Limiting factor of microscopy resolution?

[ElanHR]

Just to preface this to make sure that my understanding so far isn't totally off. Photon-microscopy works by

• shooting a laser at cells of interest through a beam splitter
• the laser hits the cell and excites an electron into a higher state
• using multiple photons allows us to impart the necessary excitation energy divided amongst multiple photons but the photons must arrive simultaneously.
• the electron returns to the relaxed state emitting a photon in the process (which we read reflected off the beam splitter)

When there are breakthroughs in resolution is this because they:

• managed to focus a higher energy/shorter wavelength laser
• found a different marker to activate
• geometric concerns
• something else?

[yaxigeigei]

Your summary is basically right. Although I am not sure about what the beam splitter you are referring to (dicoric mirror or filters?), it may not be very important. Anyway, you can find vivid comparison among bright-field, confocal, and 2-photon microscope (in general multi-photon) on Youtube which could be very helpful to understand why and under what circumstances we want to use them.

The textbook limitation of resolution is wavelength and the numerical aperture of the objective lens. http://en.wikipedia.org/wiki/Optical_resolution This simply dictates the physical limit of resolution in conventional imaging.

However, there are several ways to circumvent the limit by using some (statistical, physical or optical) tricks. I am guessing that these are the breakthroughs you would like to know? I can provide two examples.

1) STED You know pretty well how excitation works at atomic scale. Imagine that, before excitation, we slightly excite electrons just a bit using photon with longer wavelength and make them away from relaxed state, when the real excitation photon comes, there would be no electron with suitable state to excite. In another word, we "depleted" the suitable electrons in this area. Based on this phenomenon, if we create a ring of depletion using longer wavelength light and use normal excitation light (with right wavelength) shining at the center, the area can be excited will be better confined. In this way, the resolution is improved. For more info, see the intro from one of the inventor himself. https://youtu.be/YyBGiZZSslY

2) STORM If I remember correctly, this is based on "suprasampling" using very weak excitation light. As a result, we get very sporadic signal. But by using statistical tools we can recover the true location of the fluorphore. I am not very knowledgeable about this but you can see this video given by the inventor herself. https://youtu.be/w2Qo__sppcI

[akim1]

Here are my guesses: Diffraction limit is the physical limit of your spatial resolution, but shorter wavelength light starts to fry your tissue/proteins. So you can only shorten your wavelength so much especially if you want your tissue to be alive. Higher resolution means tolerance for aberrations in your optics is much lower. Higher spatial resolution also means you also need more sensitive detection (or camera).

I'm not really sure spatial resolution is the limiting factor in microscopy in general. It seems like current state-of-the-art resolution lets us see all the things in a cell you would really want to see except maybe single molecules.

It would probably be nice to see deeper than the 1 mm or so that 2-photon microscopy allows?

[yaxigeigei]

1) Short wavelength light (e.g. UV) will break chemical bonds in DNA and proteins, cause inflamatory responses and perturbation of cellular homeostasis, but not fry the cell. 2) Instead of saying more sensitive camera, I would prefer higher signal-to-noise ratio which can be improved from sample, fluorphore, laser power and so on. In practice, the limiting factor is usually not the camera or photon multiplier tube. 3) The state-of-the-art spatial resolution is enough because it uses super-resolution techniques when high resolution is required. The physical resolution limit of optic microscope is far not enough to see what we can and want to see now. That's what the Nobel Prize of the last year for. 4) We can see single molecules under optic microscope. See quantum dot, for one example. 5) Indeed, due to diffraction, the limit for two-photon in live specimen is 1mm. However, for fixed samples, one can use tissue clearing techniques to make the sample "transparent" leaving only the labeled fluorescent signals.