Image faded color issue high resolution optics

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1

Hi guys,

I have some issues with color in my microscope images. I expected this to come from a calibration issue in the software but I continue to have the same problem even after this beta fix (thanks for it !).

To explain : I’m taking pictures of algae with the high resolution optics mounted with a 40x objective. I also even glued an iris on the illumination to get finer light adjustment.
I tried multiple illumination settings and calibrations but nothing do, I get only faded green color in my images (when I get crispy green color on a commercial microscope).

2021-04-19-infinite_plan_objective_4
2021-04-19-infinite_plan_objective_43280×2464 997 KB
test 3
test 33280×2464 960 KB

On these pictures exposure is approximately around 600 in the Openflexure software, that’s the value I usually get the better results with.

I also observed that when take an image with a stock Pi camera objective, I get a green coloured image :

image test
image test3280×2464 701 KB

If anyone had some tips or fix to get images with a better color that would be really helpful !

EDIT :
I performed a white calibration with the stock Pi camera optics on a white piece of paper and managed to get some color on the pic. I did not touch the white calibration and screwed the camera on the high res optics, still no color… (see pic below for the white calibration and cells image)

White calibration green
White calibration green3280×2464 680 KB

Capture after white calibration
Capture after white calibration3280×2464 660 KB

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I think that this may be your illumination LED. The green image that you get when you use the camera to photograph the room suggests that the microscope calibration has had to enhance the green channel a lot to get the white balance in the microscope. It would have to do that if your LED is not giving much green light. With green missing from the LED it will be hard to get the algae looking green.
A ‘white’ LED is actually a blue LED with a phosphor on top that absorbs some of the blue and emits yellow. Often the blue is a rather short wavelength blue, and there is not much phosphor on top, as the conversion from blue to yellow reduces the total light output. That situation gives something that looks white, but actually emits very little green light at all. LEDs labelled ‘warm white’ generally have less of this problem, but ideally you need to look at the specified spectrum emitted by the LED.

3

Hi William and thanks for the answer !
That’s a factor I did not consider in my investigation !
I use 5mm super bright LEDs from Adafruit, I’ll look at another brand with a spectrum better fitted for my needs.
I will post new pics and hope this fixes my issue.

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