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mtorkashvand / compact-flourescent-microscope

Low-Cost Compact Flourescent Tracking Microscope
MIT License
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Improve GFP image quality #2

Closed SinRas closed 1 year ago

SinRas commented 1 year ago

Steps to improve GFP image/video quality.

@mtorkashvand are there troubleshooting steps that you have in mind to add here? e.g. what would be the steps if we want to work on this.

mtorkashvand commented 1 year ago

Jeremy sent us 3 strains. QW1602: In this strain RIM is labeled with GCaMP6. Note: it is extrachromosomal, you want to pick non-muv worms QW1657: AVA, AVD, AVE, RIM, RIV, AVB, AIB and mec-4 chrimson QW2323: Intestinal GCaMP6

we can look at the scape response in QW1657, stimulating it with the optogenetic LED we can look at QW1602 for the single neuron tracking (it is supposed to be bright) QW2323 should also be bright. this could be used to see if GFP signals need improvement

mtorkashvand commented 1 year ago

I tried all 9 combinations of the 3 excitation and 3 emission filters that we had. I chose one set that had the lowest bleed through. But there is still a possibility of not blocking the LED's output which are above 1000nm in wavelength.

we have already ordered a new excitation filter that hopefully blocks >1000nm as well.

mtorkashvand commented 1 year ago

To test the filters, we use an SM1 tube to mount only the following parts: camera <- emission filter <- excitation filter <- LED we see that some blue light passes through the filters even though we expected the blue light to be blocked. after looking at the light pattern, it seemed that the edges are defective.

img_3175_720

we added a SM1A6 adapter to block the light from the edges. this resulted in a significant reduction of light captured by the camera

but when we put everything back into the system, and tried to image some worms on a plate, we again got some background green light (as if the agar was fluorescing) and we observed that the less transparent a sample is, the more green light we receive on the camera.

To make sure it is not something that we only observe on this system. I used our confocal imaging system. I use an agar pad and in the widefield mode, turned on the 488nm laser line. I utilized GFP filter set but I observed some background green light which confirmed it does happen!

Our solution is to use more transparent samples. using agarose pads or agar pads with less peptone (we don't grow bacteria on them so it is fine)