Lab Manual Provides “Gory Detail” for Successful Imaging

Imaging in Neuroscience, the manual from our imaging series that focuses on methods for studying neurons and their circuits, is given a positive review in the current issue of The Quarterly Review of Biology.

 “The most important feature of the manual is the protocol[s] provided,” write Lynne Oland and Patty Jansma. “These are clearly written with the intent of providing the gory detail needed to actually use the protocols successfully, and most chapters include troubleshooting hints, which are most helpful.”

 Oland and Jansma feel that the protocols on glial cells and brain pathology “will make the manual especially useful.” Some of these protocols are available online from Cold Spring Harbor Protocols: For example, check out how to visualize microglia in the mouse cortex, label astrocytes with sulforhodamine 101, and study neural networks in mouse models of Alzheimer’s disease. For more information on the manual, click here.

 

Details of Development

A method called high-resolution episcopic microscopy (HREM) produces remarkably detailed images of embryos, allowing one to visualize fine structures such as the whisker pores on the snout of a 14.5-day-old mouse embryo. This month’s issue of Cold Spring Harbor Protocols features a protocol describing the first step in the HREM procedure: embedding embryos in preparation for imaging. It is written by Tim Mohun and Wolfgang Weninger, who developed the HREM technique.

In HREM, embryos are embedded in plastic that contains fluorescent dyes. Sequential images of the block face (i.e., “episcopic” images) are captured before sections are removed from the block with a microtome. This eliminates the need to align images from individual sections; furthermore, there are no distortions due to sectioning, section stretching, or section mounting. A typical HREM volume data set consists of about 1000-4000 block-face images. These images are put together using computer software to create a 3D model of the internal and external structures of the embryo.

 

3D Imaging of Embryos is Featured in CSH Protocols

Until recently, a common technique for creating a 3D image of an embryo was to slice it into hundreds of thin sections, photograph each section, and then computationally recombine the images to produce a 3D representation of the embryo.  But during this process, the specimen may become deformed, and information about the alignment of the sections – the third dimension – is lost.

Optical projection tomography (OPT) overcomes these problems, as Laura Quintana and James Sharpe (Centre for Genomic Regulation, Barcelona) explain in a featured article in the latest issue of Cold Spring Harbor Protocols.  OPT is ideal for analyzing the morphology of fixed embryos – especially for analyzing mutant phenotypes, for developing anatomical atlases, and for analyzing gene expression patterns.

 

CSH Protocols Features Methods for Imaging Kidney Development

The adult mouse kidney begins to develop at embryonic day 10.5, when the epithelial ureteric bud evaginates from the Wolffian duct and grows into adjacent metanephric mesenchyme.  Over the course of several days, the ureteric bud repeatedly branches, giving rise to the ureter, pelvis, calyces, and renal collecting ducts of the adult kidney.

The kidney can develop in culture, from the first stage of ureteric bud evagination through the first 8-10 rounds of branching.  These processes can therefore be visualized through time-lapse imaging, providing a greater understanding of normal kidney morphogenesis and how genetic perturbations affect kidney development.

This month’s issue of Cold Spring Harbor Protocols, out today, features an article that presents the general concepts of imaging kidney development and describes genetically modified mice that express fluorescent proteins useful for visualizing different cell lineages and developmental processes in these organ cultures.  A detailed step-by-step protocol for dissecting, culturing, and imaging embryonic mouse kidneys is also published in the issue.  Both articles were written by Frank Costantini (Columbia University Medical Center), Shankar Srinivas (University of Oxford), and colleagues.

 

Neuroscience Tools with Extraordinary Precision: Quantum Dots and Microbial Opsins

As a preview to the forthcoming laboratory manual Imaging in Neuroscience, due in May, the current issue of Cold Spring Harbor Protocols highlights two articles on neuroscience imaging techniques.  The articles are freely accessible here and here.

Monitoring Individual Molecules with Quantum Dots

The first article details the use of nanometer-sized quantum dots (QDs) to track the motion of individual membrane molecules over time.  QDs possess strong fluorescence and photostability, permitting extended recording times compared to other methods.  In the article, authors Sabine Lévi, Maxime Dahan, and Antoine Triller (Ecole Normale Supérieure, Paris) provide step-by-step methods to stain neurons with QDs and to track QD-labeled molecules using single-fluorophore epifluorescence, as well as guidance for interpreting the data and reconstructing the trajectory of individual QD-labeled molecules.  These methods have been successfully used to follow the diffusion of individual glycine receptors, GABA receptors, NMDA receptors, lipid raft markers, glycophosphatidylinositol-anchored green fluorescent protein (GPI-GFP), and other molecules of interest.

Studying Specific Neural Activities with Microbial Opsins

The second article describes characteristics of various microbial opsins that are used in optogenetics.  Optogenetics is a revolutionary technology that combines optics and genetics to study very specific events, such as action potentials, in their natural context—even in freely moving mammals.  Microbial opsins are light-sensing proteins that regulate ion fluxes to control biological activities, and their corresponding genes can be expressed in mammalian neurons to enable millisecond-precision optical control of neural activity.  The authors of the article, Karl Diesseroth and colleagues (Stanford) and Peter Hegemann (Humboldt-Universität, Berlin), describe the diversity of microbial opsin genes, including those for bacteriorhodopsins, proteorhodopsins, halorhodopsins, and channelrhodopsins, and the structure-function properties of their corresponding proteins.  This overview will be useful to those looking to employ optogenetics as a research tool.

 

Using μMRI to Construct Atlases of Animal Development

The cover of the March 2011 issue of Cold Spring Harbor Protocols, out today, features several striking images of mouse and quail embryos.  The method used to produce the images, microscopic magnetic resonance imaging (μMRI), is a noninvasive imaging technique that permits the visualization of regions deep within embryos that are inaccessible using optical methods.  During μMRI, the specimens remain in near-physiological conditions, remaining anatomically unperturbed.  The method is ideal, therefore, for generating developmental atlases of these organisms.

Quail embryos in a "relaxed" posture used to construct a μMRI-based developmental atlas. (©2011, CSHL Press)

In an accompanying article, the authors, Seth Ruffins and Russell Jacobs (Caltech Biological Imaging Center), describe the preparation of specimens for μMRI and appropriate applications of μMRI for developmental biology, including the construction of atlases.  Using these methods, they have successfully generated digital anatomical atlases of both quail and mouse development (see the Caltech MRI Atlases).  These atlases, and others constructed using μMRI, will be useful references for developmental biologists, providing identifiable anatomical landmarks and standards for comparison.