Microscopy at Nijmegen

Operating principle of a confocal microscope: Laser light illuminates a single pixel on the sample at a time. An APD detector picks up photons only from the focal plane of the sample and only at the wavelength of the fluorescence. The laser comes from below the sample cover slip. The sample sits on top of the cover slip. This allows the use of oil immersion lenses that touch the bottom side of the coverslip.

Images from a microarray scanner. The microarray scanner uses the same operating principle as the confocal microscope. However, the microarray scanner has a computerized x-y stage that allows large areas to be scanned. Computer software then generates images based on fluorescence intensity at each position on the sample. Microarray scanners do not use oil immersion optics, this reduces the spatial resolution but makes imaging and sample preparation easier.

Sample prep for the confocal microscope

Thin transparent glass or quartz or fused silica cover slips are required. The optimum thickness of cover slips for the confocal is 175 µm (175 +/- 25 micron thickness is perfect). Currently the only cover slip holder is for round 24 mm cover slips.

Sample prep for the microarray scanner

In general, cover slips for the microarray scanner should not we wider than 20 mm
Si wafers work okay because imaging is done from the top surface (and sample is on the top surface).
Thickness of wafer or coverslip is not critical.