Hello!
I recently decided to try and build an Open Flexure microscope. My current end goal is to build a single beam confocal laser scanning microscope, very similar to Breaking Taps build. As I’ve never built anything like it before, I decided to start simple and build the basic microscope.
I built the High-resolution microscope v7.0.0-beta1.
Printing
To print the parts, I used a Bambu Labs X1C. I printed all of the parts at 0.28mm Extra Draft, 15% fill grid, and Slice gap closing radius at 0.001. The total print time was about 10 hours and used about 350 grams of filament. Removing the brim around the main body was trivial and did not require a knife.
Parts
I ordered 3 50mm acromatic doublet lenses from Surplus Shed for $7.50 each, plus $6 shipping. It shipped out the next day (Friday) and arrived Monday (3 days later).
I bought the 30mmx34mmx2mm O-Rings on Amazon (https://www.amazon.com/gp/product/B07ZDM58S8/ref=ppx_yo_dt_b_asin_title_o00_s00?ie=UTF8&psc=1)
I bought an AmScope 40x Achromatic objective for the microscope.
I am using a Raspberry Pi 3 with an Arduino Mega and 3 28BYJ-48 stepper motors plugged into ULN2003 driver boards. I am using a standard Raspberry Pi Camera v2.
I bought these Optical Convex Condenser lenses from Amazon (https://www.amazon.com/dp/B0BR5PYPQB?psc=1&ref=ppx_yo2ov_dt_b_product_details). It just barely fits and will fall out if you bang the table so it seems 12.5mm may be too small and 13-13.5 may give a better fit.
For the LED, I printed the LED workaround and I am using a standard 5mm warm white 20mA/3-3.2V LED with a 150 ohm resistor.
I bought some 0.5mm white styrene sheets but they appear to be too thick because they block a lot of the LED’s light. Removing the sheet from under the LED did not seem to have too negative of an effect on the image.
I used 20mm instead of 25mm screws for the gears. I was not able to find any 25mm screws locally so I just ran with 20mm. They appear to work just find, but result in a slightly diminished scan range.
The nuts I own appear to be flat on both side and I could not tell one side being curved. I don’t know if this made any difference. I just stuck the two nuts together and everything worked in the end so.
Build
Putting together the microscope was pretty straight forward and for the most part it was easy to follow along.
I did need to reference the ‘livestream’ video once or twice to fully grasp what needed to be done. The main place I referenced was
## Step 5: Attaching the viton bands and foot
It is not very clear from the images what orientation the foot should be in and that the O Ring should go in the groove like.
The Arduino workaround does not specify which pins are what, only that it uses pins 2-13. I would have liked to know that X is pin 2-5, Y 6-9, etc. I had to guess and check. I am powering the motors using 5V out from the Arduino with success.
The Objective appears difficult to screw in perfectly to the optics holder so that it’s not slightly off angle. This is most likely just a flaw with 3D printing threads itself.
With mounting the optics to the body, my screw is quickly being stripped and I will probably have to replace it if I unscrew it and remount it too many more times. I think being at an angle makes it difficult. I am not sure if using a stronger metal for that screw would help much.
The long screw in the illumination appears to serve no purpose with this build. I have no issue with the illumination falling.
The sample clips appear to be a poor design for how I use them. I rotate them get a better clamp down on the slide and doing so quickly loosens the bolt. I don’t have any ideas or suggestions about how this could be better at this time.
Software
I installed the Raspbian OpenFlexure full desktop on a 256GB SD Card. I have only interacted with the microscope over the web browser so it appears I should have went with the Lite version instead.
The software is really great compared to a lot of other scientific software, but I think there is still a lot of issues and improvements that could be made. I am not sure how much of what I’m about to say has been changed or not.
The biggest issue I had was that it is not well documented what to expect from 40x images and that the focus distance to the slide is very close. It’s so close that I realized you needed to flip the slide upside down to achieve proper focus. This is not a software issue, just maybe a section on the User Your Microscope page that quickly talks about what to expect from 10x, 40x, and 100x would be greatly appreciated. Something like “Here is a sample 40x image you should expect to see. You should expect your objective to be less than 1mm from the slide. Here is a sample 10x image you should expect to see. You should expect your objective to be Xmm from the slide.”
Website
I did not read the readthedocsio page about using the website so this is my process and thoughts about using the website going in blind. I think the basic layout of the website is good.
It’s super annoying there is no “stop” button and no current position indicator. Use case: I’m at -3000 Z. I enter 3000 Z. I want to stop when the slide is in focus rather than scrolling on the mouse 1000 times. Not only is there no way to stop, once it’s finished I have to guess the position the focus or point of interest position was at.
The autofocus seems to work terribly for me. I feel like Fast misses more than it hits.
Manual Focus:
Fast Focus:
I’ve also had a lot of issues with medium and fine. Medium doesn’t seem too bad but it appears there is a limited range. So if it’s, say 1000 Z, out of focus, medium will not find focus.
For some reason, my slides are “slanted”. If you move in the X direction, it’s almost always in focus but as you move up or down in the Y, it gets more out of focus from the starting focus point. I have no idea why, other than the objective is not parallel with the body but I have no idea how to fix that.
Stack and Scan is very cool. I have had issues with some kind of timeout though. I should have written down the logs. I also hate that clicking on “Storage” does not automatically refresh captures. It has gotten me more than once scanning the same slide in the same starting position where it wasn’t updated and I tried to download an older slide.
Here is a stitched together slide:
Zea Seed L.S.
x step-size 1200
y step-size 1200
x steps 16
y steps 16
z steps 1
Autofocus Fast
Scan Style Snake
Naming Style Coordinates
I would really love to see the ability to stitch together the slides on the site itself.
I have quite a few other notes but I think this post is getting a little long and not really sure how useful they will be.
Overall, I am pretty impressed with things and look forward to seeing how things continue to develop. I am currently printing the Delta platform and look forward to getting that a try as well.
Thanks!
Additional single image scans at 40x:
someone recently posted a mod to fit the drivers and pi.