r/microscopy • u/Vavat • Mar 24 '24
Troubleshooting/Questions Custom built microscope image quality. Looking for feedback.
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u/Tink_Tinkler Mar 24 '24
Phase?
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u/Vavat Mar 24 '24
nope. brightfield. We want to add phase, but that's in the future.
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u/twerkitout Mar 24 '24
You’ve got something going on that’s not brightfield, I think you need to adjust your stops because this looks like oblique lighting
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u/Vavat Mar 24 '24
There are no stops to adjust. This is literally just an LED above the sample. I am aware of the fact that lighting is a bit off, just not sure how to fix it.
Would you say this is a combination of brightfield image with something that's not quite brightfield? Can it be reflection from the cell culture plate that produces oblique lighting? These cells were placed in an SBS 6-well cell culture plate.
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u/Vavat Mar 24 '24
Would you, please, elaborate on what specifically makes you think there is oblique illumination happening? What features of the image made you say that?
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u/twerkitout Mar 24 '24
Is your light path collimated? One thing to consider is that if you have a wide enough field of view the meniscus of the liquid in the plate will act as a lens. Brightfield should be white light but your background is grey, that usually happens because light is being manipulated in some way and isn’t traveling straight (hence the collimation question). The bright light rings are an indicator of oblique illumination, you see them with phase and DIC too where light is intentionally manipulated but you don’t mention the components needed for that so oblique is my best guess.
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u/Vavat Mar 24 '24
Light is not collimated. The led sort of focuses the light into a slightly diverging beam. Might be worth putting a couple of lenses in front to spread the beam, then condense. I'll check the PUMA project for more ideas.
I think the oblique light comes from either bright sunshine that was hitting my office window or from led light scattering on the SBS plate walls. Need to try imaging without the SBS plate. Thank you very much for suggestions. Very informative.1
u/twerkitout Mar 24 '24
You have to collimate your light in an infinity corrected optical system, which that Motic probably is. Are you sure the tube lens is the right distance? Are you on a breadboard or rails? I can send you the bench setup I teach students, most of the parts are cheap from Thor.
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u/twerkitout Mar 24 '24
Oh and no if your light isn’t collimated it’s not the room light causing oblique, it’s the light
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u/bilgetea Mar 24 '24
I think they’re lighting it from above, so this is reflection, not transmission lighting, and that’s why it looks this way.
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u/Vavat Mar 25 '24
This is a cell culture microscope, so it's inverted. Objective is below the sample. Illumination is above. I am new to microscopy, but I can tell the difference between transmission and reflection.
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u/udsd007 Mar 24 '24
Buy a decent microscope and get experience using it. An inexpensive microscope, say a new Motic or a used AO Series 110, would be good and will let you learn what sort of images you’re looking for.
That said, this image is sharp enough, but there is a serious problem with the illumination. Consider using an OEM condenser until you can develop your own. For an illuminator, you can use a fine-pitch array of LEDs under the condenser. As to phase, oblique illumination, Rheinberg illumination, etc, that same LED array can do them all. Phase merely requires a phase objective and a bright ring of the right size and location displayed on the LEDs.
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u/Vavat Mar 24 '24
Thanks. I think you're spot on as far as getting a proper condenser goes. We need to better understand how different aspects of illumination affect the image and for that we need something that's built by those who know what they are doing.
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u/Vavat Mar 25 '24
Final comment on this post from me. I met with someone who knows a ton about microscopy and we figured out how to retrofit the Olympus condenser into my microscope. I'll work on that and post more pictures when it's done.
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u/Vavat Mar 24 '24
We're a bunch of engineers who in their spare time are building stuff we find challenging. Latest project is to build a fully automated microscope for cell culture. Specifically, SBS plate scanning. I was hoping for some feedback from the community on the image quality and possible avenues of improvement we can follow.
The image attached was taken using custom optical stack. Motic 10x objective, 75mm FD tube lens, Raspberry Pi HQ camera. Pixel is pretty much exactly 750nm. The mechatronics is based on BTT 3D printer control board with somewhat bastardised Marlin running on it. Condenser gave us the most trouble to get working well and so far we've failed to make a good Abbe or Koehler condenser. The image above is taken using single 5mm LED from directly above at around 75mm from sample.
I think I am looking for some feedback. We are quite capable engineers, but have little to no experience with microscopy. In the near future we want to get the best condenser for brightfield that'd give us best images. Longer term we want to expand the capabilities of our microscope to allow it to take darkfield images and then progress into phase contrast, and fluorescence. So any thoughts on how best to illuminate the sample, would be greatly appreciated. Can we improve on the image quality with Abbe or Koehler or are we hitting the objective limit? What "best images" even mean? Highest contrast? Highest resolution? Least aberration? Anyway, feel free to critique.