Hello, I’m new to the openflexure microscope. Thanks to lots of people built up this system, I’ve made my system. Now I found that I cannot focus with my 4x objective lens(Olympus UPLFLN 4X Objective), but can focus with my 10x objective lens(Olympus UPLFLN 10X Objective). Both of their parfocalizing distance are 45mm, and I use the beamsplitter with the infinity one. How could I fix the problem? Thanks!
Hi @Appendix890421. If you can get the 10x to focus there is nothing particularly different about a 4x as you have the same series with the same par-focal distance. It might just be a question of speed and range of motion. The microscope actuators are designed with 40-100x magnification in mind. Motion feels a bit slow at 20x as the field of view in x-y and depth of field in z are so much more. With 4x it may be just that moving from out of focus to in focus is a huge number of steps.
Alternatively is the 4x fully seated in the optics module and straight, so that the par-focal distance starts from the right place? When it cannot focus, are you finding it coming to one end of the z travel? I assume that you are having to take the module out to change lenses?
A picture may help to understand what the issue might be.
@WilliamW Thanks for your reply. First, I used a 10x objective lens for focusing.
After completing the focus, I switched to a 4x objective lens but couldn’t achieve focus.
I tried raising the sample by approximately 3.5cm to get focus.
The pictures were very important! I thought 3.5cm was a typo for 3.5mm until I saw the last picture 
With 3.5 cm above the stage and at least 10mm below, that is a working distance of 45mm, rather than a parfocal distance to the lens shoulder of 45mm.
I was totally stumped, that is such a big error. However it is so big it is clearly optical not mechanical. Which optics module .STL did you use? Those lenses are infinity corrected, the standard RMS optics module is for 160mm back focal length. For a 100x lens, putting the tube lens in the wrong position moves the working distance a small amount, which is not noticeable. It does make the images less sharp than they should be for a lovely lens like those. For lower magnification the difference is bigger.
There is an optics module designed for infinity corrected optics, available in the customisations and alternatives section of the instructions. The infinity optics module is a bit longer than the standard, so you will need a taller base as well, which is available in the same place (you have the standard base, so it does look as though you have the standard optics module).
Explanation of the optics modules is in build.openflexure.org/openflexure-microscope/v7.0.0-beta1/info_pages/imaging_optics_explanation.html.
One other thing is the illumination. You have the refection optics module, but also have the standard transmission illumination for these images. The transmission illumination is designed for high NA for high magnification optics, it often is not able to fill the field of view of low magnification objectives. In your picture the condenser lens is in upside down, which may actually help in your case. The illumination spot will be more spread out and will not achieve high NA, which actually could be a better match for your low NA lens.
I did a quick calculation, and there should indeed be a substantial shift in the focal plane using an infinity corrected 4x lens with the standard optics module designed for a tube length of 160mm.
If you print the infinity corrected optics module, it would be a good idea to check the tube lens position as well, as in this thread, posts 12-14: Focusing and Chromatic Aberration Issues - #12 by j.stirling . There is a possible issue with the module design that might leave some remaining focus issue with your low magnification.
When you have the optics working, you might find it useful to try different motor gearing to speed up the motion. @MilliRowland tested a little a while ago and there did not seem to be an issue with moving from 2:1 to 1:1 or even 0.8:1 on the gearing to make it twice as quick (Faster motion, different gearing). There is a merge request to implement this option at https://gitlab.com/openflexure/openflexure-microscope/-/merge_requests/352. The gears linked from the forum post are based on the old push-fit version. Gears based on the new version, locked with screws, are untested, but these are the STLs if you wanted to try:
large_gears_ratio_0.80.stl (803.0 KB)
small_gears_ratio_0.80.stl (1.1 MB)
large_gears_ratio_1.00.stl (893.6 KB)
small_gears_ratio_1.00.stl (1.0 MB)
At the different ratios the ‘small’ gears are large, but the naming remains that the small_gears_????.stl go on the motor.
I want to know what the back focal plane is. I have also tried the 20x objective lens, which can also focus.
The difference between these three objectives is that the back focal plane values are different. The 4x objective lens is -1.3 mm, and the 10x and 20x objective lenses are - 19.1mm.
I wonder if this problem is caused by not installing an achromatic doublet lens. I saw that the objective lens had Semiapochromat and didn’t install it.
I use optics_picamera_2_rms_infinity_f50d13_beamsplitter.STL.
OK! I will try to use taller stand, thanks!
I tried transmission illumination before I got my beamsplitters and fluorescence filters. After I get them, I will use reflected illumination. Is there anything else that needs special attention in the fluorescent light path?
Thanks for your help, I will try these later!
I am not quite clear with what you mean here. Do you mean that in the printed optics module you have not installed the f=50mm 12.7mm diameter achromatic doublet tube lens? This is a necessary part in order to place the image correctly on the camera, as described in build.openflexure.org/openflexure-microscope/v7.0.0-beta1/info_pages/imaging_optics_explanation.html.
Missing it out would certainly have the effect that you are seeing. Your objectives are designed for the slide to be 45mm from the lens shoulder and the image to be an infinite distance behind the shoulder. In the microscope the camera is only ~65mm behind the lens shoulder. To get the image that close requires the object (the slide) to be further away. For a 4x lens it will be extreme as you see.
If you already have the infinity corrected optics module and it is fitting in the stand that you have then there is no particular need to change the stand to make more space.
As @WilliamW has already caught, I think this is probably an issue with the camera-objective distance and/or the lens that’s in there. If the tube lens is omitted, you can compensate to some extent by using the objective at a slightly greater working distance. At high magnifications, the change in working distance is pretty small (like a few tens of microns), but as the magnification drops, the change in working distance will be larger, by the square of the magnification (i.e. 4x will be about (10/4)^2 times bigger, or around 6 times more).
Hello, I have another problem with using 2x objective lens(PLN2X). I can’t focus on my 2x objective lens even when I touch my sample with the objective lens. Because the FOV I got from the 4x objective lens is about 3.4mm*2.55mm, I need the bigger FOV. Is there any other way to achieve a bigger FOV?
A 2x lens should focus on the microscope in the same way that a 4x lens does, as long as it is designed for 45mm parfocal distance. The nominal focal length of a 2x lens is much longer than 45mm, so I expect that quite a lot of low cost 2x lenses have a longer parfocal distance to make the design simpler.
For a large field of view you are more in the direction of macro photography or a dissection microscope. There is a design for a dissection microscope here: First prototype of the Field Dissection Microscope is ready to build.
Or something like this: Interesting (New?) Raspberry Pi Camera Microscope Lens
For that size of field of view you don’t really need the fine motion of the microscope stage.
This is the video when I make my z stage from bottom to top, it doesn’t focus on the target.
Would the 50mm achromatic doublet tube lens be the problem?
It seems to come close to focus at the end of the sequence. Is that when the lens is highest or lowest?
What is the specification of the 2x lens, and which optics module and tube lens are you using?