Hello everyone,

I'm currently working on setting up a simple total internal reflection fluorescence (TIRF) microscope using a few lenses (f1 = -50 mm, f2 = 300 mm, f3 = 400 mm) and a 532 nm laser. The lenses f1 and f2 used as a beam expander, while f3 is used to focus the laser onto the back focal plane of the objective.

This setup works well as an epifluorescence microscope. However, when I adjust the reflection mirror to change the incident beam angle to achieve TIRF, I observe some bright scattering patterns. I'm unsure of the source of this scattering and am wondering if there might be an issue with my setup.

Any insights or suggestions would be greatly appreciated.

Thank you!