S.; Bewersdorf, Joerg; Toomre, Derek K.; Schepartz, Alanna

2014-01-01

We report a lipid-based strategy to visualize Golgi structure and dynamics at super-resolution in live cells. The method is based on two novel reagents: a trans-cyclooctene-containing ceramide lipid (Cer-TCO) and a highly reactive, tetrazine-tagged near-IR dye (SiR-Tz). These reagents assemble via an extremely rapid ‘tetrazine-click’ reaction into Cer-SiR, a highly photostable ‘vital dye’ that enables prolonged live cell imaging of the Golgi apparatus by 3D confocal and STED microscopy. Cer-SiR is non-toxic at concentrations as high as 2 μM and does not perturb the mobility of Golgi-resident enzymes or the traffic of cargo from the endoplasmic reticulum through the Golgi and to the plasma membrane. PMID:25081303

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

Mitochondrial imaging in live or fixed tissues using a luminescent iridium complex.

PubMed

Sorvina, Alexandra; Bader, Christie A; Darby, Jack R T; Lock, Mitchell C; Soo, Jia Yin; Johnson, Ian R D; Caporale, Chiara; Voelcker, Nicolas H; Stagni, Stefano; Massi, Massimiliano; Morrison, Janna L; Plush, Sally E; Brooks, Douglas A

2018-05-29

Mitochondrial morphology is important for the function of this critical organelle and, accordingly, altered mitochondrial structure is exhibited in many pathologies. Imaging of mitochondria can therefore provide important information about disease presence and progression. However, mitochondrial imaging is currently limited by the availability of agents that have the capacity to image mitochondrial morphology in both live and fixed samples. This can be particularly problematic in clinical studies or large, multi-centre cohort studies, where tissue archiving by fixation is often more practical. We previously reported the synthesis of an iridium coordination complex [Ir(ppy) 2 (MeTzPyPhCN)] + ; where ppy is a cyclometalated 2-phenylpyridine and TzPyPhCN is the 5-(5-(4-cyanophen-1-yl)pyrid-2-yl)tetrazolate ligand; and showed that this complex (herein referred to as IraZolve-Mito) has a high specificity for mitochondria in live cells. Here we demonstrate that IraZolve-Mito can also effectively stain mitochondria in both live and fixed tissue samples. The staining protocol proposed is versatile, providing a universal procedure for cell biologists and pathologists to visualise mitochondria.

Live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix.

PubMed

Shih, Wenting; Yamada, Soichiro

2011-12-22

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network. By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching.

PubMed Central

Sund, S E; Axelrod, D

2000-01-01

Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about relevant chemical kinetic rates in vivo. Total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP), an established technique previously demonstrated to measure reversible biomolecular kinetic rates at surfaces in vitro, is extended here to measure reversible biomolecular kinetic rates of actin at the cytofacial (subplasma membrane) surface of living cells. For the first time, spatial imaging (with a charge-coupled device camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging produces both spatial maps of kinetic parameters (off-rates and mobile fractions) and estimates of kinetic correlation distances, cell-wide kinetic gradients, and dependences of kinetic parameters on initial fluorescence intensity. For microinjected rhodamine actin in living cultured smooth muscle (BC3H1) cells, the unbinding rate at or near the cytofacial surface of the plasma membrane (averaged over the entire cell) is measured at 0.032 +/- 0.007 s(-1). The corresponding rate for actin marked by microinjected rhodamine phalloidin is very similar, 0.033 +/- 0.013 s(-1), suggesting that TIR/FRAP is reporting the dynamics of entire filaments or protofilaments. For submembrane fluorescence-marked actin, the intensity, off-rate, and mobile fraction show a positive correlation over a characteristic distance of 1-3 microm and a negative correlation over larger distances greater than approximately 7-14 microm. Furthermore, the kinetic parameters display a statistically significant cell-wide gradient, with the cell having a "fast" and "slow" end with respect to actin kinetics. PMID:10969025

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.

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.

Tracking single mRNA molecules in live cells

NASA Astrophysics Data System (ADS)

Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

2016-06-01

mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

Communication between filamentous pathogens and plants at the biotrophic interface.

PubMed

Yi, Mihwa; Valent, Barbara

2013-01-01

Fungi and oomycetes that colonize living plant tissue form extensive interfaces with plant cells in which the cytoplasm of the microorganism is closely aligned with the host cytoplasm for an extended distance. In all cases, specialized biotrophic hyphae function to hijack host cellular processes across an interfacial zone consisting of a hyphal plasma membrane, a specialized interfacial matrix, and a plant-derived membrane. The interface is the site of active secretion by both players. This cross talk at the interface determines the winner in adversarial relationships and establishes the partnership in mutualistic relationships. Fungi and oomycetes secrete many specialized effector proteins for controlling the host, and they can stimulate remarkable cellular reorganization even in distant plant cells. Breakthroughs in live-cell imaging of fungal and oomycete encounter sites, including live-cell imaging of pathogens secreting fluorescently labeled effector proteins, have led to recent progress in understanding communication across the interface.

Effect of probe diffusion on the SOFI imaging accuracy.

PubMed

Vandenberg, Wim; Dedecker, Peter

2017-03-23

Live-cell super-resolution fluorescence imaging is becoming commonplace for exploring biological systems, though sample dynamics can affect the imaging quality. In this work we evaluate the effect of probe diffusion on super-resolution optical fluctuation imaging (SOFI), using a theoretical model and numerical simulations based on the imaging of live cells labelled with photochromic fluorescent proteins. We find that, over a range of physiological conditions, fluorophore diffusion results in a change in the amplitude of the SOFI signal. The magnitude of this change is approximately proportional to the on-time ratio of the fluorophores. However, for photochromic fluorescent proteins this effect is unlikely to present a significant distortion in practical experiments in biological systems. Due to this lack of distortions, probe diffusion strongly enhances the SOFI imaging by avoiding spatial undersampling caused by the limited labeling density.

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

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

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.

Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye.

PubMed

Sharma, Robin; Williams, David R; Palczewska, Grazyna; Palczewski, Krzysztof; Hunter, Jennifer J

2016-02-01

Although extrinsic fluorophores can be introduced to label specific cell types in the retina, endogenous fluorophores, such as NAD(P)H, FAD, collagen, and others, are present in all retinal layers. These molecules are a potential source of optical contrast and can enable noninvasive visualization of all cellular layers. We used a two-photon fluorescence adaptive optics scanning light ophthalmoscope (TPF-AOSLO) to explore the native autofluorescence of various cell classes spanning several layers in the unlabeled retina of a living primate eye. Three macaques were imaged on separate occasions using a custom TPF-AOSLO. Two-photon fluorescence was evoked by pulsed light at 730 and 920 nm excitation wavelengths, while fluorescence emission was collected in the visible range from several retinal layers and different locations. Backscattered light was recorded simultaneously in confocal modality and images were postprocessed to remove eye motion. All retinal layers yielded two-photon signals and the heterogeneous distribution of fluorophores provided optical contrast. Several structural features were observed, such as autofluorescence from vessel walls, Müller cell processes in the nerve fibers, mosaics of cells in the ganglion cell and other nuclear layers of the inner retina, as well as photoreceptor and RPE layers in the outer retina. This in vivo survey of two-photon autofluorescence throughout the primate retina demonstrates a wider variety of structural detail in the living eye than is available through conventional imaging methods, and broadens the use of two-photon imaging of normal and diseased eyes.

Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells.

PubMed

Nygate, Yoav N; Singh, Gyanendra; Barnea, Itay; Shaked, Natan T

2018-06-01

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

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.

Raman microscopy of individual living human embryonic stem cells

NASA Astrophysics Data System (ADS)

Novikov, S. M.; Beermann, J.; Bozhevolnyi, S. I.; Harkness, L. M.; Kassem, M.

2010-04-01

We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength.

Ultrasound Imaging of DNA-Damage Effects in Live Cultured Cells and in Brain Tissue.

PubMed

Tadayyon, Hadi; Gangeh, Mehrdad J; Vlad, Roxana; Kolios, Michael C; Czarnota, Gregory J

2017-01-01

High-frequency ultrasound (>20 MHz) spectroscopy can be used to detect noninvasively DNA damage in cell samples in vitro, and in live tissue both ex vivo and in vivo. This chapter focuses on the former two aspects. Experimental evidence suggests that morphological changes that occur in cells undergoing apoptosis result in changes in frequency-dependent ultrasound backscatter. With advances in research, ultrasound spectroscopy is advancing the boundaries of fast, label-free, noninvasive DNA damage detection technology with potential use in personalized medicine and early therapy response monitoring. Depending on the desired resolution, parametric ultrasound images can be computed and displayed within minutes to hours after ultrasound examination for cell death.

Trans-cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells.

PubMed

Chmyrov, Volodymyr; Spielmann, Thiemo; Hevekerl, Heike; Widengren, Jerker

2015-06-02

Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540), were first analyzed by fluorescence correlation spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics of MC540 in lipid vesicles could then also be monitored, and the influence of lipid polarity, membrane curvature, and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photoinduced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. On the basis of these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics and that the TRAST imaging technique can provide spatial maps of photoinduced isomerization as well as both photoinduced and thermal back-isomerization, resolving differences in local membrane microviscosity in live cells.

Real-time Raman and SRS imaging of living human macrophages reveals cell-to-cell heterogeneity and dynamics of lipid uptake.

PubMed

Stiebing, Clara; Meyer, Tobias; Rimke, Ingo; Matthäus, Christian; Schmitt, Michael; Lorkowski, Stefan; Popp, Jürgen

2017-09-01

Monitoring living cells in real-time is important in order to unravel complex dynamic processes in life sciences. In particular the dynamics of initiation and progression of degenerative diseases is intensely studied. In atherosclerosis the thickening of arterial walls is related to high lipid levels in the blood stream, which trigger the lipid uptake and formation of droplets as neutral lipid reservoirs in macrophages in the arterial wall. Unregulated lipid uptake finally results in foam cell formation, which is a hallmark of atherosclerosis. In previous studies, the uptake and storage of different fatty acids was monitored by measuring fixed cells. Commonly employed fluorescence staining protocols are often error prone because of cytotoxicity and unspecific fluorescence backgrounds. By following living cells with Raman spectroscopic imaging, lipid uptake of macrophages was studied with real-time data acquisition. Isotopic labeling using deuterated palmitic acid has been combined with spontaneous and stimulated Raman imaging to investigate the dynamic process of fatty acid storage in human macrophages for incubation times from 45 min to 37 h. Striking heterogeneity in the uptake rate and the total concentration of deuterated palmitic acid covering two orders of magnitude is detected in single as well as ensembles of cultured human macrophages. SRS signal of deuterated palmitic acid measured at the CD vibration band after incorporation into living macrophages. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Study of living single cells in culture: automated recognition of cell behavior.

PubMed

Bodin, P; Papin, S; Meyer, C; Travo, P

1988-07-01

An automated system capable of analyzing the behavior, in real time, of single living cells in culture, in a noninvasive and nondestructive way, has been developed. A large number of cell positions in single culture dishes were recorded using a computer controlled, robotized microscope. During subsequent observations, binary images obtained from video image analysis of the microscope visual field allowed the identification of the recorded cells. These cells could be revisited automatically every few minutes. Long-term studies of the behavior of cells make possible the analysis of cellular locomotary and mitotic activities as well as determination of cell shape (chosen from a defined library) for several hours or days in a fully automated way with observations spaced up to 30 minutes. Short-term studies of the behavior of cells permit the study, in a semiautomatic way, of acute effects of drugs (5 to 15 minutes) on changes of surface area and length of cells.

Detecting ordered small molecule drug aggregates in live macrophages: a multi-parameter microscope image data acquisition and analysis strategy

PubMed Central

Rzeczycki, Phillip; Yoon, Gi Sang; Keswani, Rahul K.; Sud, Sudha; Stringer, Kathleen A.; Rosania, Gus R.

2017-01-01

Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multi-parameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDA-approved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals. PMID:28270989

Detecting ordered small molecule drug aggregates in live macrophages: a multi-parameter microscope image data acquisition and analysis strategy.

PubMed

Rzeczycki, Phillip; Yoon, Gi Sang; Keswani, Rahul K; Sud, Sudha; Stringer, Kathleen A; Rosania, Gus R

2017-02-01

Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multi-parameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDA-approved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals.

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.

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.

Ratiometric Fluorescence Azide-Alkyne Cycloaddition for Live Mammalian Cell Imaging.

PubMed

Fu, Hongxia; Li, Yanru; Sun, Lingbo; He, Pan; Duan, Xinrui

2015-11-17

Click chemistry with metabolic labeling has been widely used for selectively imaging biomacromolecules in cells. The first example of azide-alkyne cycloaddition for ratiometric fluorescent imaging of live cells is reported. The precursor of the azido fluorophore (cresyl violet) has a fluorescence emission peak at 620 nm. The electron-rich nitrogen of the azido group blue-shifts the emission peak to 566 nm. When the click reaction occurs, an emission peak appears at 620 nm due to the lower electronic density of the newly formed triazole ring, which allows us to ratiometrically record fluorescence signals. This emission shift was applied to ratiometric imaging of propargylcholine- and dibenzocyclooctyne-labeled human breast cancer cells MCF-7 under laser confocal microscopy. Two typical triazole compounds were isolated for photophysical parameter measurements. The emission spectra presented a fluorescence emission peak around 620 nm for both click products. The results further confirmed the emission wavelength change was the result of azide-alkyne cycloaddition reaction. Since nearly all biomolecules can be metabolically labeled by reported alkyne-functionalized derivatives of native metabolites, our method can be readily applied to image these biomacromolecules.

Measuring dynamic membrane fluctuations in cell membrane using quantitative phase imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lee, SangYun; Kim, Kyoohyun; Park, YongKeun

2017-02-01

There is a strong correlation between the dynamic membrane fluctuations and the biomechanical properties of living cells. The dynamic membrane fluctuation consists of submicron displacements, and can be altered by changing the cells' pathophysiological conditions. These results have significant relevance to the understanding of RBC biophysics and pathology, as follows. RBCs must withstand large mechanical deformations during repeated passages through the microvasculature and the fenestrated walls of the splenic sinusoids. This essential ability is diminished with senescence, resulting in physiological destruction of the aging RBCs. Pathological destruction of the red cells, however, occurs in cells affected by a host of diseases such as spherocytosis, malaria, and Sickle cell disease, as RBCs depart from their normal discoid shape and lose their deformability. Therefore, quantifying the RBC deformability insight into a variety of problems regarding the interplay of cell structure, dynamics, and function. Furthermore, the ability to monitor mechanical properties of RBCs is of vital interest in monitoring disease progression or response to treatment as molecular and pharmaceutical approaches for treatment of chronic diseases. Here, we present the measurements of dynamic membrane fluctuations in live cells using quantitative phase imaging techniques. Measuring both the 3-D refractive index maps and the dynamic phase images of live cells are simultaneously measured, from which dynamic membrane fluctuation and deformability of cells are precisely calculated. We also present its applications to various diseases ranging from sickle cell diseases, babesiosis, and to diabetes.

Displacement of the mitotic apparatuses by centrifugation reveals cortical actin organization during cytokinesis in cultured tobacco BY-2 cells.

PubMed

Arima, Kengo; Tamaoki, Daisuke; Mineyuki, Yoshinobu; Yasuhara, Hiroki; Nakai, Tomonori; Shimmen, Teruo; Yoshihisa, Tohru; Sonobe, Seiji

2018-06-19

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global-local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global-local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.

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

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

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins.

PubMed

Islam, Kazi T; Shah, Dilip M; El-Mounadi, Kaoutar

2017-12-24

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro antifungal activity against a broad-spectrum of fungal pathogens and therefore have the potential to be used as antifungal agents in transgenic crops. In order to harness the full potential of plant defensins for diseases control, it is crucial to elucidate their mechanisms of action (MOA). With the advent of advanced microscopy techniques, live-cell imaging has become a powerful tool for understanding the dynamics of the antifungal MOA of plant defensins. Here, a confocal microscopy based live-cell imaging method is described using two fluorescently labeled plant defensins (MtDef4 and MtDef5) in combination with vital fluorescent dyes. This technique enables real-time visualization and analysis of the dynamic events of MtDef4 and MtDef5 internalization into fungal cells. Importantly, this assay generates a wealth of information including internalization kinetics, mode of entry and subcellular localization of these peptides. Along with other cell biological tools, these methods have provided critical insights into the dynamics and complexity of the MOA of these peptides. These tools can also be used to compare the MOA of these peptides against different fungi.

Multi-color fluorescence imaging of sub-cellular dynamics of cancer cells in live mice

NASA Astrophysics Data System (ADS)

Hoffman, Robert M.

2006-02-01

We have genetically engineered dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in the cytoplasm in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed of the cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery or heart injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse where extreme elongation of the cell body as well as the nucleus occurred. The migration velocities of the dual-color cancer cells in the capillaries were measured by capturing individual images of the dual-color fluorescent cells over time. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT)-GFP-RFP cells were injected in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, MMT-GFP-RFP cells injected into the portal vein mostly survived and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells. With the ability to continuously image cancer cells at the subcellular level in the live animal, our understanding of the complex steps of metastasis will significantly increase. In addition, new drugs can be developed to target these newly visible steps of metastasis.

Study of Fluorescent Imaging of Se (IV) in Living Cells Using a Turn-on Fluorescent Probe Based on a Rhodamine Spirolactame Derivative.

PubMed

Guan, Mingming; Mi, Hongyu; Xu, Hui; Fei, Qiang; Shan, Hongyan; Huan, Yanfu; Lv, Shaowu; Feng, Guodong

2017-03-01

A highly selective fluorescent probe 2-(2-(2-aminoethylamino)ethyl)-3',6'-bis(ethylamino)-2',7'-dimethylspiro[isoindoline-1,9'-xanthen]-3-one (ABDO) for Se (IV) had been synthesized in our earlier report. In this study, this fluorescent sensor is applied on analysis fluorescent imaging of Se (IV) in Hela cells. The experiment conditions, such as the MTT assay, different concentration of saline, incubated time of Hela cells with ABDO and Se (IV), and intracellular action position of Se (IV), are investigated. Through a series of experiments, the fluorescent image of Se (IV) in Hela cells can be observed when the cells cultured by 2 μM ABDO and 2 μM Se (IV) for 210 min. And the intracellular action position of Se (IV) is verified after the co-localization experiments are done. It is mitochondria. These experimental results show that ABDO will be an eagerly anticipated sensor for fluorescent imaging analysis of selenium ion in living cells. Besides, we also can use the complexes of ABDO-Se to observe morphology and distribution of mitochondria in cells like JG-B.

Statistical organelle dissection of Arabidopsis guard cells using image database LIPS.

PubMed

Higaki, Takumi; Kutsuna, Natsumaro; Hosokawa, Yoichiroh; Akita, Kae; Ebine, Kazuo; Ueda, Takashi; Kondo, Noriaki; Hasezawa, Seiichiro

2012-01-01

To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.

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.

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

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

Live Imaging of HIV-1 Transfer across T Cell Virological Synapse to Epithelial Cells that Promotes Stromal Macrophage Infection.

PubMed

Real, Fernando; Sennepin, Alexis; Ganor, Yonatan; Schmitt, Alain; Bomsel, Morgane

2018-05-08

During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed in vitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4 + T cells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa. Copyright © 2018. Published by Elsevier Inc.

General Staining and Segmentation Procedures for High Content Imaging and Analysis.

PubMed

Chambers, Kevin M; Mandavilli, Bhaskar S; Dolman, Nick J; Janes, Michael S

2018-01-01

Automated quantitative fluorescence microscopy, also known as high content imaging (HCI), is a rapidly growing analytical approach in cell biology. Because automated image analysis relies heavily on robust demarcation of cells and subcellular regions, reliable methods for labeling cells is a critical component of the HCI workflow. Labeling of cells for image segmentation is typically performed with fluorescent probes that bind DNA for nuclear-based cell demarcation or with those which react with proteins for image analysis based on whole cell staining. These reagents, along with instrument and software settings, play an important role in the successful segmentation of cells in a population for automated and quantitative image analysis. In this chapter, we describe standard procedures for labeling and image segmentation in both live and fixed cell samples. The chapter will also provide troubleshooting guidelines for some of the common problems associated with these aspects of HCI.

The Use of Intravital Two-Photon and Thick Section Confocal Imaging to Analyze B Lymphocyte Trafficking in Lymph Nodes and Spleen.

PubMed

Park, Chung; Hwang, Il-Young; Kehrl, John H

2018-01-01

Intravital two-photon laser scanning microscopy (TP-LSM) has allowed the direct observation of immune cells in intact organs of living animals. In the B cell biology field TP-LSM has detailed the movement of B cells in high endothelial venules and during their transmigration into lymph organs; described the movement and positioning of B cells within lymphoid organs; outlined the mechanisms by which antigen is delivered to B cells; observed B cell interacting with T cells, other cell types, and even with pathogens; and delineated the egress of B cells from the lymph node (LN) parenchyma into the efferent lymphatics. As the quality of TP-LSM improves and as new fluorescent probes become available additional insights into B cell behavior and function await new investigations. Yet intravital TP-LSM has some disadvantages including a lower resolution than standard confocal microscopy, a narrow imaging window, and a shallow depth of imaging. We have found that supplementing intravital TP-LSM with conventional confocal microscopy using thick LN sections helps to overcome some of these shortcomings. Here, we describe procedures for visualizing the behavior and trafficking of fluorescently labeled, adoptively transferred antigen-activated B cells within the inguinal LN of live mice using two-photon microscopy. Also, we introduce procedures for fixed thick section imaging using standard confocal microscopy, which allows imaging of fluorescently labeled cells deep in the LN cortex and in the spleen with high resolution.

Live-cell super-resolution imaging of intrinsically fast moving flagellates

NASA Astrophysics Data System (ADS)

Glogger, M.; Stichler, S.; Subota, I.; Bertlein, S.; Spindler, M.-C.; Teßmar, J.; Groll, J.; Engstler, M.; Fenz, S. F.

2017-02-01

Recent developments in super-resolution microscopy make it possible to resolve structures in biological cells at a spatial resolution of a few nm and observe dynamical processes with a temporal resolution of ms to μs. However, the optimal structural resolution requires repeated illumination cycles and is thus limited to chemically fixed cells. For live cell applications substantial improvement over classical Abbe-limited imaging can already be obtained in adherent or slow moving cells. Nonetheless, a large group of cells are fast moving and thus could not yet be addressed with live cell super-resolution microscopy. These include flagellate pathogens like African trypanosomes, the causative agents of sleeping sickness in humans and nagana in livestock. Here, we present an embedding method based on a in situ forming cytocompatible UV-crosslinked hydrogel. The fast cross-linking hydrogel immobilizes trypanosomes efficiently to allow microscopy on the nanoscale. We characterized both the trypanosomes and the hydrogel with respect to their autofluorescence properties and found them suitable for single-molecule fluorescence microscopy (SMFM). As a proof of principle, SMFM was applied to super-resolve a structure inside the living trypanosome. We present an image of a flagellar axoneme component recorded by using the intrinsic blinking behavior of eYFP. , which features invited work from the best early-career researchers working within the scope of J Phys D. This project is part of the Journal of Physics series’ 50th anniversary celebrations in 2017. Susanne Fenz was selected by the Editorial Board of J Phys D as an Emerging Talent/Leader.

Multimodal biophotonic workstation for live cell analysis.

PubMed

Esseling, Michael; Kemper, Björn; Antkowiak, Maciej; Stevenson, David J; Chaudet, Lionel; Neil, Mark A A; French, Paul W; von Bally, Gert; Dholakia, Kishan; Denz, Cornelia

2012-01-01

A reliable description and quantification of the complex physiology and reactions of living cells requires a multimodal analysis with various measurement techniques. We have investigated the integration of different techniques into a biophotonic workstation that can provide biological researchers with these capabilities. The combination of a micromanipulation tool with three different imaging principles is accomplished in a single inverted microscope which makes the results from all the techniques directly comparable. Chinese Hamster Ovary (CHO) cells were manipulated by optical tweezers while the feedback was directly analyzed by fluorescence lifetime imaging, digital holographic microscopy and dynamic phase-contrast microscopy. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Asymmetric rhenium tricarbonyl complexes show superior luminescence properties in live cell imaging.

PubMed

Raszeja, Lukasz J; Siegmund, Daniel; Cordes, Anna L; Güldenhaupt, Jörn; Gerwert, Klaus; Hahn, Stephan; Metzler-Nolte, Nils

2017-01-16

The synthesis and photophysical properties of a novel series of rhenium tricarbonyl complexes based on tridentate phenanthridinyl-containing ligands are described. Photophysical data reveal beneficial luminescence behaviour especially for compounds with an asymmetric ligand set. These advantageous properties are not limited to organic solvents, but indeed also improved in aqueous solutions. The suitability of our new rhenium complexes as potent imaging agents has been confirmed by fluorescence microscopy on living cancer cells, which also confirms superior long-time stability under fluorescence microscopy conditions. Colocalisation studies with commercial organelle stains reveal an accumulation of the complexes in the endoplasmic reticulum for all tested cell lines.

A novel live cell imaging system reveals a reversible hydrostatic pressure impact on cell cycle progression.

PubMed

Brooker, Holly R; Gyamfi, Irene A; Wieckowska, Agnieszka; Brooks, Nicholas J; Mulvihill, Daniel P; Geeves, Michael A

2018-06-21

Life is dependent upon the ability of a cell to rapidly respond to changes in environment. Small perturbations in local environments change the ability of molecules to interact and hence communicate. Hydrostatic pressure provides a rapid non-invasive, fully-reversible method for modulating affinities between molecules both in vivo and in vitro We have developed a simple fluorescence imaging chamber that allows intracellular protein dynamics and molecular events to be followed at pressures up to 200 bar in living cells. Using yeast we investigate the impact of hydrostatic pressure upon cell growth and cell cycle progression. While 100 bar has no affect upon viability, it induces a delay in chromosome segregation, resulting in the accumulation of long-undivided-bent cells, consistent with disruption of the cytoskeletons. This delay is independent of stress signalling and induces synchronisation of cell-cycle progression. Equivalent affects were observed in Candida albicans , with pressure inducing a reversible cell-cycle delay and hyphal growth. We present a simple novel non-invasive fluorescence microscopy based approach to transiently impact molecular dynamics to visualise, dissect and study signalling pathways and cellular processes in living cells. © 2018. Published by The Company of Biologists Ltd.

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.

Mapping Cd²⁺-induced membrane permeability changes of single live cells by means of scanning electrochemical microscopy.

PubMed

Filice, Fraser P; Li, Michelle S M; Henderson, Jeffrey D; Ding, Zhifeng

2016-02-18

Scanning Electrochemical Microscopy (SECM) is a powerful, non-invasive, analytical methodology that can be used to investigate live cell membrane permeability. Depth scan SECM imaging allowed for the generation of 2D current maps of live cells relative to electrode position in the x-z or y-z plane. Depending on resolution, one depth scan image can contain hundreds of probe approach curves (PACs). Individual PACs were obtained by simply extracting vertical cross-sections from the 2D image. These experimental PACs were overlaid onto theoretically generated PACs simulated at specific geometry conditions. Simulations were carried out using 3D models in COMSOL Multiphysics to determine the cell membrane permeability coefficients at different locations on the surface of the cells. Common in literature, theoretical PACs are generated using a 2D axially symmetric geometry. This saves on both compute time and memory utilization. However, due to symmetry limitations of the model, only one experimental PAC right above the cell can be matched with simulated PAC data. Full 3D models in this article were developed for the SECM system of live cells, allowing all experimental PACs over the entire cell to become usable. Cd(2+)-induced membrane permeability changes of single human bladder (T24) cells were investigated at several positions above the cell, displaced from the central axis. The experimental T24 cells under study were incubated with Cd(2+) in varying concentrations. It is experimentally observed that 50 and 100 μM Cd(2+) caused a decrease in membrane permeability, which was uniform across all locations over the cell regardless of Cd(2+) concentration. The Cd(2+) was found to have detrimental effects on the cell, with cells shrinking in size and volume, and the membrane permeability decreasing. A mapping technique for the analysis of the cell membrane permeability under the Cd(2+) stress is realized by the methodology presented. Copyright © 2016 Elsevier B.V. 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.

The in vivo activation of persistent nanophosphors for optical imaging of vascularization, tumours and grafted cells

NASA Astrophysics Data System (ADS)

Maldiney, Thomas; Bessière, Aurélie; Seguin, Johanne; Teston, Eliott; Sharma, Suchinder K.; Viana, Bruno; Bos, Adrie J. J.; Dorenbos, Pieter; Bessodes, Michel; Gourier, Didier; Scherman, Daniel; Richard, Cyrille

2014-04-01

Optical imaging for biological applications requires more sensitive tools. Near-infrared persistent luminescence nanoparticles enable highly sensitive in vivo optical detection and complete avoidance of tissue autofluorescence. However, the actual generation of persistent luminescence nanoparticles necessitates ex vivo activation before systemic administration, which prevents long-term imaging in living animals. Here, we introduce a new generation of optical nanoprobes, based on chromium-doped zinc gallate, whose persistent luminescence can be activated in vivo through living tissues using highly penetrating low-energy red photons. Surface functionalization of this photonic probe can be adjusted to favour multiple biomedical applications such as tumour targeting. Notably, we show that cells can endocytose these nanoparticles in vitro and that, after intravenous injection, we can track labelled cells in vivo and follow their biodistribution by a simple whole animal optical detection, opening new perspectives for cell therapy research and for a variety of diagnosis applications.

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

PubMed

Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

2018-02-13

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

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

Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.

PubMed

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 super-resolution 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.

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time.

PubMed

Bhat, Supriya V; Sultana, Taranum; Körnig, André; McGrath, Seamus; Shahina, Zinnat; Dahms, Tanya E S

2018-05-29

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

Construction of a ratiometric fluorescent probe with an extremely large emission shift for imaging hypochlorite in living cells

NASA Astrophysics Data System (ADS)

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

2018-01-01

Hypochlorite is one of the important reactive oxygen species (ROS) and plays critical roles in many biologically vital processes. Herein, we present a unique ratiometric fluorescent probe (CBP) with an extremely large emission shift for detecting hypochlorite in living cells. Utilizing positively charged α,β-unsaturated carbonyl group as the reaction site, the probe CBP itself exhibited near-infrared (NIR) fluorescence at 662 nm, and can display strong blue fluorescence at 456 nm when responded to hypochlorite. Notably, the extremely large emission shift of 206 nm could enable the precise measurement of the fluorescence peak intensities and ratios. CBP showed high sensitivity, excellent selectivity, desirable performance at physiological pH, and low cytotoxicity. The bioimaging experiments demonstrate the biological application of CBP for the ratiometric imaging of hypochlorite in living cells.

A series of terpyridine containing flexible amino diethylacetate derivatives with large two-photon action cross-sections for effective mitochondrial imaging in living liver cancerous cells

NASA Astrophysics Data System (ADS)

Jia, Ran; Zhu, Yingying; Hu, Lei; Xiong, Qiru; Zhao, Meng; Zhang, Mingzhu; Tian, Xiaohe

2018-01-01

Small molecules possess large two-photon action cross sections (Φσ) are highly demanded for biological purpose. Herein, three novel terpyridine containing flexible amino diethylacetate organic small molecules (A1, A2 and A3) were rationally designed and their photophysical properties were investigated both experimentally and theoretically. The results revealed that the three chromophores possess large Φσ and remarkable Stokes' shift in high polar solvents, which are particularly benefit for further biological imaging application. One chromophore (A1) displayed an effective intracellular uptake against lung cancerous living cells A549. Colocalization studies suggested the internalized subcellular compartment was mitochondria. Consequently, chromophore A1 provides a promising platform to directly monitor mitochondria in living cells under two-photon confocal laser scanning microscopy.

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

PubMed

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

2009-05-29

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

Multi-spectral digital holographic microscopy for enhanced quantitative phase imaging of living cells

NASA Astrophysics Data System (ADS)

Kemper, Björn; Kastl, Lena; Schnekenburger, Jürgen; Ketelhut, Steffi

2018-02-01

Main restrictions of using laser light in digital holographic microscopy (DHM) are coherence induced noise and parasitic reflections in the experimental setup which limit resolution and measurement accuracy. We explored, if coherence properties of partial coherent light sources can be generated synthetically utilizing spectrally tunable lasers. The concept of the method is demonstrated by label-free quantitative phase imaging of living pancreatic tumor cells and utilizing an experimental configuration including a commercial microscope and a laser source with a broad tunable spectral range of more than 200 nm.

Cd(2+) Triggered the FRET "ON": A New Molecular Switch for the Ratiometric Detection of Cd(2+) with Live-Cell Imaging and Bound X-ray Structure.

PubMed

Aich, Krishnendu; Goswami, Shyamaprosad; Das, Sangita; Mukhopadhyay, Chitrangada Das; Quah, Ching Kheng; Fun, Hoong-Kun

2015-08-03

On the basis of the Förster resonance energy transfer mechanism between rhodamine and quinoline-benzothiazole conjugated dyad, a new colorimetric as well as fluorescence ratiometric probe was synthesized for the selective detection of Cd(2+). The complex formation of the probe with Cd(2+) was confirmed through Cd(2+)-bound single-crystal structure. Capability of the probe as imaging agent to detect the cellular uptake of Cd(2+) was demonstrated here using living RAW cells.

Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in vivo

NASA Astrophysics Data System (ADS)

Ye, Deju; Shuhendler, Adam J.; Cui, Lina; Tong, Ling; Tee, Sui Seng; Tikhomirov, Grigory; Felsher, Dean W.; Rao, Jianghong

2014-06-01

Directed self-assembly of small molecules in living systems could enable a myriad of applications in biology and medicine, and already this has been used widely to synthesize supramolecules and nano/microstructures in solution and in living cells. However, controlling the self-assembly of synthetic small molecules in living animals is challenging because of the complex and dynamic in vivo physiological environment. Here we employ an optimized first-order bioorthogonal cyclization reaction to control the self-assembly of a fluorescent small molecule, and demonstrate its in vivo applicability by imaging caspase-3/7 activity in human tumour xenograft mouse models of chemotherapy. The fluorescent nanoparticles assembled in situ were imaged successfully in both apoptotic cells and tumour tissues using three-dimensional structured illumination microscopy. This strategy combines the advantages offered by small molecules with those of nanomaterials and should find widespread use for non-invasive imaging of enzyme activity in vivo.

A cancer cell-specific fluorescent probe for imaging Cu2 + in living cancer cells

NASA Astrophysics Data System (ADS)

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

2017-07-01

Monitoring copper level in cancer cells is important for the further understanding of its roles in the cell proliferation, and also could afford novel copper-based strategy for the cancer therapy. Herein, we have developed a novel cancer cell-specific fluorescent probe for the detecting Cu2 + in living cancer cells. The probe employed biotin as the cancer cell-specific group. Before the treatment of Cu2 +, the probe showed nearly no fluorescence. However, the probe can display strong fluorescence at 581 nm in response to Cu2 +. The probe exhibited excellent sensitivity and high selectivity for Cu2 + over the other relative species. Under the guidance of biotin group, could be successfully used for detecting Cu2 + in living cancer cells. We expect that this design strategy could be further applied for detection of the other important biomolecules in living cancer cells.

Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

PubMed

Hayashi-Takanaka, Yoko; Stasevich, Timothy J; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

2014-01-01

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

Progress in Cell Marking for Synchrotron X-ray Computed Tomography

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

Hall, Christopher; Sturm, Erica; Schultke, Elisabeth

2010-07-23

Recently there has been an increase in research activity into finding ways of marking cells in live animals for pre-clinical trials. Development of certain drugs and other therapies crucially depend on tracking particular cells or cell types in living systems. Therefore cell marking techniques are required which will enable longitudinal studies, where individuals can be examined several times over the course of a therapy or study. The benefits of being able to study both disease and therapy progression in individuals, rather than cohorts are clear. The need for high contrast 3-D imaging, without harming or altering the biological system requiresmore » a non-invasive yet penetrating imaging technique. The technique will also have to provide an appropriate spatial and contrast resolution. X-ray computed tomography offers rapid acquisition of 3-D images and is set to become one of the principal imaging techniques in this area. Work by our group over the last few years has shown that marking cells with gold nano-particles (GNP) is an effective means of visualising marked cells in-vivo using x-ray CT. Here we report the latest results from these studies. Synchrotron X-ray CT images of brain lesions in rats taken using the SYRMEP facility at the Elettra synchrotron in 2009 have been compared with histological examination of the tissues. Some deductions are drawn about the visibility of the gold loaded cells in both light microscopy and x-ray imaging.« less

Progress in Cell Marking for Synchrotron X-ray Computed Tomography

NASA Astrophysics Data System (ADS)

Hall, Christopher; Sturm, Erica; Schultke, Elisabeth; Arfelli, Fulvia; Menk, Ralf-Hendrik; Astolfo, Alberto; Juurlink, Bernhard H. J.

2010-07-01

Recently there has been an increase in research activity into finding ways of marking cells in live animals for pre-clinical trials. Development of certain drugs and other therapies crucially depend on tracking particular cells or cell types in living systems. Therefore cell marking techniques are required which will enable longitudinal studies, where individuals can be examined several times over the course of a therapy or study. The benefits of being able to study both disease and therapy progression in individuals, rather than cohorts are clear. The need for high contrast 3-D imaging, without harming or altering the biological system requires a non-invasive yet penetrating imaging technique. The technique will also have to provide an appropriate spatial and contrast resolution. X-ray computed tomography offers rapid acquisition of 3-D images and is set to become one of the principal imaging techniques in this area. Work by our group over the last few years has shown that marking cells with gold nano-particles (GNP) is an effective means of visualising marked cells in-vivo using x-ray CT. Here we report the latest results from these studies. Synchrotron X-ray CT images of brain lesions in rats taken using the SYRMEP facility at the Elettra synchrotron in 2009 have been compared with histological examination of the tissues. Some deductions are drawn about the visibility of the gold loaded cells in both light microscopy and x-ray imaging.

Intravital microscopy of biosensor activities and intrinsic metabolic states

PubMed Central

Winfree, Seth; Hato, Takashi; Day, Richard N.

2018-01-01

Intravital microscopy (IVM) is an imaging tool that is capable of detecting subcellular signaling or metabolic events as they occur in tissues in the living animal. Imaging in highly scattering biological tissues, however, is challenging because of the attenuation of signal in images acquired at increasing depths. Depth-dependent signal attenuation is the major impediment to IVM, limiting the depth from which significant data can be obtained. Therefore, making quantitative measurements by IVM requires methods that use internal calibration, or alternatively, a completely different way of evaluating the signals. Here, we describe how ratiometric imaging of genetically encoded biosensor probes can be used to make quantitative measurements of changes in the activity of cell signaling pathways. Then, we describe how fluorescence lifetime imaging can be used for label-free measurements of the metabolic states of cells within the living animal. PMID:28434902

Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment.

PubMed

Ricard, Clément; Debarbieux, Franck Christian

2014-01-01

The majority of intravital studies on brain tumor in living animal so far rely on dual color imaging. We describe here a multiphoton imaging protocol to dynamically characterize the interactions between six cellular components in a living mouse. We applied this methodology to a clinically relevant glioblastoma multiforme (GBM) model designed in reporter mice with targeted cell populations labeled by fluorescent proteins of different colors. This model permitted us to make non-invasive longitudinal and multi-scale observations of cell-to-cell interactions. We provide examples of such 5D (x,y,z,t,color) images acquired on a daily basis from volumes of interest, covering most of the mouse parietal cortex at subcellular resolution. Spectral deconvolution allowed us to accurately separate each cell population as well as some components of the extracellular matrix. The technique represents a powerful tool for investigating how tumor progression is influenced by the interactions of tumor cells with host cells and the extracellular matrix micro-environment. It will be especially valuable for evaluating neuro-oncological drug efficacy and target specificity. The imaging protocol provided here can be easily translated to other mouse models of neuropathologies, and should also be of fundamental interest for investigations in other areas of systems biology.

Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos.

PubMed

Esain, Virginie; Cortes, Mauricio; North, Trista E

2016-01-01

Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.

Live-cell MRI with xenon hyper-CEST biosensors targeted to metabolically labeled cell-surface glycans.

PubMed

Witte, Christopher; Martos, Vera; Rose, Honor May; Reinke, Stefan; Klippel, Stefan; Schröder, Leif; Hackenberger, Christian P R

2015-02-23

The targeting of metabolically labeled glycans with conventional MRI contrast agents has proved elusive. In this work, which further expands the utility of xenon Hyper-CEST biosensors in cell experiments, we present the first successful molecular imaging of such glycans using MRI. Xenon Hyper-CEST biosensors are a novel class of MRI contrast agents with very high sensitivity. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry. Through the use of a state of the art live-cell bioreactor, it was demonstrated that xenon MRI biosensors can be used to image cell-surface glycans at nanomolar concentrations. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Examining live cell cultures during apoptosis by digital holographic phase imaging and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Khmaladze, Alexander

2017-11-01

Cellular apoptosis is a unique, organized series of events, leading to programmed cell death. In this work, we present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy measurements of live cell cultures yield information about cell shape and volume, changes to which are indicative of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopic measurements provide complementary information about cells, such as protein, lipid and nucleic acid content, and the spectral signatures associated with structural changes in molecules. Our work indicates that the chemical changes in proteins, which were detected by Raman measurements, preceded morphological changes, which were seen with digital holographic microscopy.

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

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

PubMed

Zhao, Ming; Li, Yu; Peng, Leilei

2014-05-05

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

Fluorescent kapakahines serve as non-toxic probes for live cell Golgi imaging.

PubMed

Rocha, Danilo D; Espejo, Vinson R; Rainier, Jon D; La Clair, James J; Costa-Lotufo, Letícia V

2015-09-01

There is an ongoing need for fluorescent probes that specifically-target select organelles within mammalian cells. This study describes the development of probes for the selective labeling of the Golgi apparatus and offers applications for live cell and fixed cell imaging. The kapakahines, characterized by a common C(3)-N(1') dimeric tryptophan linkage, comprise a unique family of bioactive marine depsipeptide natural products. We describe the uptake and subcellular localization of fluorescently-labeled analogs of kapakahine E. Using confocal microscopy, we identify a rapid and selective localization within the Golgi apparatus. Comparison with commercial Golgi stains indicates a unique localization pattern, which differs from currently available materials, therein offering a new tool to monitor the Golgi in live cells without toxic side effects. This study identifies a fluorescent analog of kapakahine E that is rapidly uptaken in cells and localizes within the Golgi apparatus. The advance of microscopic methods is reliant on the parallel discovery of next generation molecular probes. This study describes the advance of stable and viable probe for staining the Golgi apparatus. Copyright © 2015 Elsevier Inc. All rights reserved.

Multicolor Fluorescence Imaging of Traumatic Brain Injury in a Cryolesion Mouse Model

PubMed Central

2012-01-01

Traumatic brain injury is characterized by initial tissue damage, which then can lead to secondary processes such as cell death and blood-brain-barrier disruption. Clinical and preclinical studies of traumatic brain injury typically employ anatomical imaging techniques and there is a need for new molecular imaging methods that provide complementary biochemical information. Here, we assess the ability of a targeted, near-infrared fluorescent probe, named PSS-794, to detect cell death in a brain cryolesion mouse model that replicates certain features of traumatic brain injury. In short, the model involves brief contact of a cold rod to the head of a living, anesthetized mouse. Using noninvasive whole-body fluorescence imaging, PSS-794 permitted visualization of the cryolesion in the living animal. Ex vivo imaging and histological analysis confirmed PSS-794 localization to site of brain cell death. The nontargeted, deep-red Tracer-653 was validated as a tracer dye for monitoring blood-brain-barrier disruption, and a binary mixture of PSS-794 and Tracer-653 was employed for multicolor imaging of cell death and blood-brain-barrier permeability in a single animal. The imaging data indicates that at 3 days after brain cryoinjury the amount of cell death had decreased significantly, but the integrity of the blood-brain-barrier was still impaired; at 7 days, the blood-brain-barrier was still three times more permeable than before cryoinjury. PMID:22860222

Current State-of-the-Art 3D Tissue Models and Their Compatibility with Live Cell Imaging.

PubMed

Bardsley, Katie; Deegan, Anthony J; El Haj, Alicia; Yang, Ying

2017-01-01

Mammalian cells grow within a complex three-dimensional (3D) microenvironment where multiple cells are organized and surrounded by extracellular matrix (ECM). The quantity and types of ECM components, alongside cell-to-cell and cell-to-matrix interactions dictate cellular differentiation, proliferation and function in vivo. To mimic natural cellular activities, various 3D tissue culture models have been established to replace conventional two dimensional (2D) culture environments. Allowing for both characterization and visualization of cellular activities within possibly bulky 3D tissue models presents considerable challenges due to the increased thickness and subsequent light scattering features of such 3D models. In this chapter, state-of-the-art methodologies used to establish 3D tissue models are discussed, first with a focus on both scaffold-free and scaffold-based 3D tissue model formation. Following on, multiple 3D live cell imaging systems, mainly optical imaging modalities, are introduced. Their advantages and disadvantages are discussed, with the aim of stimulating more research in this highly demanding research area.

Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye

PubMed Central

Sharma, Robin; Williams, David R.; Palczewska, Grazyna; Palczewski, Krzysztof; Hunter, Jennifer J.

2016-01-01

Purpose Although extrinsic fluorophores can be introduced to label specific cell types in the retina, endogenous fluorophores, such as NAD(P)H, FAD, collagen, and others, are present in all retinal layers. These molecules are a potential source of optical contrast and can enable noninvasive visualization of all cellular layers. We used a two-photon fluorescence adaptive optics scanning light ophthalmoscope (TPF-AOSLO) to explore the native autofluorescence of various cell classes spanning several layers in the unlabeled retina of a living primate eye. Methods Three macaques were imaged on separate occasions using a custom TPF-AOSLO. Two-photon fluorescence was evoked by pulsed light at 730 and 920 nm excitation wavelengths, while fluorescence emission was collected in the visible range from several retinal layers and different locations. Backscattered light was recorded simultaneously in confocal modality and images were postprocessed to remove eye motion. Results All retinal layers yielded two-photon signals and the heterogeneous distribution of fluorophores provided optical contrast. Several structural features were observed, such as autofluorescence from vessel walls, Müller cell processes in the nerve fibers, mosaics of cells in the ganglion cell and other nuclear layers of the inner retina, as well as photoreceptor and RPE layers in the outer retina. Conclusions This in vivo survey of two-photon autofluorescence throughout the primate retina demonstrates a wider variety of structural detail in the living eye than is available through conventional imaging methods, and broadens the use of two-photon imaging of normal and diseased eyes. PMID:26903224

Live-cell imaging by confocal Raman and fluorescence microscopy recognizes the crystal structure of calcium carbonate particles in HeLa cells.

PubMed

Abalymov, Аnatoly A; Verhovskiy, Roman A; Novoselova, Marina V; Parakhonskiy, Bogdan V; Gorin, Dmitry A; Yashchenok, Alexey M; Sukhorukov, Gleb B

2018-06-19

Porous calcium carbonate (CaCO 3 ) vaterite particles are very attractive templates for the encapsulation of pharmaceuticals and for the construction of hollow polyelectrolyte capsules, sensors, and enzyme-catalyzed reactors. Although CaCO 3 is biocompatible and biodegradable, little is known about the intercellular behavior and properties of vaterite particles in the cytoplasm of cells. In this work, we combined confocal Raman and fluorescent microscopy for the imaging of porous CaCO 3 vaterite particles in HeLa cells to study the uptake and status of the particles inside the cells in real time. Analysis of the fluorescence images showed that the particles penetrated the plasma membrane 3 h after being added to the cell culture and that the internalization of the particles continued up to 48 h. The crystal structure of individual vaterite particles in the cytoplasm of HeLa cells did not obviously change for 144 h. For clusters of particles, however, we identified Raman spectroscopic signatures of the stable calcite phase after 72 h of incubation, confirming an ion-exchange mechanism of vaterite transformation to calcite. The results indicate that our imaging approach to examining inorganic particles in living cells may have theranostic applications. This article is protected by copyright. All rights reserved.

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

PubMed Central

Lu, Fa-Ke; Basu, Srinjan; Igras, Vivien; Hoang, Mai P.; Ji, Minbiao; Fu, Dan; Holtom, Gary R.; Neel, Victor A.; Freudiger, Christian W.; Fisher, David E.; Xie, X. Sunney

2015-01-01

Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time. PMID:26324899

Fluorescent molecularly imprinted polymers as plastic antibodies for selective labeling and imaging of hyaluronan and sialic acid on fixed and living cells.

PubMed

Panagiotopoulou, Maria; Kunath, Stephanie; Medina-Rangel, Paulina Ximena; Haupt, Karsten; Tse Sum Bui, Bernadette

2017-02-15

Altered glycosylation levels or distribution of sialic acids (SA) or hyaluronan in animal cells are indicators of pathological conditions like infection or malignancy. We applied fluorescently-labeled molecularly imprinted polymer (MIP) particles for bioimaging of fixed and living human keratinocytes, to localize hyaluronan and sialylation sites. MIPs were prepared with the templates D-glucuronic acid (GlcA), a substructure of hyaluronan, and N-acetylneuraminic acid (NANA), the most common member of SA. Both MIPs were found to be highly selective towards their target monosaccharides, as no cross-reactivity was observed with other sugars like N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucose and D-galactose, present on the cell surface. The dye rhodamine and two InP/ZnS quantum dots (QDs) emitting in the green and in the red regions were used as fluorescent probes. Rhodamine-MIPGlcA and rhodamine-MIPNANA were synthesized as monodispersed 400nm sized particles and were found to bind selectively their targets located in the extracellular region, as imaged by epifluorescence and confocal microscopy. In contrast, when MIP-GlcA and MIP-NANA particles with a smaller size (125nm) were used, the MIPs being synthesized as thin shells around green and red emitting QDs respectively, it was possible to stain the intracellular and pericellular regions as well. In addition, simultaneous dual-color imaging with the two different colored QDs-MIPs was demonstrated. Importantly, the MIPs were not cytotoxic and did not affect cell viability; neither was the cells morphology affected as demonstrated by live cell imaging. These synthetic receptors could offer a new and promising imaging tool to monitor disease progression. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Quantitative measurement of permeabilization of living cells by terahertz attenuated total reflection

NASA Astrophysics Data System (ADS)

Grognot, Marianne; Gallot, Guilhem

2015-09-01

Using Attenuated Total Reflection imaging technique in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of epithelial cells by saponin. The terahertz signal is mostly sensitive to the intracellular protein concentration in the cells, in a very good agreement with standard bicinchoninic acid protein measurements. It opens the way to in situ real time dynamics of protein content and permeabilization in live cells.

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.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

PubMed

Görlitz, Frederik; Kelly, Douglas J; Warren, Sean C; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J; Stuhmeier, Frank; Neil, Mark A A; Tate, Edward W; Dunsby, Christopher; French, Paul M W

2017-01-18

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

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

PubMed

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

2005-02-01

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

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

PubMed Central

Warren, Sean C.; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A.; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J.; Stuhmeier, Frank; Neil, Mark A. A.; Tate, Edward W.; Dunsby, Christopher; French, Paul M. W.

2017-01-01

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set. PMID:28190060

Revealing dynamically-organized receptor ion channel clusters in live cells by a correlated electric recording and super-resolution single-molecule imaging approach.

PubMed

Yadav, Rajeev; Lu, H Peter

2018-03-28

The N-methyl-d-aspartate (NMDA) receptor ion-channel is activated by the binding of ligands, along with the application of action potential, important for synaptic transmission and memory functions. Despite substantial knowledge of the structure and function, the gating mechanism of the NMDA receptor ion channel for electric on-off signals is still a topic of debate. We investigate the NMDA receptor partition distribution and the associated channel's open-close electric signal trajectories using a combined approach of correlating single-molecule fluorescence photo-bleaching, single-molecule super-resolution imaging, and single-channel electric patch-clamp recording. Identifying the compositions of NMDA receptors, their spatial organization and distributions over live cell membranes, we observe that NMDA receptors are organized inhomogeneously: nearly half of the receptor proteins are individually dispersed; whereas others exist in heterogeneous clusters of around 50 nm in size as well as co-localized within the diffraction limited imaging area. We demonstrate that inhomogeneous interactions and partitions of the NMDA receptors can be a cause of the heterogeneous gating mechanism of NMDA receptors in living cells. Furthermore, comparing the imaging results with the ion-channel electric current recording, we propose that the clustered NMDA receptors may be responsible for the variation in the current amplitude observed in the on-off two-state ion-channel electric signal trajectories. Our findings shed new light on the fundamental structure-function mechanism of NMDA receptors and present a conceptual advancement of the ion-channel mechanism in living cells.

Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early

DTIC Science & Technology

2013-07-01

biology, nanotechnology, and imaging technology, molecular imaging utilizes specific probes as contrast agents to visualize cellular processes at the...This reagent was covalently coupled to the oligosaccharides attached to polypeptide side-chains of extracellular membrane proteins on living cells...website. The normal tissue gene expression profile dataset was modified and processed as described by Fang (8) and mean intensities and standard

Fluorescence image-guided photodynamic therapy of cancer cells using a scanning fiber endoscope

NASA Astrophysics Data System (ADS)

Woldetensae, Mikias H.; Kirshenbaum, Mark R.; Kramer, Greg M.; Zhang, Liang; Seibel, Eric J.

2013-03-01

A scanning fiber endoscope (SFE) and the cancer biomarker 5-aminolevulinic acid (5-ALA) were used to fluorescently detect and destroy superficial cancerous lesions, while experimenting with different dosimetry levels for concurrent or sequential imaging and laser therapy. The 1.6-mm diameter SFE was used to fluorescently image a confluent monolayer of A549 human lung cancer cells from culture, previously administered with 5 mM solution of 5-ALA for 4 hours. Twenty hours after therapy, cell cultures were stained to distinguish between living and dead cells using a laser scanning confocal microscope. To determine relative dosimetry for photodynamic therapy (PDT), 405-nm laser illumination was varied from 1 to 5 minutes with power varying from 5 to 18 mW, chosen to compare equal amounts of energy delivered to the cell culture. The SFE produced 500-line images of fluorescence at 15 Hz using the red detection channel centered at 635 nm. The results show that PDT of A549 cancer cell monolayers using 405nm light for imaging and 5-ALAinduced PpIX therapy was possible using the same SFE system. Increased duration and power of laser illumination produced an increased area of cell death upon live/dead staining. The ultrathin and flexible SFE was able to direct PDT using wide-field fluorescence imaging of a monolayer of cultured cancer cells after uptaking 5-ALA. The correlation between light intensity and duration of PDT was measured. Increased length of exposure and decreased light intensity yields larger areas of cell death than decreased length of exposure with increased light intensity.

3D/4D multiscale imaging in acute lymphoblastic leukemia cells: visualizing dynamics of cell death

NASA Astrophysics Data System (ADS)

Sarangapani, Sreelatha; Mohan, Rosmin Elsa; Patil, Ajeetkumar; Lang, Matthew J.; Asundi, Anand

2017-06-01

Quantitative phase detection is a new methodology that provides quantitative information on cellular morphology to monitor the cell status, drug response and toxicity. In this paper the morphological changes in acute leukemia cells treated with chitosan were detected using d'Bioimager a robust imaging system. Quantitative phase image of the cells was obtained with numerical analysis. Results show that the average area and optical volume of the chitosan treated cells is significantly reduced when compared with the control cells, which reveals the effect of chitosan on the cancer cells. From the results it can be attributed that d'Bioimager can be used as a non-invasive imaging alternative to measure the morphological changes of the living cells in real time.

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.

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.

Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

NASA Astrophysics Data System (ADS)

Rietdorf, Jens; Stephens, David J.; Squire, Anthony; Simpson, Jeremy; Shima, David T.; Paccaud, Jean-Pierre; Bastiaens, Philippe I.; Pepperkok, Rainer

2000-04-01

Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.

Fungicidal mechanisms of cathelicidins LL-37 and CATH-2 revealed by live-cell imaging.

PubMed

Ordonez, Soledad R; Amarullah, Ilham H; Wubbolts, Richard W; Veldhuizen, Edwin J A; Haagsman, Henk P

2014-01-01

Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides kill C. albicans rapidly. CATH-2 is the most active peptide and kills C. albicans within 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to the C. albicans cell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreases C. albicans cell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37.

In vivo imaging: shining a light on stem cells in the living animal.

PubMed

Nguyen, Phong Dang; Currie, Peter David

2018-03-28

Stem cells are undifferentiated cells that play crucial roles during development, growth and regeneration. Traditionally, these cells have been primarily characterised by histology, cell sorting, cell culture and ex vivo methods. However, as stem cells interact in a complex environment within specific tissue niches, there has been increasing interest in examining their in vivo behaviours, particularly in response to injury. Advances in imaging technologies and genetic tools have converged to enable unprecedented access to the endogenous stem cell niche. In this Spotlight article, we highlight how in vivo imaging can probe a range of biological processes that relate to stem cell activity, behaviour and control. © 2018. Published by The Company of Biologists Ltd.