DIY Epifluorescence Macroscope

[llamero]

Hey Microscopists,

I recently designed and built an epifluorescence macroscope centered around the Olympus XLFLUOR 4x/0.28NA objective and Kinetix camera for both fixed and live imaging. It uses the high power CBT-90 LEDs driven at 27 amps as the excitation source to get up to 690 mW (29 mW/mm^2) of excitation light onto the sample plane for very high temporal resolution imaging.

Preliminary tests showed that the system is working very well for us so I figured I would share the CAD files with anyone who may need a similar setup: https://github.com/Llamero/DIY_Epifluorescence_Macroscope

Specifically, this macroscope has a 4.7 mm x 4.7 mm FOV and the optical resolution is pixel limited with the 10 MP Kinetix camera; having pixels that are equal to 1.5 µm x 1.5 µm at the sample plane. The end result is a nearly 5 mm FOV with subcellular resolution!

If high temporal resolution is not needed, the custom LED source can be replaced with any standard microscope illumination source that uses a 3 mm liquid light guide, and the Kinetix camera could be replaced with an astronomy camera with a sensor that is at least 22 mm x 22 mm, such as this camera: https://www.bhphotovideo.com/c/product/1417197-REG/starlight_xpress_trius_sx_46_usb_hub.html

If you have any questions, please feel free to ask.

Cheers,
Ben Smith

P.S.
The development of this scope and illuminator was funded by the following grant: NIH P30EY003176

Also, definite shout-out to Thorlabs for selling screwball parts such as the 60 mm filter cube and tube lenses with massive apertures, which make this sort of system even possible.

[llamero]

Here is the first test image we got off the scope; this is an image of
endogenous tdTomato fluorescence in a PFA fixed Opn4-Cre/+; floxed
tdTomato/+ P4 mouse retina (link to full resolution raw tiff): Opn4-Cre - tdTom P4 retina.tiff - Google Drive

The image was taken in dynamic range mode (16-bit) with a 50 ms exposure.
The retina wasn’t perfectly flat, so some parts are in focus while others
are a bit out of focus, which is a testament to the high axial resolution
of the 4x objective.

[sarah]

Hi Ben,

Thanks for sharing this - I know it’s been a few years, but really appreciate the CAD files and the detail in your writeup.

I’ve been trying to reproduce the design and I’m running into some conceptual confusion around the lens train. My assumption going in was that the excitation light would be focused at the back focal plane of the objective (as in a standard epi configuration for Köhler-style illumination), but looking at your layout it seems like the light is entering the objective collimated rather than focused at the BFP. Could you clarify the intent there? Specifically, what motivated that choice, and is the BFP being deliberately bypassed or am I misreading the optical path?

Thanks in advance!

Sarah