Hello I recently built the v7 high resolution microscope as a gift for my son, I bought the Achromatic lens and board from iorodeo along with other parts. I tried using the “plan ASC 40/0.65 160/0.17 objective and a 100x objective. My problem is that I cannot get it into focus and auto calibration does not seem to work either. The motors move but it just shifts the x axis and then says it did not detect movement. I can see movement but it never seems to notice and never bothers to move z axis. So I have done the following steps.
1: I removed motors and tried to focus manually. This never produces a good image. I went from objective practically touching the slide to as far away as it can go.
2: I have tried both 100x and 40x objective and it does not focus.
3: I have remounted the objective, I placed it further away and this does not work either.
4: I have seen suggestions in the forum that say flip the slide upside down, this also does not work and is messy cause sometimes the sample will fall off the glass, not sure how this is done.
I am just using a slice of red onion I can see just a red blurry blob that gets deformed as I move the objective. I also have no idea how far away the light should be, I have experimented with various lengths and it produces just a blur to complete white out. I also do not know what the starting position should be, I have tried moving gear in both directions and nothing seems to work.
Any help would be appreciated,
Thanks in advance
Hi @jdesgarennes. With a normal 40× or 100× objective the sample needs to be very close to the lens. There is enough space for a 0.17mm coverslip, but not much more. (The ‘0.17’ lens marking is the design coverslip thickness). A 40× lens might have an additional 0.4mm working distance, but certainly not enough to be able to focus through a 1mm thick microscope slide. This is why the sample usually needs to be on the underside of the slide in an inverted microscope like this. To stop your sample falling off you need to have a coverslip, and possibly fix that in place if it is a relatively thick sample.
At this stage it would be useful to make a dry test sample to check that your microscope is performing correctly. The best way to do that is to get a marker pen and scribble on a blank slide. A dry-wipe board marker can be best as you can put your finger on it to make structure in the area. You can sometimes do that with other types of marker before they dry, or use a thin line marker to get structure. Then place this with the marker side facing the lens and see whether you can get focus and calibration.
Going back to your samples, with things like onion skin a 20× objective is usually sufficient to see the detail. That has a longer working distance and will usually be able to focus through a slide with the sample on top. The Low Cost optics version of the OpenFlexure Microscope will do the same, with about the same resolution. If you are able to get the two printed parts, you already have the lens from the Pi Camera v2.
For higher magification, there are 40× lenses that exist which are designed to view through a 1mm microscope slide. They are usually fairly expensive. It is also possible to modify the standard inverted OpenFlexure Microscope into the Upright version with a couple of printed parts and a few more screws.
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Hi William, thank you for the reply, I have tried flipping the sample with both the onion skin and using marker method both seem to just yield a blurry mess. I started from as close as you can get without touching sample and slowly moved back, but it just stays blurry. I have also tried moving the light source and it seems to make no difference, When I do the calibration it just states that it moved and no movement was observed even though it did move. I attached what I am seeing it does not ever really get much better than this. This is while using the 40x objective.
The stage motion calibration will need a good image, so that is not going to work yet.
It looks as though the illumination might need adjustment as well. Could you take the slide off and go into the settings tab and disable flat field correction, then auto gain and shutter speed and post that image?
In the optics module, could you remove the objective and check that the tube lens is firmly seated in its mount.
