Epi-Illumination
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- Опубликовано: 13 фев 2022
- PUMA is a DIY open source portable 3D printed microscope with augmented reality, fluorescence, polarisation, dark ground, phase contrast, epi-illumination and other advanced features. Its illumination system (called 'Dominus') has many configurations and in this video I show you how to build and use the coaxial epi-illuminator for epi-fluorescence and reflected light microscopy including metallurgical analysis. I also demonstrate these features with many images taken with PUMA of a fluorescent test slide and a silicon chip die imaged up to the limit of standard optical microscopy resolution with oil immersion. I also show a comparison to a professional epi-illumination microscope - the Olympus BH2 BHT-ML metallurgical microscope.
CONTENTS
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00:08 Overview of epi-illumination microscopy
01:34 The PUMA epi-illumination system
06:35 Black holes and the Event Horizon
13:13 Types of photomicrography used in this video
15:26 Epi-fluorescence example: plant histology
19:03 Reflected light low power: PCB
20:53 Afocal photography: Silicon chip die with Olympus BH2 comparisons
22:24 Direct chip projection: Silicon chip die (PUMA only)
26:58 Building the Epi-illuminator
30:18 The clip lock and stay thumb wheel
34:10 Concluding remarks
SUPPORT PUMA
============
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
===============
For more 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.
Models revised since the v1.0 release of PUMA:
Monocular
- Occular_Extn_CM - New model ocular tube for direct C-mount connector
- Monocular_tube_CM - New model direct C-mount connector.
Filterblock
- Side_Port_Separate_EpiStop - New model optional AR side port with baffles.
Models covered in previous videos (not repeated here):
LED and Lower Collector
• The LED Illuminator an...
Trans-Polarisation Illuminator
• The Trans-Polarisation...
Köhler Illuminator
• Köhler Illumination - ...
Condenser Upgrade
• The Abbe Condenser wit...
Beam Splitters and Advanced Filter Block
• Beam Splitters and the...
3D Printed Models (quantity)
----------------------------
DI_Cnd_Adj_thumbwheel.stl (4)
DI_Cnd_gripper.stl (1)
DI_M3_Adjustment_ring.stl (1)
DI_Epi_attachment.stl (1)
DI_Epi_black_body.stl (1)
DI_Epi_condenser.stl (1)
DI_Epi_Cnd_Aperture_04.stl (1)
DI_Epi_Cnd_Aperture_10.stl (1)
DI_Epi_Cnd_Aperture_13.stl (1)
DI_LC_Adjust_collar.stl (1)
FB_Side_Port_Separate_EpiStop (1)
DI_Epi_pol.stl (1)
AR_Clip_lock.stl (1)
AR_Clip_c_adhesin.stl (1)
AR_Stay_thumbwheel.stl (1)
FB_Filter_F17_slider.stl (1) - for fluorescence only
FB_Filter_slider.stl (1) - for analyser
FB_Filter_collar.stl (1) - for analyser
Parts for the advanced filter block
Additional filter sliders and filter collars as required.
Protective caps as required.
Illuminator parts as required.
Non-Printed Parts and Tools
---------------------------
M3_Hex_Head_Set_Screw_10mm_long (1 for each M3 thumbscrew)
M3_Full_Nut (1 for each M3 thumbscrew, 3 for each illuminator adjustment ring and 1 for the clip lock)
M4 Screw 14 mm long (1 - for the front of the focus plate to take the stay thumb wheel)
M4_Full_Nut (1)
M2_Allen_Bolt_Socket_Cap_DIN912_12mm_long (4) for epi-polariser
For fluorescein fluorescence:
Filter fluo yellow (510nm long pass 17 mm diameter 1.5 mm thick)
Filter fluo blue (450nm narrow band pass 14 mm diameter x 1 mm thick)
Allen keys (for the screws)
Tools for cleaning up 3D printed parts (e.g. craft knife, wire brush)
FOR FURTHER INFORMATION
=======================
1. Open access write-up about PUMA on the FreeCAD blog:
blog.freecad.org/2023/02/13/a...
2. The official PUMA GitHub page where you can download the specs and source files to build or customise your own PUMA system:
github.com/TadPath/PUMA
3. The scientific peer reviewed publication on PUMA that was published in 2021 in the Journal of Microscopy (a journal of the Royal Microscopical Society) available here:
pubmed.ncbi.nlm.nih.gov/34151...
onlinelibrary.wiley.com/doi/1...
Thanks for your interest in the PUMA microscope system.
PJT 14/02/22 
Наука
If you like this, see my series on Photology - latest episode here: ruclips.net/video/u_0xczp4210/видео.html
Absolutely amazing. Thank you for sharing this with the world :)
Thanks for your support.
Wow! I'm glad I stumbled onto this channel even though I've been collecting and assembling microscopes from parts for decades. I am getting the impression that your project may be just what I need to build some of the components I've only dreamed about. I currently don't own a 3d printer. I think this is one of the few projects I've seen that warrants the investment of time and money in 3d printing that doesn't involve toys or trinkets. I'm going to have to watch your other videos for further study - I'm not sure if I am up to the task for this project but I can see it's potential. Sincere thanks for all your efforts.
Thanks for your interest. There is a lot more to come - this 'first wave' of videos is just the beginning phase where I show people the basics of how to build the more common modules. More specialist modules and practical experiments will follow. Please help get the word out by telling others about PUMA and posting about it on social media. Although you could build PUMA systems using an online 3D print service (so avoid buying a printer) - in the long run if you are interested in the project you will find that having your own printer is definitely preferable. The next video I am working on now is about the 'Ocular Heads' modules - there are many options besides the fixed monocular, as you will see.
@@PUMAMicroscope Possibly you could do a video on taking part from the FreeCAD file and printing it.
The 3D printing guide PDF on Github explains how to make and save an STL mesh from any part in any FreeCAD file - that is a step-by-step illustrated guide. Once you have the STL mesh file you just load that into Cura and slice it using the Cura profile and any supports specified for that part in the same 3D printing guide document under the section for that part. I intend to make a video with 3D printing tips - how I set up my printer, etc. so perhaps I can cover those steps in that video. I still have several basic 'HowTo' videos to do before that - next up will be the video on the Trinocular Camera Port and an Intro to Augmented Reality Microscopy due out later this month.
7:17 May I suggest a more efficient and simpler Epi_Black_body Module?
Your current design probably work just fine but.... As you know, there is always a better alternative, if anybody want to pursue that route.
The main issue is the perpendicular surface (the closer we get to the center) that bounce the input light back to the source. Fortunately, the fix is quite easy.
I worked with colorimeter for paint analysis. We use a certified zero reflectance black standard to calibrate the instrument.
⚫It was a cylinder (4 cm diameter 10 cm tall) with a cone inside (4 cm diameter, 8 cm tall). The opening was a simple 4 cm diameter disk with 3 cm diameter opening in the middle (so, just a 0.5 cm ridge at the top of the cylinder). The tip of the cone was on the opening side (if you put your finger inside the device, you would touch the tip and ruin the black standard! Ask me how I know.).
The inside surfaces are coated with a black velour fabric with fiber oriented toward the bottom of the cylinder.
It was a strange experience to look at that perfect black hole. Impossible to see any reflection, even with strong light directly shining toward it.
For microscope, velour can be replace with paint.
So, short story, if anybody want to improve the already functional design, I hope this info help to get inspiration to do so.
Yes, there are ways to improve it (I did try with a cone at the back but this did not work well because you get a reflection from the tip of the cone - but I note that your cone has a hole on top so that might help!). However if I were improving this I would simply replace the flat spot at the back with a hole to a smaller cavity and repeat this once or twice. Having thought about all those things, I realised through experiment that the current design, with good flocking paint, does such a good job that any improvements would add very little to the overall effect. So, as ever, the final design was a trade-off between perfection of function and ease of build. However these are good discussions for anyone whose particular experiments need that little bit extra.
9:00 A DIY alternative for specialized black paint might be coating the parts with glue (or regular dark paint) and then spreading some black toner from a laser printer.
There is probably just enough toner from an old 'empty' cartridge.
Fine charcoal dust (activated carbon for filtration or crushed coal from fire pit) can be sleeve with a pantyhose to get the finer stuff.
All the above can also be mixed with regular black paint and spread.
Once completely dry, shake and brush off the excess to avoid dust contamination in optics.
Note that I have not done any testing; theses are just some suggestions to explore.
These videos are a wonderful combination of assembly instructions, explaining the microscope's design choices, and even explaning important microscope functionality!
Thank you a lot for the detailed work.
I do have one question: What is the source of the lenses used here?
I might want to eventually build my own as learning experience - and I might want to use the lensing for a project requiring a focused beam of IR light, for which I would like a source of lensing that is repeatable, (i.e. not Aliexpress or similar, as it's not clear what exact lens you might get)
Thanks for the support. For standardisation you best go with lenses from Zeiss (Zeiss Plan), Olympus (SPlan - not MSPlan) or Leitz. In this video I use lenses from OptArc (.co.uk - not .com, that site belongs to someone else) - these are QC'ed in the UK but I do not manufacture them so cannot guarantee they will never change. I do not know the performance of any of these lenses from any manufacturer for IR in terms of focussing ability and resolution. IR tends to be a problem for my work not a goal so I have not studied IR imaging with these lenses. For Zeiss Plan, Olympus SPlan and Leitz you may find good deals in the second hand market (e.g. eBay) but also pitfalls - like buying a used car. As you see from my other videos I have objectives from all these manufacturers - all very good and all can be used with PUMA as long as they are standard RMS thread, 160 mm tube length, 195 conjugate focus and 45 mm par focal length (for Leitz objectives with 170 mm tube length you must use PUMA's 10 mm extended ocular holders).
@@PUMAMicroscope Thank you for the detailed answer!
I should look into sourcing lenses from the manufacturer directly.
My application would not need a very finely focused beam, so the exact quality wouldn't matter. The main point is having somewhat reliable lenses, rather than telling people who would like to replicate my work "Just look on eBay for something that looks vaguely similar".
Great video, and you really have the perfect voice for explaining stuff, very pleasant ;)
About the large "dark" areas on the chip, those are usually caches, so very fast but limited capacity volatile memory for (temporarily) storing values. Actual "storage" memory usually has its own chip and doesn't have to reside so close to the computing parts.
And interesting that you mention that ~200nm are very fine structures on chips. Current high-performance chips like CPUs/GPUs already border on using single-digit nm scales for their functional "building blocks". AFAIK the chip manufacturers have more or less reached the limit of what is possible with "normal" lithography equipment (using UV light sources below 200nm wave length), even with all the optical/physical tricks they're already using. The next step down will require switching to EUV (extreme ultra-violet) lithography, but currently the yield of working chips is (AFAIK) still too low for using it in mass production.
Thanks. That's really interesting info. I intend to demonstrate some super-resolution microscopy techniques with PUMA eventually (and share them here of course so others can follow the experiments at home) - and I think microchips may be a good test object because the 'ground truth' is known quite well (as opposed to the biological tissues I normally work with - where the ground truth is what we are trying to find out).
Hi, great video!
The image quality is pretty impressive, and it's really cool to have the comparison with the Olympus microscope.
For the integrated circuit, the only thing I can recognize is the standard cells logic at 0:24:48
The long horizontal traces alternate between VCC and VDD, and between you have standard cells that are boolean logic functions of variable width but fixed height (so they can be stacked automatically).
Thanks for the feedback - very interesting. I am learning new things here.
Hi Paul, I noticed you have a cap on the C-RMS side of the advanced filter block (29:31). Did you design a separate stopper with C-RMS thread or you're just using the same stopper as the top and side ports? Thanks
Well-spotted. That is just the standard FB_Stopper used elsewhere on the filter block. It doesn't actually 'thread in' to the RMS hole properly but I found if I place it there and just turn it a little it will 'catch' well enough to act as a protective dust cap to protect the beam splitter when this filter block is not being used. So no, I haven't made a cap with a male RMS thread.
fantastic, will the Epi-Illumination components be available for purchase?
Thanks. Possibly - although I can't give specifics on that yet, it depends on various logistical factors and the amount of interest shown in the project as a whole. While the advanced filter block is amongst the simplest upgrade to do DIY - there is only one optical component (the splitter plate) and the rest is 3D printing and screws - you will also need the LED illuminator and collector lens assemblies including the LPC and power supply controller and polariser and analyser (or fluorescence filter set) modules. Although the parts are not expensive, there is a lot of complex manual work involved. So unless a reasonably good-sized batch could be made up with expectation to sell, it would be difficult to achieve at a consumer-friendly price point. It is the curse of highly specialised niche equipment (and also the reason why I made PUMA DIY-able and open source). If you want to DIY but don't have a printer or printing experience, there are online 3D print services that can make the printed parts for you - although I have not used them so can't comment on the print quality!
I will order 3 Epi-Illumination kits, if it is avaiable.
Is bigger cap that goes into Pol_Adjustment_ring at 28:38 name LC_Cap? It has a thread that is not present in Pol_Adjustment_ring.
Yes it is the LC_Cap (p.142 of current 3DP guide PDF on GitHub). In the video clip the Pol_Adjustment_ring has an LC_Adjust_collar fitted into it (p.111 of the PDF) held by the three thumbscrews and this LC_Adjust_collar provides the female thread that the LC_Cap threads into.
@@PUMAMicroscope Thank you very much, that makes a lot of sense. Great work btw! One more question both monocular tubes (CM and for regular ocular) have very thin thread wall that screws into filter block, but there is a lip inside that makes this hole narrower anyway. Is it done on purpose, or could it be thickend? (I just broke off the thread off one of the tubes :()
@@lextorn92 This is a potential weak spot - at least one other builder has had the same misfortune as you. The lip is a deliberate design feature - it is part of the anti-reflective baffle system and it needs to jut out like that to work - so just making the wall thicker is not a solution. It might be that I need to adjust the infill density (or make it 100%) for this part of the tubes. In the mean time, the most practical solution is just not to tighten the tube too much - you will have gotten a feel for how much tension it can take from your unfortunate experience.
Hi. Sorry your latest comments were filtered out by the YT auto-filter due to containing a link (my channel is set to disallow links in comments other than my own) but your suggestion of buttressing the inner diaphragm may work - I would need to try it. Thanks for the suggestion.
@@PUMAMicroscope No worries, thank you!
please tell me how can i get a 3d model with this microscope
Hello. In principle all you need to do is use the Z-stage motor and a camera to capture a Z-sweep sequence of images at mutliple depth planes then perform a 3D deconvolution. You then need to segment the model if you intend to make a 3D print of it. Not all specimens will be suitable. Alternatively you can make a 3D model using the SLM to effect 3D microtomography. Another three methods are: reconstruction from interference microscopy data, direct micro CT reconstruction and light sheet illumination - but I haven't got those modules ready for release yet - announcements will be made as they become available. Of course there is a lot of detail involved here but I intend to cover these topics in later 'applications' videos so you can work along to those. If you subscribe to the channel you can get notifications of when new videos come out.
@@PUMAMicroscope Thank you!!