Evaluation of Confocal Microscopy System Performance: Applications for Imaging Morphology and Death in Embryos and Rerpoductive Tissue/Organs

Abstract

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. It is remarkable that procedures to test the performance of these machines are not done routinely by most investigators and thus many of the machines in operation are working at levels of sub optimal performance. When these machines are checked, it is usually a subjective assessment of performance accomplished by primarily evaluating the system using a specific test slide provided by each user’s laboratory. We have devised test methods on the Leica TCS-SP and TCS-4D systems to ensure that these machines were working properly and delivering their correct performance. Tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, overall machine stability, and system noise. It is anticipated this type of data will help to set performance standards for confocal microscopes and eliminate the current subjectivity in evaluating the CLSM. These tests will also serve as a guide for other investigators to insure that their machines are working correctly and providing data that is accurate and having the necessary resolution, sensitivity and precision.