Sourcing non-printed parts

General
42

Ok thanks. I finally found it. Too bad the shipping is just as expensive as the kit…

43

oh? I guess that will vary depending on where you are - I didn’t think it was crazy from the UK, but I’ve not checked from elsewhere…

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[Posting Deleted]

See answer about camera compatibility below

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Currently only the Pi Camera V2.1 is compatible with the software of the microscope.

There are optics modules that are designed to fit M12 cameras and the Logitech C270, but the camera feed can only be used through other general camera software, not integrated in the microscope control. These are listed towards the bottom of eth customisations page https://build.openflexure.org/openflexure-microscope/v7.0.0-beta1/customisation.html

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46

Thank you. So for my first build i want to keep things simple and use the stock cam. Even the resolution is rel. low compared to newer models. Should i obtain the regular Pi Camera V2.1 with IR filter or the one without IR filter (NoIR)?

Then, can someone recommend a lense for a High-resolution build aimed at doing blood, dark field imaging?

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It’s worth noting that, while the newer cameras have more pixels than v2.1, the optical resolution (i.e. the smallest detail you can see) is generally limited by the microscope objective: adding more pixels to the camera will give you larger images, but won’t necessarily enable you to see anything more.

The LEDs we use for illumination don’t have much IR, so you ought not to notice much difference between the regular and NoIR versons, but I usually get the regular ones (the IR filter makes it a bit easier to clean if needed). For dark field blood imaging you probably want a 40x or 60x air objective - and I recommend the slightly more expensive “plan corrected” type. AmScope is a brand that strikes a decent compromise between quality and price in the £100 region. For our blood imaging we typically use a 100x oil immersion lens, but it’s harder to get dark field imaging working with this setup due to the very high numerical aperture of the objective.

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Thank you, this answers my questions. I will start with this 60 lense here and will probably upgrade to a 100 with oil immersion after i have some practice using the microscope.

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Sourcing the non printed parts will still take some time. I will report my results as soon as the microscope is ready.

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Super, let us know how you get on :slight_smile: As with the 40x vs 100x, the 60x lens will need your dark-field light to be coming in at a shallower angle in order to miss the objective - an NA of 0.85 means your illumination has to make an angle of less than 30 degrees to the microscope slide, whereas 0.65 means you can get away with 50 degrees, meaning you can use a smaller illumination ring, or place it further from the sample. Either should be possible though, depending on how you build it.

It’s also worth saying that the standard build is just for bright field - though there’s the LED array version that will do dark field, and also a forum thread from about a week ago doing dark field with a modified condenser.

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Hi All

I added this to the relevant gitlab issue but cross-posting to forum Add complete instructions for reflection illumination and fluorescence (#243) · Issues · OpenFlexure / openflexure-microscope · GitLab

I am trying to source filters equivalent to the epifluorescence set up in the 2022 paper “Multi-modal microscopy imaging with the OpenFlexure Delta Stage” (DOI: https://doi.org/10.1364/OE.450211)

Methods section:

A blue LED (LZ1-10B202-0000, nm) was used, with a short pass excitation filter with 510 nm transition (Comar optics 510 IK 50), long pass dichroic beamsplitter with 506 nm transition at 45° (Comar optics 550 IY 16), and long pass emission filter with 510 nm transition (Comar optics 510 IY 50).

Comar have only one of these in stock and do not plan to restock. I found the following at Knight Optical which seem equivalent but I am not an optics person…

Sorry for lack of URLs for instant clicking through to the product but I haven’t posted before with this account so I’m limited to two per post. You can find them in the gitlab issue :slight_smile:

Short pass excitation filter with 510 nm transition (Comar optics 510 IK 50)

  • Knight Optical SKU: 510FDS50
  • Shortpass Dichroic Filter 510nm 50x50x1.1mm
  • £37.95 at time of writing

Long pass dichroic beamsplitter with 506 nm transition at 45° (Comar optics 550 IY 16)

  • Knight Optical SKU: 530FDL50
  • Dichroic Longpass Filter, 530nm, 50mmx50mmx1mm (note different advertised 0° wavelength to the Comar one but this is has the closest equivalence to 506 nm transition at 45°: it is 505 nm transition at 45°)
  • £39.60 at time of writing

Long pass emission filter with 510 nm transition (Comar optics 510 IY 50)

  • Knight Optical SKU: 510FDL50
  • Longpass Dichroic Filter 510nm 50x50x1.1mm
  • £37.95 at time of writing

If anyone thinks these will not work, I’d be grateful for feedback on either the filter choices or Knight Optical as a supplier. Otherwise I hope this is useful - at least for those in or near the UK!

Jenny

Update - suggested mCherry filters

**Short pass excitation filter with 550 nm transition **

  • Knight Optical SKU: 550FDS50
  • Shortpass Dichroic Filter 550nm 50x50x1.1mm
  • £37.95 at time of writing

Long pass dichroic beamsplitter with 506 nm transition at 45° (Comar optics 550 IY 16)

  • Knight Optical SKU: 530FDL50
  • Dichroic Longpass Filter, 530nm, 50mmx50mmx1mm (note different advertised 0° wavelength to the Comar one but this is has the closest equivalence to 506 nm transition at 45°: it is 505 nm transition at 45°)
  • £39.60 at time of writing

Long pass emission filter with 615 nm transition

  • Knight Optical SKU: 615FDL50
  • Longpass Dichroic Filter 615nm 50x50x1.1mm
  • £37.95 at time of writing
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I know @gerrit and @jorodeo have been looking at sourcing options for fluorescence. It is exciting that we are seeing renewed interest in Fluorescence, it would be great to have a solution that is more robust and more turnkey

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What excitation wavelength are you planning to use, and what fluorescent wavelength? The filter set will need to match the wavelengths that you have. I have not used the Microscope for fluorescence yet, but probably the most important part is to look at the regions that are blocked well by the filters.

You don’t want any of the excitation light to get to the camera. If you are using a broadband LED this means that the excitation and fluorescence filters need to have reflection regions that meet. If you have a coloured excitation LED, then this requirement may be less stringent, depending on how far the LED emission spreads to long wavelength. The RGB filters on the colour Raspberry Pi camera v2 may also help in some cases.

Looking at the specific short-pass filters that you mention, the Comar one blocks well from 550-650nm, whereas the Knight Optical one still has a few % transmission at 550nm. It may be that a shorter wavelength cut-off filter from Knight Optical will match better, as long as it still substantially transmits the excitation wavelengths.

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Thanks @jenny_molloy for sharing your finds!

Currently we are testing the epi-fluorescence version using a GFP fluorescent slide. We purchased these filters (we are not in the UK) for GFP imaging so these filters are applicable for GFP ex/em:

In addition, we are using the following parts:

We haven’t had much time to do testing yet, but we will post updates in the other thread here: Fluorescence optics for V7? as we go. @gerrit in the UK is also working on sourcing filters and testing so we have a nice group starting to work on this project. Hopefully we can collectively share experiences on the forum!