Hi uForum! Longtime lurker, firsttime poster, etc. etc.
I’ll preface this by saying this is more of a thought and educational exercise for me, at least until I convince someone to throw some grant money my way
In my pursuit of automated, high-throughput, high-plex (>5 colors) widefield fluorescent imaging of slides, I continue to run into a rate limiting step of the long switching times (250-1000ms) for dichroic filter turrets or linear sliders. The rest of the equipment on the TTL chain (excitation, filter wheels, xy(z) stage, camera) sit waiting for that cha-chunk of the dichroic changer.
Removing two dichroic changes per field saves about 1 second, multiplied by however many fields are being acquired per sample, so time savings scale geometrically with magnification.
My question is this: cost being no object, are there any fundamental issues precluding the creation of useful transmitted fluorescent scopes?
There are two key assumptions that make this hypothetical instrument temporally superior to epiilluminated scopes:
- Each field/tile must be acquired with all colors before the next field/tile is imaged.
- The instrument/user is already using a fast ‘emission cleanup’ filter wheel in conjunction with the multipass dichroic. Meaning each color requires a 30-50ms wheel change.
I understand that phosphor-coated LED light sources are susceptible to secondary emission via emitted fluorescence of the sample propagating back into the excitation source. I’ve only seen this with ‘white light’ LEDs and am unsure if it occurs in monochromatic light sources using light pipes. This should be solvable with ND filters in the excitation path or using non-excitable light sources such as a lamp.
Additional benefits would include the availability of all emitted light. I dream of using two filter wheels (one populated with shortpass filters, the other with longpass) for flexible emission selection, useful for broad wavelength collection with dim fluors, or for separating short and long stokes-shift fluors on the same excitation wavelength from each other e.g. 488 and PerCP.
Happy to hear how silly of a thought this is, or if it’s already out there somewhere and I missed it.