Evaluation of confocal microscopy system performance.
The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. When tests are made to evaluate the performance of a CLSM, the usual subjective assessment is accomplished by using a histological test slide to create a "pretty picture." Without the use of functional tests, many of the machines could be working at suboptimal performance levels, delivering suboptimum performance and possibly misleading data. To replace the subjectivity in evaluating a confocal microscope, tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, scanning stability, photomultiplier tube quality, overall machine stability, and system noise. These tests will help serve as a guide for other investigators to ensure that their machines are working correctly to provide data that are accurate with the necessary resolution, sensitivity, and precision. Utilization of this proposed testing approach will help eliminate the subjective nature of assessing the CLSM and allow different machines to be compared. These tests are essential if one is to make intensity measurements.
Zucker
Robert M
RM
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC, USA.
D000818:Animals; D002138:Calibration; D002460:Cell Line; D005456:Fluorescent Dyes; D007834:Lasers; D008029:Lighting / Q000295:instrumentation; D018613:Microscopy, Confocal / Q000191:economics / Q000295:instrumentation / Q000379:methods; D008863:Microspheres; D055095:Optics and Photonics / Q000295:instrumentation; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012984:Software
D005456:Fluorescent Dyes