Imaging/Microscopy


Improvements in automation and acquisition time have made the microscope a viable platform for performing hundreds of concurrent parallel experiments. Using these sorts of tools, it is now possible to run high-throughput screens for protein function and interaction in living cells, examining dynamic cellular processes to distinguish between primary and secondary phenotypes, and to study the phenotype kinetics. In the August issue of Cold Spring Harbor Protocols, Jan Ellenberg and colleagues from the EMBL present High-Throughput Microscopy Using Live Mammalian Cells, an overview of how to screen live cells using imaging technologies. The article examines each aspect of the general screening process and considers specific examples in the processing of time-lapse experiments. The techniques discussed are based on the use of cultured mammalian cells, but the concepts are easily transferred to cultured cells from other species like Drosophila and small organisms such as C. elegans.

While it is possible to analyze the global lipid composition of a cell, a deeper understanding of what lipids are doing within that cell is more difficult to come by. Though the lipid components may be known, finding their exact position, how dynamically they change location, and how rapidly they are metabolized presents an experimental challenge. The obvious approach would be the addition a fluorescent tag, which would allow for imaging of lipids in cells. Unfortunately, most commonly used fluorescent tags are as large as the lipid itself and are likely to have a strong effect on lipid location and metabolism.

In the July issue of Cold Spring Harbor Protocols, Joachim Goedhart and colleagues present a suite of protocols to get around these problems and allow for live imaging of lipids in cells. Their introduction to the topic explains the approach:

To circumvent this problem, two solutions have been developed–namely, the use of fluorescently labeled proteins that specifically recognize lipids and a chemical method to introduce the fluorescent tag inside the cell.

Protocols are provided for Transfection of Cells with DNA Encoding a Visible Fluorescent Protein-Tagged Lipid-Binding Domain, Labeling Lipids for Imaging in Fixed Cells, and Labeling Lipids for Imaging in Live Cells.

The zebrafish (Danio rerio) has rapidly become a favored model organism for studying developmental biology. One of the most commonly used methods for genetic manipulation in the zebrafish is the delivery of plasmids or oligonucleotides to cells within the living embryo via electroporation. When cells are exposed to brief electrical fields, transient membrane destabilization occurs and nucleic acids can cross the plasma membrane. When the electrical field is removed, the membrane seals and the nucleic acids are trapped inside the cell. In vivo electroporation has proven particularly effective for delivering fluorescent protein expression vectors for imaging and loss-of-function reagents such as morpholinos or RNA interference (RNAi) constructs for the knockdown of gene function. In the July issue of Cold Spring Harbor Protocols, Jack Horne and colleagues present Targeting the Zebrafish Optic Tectum Using In Vivo Electroporation, a modification of the technique that can be used to specifically target the developing optic tectum, the midbrain’s visual processing center. Instructions are given for the construction of electroporation electrodes, preparation and injection of DNA, and electroporation of the DNA into the embryonic brain.

The dynamic nature of biological processes has long been difficult to document, as researchers have been limited to static studies based on fixed specimens. Methods like immunocytochemistry or in situ hybridization can only provide accurate information on one organism at one particular time point. As Scott Fraser has remarked, it’s akin to trying to figure out the rules of football from looking at a set of still photographs taken during a game. But recent developments in imaging techniques, particularly the use of Green Fluorescent Protein (GFP) and its variants, have provided nondestructive ways to study dynamic processes over time, taking our understanding into the fourth dimension.

These new imaging techniques generate an enormous amount of digital image data, which can be difficult to cope with as it builds up over time. Computer-based image analysis is required for the extraction of reproducible and quantitative information. Previously, Cold Spring Harbor Protocols has featured Khuloud Jaqaman and Gaudenz Danuser’s case study using particle tracking to study cellular dynamics. In the June issue of the journal, Roland Eils and colleagues present Tracking and Quantitative Analysis of Dynamic Movements of Cells and Particles. The article sketches a general workflow for quantitative analysis of live cell images and details automated methods for image analysis including preprocessing, segmentation, registration, tracking and classification.

The rapid pace of technological progress in biological imaging has provided great insight into the processes of embryonic development. But for higher organisms with opaque eggs or internal development, optical access to the embryo is limited. While various embryonic culture methods are available, vertebrate development is best studied in an intact embryo model, one in which the natural environment has not been disrupted. In the June issue of Cold Spring Harbor Protocols, Paul Kulesa and colleagues from the Stowers Institute for Medical Research present In Ovo Live Imaging of Avian Embryos, a detailed set of instructions for time-lapse imaging of fluorescently labeled cells within a living avian embryo. During the procedure, a hole is made in the shell, and a Teflon membrane that is oxygen-permeable and liquid-impermeable is used to provide a window for visualization of the embryo via confocal or two-photon microscopy. Imaging can take place for up to five days without dehydration or degradation of the normal developmental environment. As one of June’s featured articles, the protocol is freely available to subscribers and nonsubscribers alike. Kulesa’s group also supplies a second protocol in the issue, covering Multi-Position Photoactivation and Multi-Time Acquisition for Large-Scale Cell Tracing in Avian Embryos, a technique that produced June’s cover image.

The large size and external development of the frog Xenopus laevis make it an ideal system for in vivo imaging of dynamic cellular activity. Xenopus embryos are amenable to simple genetic manipulation techniques including knockdowns and misexpression, as well as transgenesis. The ease of collecting large numbers of embryos and the larger size of individual cells within an embryo as compared with other vertebrate model systems provides an excellent platform for the observation of cellular behavior and subcellular processes. In the May issue of Cold Spring Harbor Protocols, John Wallingford and colleagues from the University of Texas provide a suite of articles detailing live imaging of Xenopus laevis at low magnification, confocal imaging of fixed tissues, and in one of May’s featured articles, High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology. This protocol describes methods for labeling and high-magnification time-lapse imaging by confocal microscopy. Like all of our featured articles, it’s freely available to subscribers and non-subscribers alike.

Neurons are organized into anatomical and functional groups called “circuits”. The activity of these circuits is traditionally monitored using conventional electrophysiological techniques. But some cells, such as the submandibular ganglia, are difficult to impale for intracellular recordings. Instead, viral vectors can be used to deliver fluorescent calcium sensors for detecting activity in a living animal. Calcium Imaging of Neuronal Circuits In Vivo Using a Circuit-Tracing Pseudorabies Virus, from Lynn Enquist and colleagues at Princeton University, provides detailed instructions for the use of the pseudorabies virus (PRV) as a vector for imaging connectivity and activity of neuronal circuits. PRV has a broad host range but does not infect higher-order primates, and it travels along chains of synaptically connected neurons. The PRV strain used in this procedure encodes G-CaMP2, a sensitive fluorescent calcium sensor protein. Available in the April issue of Cold Spring Harbor Protocols, the method allows for reliable detection of endogenous circuit activity at single-cell resolution. As one of April’s featured articles, it is freely available to subscribers and nonsubscribers alike.

The goal of tissue engineering is to recapitulate healthy human organs and tissue structures in culture, and then transplant them into patients, where they are fully integrated. This is a complicated process, and the use of high-throughput imaging systems that allow researchers to directly monitor transplanted tissues in live animals over time is important for improving the culturing and implantation techniques, as well as the design of artificial tissue scaffolds. By using transgenic animals with cell-specific fluorescent reporters, parameters such as tissue perfusion, donor cell survival, and donor-host cell interaction/integration can be observed. In the April issue of Cold Spring Harbor Protocols, Mary Dickinson and colleagues from the Baylor College of Medicine present a protocol for the use of The Mouse Cornea as a Transplantation Site for Live Imaging of Engineered Tissue Constructs. This is a modified version of the classical corneal micropocket angiogenesis assay, which employs it as a live imaging “window” to monitor angiogenic hydrogel tissue constructs. As one of April’s featured articles, it is freely available to subscribers and nonsubscribers alike.

The recent explosion in the availability and variety of fluorescent proteins, new organic dyes and quantum dots has been a driving force in the growing use of Total Internal Reflection Fluorescence Microscopy (TIRFM). TIRFM only illuminates molecules that are within a thin volume near the coverslip surface of a specimen and not those deeper in solution. This allows for an unparalleled signal-to-noise ratio and tremendous resolution. In the March issue of Cold Spring Harbor Protocols, Samara Reck-Peterson, Nathan Derr and Nico Stuurman present Imaging Single Molecules Using Total Internal Reflection Fluorescence Microscopy (TIRFM), which includes an overview of the theory behind TIRFM, considerations for TIRFM setup and purification/labeling of proteins, and a discussion of new techniques for imaging single molecules with super-resolution localization. In addition, the group offers step-by-step protocols for Determining Single-Molecule Intensity as a Function of Power Density and Imaging Single Molecular Motor Motility with TIRFM. An example of TIRFM imaging of single dynein molecules labeled with TMR (green) moving along axonemal microtubules labeled with Cy5 (red) can be seen here.

Cold Spring Harbor Protocols is hosting the movie figures that accompany the new lab manual, Live Cell Imaging, Second Edition, edited by Robert Goldman, Jason Swedlow and David Spector, . These movies are freely accessible to all, and worth a look if you’re interested in seeing the state of the art in time lapse imaging.

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