Illuminator: A “high CRI” white LED and an aspheric collector lens to roughly collimate the beam. I also made a little bayonet mount where I can fit field stops.

For the tube lenses, I would have expected the Thorlabs achromats to be best when operated in their design configuration with the image at the focal distance from the lens - which you get with an infinity corrected objective. But that is moving in the opposite direction from your observation - a larger distance of 75mm for your lens.

It is really interesting to see in this and other threads people really looking into the detail of the performance of their microscopes. The overall system works well enough that the optical quality of different objective lenses becomes apparent. With the motorized focus and scanning the microscope has access to all of the information that is necessary to characterise its own performance. A self-check routine is a goal of current developments of the software. I don’t think that evaluation of field curvature is planned initially, but it should be possible to quantify field curvature and tilt, and even identify the presence of aberration at the edges that is not just a different focus but blurring at any focus.

I’m not sure what the best way to ensure alignment is, but one way I spotted the issue was removing the camera from the optics module and pointing the objective directly at a bright, small light source like a surface mount LED. I put a peice of paper on the back of the optics module so I could see the projected image when the room lights were dimmed. This showed me that despite centering the LED relative to the objective the image was not centered where the sensor should be. That clued me in that the objective was misaligned.

Results Raspberry Pi Camera v2 with two-lens tube lens (I manually flat-fielded these images with varying degrees of success) reticle_flatfield1920×1439 135 KB flat_field smear1920×1439 315 KB flat_field pinworm1920×1439 267 KB

I went a bit off the deep end after building my first OFM, trying to see how I could modify the optics a bit. I wanted to answer these questions:

Raspberry Pi HQ camera with 75 mm achromatic doublet tube lens (no postprocessing) 2024-02-07T14-20-27Z1920×1439 72.9 KB 2024-02-07T14-21-39Z1920×1439 364 KB 2024-02-07T14-22-30Z1920×1439 393 KB

Well, the colors looking better has nothing to do with the optics; that’s down to the camera sensor and its bayer filter/microlens array. Using the HQ camera alleviates this but it’s not compatible with the OFM software yet.

The softness is worse to one side (you say it’s consistently the left) which does suggest either sensor tilt, objective tilt, or another other misalignment somewhere between objective and sensor. I had a similar problem before and the problem was that the objective was not seated exactly straight on it’s mount.

I guess the “rolls Royce” of optics would be a purpose-built microscope tube lens like the Thor labs ttl200, but they are real expensive (much more than the raynox). You can also use any “long” camera lens like a telephoto lens. I’m not suggesting any of this would be appropriate for the OFM since these optics are big and chunky too.

The setup: I wanted a basic upright brightfield microscope that would use infinite-conjugate objectives to evaluate different tube lenses, sensors, and illumination conditions. I ended up building a simple setup that slides along a vertical “2020” aluminum extrusion. Alignment and vibration are problems with this “minimal viable microscope” consisting of illuminator, condenser, stage, objective, tube lens, and sensor, but it works and makes some nice images when I can get everything lined up and not shaking. I didn’t use OpenSCAD for modeling this but I can share models as STL or STEP if any are interesting. image1920×2957 408 KB

Very nice work and thanks for sharing. This explains the reason of the out of focus areas in all of my builds. I noticed the same artifact mostly in the upper left corner of the field. I was under the impression that could be a problem with an uneven stage due to a problem with my printer, or something with the doublet lens position inside the tube. However, neither tilting the slide, sanding the stage, nor repositioning the lens improved the image.

Just wanted to chime in here to say that people in the macro photography world swear by the raynox DCR 150. Not sure the 200mm focal length is what you want for a rpi camera though.

My application is transmitted light for which those issues wouldn’t crop up. You might want to check out people disussing the pi cameras for astrophotography as they have detailed knowledge of how the processing affects low light and long exposure captures. I know they disable some of the dead pixel correction as it interferes with some captures.

3) # 185 Objective Lens 60X https://www.amazon.com/gp/product/B0C55VKJYN/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1

Absolutely. I think I might explore C-mount photo/video lenses with 50-60mm focal lengths eventually - they might work well, are often inexpensive, and would be a lot more compact than my current system

Stage: Just a (reasonably) flat surface that goes up and down. I’m using a bolt and captive nut to raise and lower it, and I built a simple double-paralellogram flexure to hold it in place. I also put some captive nuts in the stage that hold screws to adjust a platform above the stage, if I need a really flat field. This only sort of worked and probably isn’t worth the trouble.

DIN standard ------------------------------------ Yes Magnification ------------------------------------ 40x Numerical Aperture ------------------------------ 0.65 Working Distance -------------------------------- 0.6 Field of View...

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These last images look amazing!. No out of focus areas and the colors are perfect. Please tell me your secret! I am use finite conjugate objectives: 160/0.17 1) Parco Scientific 58A-0445 20X and 40x Semi-Plan Achromatic DIN Objective Lens

In term of alignment, I used a reasonably stable laser and the two-pinhole “alignment” tool from Thorlabs. Lens tubes from Thorlabs were OK, to make sure the obj and the tube lens were “mostly OK”.

In terms of stage Z adjustment etc: for a limited budget one can find used spring-loaded translation stages on ebay (Standa, Thorlans, Newport etc). Also, ebay has “aliexpress-grade” stages which cost 5x+ less than Thorlabs ones. I ordered a few and I used them for laser alignment; they seemed OK, but I haven’t characterized drift systematically. Rack-and-pinion stages are very drifty.

Your 40x has less severe softness but it’s still noticeable. It’s supposed to be a semi-plan objective which means that 80% of the field of view is supposed to be in focus at the same time. This doesn’t look like it’s performing as designed but where the issue is isn’t clear to me.

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The optical fibre works on the principle of total internal reflection. Light rays can be used to transmit a huge amount of data, but there is a problem here – the light rays travel in straight lines. So unless we have a long straight wire without any bends at all, harnessing this advantage will be very tedious. Instead, the optical cables are designed such that they bend all the light rays inwards (using TIR). Light rays travel continuously, bouncing off the optical fibre walls and transmitting end to end data. Although light signals degrade over progressing distances, depending on the purity of the material used, the loss is much less than using metal cables. A Fibre Optic Relay System consists of the following components:

Raspberry Pi HQ camera with two-lens tube lens (no postprocessing) 2024-01-28T04-23-12Z1920×1439 75.3 KB 2024-02-07T14-05-14Z1920×1439 410 KB 2024-02-07T13-59-08Z1920×1439 480 KB

The mode of propagation and refractive index of the core is used to form four combination types of optic fibres as follows:

These aren’t high sensitivity monochrome cameras though. I don’t know how the Bayer filter affects resolution especially with regard to repeatability of a stage

Because I was using the HQ camera, I didn’t use the OFM software and couldn’t leverage its automatic exposure, white balance, and flat fielding. I used RaspiCam, which worked quite well otherwise.

This thread also brings up the quality of the tube lens as a weak point when using the best objectives. Working out the best price/performance for doing something about that is a substantial investigation.

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May I ask if you have tried imaging at low light, to get an idea for pixel-to-pixel heterogeneity, “hot pixels” etc? I wonder if these artefacts, if present, can be handled by libcamera which Raspi comes with. My application is 3D real-time tracking of microparticles, with each particle image occupying 10-20 pixels. I’ve done it with high-end cameras, NA1.40+ objectives and nano-stages on “proper” microscopes, and I wonder if the setup can be miniaturized w/o losing too much performance.

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Optical fibre is the technology associated with data transmission using light pulses travelling along with a long fibre which is usually made of plastic or glass. Metal wires are preferred for transmission in optical fibre communication as signals travel with fewer damages. Optical fibres are also unaffected by electromagnetic interference. The fibre optical cable uses the application of total internal reflection of light. The fibres are designed such that they facilitate the propagation of light along with the optical fibre depending on the requirement of power and distance of transmission. Single-mode fibre is used for long-distance transmission, while multimode fibre is used for shorter distances. The outer cladding of these fibres needs better protection than metal wires.

As you say, the filter/microlens array of the PiCamera 2 gives problems that are hard to correct completely (discussed in other threads about colour saturation) the High-Q camera does not have that issue and so should be a lot better. Unfortunately until now the Pi Camera V2 has been the only one that could be integrated into the software to give the live illumination and flat field correction and fast autofocus, which are necessary for scanning and tiling. The upcoming Openflexure server V3 should allow us to use the other Pi cameras.

The OFM software is really doing a tremendous job improving the camera’s output as I found it nearly impossible to work with the unprocessed output from the standard raspberry pi camera. That said, the photos from the HQ camera looked really good without post processing beyond a simple white balance.

Condenser: Two achromatic doublets (50 mm FL and 19 mm FL) to create a condenser with an approximate NA of .57 - This is hugely overkill for the objective I’m using but I plan to reuse this later since it appears to work well. In the future I think an Abbe style condenser would be a better and cheaper choice but it’s not easy to find large ball lenses like they tend to use. Anyway, using achromats here seems like overkill and I could definitely have gotten away with singlet lenses. There’s also a bayonet mount on the bottom of the condenser that I can mount aperture stops to. I haven’t tested that yet so all these images are with the condenser “wide open”

In my digital photography classes I learned that optical magnification is always superior to digital magnification. But the former is way more expensive than the latter. Adding $70 USD is a significant amount when you look at the total cost of the current microscope. Could this image aberration be programmatically fixed? Can z stacking be used to obtain a sharper image? If money was not an issue, what would it take to build the OFM optics “Rolls Royce” ?

The HQ cam is a very substantial increase in quality. The issue isn’t the detector size, resolution, or the mounting hardware (which is nice to have), but the CRA mismatch between the bayer filter/microlens array designed for webcam/cellphone lenses and the lenses used in microscopy really compromises the quality achievable from the standard camera. Some of this can be post-processed out, but the images I took today were saturated in one or more channels in part of the image, which makes correction not worth it.

Objective: I tested a used Olympus 10X Plan achromat from Ebay. These have field numbers of 22 with their native tube lens focal length (180 mm) and are pretty easy to find used for cheap. I use similar objectives on commerical scopes and the quality is fine for a low-power objective. Most importantly, the field is quite flat across the whole field of view.

I wouldn’t discourage this type of exercise. In fact, I learned a lot by exploring how different objectives affect the quality of the image. I realized that I was mistakenly using a semi-plan when I should have been using a plan objective.

I was not intending to discourage it at all. It is very useful to understand all the things that affect overall performance and I have learnt a lot from what you and others are doing.

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As for the condenser, it looks good but with such a low-NA objective it’s hard to tell if it’s actually doing a good job. Once I get a higher NA objective to test it with, we’ll see how it performs.

The Thor labs achromatic doublets don’t seem to have flat fields and the lens I used produced a pretty noticable field curvature when used alone. I think this is actual field curvature and not a tilted slide based on focusing up and down and seeing a pronouced circumferential difference in focus. It’s probably not an aberration, per se, since I could focus the edges of the image nicely, just not the center and the edges at the same time. I guess this is expected as these lenses aren’t really meant for imaging. Interestingly, when combined with the Raynox (which is intended for photography) the issue was not really noticable. I think this is due to the distance between the achromatic doublet and the detector. Longer distance = more noticeable curvature.

Field curvature is definitely something that can be compensated for by focus stacking. It’s actually an ideal use-case for it! You mentioned that you’ve experienced this; are you using infinite conjugate objectives? I am curious if the current tube lenses works better with finite conjugate objectives.

I tested a new Plan 40x/0.65 objective. It looks better and the out of focus area on the left is less evident but still there. Besides the color problems, it looks overall better, sharper, and more magnified . OFM_2024-02-22T16_41_26.212Z1920×1442 227 KB

As for the focus, I’m not 100% sure what the source is - but it could be the objectives themselves. Your 20X doesn’t purport to be a plan objective which could account for the very soft edges.

As to your question about magnification, the first set of 3 images use the Raspberry Pi v2 Camera, which has a smaller sensor than the HQ camera. The smaller sensor produces larger apparent magnification (crop factor).

DIN standard ------------------------------------ Yes Magnification ------------------------------------ 40x Numerical Aperture ------------------------------ 0.65 Working Distance -------------------------------- 0.6 Field of View...