Microscope alignment for optimal image quality: Koehler Illumination
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- เผยแพร่เมื่อ 29 มิ.ย. 2024
- This video is a tutorial on how to align the illumation optics in the microscope for Koehler illumation. Koehler illumination provides even illumination across the field of view, and is critical for obtaining high quality phase and DIC images.
A video on the role of condenser aperture diaphragm in transmitted light microscopy will be coming soon!
A nice review on Koehler illumination: doi.org/10.1002/0471143030.cb...
0:00 Introduction
0:17 What is Koehler illumination?
1:29 Why should I care about Koehler illumination?
2:54 Optics 101
4:50 How does a lens form an image?
6:08 The critical parts & pieces
7:50 The parts you need to align
8:32 Uneven illumination
9:47 What are conjugate focal planes?
10:54 Conjugate illumination planes
12:32 Conjugate image planes
13:32 All the conjugate focal planes
14:50 Koehler illumination step-by-step
16:44 Finding the parts
17:59 What you should see when looking in the scope - วิทยาศาสตร์และเทคโนโลยี
![[HIGHLIGHT] หมีLIVEปะ? | EP.171 เห็นแต่ในจอ วันนี้มาต้องขอคารวะ](http://i.ytimg.com/vi/gPWLxa0e_Z8/mqdefault.jpg)
The best explanation ever found. Koehler Illumination is no longer a mystery to me. This not only helps me with the microscope, but it's also an enlightening optics lesson.
Yay!
I was struggling to understand the whole concept for the last 3 days, until watching this 20 minute-video!
Extremely thorough, yet never confusing. Much appreciated!
Couldn't be clearer than this!! thank you.
The video is much detailed as needed to know. Thank you for sharing such great information with Animation. Before it was full of confusion, but after watching this video it is really in focus. Thanks again.
I'm so glad to hear that!! Thank you.
This video is excellent. All you need to know about Kôhler illumination presented in a very clear manner. Thank you so much for making this public.
Thank you very much for this detailed explanation. It was just perfect with all the background information, animation, and examples. :)
This is amazing!!! Excellent tutorials on this channel, I wish there were more information sources like this in the world
Thank you so much!
This is an amazing and super clear explanation, thanks so much for you effort!!! :)
This was more useful than I thought it would be. This deep dive into the optics of a microscope really gave me a whole new level of understanding. wow!
Feels good to know what all those parts are for, right? :)
deep dive?!
Thank you very much. This video really helped me going through the optical microscopy.
This is so useful. Exactly what I was looking for. You explained it so well. The productive 20 minutes of my day :).
Glad it was helpful!
Thanks for this amazing video.. i like the extra info about optics didn't found a better explained video on this topic
Superb video... comprehensive yet very simply explained. Thanks :o)
This was so helpful. My background is in electrical, so I had to watch it at 75% speed and take notes. I now understnad the basics of microscope optics.
Very clear explanation. Thank you for putting in so much effort!!
Glad it was helpful!
Excellent and very short precise with concept as well as how to do yourself explanation. This is how tutorials should be. Please make more like phase contrast and Fluorescent FRET FLIM etc. Would be great if same way DIC and BF can be explained and elaborated first showing the light path explanation difference side by side then inverted microscope components and how they work and then how to take images in DIC and BF. This will help to understand microscope in details that what exactly happening when we take DIC and BF inside microscope in context of light path.
Thanks a ton for such a comprehensible video.
Finally understood it from this video .... Well explained !
Super useful! Thank you so much for this incredible resource.
Glad it was helpful!
Thank you so much!!! Really clear the explanation!
Perfectly explained. Thank you so much !!!
fantastic tutorial!
Comprehensive and simple. Nice
thanks a lot for the effort for this video. It is complete!
Thanks. This was a good tutorial.
Very nice video. It will be very useful for my students' training..
Thanks so much for this clear explanation!
You're very welcome!
Great video, thanks
Your videos are excellent. Exactly what I was looking for! Do you intend to publish more in the future?
That's the plan!
Just wow
Great explanation
Thank you very much :)
Glad you liked it!
I found it very useful!
Thanks a lot for this! :)
I'm in my senior level Criminalistics class in the major of Forensic Science. We are setting up polarized light microscopes for Koehler illumination. With this video, I can understand the necessity for the ideal spacing between the different lenses. I just wish I could find a video that includes the stage rotation, aperture diaphragm centering, objective centering, and polarizer and analyzer adjustments.
I don't know of a video either, but Shinya Inoue's book Video Microscopy is an excellent resource for polarized light microscopy.
thanks. this was very useful. greetings from germany :)
yeah this was actually incredible.
So great.
Very concise and ell explained! Extra brownie points for kitty cats!
thanks!!
nice job
This was not just useful, it was superusefull.
Tks,great video！I have subscribed
Thanks for the sub!
So, in a nutshel: Koehler is a fancy contraceptive method invented in Harvard 4 years ago.
You should not use it until you are on your second cycle.
Thank you very much for sharing such precious information for free.
It’s not that fancy.
@@Microcourses Lol!
I'm sorry, I did not want you to waste your time with an answer to my ridiculous comment!
I've put a thumbs up and just wanna give a little boost on YT algo on that topic with that silly comment.
Have a great day, love from Montréal.
I appreciated the laugh!
In theory, can’t you just use a single lens to defocus the light source? Is the reason that we use two lenses, collector and condenser, is to maintain sufficient brightness?
Great video by the way!
Excellent explanation! Now I'm wondering how I'm going to keep my cat still under the objective, to get it into focus.... 🤔
nice video! a quick question: in principle, you could use a single lens (rather than two lenses, colletor + condenser) to collimate the light from the source. If there is indeed a reason to do that? I guess would be using the image of the field diaphragm as a reference in the image captured by the retina(?)
A single lens could work but a series of lenses gives greater flexibility, and it’s good to keep the heat of the light source far away from the sample
great, i just wondering how the image in the is so good, and when in the lab no one can achieve it, it took me 1 year to know this term
So we are adjusting the position of the condenser lens based on the position of the field diaphragm but how is the position of the field diaphragm determined?
Very helpful. How do you do this for higher magnifications, though? Using the 40X objective the image of the field diaphragm (fully closed) is bigger than the field of view.
In this case, close the FD all the way then move an edge of it into the field of view. Focus on that edge, then do your best to center it.
@@Microcourses Very helpful. Thanks!
Thank you very much for the detailed explanation!
I have a doubt though. Why is required for the Field Aperture to be in the sample plane as a conjugate? The de-focused light is already illuminating the sample. Or in case the Field Aperture is illuminating the sample for the correct Numerical Aperture, then is the defocused light bleaching the image?
The purpose of the field diaphragm is to limit the illumination to the field of view (in XY) of the objective lens.
@@Microcourses Thank you very much for the answer!
In that case, when designing the microscope, one would first create the lamp-collector-condenser with infinity focus and then place the field diafram at any position and lock it in place? It would then imply that if one focuses the condenser to get an image of the field diafram in the sample plane, then the light is defocused. Is that right? Also, is there a link which i could read in depth about this?
I agree, with everyone else--nice and concise explanation. One thing I don't understand though, that fact of it being called "Koehler illumination" seems to imply there might be other types of illumination set ups that don't follow this procedure. Is that true? Or is Koehler illumination basically the only kind of lens set up there is? And/or are there cases where you just don't care about condenser z-alignment that much?
Thank you for the kind comments. There is another type of alignment, but it is rarely used anymore: en.wikipedia.org/wiki/Critical_illumination. You can probably get away with ignoring the condenser for low resolution brightfield. But if you always need it for phase & DIC, and to obtain decent resolution.
I'm an amateur microscopist (well I cannot even call myself microscopist, that's a bold claim) and I got myself a new upright microscope with Köhler illumination.
Following the procedures demonstrated in this video and any other literatures, I find that I have to re-center the condenser each time I switch to the next objective lens. Since I don't have access to higher end microscopes to evaluate this particular behavior, I cannot tell this is normal or not. Would you please me do I have to re-center the condenser with each objective or there's a misalignment in my microscope nosepiece?
It's expected that you would need to realign between objectives - in fact, that is why the condenser lens and apertures are adjustable. It's not possible for all of the different objectives on the nosepiece to be aligned perfectly relative to the optical axis (ie, light path) of the microscope. Feel free to post microscopy questions on the discussion forum at forum.microlist.org.
Video tutorials don't come any clearer that this. Great job! Why can't you just place the light source at the focal plane of the Field lens to create a collimated beam of even light that could be passed to the Condenser lens?
Field lens? Do you mean field diaphragm?
@@Microcourses Sorry, using your terminology then I should have said "Collector" lens. Tell me if I'm wrong but for a biconvex lens then if you put a light source at the focal point of the lens then doesn't it produce a collimated beam of light? If so then why not put the light source at the focal point of the Collector lens and create a collimated beam of light that shines straight into the Condenser lens. Wouldn't this provide the even illumination that Kohler illumination is trying to achieve?
@@edward_grabczewski Got it. If you put the light source in the focal plane of the collector lens, the condenser would focus the image of the light source onto the specimen. In principle you could place the light source at the focal plane of the condenser. However, using two lenses in serial allows for optimal alignment for different objective lenses, and minimizes the amount of heat (from the light source) that makes it to the sample. The collector lens can also be used to magnify the image of the light source, allowing use of higher NA condenser lenses (I didn't get into the benefit of using a high NA condenser in this video).
@@Microcourses Good point! The Condenser lens would focus the collimated beam back into a point! And the other good point about heat was no doubt very important up until recently when LEDs became popular. Thanks for putting me right 🙂
Thank you.
And not to be picky, at 4:46, it would be helpful to explain why would the object itself gives out lights going in different directions. I know it now. Because in an environment where natural light presents, the object will reflect the lights. If the object reflects nothing, then we can neither see the object nor the object can image behind a lens.
Also, at 4:46, why is it that the lights from the object on the focal plane defocused to be parallel lights to the lens axis? Shouldn't this be the behavior of only those lights from the object that passes the focal point? I ask because I am confused about why the concept of the focal plane is introduced here. Why is the focal plane useful to us?
Then at 6:03, it is said we put an object at a focal point. How do we do this, since the focal point is just a point and the object is much bigger? What would happen to the points on the object which are not on the focal point but on the focal plane?
Good video however, you did not mention the NA (numercial aperture) which is the optimal setting of the condenser aperture diameter for the objective in use.
I have a separate video on NA, and plan to make a video on adjusting the condenser aperture and it's effect on resolution.
Hi, I'd like to know how to adjust the condenser for darkfield illumination, thanks!
Same method - just move/rotate the darkfield stop out of the light path while aligning.
@@Microcourses thank you
My micoscopic view under 10x is completely okay but when ever i turn 40x, and 100x, the view is almost out of the light. So whats the solution mam!!
Damn... that cat is small AF!... jokes aside, great video.
isn't this el Hazen's theory and invention ? why do they call it koehler's?
20 minutes of my life I will never get back boring and overcomplicated glad your not my lecturer