Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 μm) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.     

Going live: A comparative analysis of the suitability of the RFP derivatives RedStar,mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites

Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.     

Morphological analysis of rat ureteric terminal arterioles in situ

Confocal imaging of Fluo‐4, Propidium iodide, and di‐8‐Anepps loaded ureter were used to study the morphology of terminal arterioles with an inner diameter <50 μm in intact rat ureter. Optical sectioning showed that the muscle coat of the terminal arterioles consisted of a monolayer of highly curved smooth muscle cells which run circumferentially around the endothelium. This technique allowed not only to measure the inner diameter of the terminal arterioles but also to define the orientation and number of revolutions an individual smooth muscle cell made around the endothelium. We measured thickness, width, length, and morphological profile of the myocytes and endothelial cells. Propidium iodide staining showed nuclei of individual cells by continuous imaging at high resolution in serial optical sections. Conventional haematoxylin‐eosin, Masson's tri‐chrome staining, and transmission electron microscopy were also used in this study to compare the measurements obtained from live confocal imaging with histological standard methods. Parameters obtained from live imaging were significantly different. This technique of live staining allowed measuring the cellular and nuclear dimensions of the terminal arterioles in their natural environment which are important in studying the effects of vascular disease or aging on vascular structure. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.     

Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging microscopy (FLIM) is a quantitative microscopy technique for imaging nanosecond decay times of fluorophores. In the case of frequency-domain FLIM, several methods have been described to resolve the relative abundance of two fluorescent species with different fluorescence decay times. Thus far, single-frequency FLIM methods generally have been limited to quantifying two species with monoexponential decay. However, multiexponential decays are the norm rather than the exception, especially for fluorescent proteins and biological samples. Here, we describe a novel method for determining the fractional contribution in each pixel of an image of a sample containing two (multiexponentially) decaying species using single-frequency FLIM. We demonstrate that this technique allows the unmixing of binary mixtures of two spectrally identical cyan or green fluorescent proteins, each with multiexponential decay. Furthermore, because of their spectral identity, quantitative images of the relative molecular abundance of these fluorescent proteins can be generated that are independent of the microscope light path. The method is rigorously tested using samples of known composition and applied to live cell microscopy using cells expressing multiple (multiexponentially decaying) fluorescent proteins.     

Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle α‐actin gene

Smooth muscle α actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells (vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein (mCherry), driven by the full‐length SMA promoter and intronic sequences. We determined that the founders and F1 progeny of five independent lines contain 1–3 copies of the mCherry‐substituted BAC vector. Furthermore, we characterized the expression of SMA‐mCherry in relation to endogenous SMA in the embryo and in adult tissues, and found that the transgenic reporter in each line recapitulated endogenous SMA expression at all time points. We were also able to isolate SMA expressing cells from embryonic tissues using fluorescence‐activated cell sorting (FACS). We demonstrated that this marker can be combined with other vital fluorescent reporters and it can be used for live imaging of embryonic cardiodynamics. Therefore, these transgenic mice will be useful for isolating live SMA‐expressing cells via FACS and for studying the emergence, behavior, and regulation of SMA‐expressing cells, including vascular smooth muscle cells and pericytes throughout embryonic and postnatal development. genesis 48:457–463, 2010. © 2010 Wiley‐Liss, Inc.     

Dynamics of the PI3K-like protein kinase members ATM and DNA-PKcs at DNA double strand breaks

The protein kinases ATM and DNA-PKcs play critical roles in the cellular response to DNA double strand breaks (DSBs). ATM and DNA-PKcs are activated in response to DSBs and play several important roles in propagation of the damage signal and for the repair of DNA damage. Recent work from several groups, including ours, has focused on studying the dynamics of each of these proteins at DSBs and the requirements and factors which play a role(s) in this process. The use of live cell imaging of fluorescently-tagged ATM and DNA-PKcs has allowed us to study the real-time response of these proteins to laser-generated DNA damage in vivo. Here, we will extensively discuss the behavior of the ATM and DNA-PKcs proteins at DSB sites.Key words: ATM, DNA-PKcs, DNA double strand breaks, autophosphorylation, live cell imaging     

Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells

Live-cell imaging technology using fluorescent proteins (green fluorescent protein and its homologues) has revolutionized the study of cellular dynamics. But tools that can quantitatively analyse complex spatiotemporal processes in live cells remain lacking. Here we describe a new technique--fast multi-colour four-dimensional imaging combined with automated and quantitative time-space reconstruction--to fill this gap. As a proof of principle, we apply this method to study the re-formation of the nuclear envelope in live cells. Four-dimensional imaging of three spectrally distinct fluorescent proteins is used to simultaneously visualize three different cellular compartments at high speed and with high spatial resolution. The highly complex data, comprising several thousand images from a single cell, were quantitatively reconstructed in time-space by software developed in-house. This analysis reveals quantitative and qualitative insights into the highly ordered topology of nuclear envelope formation, in correlation with chromatin expansion - results that would have been impossible to achieve by manual inspection alone. Our new technique will greatly facilitate study of the highly ordered dynamic architecture of eukaryotic cells.     

Using electroporation and lipid-mediated transfection of GFP-expressing plasmids to label embryonic avian cells for vital confocal and two-photon microscopy

Fluorescent proteins have emerged as an ideal fluorescent marker for studying cell morphologies in vital systems. These proteins were first applied in whole organisms with established germ-line transformation protocols, but now it is possible to label cells with fluorescent proteins in other organisms. Here we present two ways to introduce GFP expressing plasmids into avian embryos for vital confocal and two-photon imaging. First, electroporation is a powerful approach to introduce GFP into the developing neural tube, offering several advantages over dye labeling. Second, we introduce a new lipid-based transfection system for introducing plasmid DNA directly to a small group of injected cells within live, whole embryos. These complementary approaches make it possible to transfect a wide-range of cell types in the avian embryo and the bright, stable, uniform expression of GFP offers great advantages for vital fluorescence imaging.     

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

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.     

Sample Preparation and Imaging Conditions Affect mEos3.2 Photophysics in Fission Yeast Cells

Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a three-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate, and photobleaching rate of mEos3.2 sensitive to the buffer conditions likely by permeabilizing the yeast cell membrane. Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that 405-nm irradiation drove some red-state mEos3.2 molecules to enter an intermediate dark state, which can be converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to quantitatively compare conditions for imaging mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells.     

Visualisation of PCNA Monoubiquitination In Vivo by Single Pass Spectral Imaging FRET Microscopy

Monoubiquitination of the DNA sliding clamp, PCNA, plays a central role in the control of damage bypass during replication. By combining a widely-spaced FRET donor/acceptor pair (CFP and mRFP) with spectral imaging, we have developed a simple method for the visualisation of PCNA monoubiquitination in both fixed and live cells with a single imaging pass. We validate the method with genetic controls in the avian cell line DT40 and use it to examine the intracellular dynamics of PCNA ubiquitination following subnuclear UV irradiation. This general approach is likely to be of utility for live imaging of post-translational modifications of a wide range of substrates in vivo.     

Dynamics of the PI3K-like protein kinase members ATM and DNA-PKcs at DNA double strand breaks

The protein kinases ATM and DNA-PKcs play critical roles in the cellular response to DNA double strand breaks (DSBs). ATM and DNA-PKcs are activated in response to DSBs and play several important roles in propagation of the damage signal and for the repair of DNA damage. Recent work from several groups, including ours, has focused on studying the dynamics of each of these proteins at DSBs and the requirements and factors which play a role(s) in this process. The use of live cell imaging of fluorescently-tagged ATM and DNA-PKcs has allowed us to study the real-time response of these proteins to laser-generated DNA damage in vivo. Here, we will extensively discuss the behavior of the ATM and DNA-PKcs proteins at DSB sites.     

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.     

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.     

Zebrafish lines expressing UAS‐driven red probes for monitoring cytoskeletal dynamics

Actin filaments and microtubules are principal components of the cytoskeleton that regulate the basic cellular phenomena underlying many fundamental cellular processes. Therefore, analyzing their dynamics in living cells is important for understanding cellular events more precisely. In this article, we report two novel transgenic zebrafish lines expressing red fluorescent proteins tagged with Lifeact or EB1 that interact with actin filaments and microtubule plus ends, respectively, under the control of the GAL4‐UAS system. Using these transgenic lines, we could detect F‐actin and microtubule plus end dynamics in specific tissues of living zebrafish embryos by crossing with GAL4 driver lines. In addition, we could achieve multi‐color imaging using these transgenic lines with GFP‐expressing transgenic lines. Therefore, our transgenic lines that carry UAS‐driven red fluorescent cytoskeletal probes are useful tools for analyzing spatiotemporal changes of the cytoskeletal elements using multicolor live imaging.     

Three-dimensional segmentation of nuclei and mitotic chromosomes for the study of cell divisions in live Drosophila embryos

Drosophila embryogenesis is an established model to investigate mechanisms and genes related to cell divisions in an intact multicellular organism. Progression through the cell cycle phases can be monitored in vivo using fluorescently labeled fusion proteins and time-lapse microscopy. To measure cellular properties in microscopic images, accurate and fast image segmentation methods are a critical prerequisite. To quantify static and dynamic features of interphase nuclei and mitotic chromosomes, we developed a three-dimensional (3D) segmentation method based on multiple level sets. We tested our method on 3D time-series images of live embryos expressing histone-2Av-green fluorescence protein. Our method is robust to low signal-to-noise ratios inherent to high-speed imaging, fluorescent signals in the cytoplasm, and dynamic changes of shape and texture. Comparisons with manual ground-truth segmentations showed that our method achieves more than 90% accuracy on the object as well as voxel levels and performs consistently throughout all cell cycle phases and developmental stages from syncytial blastoderm to postblastoderm mitotic domains.     

Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor

Fluorescent Ca(2+) indicator proteins (FCIPs) are attractive tools for studying Ca(2+) dynamics in live cells. Here we describe transgenic mouse lines expressing a troponin C (TnC)-based biosensor. The biosensor is widely expressed in neurons and has improved Ca(2+) sensitivity both in vitro and in vivo. This allows FCIP-based two-photon Ca(2+) imaging of distinct neurons and their dendrites in vivo, and opens a new avenue for structure-function analysis of intact neuronal circuits.     

Gene expression and immune response kinetics using electroporation-mediated DNA delivery to muscle

BACKGROUND: Injection of DNA encoding exogenic proteins into muscle tissue combined with electroporation often results in a transient increase of the encoded protein concentration in the muscle and the blood. The reduction is normally due to an immune response against the exogenic protein but other factors may also be involved. How various electroporation parameters affect the concentration kinetics of syngenic and exogenic proteins is studied in relation to immune response and muscle damage after electroporation-mediated DNA transfer to muscle. METHODS: Electroporation was applied to mouse quadriceps and rat tibialis anterior muscles after injection of DNA encoding either secreted alkaline phosphatase (SEAP), beta-galactosidase (beta-gal), luciferase or a mouse IgG molecule. Protein concentrations in blood or muscle and antibody responses were measured for a period up to 3 months. Tissue inflammation and muscle cell damage were studied on muscle cross-sections and assessed by measuring the concentrations of creatine phosphokinase (CPK) in blood. RESULTS: Mice with the highest SEAP concentration in blood at day 7 also had the highest rate of decrease afterwards, the strongest antibody responses against SEAP and the highest acute levels of CPK in blood. DNA-transfected muscle fibers were significantly reduced in number from days 7 to 14. Mononuclear cells surrounded the reporter gene expressing muscle fibers, thus indicating a cellular immune response. When using DNA encoding a syngenic protein the protein concentration in blood was relatively stabile over a 3-month period, but showed different kinetics for various electroporation parameters. CONCLUSIONS: Our findings suggest that the optimal electroporation parameters for DNA vaccination may be different from the optimal parameters for long-term expression of genes encoding syngenic proteins.     

Super-resolution Microscopy Approaches for Live Cell Imaging

By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells.