Practical Confocal Microscopy and the Evaluation of System Performance

• Published in: Methods (San Diego, Calif.) Vol. 18; no. 4; pp. 447 - 458
• Main Authors: Zucker, Robert M., Price, Owen T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.1999
• Subjects: Boron Compounds; Fluorescent Dyes; Microscopy, Confocal - instrumentation; Microscopy, Confocal - methods; Microscopy, Confocal - standards; Microscopy, Fluorescence - instrumentation; Microscopy, Fluorescence - methods; Microscopy, Fluorescence - standards; Microspheres; Neurosciences - instrumentation; Optics and Photonics; Reference Standards
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1006/meth.1999.0812

The laser scanning confocal microscope has enormous potential in many fields of biology. Currently there is a subjective nature in the assessment of a confocal microscope's performance by primarily evaluating the system with a specific test slide provided by the user's laboratory. To achieve better performance from the equipment, it is necessary to run a series of tests to ensure that the optical machine is functioning properly. We have devised these methods on the Leica TCS-SP and TCS-4D systems. Tests measuring field illumination, lens clarity, laser power output, dichroic functioning, spectral alignment, axial resolution, laser power stability, machine performance, and system noise were derived to test the Leica laser scanning confocal microscopy system. These tests should be applicable to other manufacturers' systems as well. The relationship between photomultiplier tube (PMT) voltage, laser power, and averaging using a 10-μm-diameter test bead has shown that the noise (coefficient of variation of bead intensity, CV) in an image increases as the PMT increases. Therefore increasing the PMT setting results in increased noise. For ideal image quality, it appears that it is better to decrease the PMT setting and increase laser power, as noise generated by high PMT settings will reduce the image quality far more than the bleaching caused by higher laser power. Averaging can be used to improve the image at high PMT values, provided the sample is not bleached by repeated passes of the laser.