Focus On Light Microscopy.

It would be hard to argue that light microscopy has not played a highly influential role in science since its invention in 1595. However, the past 35 years have seen impressive developments in light microscopy and its associated analytical techniques. The modern light microscope is now a highly evolved opto-electromechanical device. The “hottest” developments have occurred over a relatively short time frame, the most notable being the development of several new “flavors” of super-resolution microscopy for which the Nobel Prize was awarded recently. These advances have allowed the light microscope to be used as a powerful quantitative research tool. In this issue, there are 4 articles that give readers a sense of the breadth of modern light microscopy. These range from a mostly mathematical paper, A Quantitative Measure of Field Illumination, to a review of the current state of confocals, Any Way You Slice It—A Comparison of Confocal Microscopy Techniques. There is a research paper on spindle microtubules called Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes. Rounding out the group is a paper on genetics that uses imaging in a novel way: Angiogenesis QTL on Mouse Chromosome 8 Colocalizes with Differential β-Defensin Expression. Brief summaries of the papers are indicated as follows: A Quantitative Measure of Field Illumination is a paper that produced the first mathematical algorithm for scoring the evenness of the illumination in any imaging device. Adaptive learning was used to construct the algorithm that was coded in MatLab. There were 89 test scopes, which were collected during an Association of Biomolecular Resource Facilities-sponsored project. To verify the algorithm, 33 additional scopes of various modalities were used. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes addresses the quantitative characterization of kinetochore microtubule flux during metaphase and anaphase A in Drosophila male meiois. The method includes transgenic expression of GFP-tubulin, Drosophila melanogaster spermatocytes primary culturing, confocal laser scanning-based photobleaching/image acquisition, and image analysis using the open source platform NIH ImageJ. Any Way You Slice It—A Comparison of Confocal Microscopy Techniques provides an up-to-date guide detailing the various modes of single photon confocal microscopes, which currently are available commercially. Newbies to confocal microscopy, who are in the market for a system or are trying to determine which platform is most suitable for their project, will find this review most useful in guiding their decision. The “New Developments” section brings the reader quickly up to date with the latest technological innovations and improvements. Angiogenesis QTL on Mouse Chromosome 8 Colocalizes with Differential β-Defensin Expression used a novel method to follow angiogenesis in mice. Three-dimensional fluorescently (nuclei)-labeled Matrigel-embedded explants were collected and analyzed. Composite images were processed with large spectral filters and a watershed routine to distinguish overlapping nuclei. Semiautomatic image processing and object tracking procedures were developed, which are applicable to other explant assays. This assay was chosen with the aim of increasing sensitivity to detect angiogenesis response alleles by decreasing the number of genetic factors that might affect the measured phenotype.