Bench Marks

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.

4-dimensional live cell imaging has gone from being a rare technique used only by cutting-edge laboratories to a mainstream method in use everywhere. While more and more labs are becoming comfortable with the equipment and protocols needed to collect imaging data, performing detailed analyses is often problematic. The application of computational image processing is still far from routine. Researchers need to determine which measurements are necessary and sufficient to characterize a system and they need to find the appropriate tools to extract these data. In Computational Image Analysis of Cellular Dynamics: A Case Study Based on Particle Tracking, Gaudenz Danuser and Khuloud Jaqaman introduce the basic concepts that make the application of computational image processing to live cell imaging data successful. As one of the featured articles in December’s issue of Cold Spring Harbor Protocols, it is freely accessible for subscribers and non-subscribers alike.

The article is adapted from the new edition of Live Cell Imaging: A Laboratory Manual, now available from CSHL Press.

With the recent progress in understanding epigenetic mechanisms, methods for profiling patterns of DNA modification have become important tools for analysis of gene regulation. DNA methylation, in which cytosine is modified to form 5-methylcytosine, is a well-characterized epigenetic modification essential for normal development in plants and mammals. In the December issue of Cold Spring Harbor Protocols, Jon Reinders presents Amplification of Bisulfite-Converted DNA for Genome-Wide DNA Methylation Profiling. This method utilizes the treatment of DNA with sodium bisulfite, which converts unmethylated cytosine to uracil (5-methylcytosine is not converted). This is followed with PCR amplification, where the uracil amplifies as thymine, creating a C-to-T transition. The genome can then be analyzed for these transitions using an array-based platform. Reinders protocol mitigates the major issues with bisulfite conversion (DNA fragmentation and poor reproducibility) and reduces bias during the amplification step. While the protocol is optimized for use in Arabidopsis, it can potentially be adapted for use in other organisms.

Live cell imaging techniques are driving a revolution in biological research. Instead of viewing dead tissues and cells fixed at a particular stage of activity, scientists can now visualize dynamic changes as they happen, permitting a better understanding of complete processes. The revolution has been fueled by the implementation of genetically encoded fluorescent proteins, the subject of the 2008 Nobel Prize in Chemistry.

The diverse array of applications benefiting from fluorescent proteins ranges from markers targeted at organelles and protein fusions designed to monitor intracellular dynamics to reporters of transcriptional regulation and in vivo probes for whole-body imaging and detection of cancer. Fluorescent proteins have enabled the creation of highly specific biosensors to monitor a wide range of intracellular phenomena, including pH and metal-ion concentration, protein kinase activity, apoptosis, membrane voltage, cyclic nucleotide signaling, and tracing neuronal pathways. In the December issue of Cold Spring Harbor Protocols, David Piston and colleagues present Fluorescent Protein Tracking and Detection: Fluorescent Protein Structure and Color Variants, a comprehensive overview of the wide variety of fluorescent proteins that are currently available. The article features more than twenty movies of different fluorescent proteins in action and is a great primer for planning imaging experiments. As one of December’s featured articles, it is freely available to subscribers and non-subscribers alike.

In addition, the same authors have also contributed Fluorescent Protein Tracking and Detection: Applications Using Fluorescent Proteins in Living Cells. This article provides some general tips for the practical aspects of using and imaging enhanced green fluorescent protein (EGFP) and newer members of the color palette, as well as quantitative imaging of fluorescent proteins and imaging of several fluorescent proteins at the same time. Finally, an overview is provided for the different types of biosensors that have been derived from flourescent proteins.

Both articles are adapted from the spectacular new manual, Live Cell Imaging: A Laboratory Manual, Second Edition which is due out by month’s end.

CSH Protocols December Cover