Imaging/Microscopy


Tumors contain many components in addition to the cancer cells, including blood vessels, fibroblasts and immune cells. Understanding the dynamic interactions of these populations in the tumor microenvironment is an important key for understanding cancer progression. While genetic studies and tumor biopsies have generated insights, direct time lapse imaging adds much to our understanding of the importance of these stromal components. In the February issue of Cold Spring Harbor Protocols, Andrew Ewald, Zena Werb and Mikala Egeblad provide Dynamic, Long-Term In Vivo Imaging of Tumor-Stroma Interactions in Mouse Models of Breast Cancer Using Spinning-Disk Confocal Microscopy. In addition to this overview of the technique, related protocols for Preparation of Mice for Long-Term Intravital Imaging of the Mammary Gland and Monitoring of Vital Signs for Long-Term Survival of Mice Under Anesthesia are also available.

Immunoimaging is rapidly developing from a merely descriptive technique into a set of methods and analytical tools that can be used to quantify and characterize an immune response at the cellular level. In the February issue of Cold Spring Harbor Protocols, Ian Parker and colleagues from the University of California, Irvine present Immunoimaging: Studying Immune System Dynamics Using Two-Photon Microscopy. The article outlines the hardware required for immunoimaging and discusses methods for quantitative analysis of multidimensional image stacks. As one of our featured articles for February, this overview is freely available to subscribers and nonsubscribers alike.

The issue also contains protocols from the same authors for a General Approach to Adoptive Transfer and Cell Labeling for Immunoimaging, Induction of an Immune Response for Imaging Antigen-Presenting Cell/T-Cell Interactions, In Situ Lymph Node Imaging and In Vivo Lymph Node Imaging.

The ability to image living cells and tissues has revolutionized our understanding of many biological processes. Being able to visualize what’s happening in these time lapse movies at an ultrastructural level would make them even more informative. But commonly used fluorescent proteins like GFP are not directly visible by electron microscopy (EM). Fluorescent nanoparticles or quantum dots can be visualized by EM, but targeting these to cytoplasmic proteins in living cells remains difficult. One method which does allow correlation between light microscopy and EM is fluorescence photoconversion, where observation of the fluorescent label causes the deposition of a reaction product that can be rendered electron-dense and directly visualized by EM. In the January issue of Cold Spring Harbor Protocols, Mark Ellisman and colleagues from UCSD’s National Center for Microscopy and Imaging Research provide a set of articles for this method in combination with a class of genetically encoded peptide tags that can be labeled in living cells by fluorophores bearing two appropriately spaced arsenic atoms (biarsenicals).

Correlated Live Cell Light and Electron Microscopy Using Tetracysteine Tags and Biarsenicals provides an overview of the technique, and protocols are provided for Labeling Tetracysteine-Tagged Proteins with Biarsenical Dyes for Live Cell Imaging and Fluorescence Photoconversion of Biarsenical-Labeled Cells for Correlated Electron Microscopy (EM).

New technologies and methods are spurring a renaissance in the study of organogenesis. Organogenesis, essentially the process through which a group of cells becomes a functioning organ, has important connections to biological processes at the cellular and developmental levels, and its study offers great potential for medical treatments through tissue engineering approaches. The January issue of Cold Spring Harbor Protocols features a method from Washington University’s Hila Barak and Scott Boyle for Organ Culture and Immunostaining of Mouse Embryonic Kidneys. The kidney is particularly interesting as it also serves as a model for branching morphogenesis. The protocol describes the isolation, culture and fluorescent immunostaining of mouse embryonic kidneys. As one of January’s featured articles, the protocol is freely available to subscribers and nonsubscribers alike.

The “Brainbow” strategy was originally developed in mice, as a system for labeling neurons in a variety of different colors, allowing one to follow multiple cells regardless of their proximity. Brainbow uses a construct that carries sequences for red, blue and green fluorescent proteins in tandem array, with two pairs of lox sites flanking the first two fluorescent proteins. Recombination occurs in the presence of the Cre recombinase, and one gets a variety of outcomes, resulting in the production of a red, blue or green label. When more than one copy of the Brainbow cassette exists within a cell, the primary tones can be mixed, providing more possible color combinations. This provides a powerful platform for studying neuronal morphology and cell movements. In the January issue of Cold Spring Harbor Protocols, Alex Schier and colleagues offer Multicolor Brainbow Imaging in Zebrafish. This protocol translates the system for use in zebrafish, which offer the advantages of easy visualization of transparent embryos and efficient generation of labeled subjects.

For those looking to add to their arsenal of laboratory techniques, Cold Spring Harbor Laboratory Press has just released a new series of Imaging manuals.

I had a hand in putting these books together, and I’m always pleased when we manage to publish books that I know I would have found incredibly helpful in my previous incarnation as a bench scientist. These two hit home as I was a postdoc in an imaging lab. While that was ten (10!!!) years ago, it’s almost shocking to realize that there weren’t any comprehensive lab manuals out there that really covered the whole of bio-imaging, from the basics of optics to the most current, bleeding-edge techniques. Consider that problem solved, courtesy of series editor Rafa Yuste.

The new series spins off from a previous set of publications. In 2000, CSHL Press published Imaging Neurons, based on a CSHL laboratory course. The book was a few years ahead of its time, and the methods had really caught on by the time the sequel, Imaging in Neuroscience and Development was released in 2005. Five years later, and there’s been far too many new applications developed to fit into one volume, hence the release of the new series.

Imaging: A Laboratory Manual is the flagship of the series. It offers all the basics: optics, confocal, multi-photon, lasers, cameras, staining cells, etc. The manual goes on from there though, through labeling and indicators to advanced techniques like photoactivation, light sheet imaging, array tomography, fast imaging, molecular imaging, superresolution imaging and every acronym you can think of (FRET, FLIM, FRAP, FIONA, PALM, STORM, BiFC, AFM, TIRFM to name a sampling). If you have a microscope in your lab or if you spend any time in your local imaging center, this is the book you need.

Imaging in Developmental Biology: A Laboratory Manual is the second book in the series, just released. We old-school developmental biologists used to have to look at fixed sectioned specimens taken from different time points, and try to piece together the big picture of what was really happening as an embryo developed. New techniques have revolutionized our understanding of dynamic processes, as they allow for real-time imaging, often over the entire course of an organism’s development. Like the preceding volume, the book starts with the basics, methods for visualizing development in laboratory standard model organisms (C. elegans, Drosophila, zebrafish, Xenopus, avians and mouse) and then step by step brings the reader to the cutting edge of imaging technology.

The supplemental movies from both books are freely available through Cold Spring Harbor Protocols. Look for a third volume in March, on Imaging in Neuroscience, which will offer an astounding 90-plus chapters for analyzing every aspect of the nervous system in detail.

Visualizing mammalian development presents an obvious problem: embryos must develop in utero. That makes them a lot more difficult to see under a microscope than a zebrafish or a frog that develops as a free-standing egg. Extensive work has been done to develop embryonic culture techniques for external development of mouse embryos, allowing imaging approaches to be applied. Early efforts by members of Scott Fraser’s lab (including myself) provided a protocol for growing d 6.5-9.5 mouse embryos on the microscope stage. The December issue of Cold Spring Harbor Protocols features Imaging Cell Movements in Egg-Cylinder Stage Mouse Embryos from Oxford University’s Shankar Srinivas. The article describes a method for isolating and culturing much earlier mouse embryos, as well as an approach for time-lapse imaging as those embryos develop. While cell movements can be followed using light microscopy alone, the increasing variety of transgenic fluorescent reporter mice makes studies of cell movement easier and more informative. As one of December’s featured articles, the protocol is freely available to subscribers and non-subscribers alike.

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