TissueCyte Beam Alignment: Primary Dichroic

The TissueCyte imaging dichroic is the final reflective element in the excitation path. Infrared laser light is reflected into the objective by the imaging dichroic. The fluorescence emitted from the sample passes through the imaging dichroic and onwards to the PMTs. The dichroic needs to be 45 degrees with respect to the objective back aperture. Should the imaging dichroic need alignment, do the following:

• Place a microscope slide on the metal piece into which the PIFOC is coupled. The slide should cover the aperture in the metal plate.
• The front surface of the slide will act as a reflective surface and you will use the light coming off it to adjust the imaging dichroic. If the reflected beam is too dim, you increase the laser power or even place a 1 inch mirror on top of the slide to reflect all the light.
• Remove the left panel of the enclosure with a 4 mm hex key.
• Unplug the X and Y mirror output cables from the scan control box. This ensures the scanners are positioned at the middle of their range.
• Set the laser to visible wavelength (e.g. 780 nm) and set the power to a fairly low value using the LCVR.
• Open the laser shutter.
• The beam will strike the slide or mirror placed upon it and then be reflected back along the path.
• If the imaging dichroic is aligned, the return beam will coincide with the out-going beam. You can judge this by observing the location of the return beam on the mirrors in the path.
• If the dichroic needs alignment, unlock the adjustment knobs on it by loosening the small set screws. Adjust the dichroic. Lock the setscrews.
• Replace the scanner control cables, replace the left panel, remove the slide from the light path.