Interference Reflection Microscopy (IRM) uses reflected polarised light is normally used to study cellular interfaces contact sites on a glass surface without using fluorescent labels. Confocal microscope lasers are polarised, which makes them ideal for IRM. You can acquire fluorescence images at the same time as reflection images but with the White Light Laser (WLL) on the LMCB SP8 a notch filter will be needed to stop reflected light being detected in the fluorescence channels.
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Setting up reflection imaging on the SP8
Select a detector for your reflection image. It would probably be best to use a PMT since the laser light reflected off the coverslip could well be too bright for a HyD. Keep the gain reasonably low to avoid saturation
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You must set the Sequential Scan to Between Frames or Between Stacks as the Fluorifer Disc cannot rotate quickly enough to switch between channels between lines
You should end up with a reflection and a confocal fluorescence image like the ones below.
