Köhler Illumination - How to build, collimate and use the Köhler Illuminator for the PUMA microscope
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- Опубликовано: 8 июл 2024
- Here I present Köhler's original paper from 1893 (with an English translation from the original German) to show you WHY and how he did it. PUMA is a DIY open source portable 3D printed microscope with augmented reality, fluorescence, phase contrast, polarisation, epi-illumination and other advanced features. This video will show you how to build the Köhler illuminator module, align it and use it to make high quality observations and photomicrographs.
For more information about PUMA see:
• Introduction to PUMA M...
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CONTENTS
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00:08 What Köhler Achieved, How and Why
13:39 Design of the PUMA Köhler Illuminator
19:36 Comparing PUMA to the Olympus BH2 Microscope
22:50 Diffusers in a Köhler Illuminator
23:50 Parts and Tools Needed for the Build
25:43 How To Assemble the PUMA Köhler Illuminator
38:06 How to Fit and Collimate the System
49:36 Further Information
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SUPPORT PUMA
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PUMA is an open source microscopy project. You can help support the project by:
1. Become a Patron: / pumamicroscope
2. Donate via PayPal: www.paypal.com/donate/?hosted...
3. Simply subscribing to this RUclips channel, like, comment and share these videos.
4. Spread the word - post the news about PUMA and link to this RUclips channel on your social media sites and any other outreach method (tell your friends, colleagues, students or teachers and lecturers about PUMA, for example).
5. Consider purchasing your optics and related supplies from our affiliated online optics store, OptArc.co.uk ( www.optarc.co.uk/ )
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PARTS AND TOOLS
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For details see the 'Bill_of_Materials' on the PUMA GitHub page ( github.com/TadPath/PUMA ) which also gives examples of where you can buy some of the more specialised items.
3D Printed Models
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* Update note for users of v1.0 of PUMA: The parts for the LPC in the v.1 release of PUMA were based on a thin lens of focal length 63 mm but this was only a temporary stop-gap solution to test the LPC which was still at an experimental stage of development compared to the rest of the illumination system. That lens was plastic and obtained from an unsustainable source and so has not been transferred into the current PUMA standard. Instead an f=60 mm glass lens has been source which is a sustainable generic component and the lens mounts at both ends of the LPC have been modified to ensure the system works well with this (and, in fact, it works even better than the prototype). For anyone who has printed the (now defunct) v.1 components, the changes are as follows (all modern components are on the main branch of the GitHub repository):
- LPC_S23_Lens_retainer - abolished, use a standard LC_Collar_2 instead
- LPC_Single_23 - modified to house the lens close to LED
- LPC_Lens_retainer - elongated by 1 mm to both retain the new thin glass lens and to leave a gap to easily remove it. Edges and gap also modified to fit the new lens holder.
- LPC_Lens_holder - Reduced lumen to fit glass lens. Also, the spanner flats extend the whole length.
- LPC_Lenseless_23 - New. Optional. A lensless version of the LPC_Single_23 which can retain membrane filters.
- Ferrule_internal_ring - abolished
- The 'AR_Cc_locknut' was previously used to adjust the spacing of the proximal lens from the LED but this is no longer needed for the LPC because the new single lens holder presses the lens right up against the LED which is the optimum position and no spacing should be used hence this component is now redundant for the LPC (although it remains part of the AR projector module so it is not 'abolished' from the PUMA project).
* All the above updates have already been made to the documents and models on the main branch of the PUMA GitHub repository just prior to this video being released and will form part of the next version release of PUMA.
Non-Printed Parts and Tools
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See Bill_of_Materials. Also:
Allen keys 1.5 mm and 2.5 mm (for the screws)
Tools for cleaning up 3D printed parts (e.g. craft knife, wire brush)
High quality drafting paper (for diffusers)
Membrane neutral density filter (see LED Illuminator video)
Lens paper, paper towels (for handling the lenses)
Scissors
Glue for mirror (e.g. epoxy)
Glue for collimator screen (e.g. PVA)
LINKS
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1. Köhler's paper
archive.org/details/cbarchive...
2. Video on the Abbe Condenser
• The Abbe Condenser wit...
3. Video on the LED Illuminator and 2x23 mm Lower Collector
• The LED Illuminator an...
4. Video on the Polarising Condenser / Illuminator
• The Trans-Polarisation...
5. Document on the Usage of the Köhler Illuminator
github.com/TadPath/PUMA/blob/...
6. Open access write-up about PUMA on the FreeCAD blog:
blog.freecad.org/2023/02/13/a...
Thanks for your interest in the PUMA microscope system.
PJT 27/11/21 
Наука
See the sequel video here: ruclips.net/video/gDGqXsudmgE/видео.html
and my video which explains about Köhler's conjugate planes: ruclips.net/video/Ai86SMBJqr8/видео.html
Find details of the lenses and the optical paths on GitHub here: github.com/TadPath/PUMA/blob/main/docs/PUMA_Kohler_Illuminator_Specs.pdf
Thank you so much for translating the paper, and sharing that translation with us!
Glad it helps. I'll be doing a lot more on classical microscopy theory and image processing, as well as videos explaining the recent advances in some of my original published research - so stay tuned for that.
I look forward to the future content!@@PUMAMicroscope
Thanks for making this video. This project is amazing and looks like so much work went into it. Everything looks very well designed and clearly explained.
Thanks.
Thanks! A beautifully clear explanation, spoken with great care.
Thanks for your support.
I just built the openflexure microscope. Cant wait to build this scope to its full potential. Amazing work!!
Hope it goes well. Perhaps you can do a compare / contrast video when you've had a chance to use both!
Finally, someone explained kohler illumination clearly. I've looked up so many sources on it over the years, but never fully got it until watching this video. The diagrams and reflection illustrations are perfection😘🤌
Thanks. You may also like this video where I explain in a little more detail the 'conjugate planes' aspects of Köhler and how it relates to what you have just seen: ruclips.net/video/Ai86SMBJqr8/видео.html
Thanks for sharing, very good information
Thanks. The PUMA project is also about education in microscopy optics and mechanics. So even those who have no intention of building or using the scope can still benefit by following the project. A new video will be out soon with even more educational content.
Remarkable explanation! Thanks a lot..
Thanks for the feedback. There is additional info in this sequel you might also like: ruclips.net/video/Ai86SMBJqr8/видео.html
could we redesign the abbe condenser and kohler illumination to use aspheric lenses to remove some abbarations?
Hello. Sure, if you need real precision then using a matched microscope objective as condenser would seem to be the simplest solution (this is effectively what happens when you do epi-illumination, in that case the very same objective acts as both condenser and imager). When it comes to imaging the field stop in transillumination mode that would need to be designed separately but should not be a problem. This is one reason why I release the project parts as parametric models instead of STL - so that people can experiment with such things as per their needs.
This is great, the concept of a professional 3d printed microscope with all the capabilities and features is beyond anything I've seen yet. I'm working to build one of these and was thinking a 90 degree off-axis parabolic mirror would suit it perfectly to reduce parts and improve image quality. The sort I'm considering are offered by Edmund Optics at the best quality and price I've been able to find. Could you make a modified version that implements the parabolic mirror which replaces the mirror and upper collector lens? Due to price I'm planning to use a 25.4mm diameter mirror. Also, what are the focal lengths of each of the lenses used in this design?
Hello. Thanks for the comment. The infinity tube lens must be 100 mm focal length and fit in the cavity for it but I did not give specifics for that because it is not ideal (infinity optics with the current setup is a concession not a recommendation - you get uneven field and lack of optimum corrections with this, even with expensive professional infinity objectives, but I made it for those who only have infinity objectives spare and want to use them with PUMA - and also to teach people about infinity optics - microscopy education is part of the PUMA project as well) so I plan to make a higher quality dedicated infinity filter block at some later stage - it will not be easy. Currently the best image quality is without the infinity lens for 160 / 170 mm tube length 195 objectives. Also the infinity tube lens prohibits the use of the trinocular port and the AR HUD.
Regarding other lenses, all specs and examples are on the GitHub page Bill of Materials. Please read the ReadMe.md on GitHub before attempting a build and only use models that are on the main branch of the repository - not the 'v1 release' download because that is now out of date.
I have deliberately avoided using any optics from Edmund, Thorlabs, etc. - not because there is anything wrong with them but because they are too expensive for PUMA and can be difficult for some people to get hold of. This means I would not make any PUMA systems to work with those BUT it does not stop you from making your own modified versions - this is why I give away the editable CAD files and not just STL meshes. I encourage people to experiment with the design and customise the scope to their needs.
can one build the PUMA without any prerequisite understanding, or are there fundamental primers one should visit first?
I try to give all the specific knowledge and understanding required in my videos - so you can consider watching my videos as an essential primer before you start.
I assume builders will have a basic high school level of knowledge of maths and physics as background education as a minimum prerequisite to understand the theory parts of my videos but you can self-learn those things from online resources.
You also need to be familiar with hobby-level 3D printing and have some experience there. A complete newbie to 3D printing would struggle so they should get some experience printing and building simpler projects first - but it is not difficult to do that. I only started 3D printing in 2018 and I only ever used my Ender 3 - so definitely no high powered knowledge required there.
I am currently making videos about microscopy optics theory to explain how a microscope works in detail (see my photology series - first video here: ruclips.net/video/MbPod24Ta8o/видео.html ). The more you know, the more you will get out of the project and the microscope and the more you will be able to customise and innovate with it and go on your own voyage of discovery - maybe even find out new knowledge and publish it!
What size is the larger condenser lens?
This document gives the lens specs: github.com/TadPath/PUMA/blob/main/docs/PUMA_Kohler_Illuminator_Specs.pdf
Hello,
I'm trying to add the LPC to my PUMA microscope. Any idea were I can source the f=60 mm glass lens for the LPC? The aliexpress link on the BoM xls expired.
Do you think I can adapt a 30mm f=50mm lens instead? This is the only lens I have that seems to be not too different... and I can source them for cheap (~1€/piece) so it may be interesting. How much did you pay for yours (approximately)?
Thank you!
Hello. Yes, I noticed that link was out recently too and I didn't find a direct replacement doing a quick search - it only cost a couple of pounds. Sure you can use an alternative and just alter the parameters of the parts, I think f=50 mm should work. However, first please see my Köhler update video from about timestamp 05:50 ( ruclips.net/video/B0DOYpfdsyg/видео.html ) because this shows how to make a better lower collector that makes the LPC redundant for Köhler transillumination. The standard LPC is still useful for making the PWG (Plane Wavefront Generator) - but I haven't released that video yet (it's coming in a few months time after my series on Photology is complete).
@@PUMAMicroscope I see, thank you for the fast answer.
I want to make a LPC for epi-illumination, especially to compare with the smallest one and using a 10x objective.
Do you think I have to reduce the length of the Epi-attachment by 10mm to compensate?
I can't visualise the light path you are trying to achieve. Are you using the standard PUMA LED illuminator with 2x23 LC lens system and getting some issues with uneven field illumination or something? If so you could try various things like different spacers (if +/- the standard 5 mm spacer doesn't work), or +/- diffuser at the back of the LC or use only one 23 mm lens in the standard LC - or maybe even try to adapt the new design LC (described in the Köhler update video) for the epi-attachment. Just some ideas. [edited to remove irrelevant stuff].
@@PUMAMicroscope Interesting, good ideas, I will probably try that.
For the LPC it's just because of this segment in the Epi-Illumination video: ruclips.net/video/cAEB10K8PqI/видео.html
I wanted to see the difference, if there was any (I have some contrast issues with some of my 10x objectives, maybe that would change something).
I use your microscope to learn optics, and for experimentation. It's very interesting and I am very impressed by the possibilities.
Recently I mounted the PUMA on a 3d printer and made a CNC microscope with it, it's very fun.
I should add, to be clear, the most important thing for even, Köhler-like, epi-illumination is to realise that your imaging objective is also your condenser. So, as with transillumiation, your ultimate aim it to get a focussed image of your light source placed at the back focal plane (BFP) of your objective and that image should fill the BFP for maximum resolution imaging. The other stuff I was talking about re angles of beams etc. are actually a red herring in that regards - so apologies for that. I edited my previous post to remove that stuff.