Super resolution microscopy | Stimulated emission depletion (STED) microscopy
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- เผยแพร่เมื่อ 21 ก.ค. 2024
- Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimising the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system.
It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development.
Two very important things that I think are wrong here:
1- the smallest spot that an optical system can resolve is not the wavelength of light (lambda) but lambda/(2NA), NA being the numerical aperture of the optical system. That is the diffraction limit, also defined as the Abbe limit. This can be explained when studying a single slit diffraction: the position of the first minima in the diffraction pattern = diffraction limit.
2- It is breaking the diffraction limit not the PSF. PSF is the diffraction pattern that is imaged through the optical system but diffraction limit is the first minima position of the PSF pattern. So high resolution is breaking this diffraction limit not breaking the PSF.
I believe you are completely right, I thought the same when watching the video
Thank you!
Exactly, but to be precise, by using depletion laser onto excitation laser (= excitation PSF) you shape it's PSF to a more narrow point (referred to the middle spot of usual PSF surrounded by the first minimum). It is PSF-shaping, which allows you to sum up point records of the object by scanning mode into high res image.
you explained very difficult concept in simply way. Thank you
great job; making it so easy to understand!
Thank you for this informative video!
Super helpfull, thank you for the clear explanations !
Your effort is appreciated, but you left out the ring-shaped illumination, the essence of STED besides the depletion laser! Your slide with the orange laser somehow illuminating a series of spots around the green spot is thus incorrect. You are also mixing up STED and single molecule localization techniques (e.g. STORM and PALM) when you mix in the spot-fitting software algorithm into the presentation at the end.
10/10 very helpful, thank you!
Excellent, thank you for a clear and concise explanation!
very helpful. Thank you
I think STED technique doesn’t have to be confocal, as the later is only defined by a notch filter, aiming to improve vertical resolution, and doesn’t necessarily contain Raster scanning ( scann pixel by pixel). I’m just a student and could be wrong, hope someone could confirm my point.
OMG thank you very much!!!!
thanks a lots KING
you're the best!
Why are you whispering
I wonder how hard it would have been to get a speaker of regular English? Too hard apparently.
imagine knowing only English.
what a sad life.
@@nelsonfavedraDon't be so hard on him. 80% of his time in school is taking classes on how to avoid mass shootings. He does not have time for such simple things as learning a new language or geography