that is not the prolem of FM. The problem is that the images ar so unsharp. DIC is much better
@PunmasterSTP4 ай бұрын
PSF? More like "Perfect videos; you're the best!"
@PunmasterSTP4 ай бұрын
I came here after hearing about the high-NA EUV lithography, and this was a great explanation of the core concept. Thank you so much!
@gugok14 ай бұрын
Excellent explanation! Now I'm wondering how I'm going to keep my cat still under the objective, to get it into focus.... 🤔
@conehenge774 ай бұрын
Thank you so much!! That was very helpful.
@uzihadad11484 ай бұрын
This is one of the best lectures I have listen too about microscopy. Brilliant!
@henriquesouza87046 ай бұрын
Damn... that cat is small AF!... jokes aside, great video.
@lotharmayring60637 ай бұрын
much blah-blah and no results. DIC is the better solution because fluorescence-microscopy still gives unsharp images
@testboga59916 ай бұрын
Dude your words make no sense. How do you label a specific protein in DIC?
@lotharmayring60636 ай бұрын
@@testboga5991 why should i label proteins, only Dudes label proteins
@lotharmayring60636 ай бұрын
ok, some people at universties may label proteins but not me. You can also label proteins with radioactiv substances or normal dies specific. Fluorescence is not nessesary
@hosseinghasemi16599 ай бұрын
awesome
@tinglianyuan30989 ай бұрын
Could not find a better instructional video on spherical aberration than this! Reach Brilliant when deciphering the aberration at the imaging plane.
@tinglianyuan30989 ай бұрын
Lovely to see the pink heart picture flashing out when doing the axial scanning (around 3'30") and she says "the PSF is beautiful!".
@nickpaige81789 ай бұрын
thanks for posting!
@damiancavazos70439 ай бұрын
Thank you for your videos
@richardred43969 ай бұрын
you were able to make me understand these concepts with a 12 minute video
@RadhikaSharma-oi4ne10 ай бұрын
Wow. I am a phd student learning fluorescence microscopy at the moment for some of my experiments. This totally blew my mind away and cleared most of my misconcepts. Thank you!
@Microcourses10 ай бұрын
Yay!
@alexandrevaliquette3883Ай бұрын
Whaaaat? In 2024, how a PhD student in microscopy not know this very basic stuff?
@alexandrevaliquette388310 ай бұрын
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.
@Microcourses10 ай бұрын
It’s not that fancy.
@alexandrevaliquette388310 ай бұрын
@@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.
@Microcourses10 ай бұрын
I appreciated the laugh!
@dummag412611 ай бұрын
increase the subject illumination!!!
@Microcourses10 ай бұрын
Sure, with the trade-off of increased photobleaching, and phototoxicity in live samples.
@RossOCarroll330111 ай бұрын
yeah this was actually incredible.
@dsasmalmnp11 ай бұрын
Fantastic.. I just taught a PG class on PSF.. This helped me a lot to prepare materials.
@Roman-vy6po11 ай бұрын
Geting prepared for an interview with this course (I was far away from the miscroscopy before). Well, that's a masterpiece. I absolutely love it. Please don't stop!
@Microcourses11 ай бұрын
Good luck with your interview!
@kochipj Жыл бұрын
Pretty neat explanation! What is missing for a nerd like me are some remarks on the differences between coherent and incoherent imaging and Abbe theory of image formation. What was really enlightening to me was when I read the treatment of Goodman on Fourier Optics that shows that in the coherent case, Abbe theory based on the collection of diffraction orders from the object interfering in the image plane and the convolution approach using the Amplitude PSF are equivalent, because the amplitude transfer function involves a second fourier transform of the Airy Disc PSF that gives a circ while the incoherent case (e.g. from a self-luminous object) yields the autocorrelation of the aperture function because of the square operation in the spatial frequency domain (convolution theorem).
@perspectivex Жыл бұрын
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?
@Microcourses Жыл бұрын
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.
@christianforrer2332 Жыл бұрын
I kindly suggest you to do a video about the entrance and exit pupil, and the aperture and field stop as well, of an optical system. Might be presented in a more linear and clearer way than how I was taught those concepts in the first place.
@govindanunnipadmakumar1959 Жыл бұрын
This is so useful. Exactly what I was looking for. You explained it so well. The productive 20 minutes of my day :).
@Microcourses Жыл бұрын
Glad it was helpful!
@jrahm111 Жыл бұрын
Very clear explanation. Thank you for putting in so much effort!!
@Microcourses Жыл бұрын
Glad it was helpful!
@akatsuki6371 Жыл бұрын
Thanks for uploading! What programs do you recommend for image analysis?
@Microcourses Жыл бұрын
ImageJ/FIJI is a great place to start. You can get support for image analysis at the forum image.sc.
@CharlesGervasi Жыл бұрын
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.
@WastedElephant Жыл бұрын
On my regular old compound microscope, I would love to only move the high power lenses away from the slide when focusing. The problem is that I am unable to see the tiny distance from the the slide to the lens. This means I can't safely bring the lens to the slide in order to move it only away while focusing. 🫤
@huguesbitault7242 Жыл бұрын
Thanks a lot for the video. The explanation is crystal clear and easy to follow! Thanks for sharing.
@agnieszkadyrda9918 Жыл бұрын
Thank you so much for this explanation! It is much much clearer now. I am so happy that I found this video.
@DP-qb1zw Жыл бұрын
I covered the bottom of my stage petri dish holder with tape so the objective would hit a softer surface if accidentally touching it, plus, it is not fixed to the stage, so it can lift up, too, if touched by the objectives. But, best practice is lowering objectives before changing them.
@Bretaxy Жыл бұрын
Very useful information.
@mp3lwgm Жыл бұрын
Your equation for d makes it larger than the Abbe diffraction equation. According to Abbe the correct number in the numerator os 0.5 and not 0.61.
@Microcourses Жыл бұрын
The equation I’ve given is not mine. It’s the Rayleigh equation, which uses a different criterion than Abbe for defining resolution. Rayleigh criterion is the most commonly used and accepted.
@goombaboomba593 Жыл бұрын
Shiiiiiii tally with the clutch
@yaxinchen3507 Жыл бұрын
I was struggling to understand the whole concept for the last 3 days, until watching this 20 minute-video!
@MelHugs Жыл бұрын
This is a really clear and crisp explanation of why image quality can often be non-optimal in one sample, and completely non-standardised from sample to sample. Thank you! The partial solutions described are all good when considering the standard microscope slide system we have to work with. I would be very interested in talking to you about a way in which we can consider improving the sample position, during preparation, on the microscope slide rather than the partial solutions using the microscope.
@allenshaw9996 Жыл бұрын
Are there any downsides to using higher numerical apertures?
@allen8376 Жыл бұрын
If the pinhole at the image plane is what rejects the out-of-focus fluorescence, then why does focused illumination matter? (I.e., why laser instead of widefield?) Is it to reduce interference from neighboring fluorescence?
@roadrunner665310 ай бұрын
You want to put your light in the place where it can be used. If you illuminated everything, very little light would return and you'd have to sit on each voxel for a long time.
@allen837610 ай бұрын
@@roadrunner6653 so it’s for the intensity of light at the point of interest?
@roadrunner665310 ай бұрын
Yes, all the other light wouldn't be useful (not collected through the aperture) and you'd like to maximize the signal from the point of interest. That part of your sample might only return 1 in a million photons (low reflectivity). @@allen8376
@amribrahim7850 Жыл бұрын
Thanks for your informative video. I'd like to ask about the correlation cofficients used in co-localization between Magenta and Green, could you please elaborate how do you calculate that correlation between the two colors?
@Microcourses Жыл бұрын
This is an excellent question for the image analysis experts at image.sc discussion forum!
@MRchalkie666 Жыл бұрын
Nice instructional video! Perhaps we can say that spherical shaped lenses are easier to manufacture and polish, and so they are used, but they are the wrong shape to focus to a point. This is why it's called spherical aberration. Large aspherical lenses are possible to make and are the right shape, but the tiny ones needed on objective lenses are super hard to make and so are not used, and so extra lenses in the objective are used to cancel out the spherical aberration from the spherical top lens.
@Microcourses Жыл бұрын
Thank you for your comment and clear explanation!
@manishsuthar4002 Жыл бұрын
I've never come across lateral and axial in these waves... Thanks...
@vijaybenz9741 Жыл бұрын
Jenifer Walters attorney at law
@edward_grabczewski Жыл бұрын
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?
@edward_grabczewski Жыл бұрын
@@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?
@jacquescousteau4612 жыл бұрын
Thank you very much. This video really helped me going through the optical microscopy.
@davidtaylor35442 жыл бұрын
Extremely thorough, yet never confusing. Much appreciated!
@diatomsaus2 жыл бұрын
This is an exceptional video, but the diatom at 1:00 is certainly not Pleurosigma angulatum. Not trying to nit-pick or act smug, this is one of the best PSF videos I have watched so far.
@Microcourses2 жыл бұрын
Thank you! My records may be incorrect. What species is it?
@diatomsaus2 жыл бұрын
@@Microcourses I think it should be Frustulia rhomboides.
@LIght-lj5fx2 жыл бұрын
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(?)