Laser scanning confocal microscopy uses focused laser light to scan an image, and record signals from fluorescent probes in cells and tissues. When fluorescent probes are illuminated by a specific wavelength of light, they absorb some of this light, and re-emit it at a longer wavelength. For example, jellyfish green fluorescent protein absorbs blue light, and fluoresces green. There are many variants of fluorescent probes in the visible spectrum, which can be genetically encoded or used as stains against specific proteins, and these can be combined to define the location and size of structures within cells and tissues. Confocal microscopy has a major advantage over standard microscopy, since the illuminating light is limited to a small spot at a time, reducing noise by eliminating the detection of light that is out-of-focus, e.g. not coming from the spot that is being illuminated. Using mirrors, the microscope focuses specific wavelengths of laser light onto a very small section of the sample, and detects photons emitted from the fluorescent probes at that location for a very short period of time (a few microseconds), before moving to an adjacent location on the sample. By scanning the sample, an image is generated.
Researchers at the University of Nevada, Reno are using laser scanning confocal microscopy to detect and measure very small subcellular structures in tissues, as well as follow changes in cellular dynamics over time, using fluorescent probes to detect specific biological signals. The upright Leica SP8 confocal microscope is capable of simultaneous collection from up to 3 detection channels (two PMTs, one HyD) using multiple laser lines (diode 405, blue 488, green 552, red 638 nm). The confocal is configured with a conventional tandem scanner (8 kHz), a super Z-galvo stage type S, multiple objectives (10x, 20x, 25x, 40x, 60x), and stands on an anti-vibration table. The confocal is connected to a computer workstation with 30 inch monitor and Volocity imaging software, which provides high-performance cellular and imaging analysis.