Confocal Microscopy System Performance: Laser Power Measurements

Abstract

The reliability of the confocai laser-scanning microscope (CLSM) to obtain intensity measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. The laser power test appears to be one of the most useful tests to quickly evaluate if a system is misaligned or functioning sub optimally by recording insufficient laser power readings. The test using a power meter can indicate if the system is aligned properly up to the plane of excitation on the stage, or if the machine has a defective component (i.e. a dying laser, or a defective fiber). In our experience, without sufficient power throughput in the system, the PMT voltages will have to be increased to high values to visualize fluorescence derived from specimens, which will introduce reduced image quality. In addition the cause of the decreased laser power may result in other problems i.e. laser instability, loss of axial resolution, and increased noise.