I have to image some cells with Green Fluorescent Protein for my project.
I’m confused, can the fluorescence optics module work with the openFlexure regular microscope. Are there instructions and sources for the parts? I just built this one I think it’s v7: OpenFlexure Microscope Kit – IO Rodeo
I found this in another thread
<<Instructions for assembling the reflection/fluorescence optics module are most complete for the Delta stage [OpenFlexure Delta Stage]>>
Hi @techdog, coincidentally I am currently looking into this for a commission job.
Long story short it would work in principle but the v7 hasn’t been tested with a fluorescence setup so there are some quirks and might need some tweaking to get working. It’s not an out of the box solution like regular light microscopy.
I’m still waiting for a glass manufacturer to get back to me with some filters so I haven’t got a complete one yet. But I can try to point you in the right direction if you need some general guidance.
The necessary parts are linked in the v7 microscope instructions. As @malkanicus says, that imaging mode has not been tested much recently. The assembly instructions for the Delta stage give a few more details on how to build the optical system, but you do need the STLs from the v7 microscope not from the Delta stage -the camera is rotated 45 degrees between the two systems.
Where would I get the optical filters and beam splitter that I would cut, and once in place it seems like you can’t switch it out with the regular camera very well for example how would I switch between regular imaging and fluorescent imaging.
Hi @techdog sorry I didn’t get an email notification about your reply so missed it.
Getting filters is basically the tricky part. There are a handful of companies that I know who could supply the filters (Comar Optics, Thor Labs & Edmund Optics) though from personal experience they can be a bit of a headache to get in touch with and aren’t really set up for small bespoke orders.
Out of interest what dyes are you using and do you know what excitation & emission wavelengths you are targeting? If by luck it’s the same that I am currently enquiring about I might be able to help but that is likely a long shot.
As for your second question the short answer is it’s a bit complicated. In an ideal world I would suggest separate microscopes for LM and Fluro. As an alternative you could have two separate optical assemblies and swap them out as required. This would require partial disassembly and you would need 2 doublet tube lenses at the very least but it is theoretically doable. Though I wouldn’t say I recommend it.
I bought some filters on ebay they are cheap in old used filter cubes. I got one for 54 dollars because it wasn’t in good shape. Most are 139. I’m using Rhodamine so the filter had to be 530-550 emission and 505 excitation. I’m not sure how I’m going to cut the filters out yet or make them into rectangles first I have to get them out of their filter holder.
Honestly a pretty creative solution. I’m not an expert of glass cutting so I don’t really have any advice on that front but I will say good luck in getting it working.
The filters are easily removed from the filter cube and then again from their screw-on rings but now they are just 1 inch circular filters in metal rings. The filter cube comes with a flat dichroic beam splitter that is flat so that is easy to work with because it’s already rectangular and could be cut and this maybe the hardest part to find. I’m wondering if a better solution would be to modify the epi-fluoresence assembly plastic to accept these common sized rings. Then it would be an easy solution for people to make these. The rings would slide into slots, The ring looks like it’s about 3mm and the diameter is about an inch. I don’t think I can successfully remove the filter from the ring and cut it into 15x18mm. For people who need to do this type of thing it would be a good solution. The filter cube itself is pretty big like 1.5 inches on each side so I don’t think it would fit in the assembly but maybe that would be even easier. Since you can find any filter set for about $139 maybe someone who designs the microscope parts could think of a solution where you could just insert a standard filter cube.
Hi all, I’m joining this thread to see how I can help with this project long-term. We (IO Rodeo) have gotten a few requests for the epi-fluorescence version of the microscope in the past too, so it’s really cool to see people working on it.
I’m going to be printing some parts for @techdog to test their filters. I’m planning to print the parts listed here but I’m not very familiar with this build.
@malkanicus is there anything you have learned or can share about your experience with the build so far, especially to confirm the parts needed to print for converting the microscope for this application?
H @jorodeo. My commission job got a little delayed looking for the correct filter glass for the researcher’s needs. Found the parts but the quote is currently being considered so haven’t built it yet.
I did do some rudimentary mock-ups while waiting though. With a finite objective the assembly is a little snug against the main body but without having all the parts to assemble it, it’s hard to tell if that’s an actual issue or not. Either way it would be fixable with a bit of tweaking of the supporting arch. This doesn’t seem to be an issue with the infinite assembly which sits a little lower.
Filter glass is going to be a tricky one I think. Unlike the other glass items it’s very use-case specific so harder to buy in bulk without knowing what the demand will be. And second-hand is a little dicey in a sold kit. Especially with the new GPSR regulations that I’m in the process of getting compliant with. I think the various vendors might end up trading filters back and forth depending on who already has surplus glass.
It’s definitely in the finicky stage but if there is a reasonable amount of interest and demand (which it’s starting to look like) it should polish out fairly quickly.
Thanks @malkanicus, it does seem like there is interest. We spoke to another researcher today also looking for this application. Yeah, the filter is going to be the tricky part, especially as the applications will vary with different filters needed I guess. If we could identify the most commonly requested filters, then I could see some bulk purchase of a large glass and getting it cut to size might bring the cost down.
Often glass cutting can be achieved with with a glass cutting knife. Using the knife you can score a straight line. You can then tap the glass until a crack starts to form, and then snap it by hand.
The epi-illumination set up probably needs some love from a design side too!
We did some glass cutting a long time ago on some very thick UV transilluminator glass. I think it was 0.25" thick. We ended going to a professional glass cutter! He only charged us $20 though.
We still have the glass cutter somewhere, so will give it a go!
We have ordered a large-ish piece of beamsplitter glass which should yield 4 x OFM size pieces in theory: Optical Glass Beam Splitter Plate Prism Size 40x30x1.1mm 50T/50R for Laser Spectrum Analysis Instruments Accept Customization: Amazon.com: Industrial & Scientific
In the meantime, we did some testing with the 3D-printed parts and LED. We bought a blue STAR LED board but it was abit tricky to solder because of the aluminum PCB.
Attached are some images of testing the LED today with the Sangaboard and the optics assembly so far.
We made a little bit more progress … We were able to pretty easily cut the beamsplitter filter we bought into a 12mm x 18mm size using a glass cutting tool to score the filter and snapping.
Right now we don’t have any GFP excitation/emission filters or samples (next step), so while we wait we just did a simple check that this optics set-up fits into the microscope and can be used with the standard microscope and white LED illumination:
So far so good! Next step is getting our hands on the filters and slides.
