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that is not the prolem of FM. The problem is that the images ar so unsharp. DIC is much better

PSF? More like "Perfect videos; you're the best!"

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!

Excellent explanation! Now I'm wondering how I'm going to keep my cat still under the objective, to get it into focus.... 🤔

Thank you so much!! That was very helpful.

This is one of the best lectures I have listen too about microscopy. Brilliant!

Damn... that cat is small AF!... jokes aside, great video.

much blah-blah and no results. DIC is the better solution because fluorescence-microscopy still gives unsharp images

awesome

Could not find a better instructional video on spherical aberration than this! Reach Brilliant when deciphering the aberration at the imaging plane.

Lovely to see the pink heart picture flashing out when doing the axial scanning (around 3'30") and she says "the PSF is beautiful!".

thanks for posting!

Thank you for your videos

you were able to make me understand these concepts with a 12 minute video

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!

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.

increase the subject illumination!!!

yeah this was actually incredible.

Fantastic.. I just taught a PG class on PSF.. This helped me a lot to prepare materials.

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!

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).

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?

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.

This is so useful. Exactly what I was looking for. You explained it so well. The productive 20 minutes of my day :).

Very clear explanation. Thank you for putting in so much effort!!

Thanks for uploading! What programs do you recommend for image analysis?

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.

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. 🫤

Thanks a lot for the video. The explanation is crystal clear and easy to follow! Thanks for sharing.

Thank you so much for this explanation! It is much much clearer now. I am so happy that I found this video.

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.

Very useful information.

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.

Shiiiiiii tally with the clutch

I was struggling to understand the whole concept for the last 3 days, until watching this 20 minute-video!

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.

Are there any downsides to using higher numerical apertures?

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?

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?

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.

I've never come across lateral and axial in these waves... Thanks...

Jenifer Walters attorney at law

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?

Thank you very much. This video really helped me going through the optical microscopy.

Extremely thorough, yet never confusing. Much appreciated!

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.

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(?)

Best explantion out there. Congratulations.

It’s also a property optical fiber, how does the definition vary in this as compared to lens? Why doesn’t the diameter matter

Thank you ♡