Optimizing laser source operation for confocal and multiphoton laser scanning microscopy.

Confocal laser scanning microscopy (CLSM) and multiphoton laser scanning microscopy (MPLSM) are methods both widely used by life-sciences researchers for imaging fluorescently labeled live cells and fixed tissue specimens. Key to the success of both CLSM and MPLSM is the application of a suitable laser source, namely one that provides sufficient average or peak power at the correct wavelength to excite fluorescence. High stability of the laser source output is required for three-dimensional imaging, time-lapse studies of live cells, and quantitative studies and inter-experiment comparisons. The laser technology associated with the design of such lasers is mature, yet is unfortunately rather complex. This complexity can be off-putting for the life-sciences researcher who needs to optimize the system for the best possible images, but this apprehension can be overcome by understanding the function of the system components. This unit summarizes the optimization of the most commonly used laser sources for CLSM and MPLSM, including power and wavelength tuning and methods for cleaning optical components.