I just wanted to post about a pitfall with widefield LED light sources here that I haven’t noticed much discussion around.
The issue is bleedthrough from the widefield illuminiation path into the fluorescence images due to stokes-shifted re-emission from the widefield illumination LED.
When taking epi-fluorescence images, some of the fluorescence excitation light travels through the widefield lightpath and hits the widefield illumination LED (if the light path is not shuttered). Due to the phosphors on the LED there will be a stokes-shifted emission that travels back through the transmitted light lightpath and will pass through the emission filter. As a result, one will get an additive mixture of the desired fluorescence image and a transmitted light image. The degree of how strong this is depends on a number of factors (condenser and field stops, wavelength, additional filters) and can range from very subtle to glaringly obvious.
I first noticed this at my previous job and it was not something I was aware of (neither were most of my colleagues). To alleviate this, we placed neutral density and low-pass filters into the transmitted light path. The ND filters will attenuate the light getting to the LED and the reemiited light. If the ND filters are strong enough (we used several), the bleedthrough can be reduced to a level were it becomes insignificant (the LED brightness will have to be cranked up for transmitted light images).
The idea with the low-pass filter was that longer wavelengths may not excite the phosphors in the LED (incoclusive, would need more testing).
The reason I wanted to bring this up and create awareness of it is that I just communicated with someone who returned from a multi-week research visit elsewhere where she spent many weeks acquiring images that have this same issue.
At the facility I work at currently we don’t have a microscope with such a setup, but I’d still be curious to hear about workarounds other than ND filters.