Spread out bates resolution easily

Aug 6th, 2022
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How to spread out bates resolution

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Hello everyone! Welcome to the microscopy lecture series. My name is Xiaowei Zhuang. And I am a professor at Harvard University and an investigator of Howard Hughes Medical Institute. And today what I am going to tell you about is a brief introduction of super-resolution fluorescence microscopy, which breaks the diffraction limit. And such imaging methods allow us to look at cells with much better clarity. For example, here we see a conventional image of mitochondria. And here is a three dimensional super-resolution image of mitochondria. So Ill tell you a few methods that allow us to get there. But first let me tell you why we like fluorescence microscopy so much. As we all know, this is one of the most widely used imaging modality for biological research. And two of the advantages of fluorescence microscopy is really very well recapitulated by this movie that I downloaded from the book . by Bruce Alberts et al. So first right away jumping into the scene even without even knowing w

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Thus, the only mechanism for optimizing spatial resolution and image contrast is to minimize the size of the diffraction-limited spots by decreasing the imaging wavelength, increasing numerical aperture, or using an imaging medium having a larger refractive index.
STED microscopy allows super-resolution imaging in the 50nm range. Unfortunately, this increased optical resolution also leads to a drawback: because many fluorophores are depleted by the depletion laser, this also results in a lower signal (fewer photons) being captured by the detector.
Defining super resolution This limit, also called the point-spread function (PSF), is the fixed size of the spread of a single point of light that is diffracted through a microscope; it is also a measure of the minimum-size point source or object that can be resolved by a microscope.
STED microscopy surpasses this limit by taking advantage of the saturated response of fluorophores: once the depletion laser intensity is above the saturation level, the number of fluorophores remaining in the excited state (and thus capable of generating fluorescence) approaches zero.
Stimulated Emission Depletion Microscopy (STED) is a method to resolve structures below the limits of optical resolution and is therefore attributed to super-resolution. STED uses a differential method of two different diffraction patterns, where one pattern excites and the second pattern de-excites fluorochromes.
In order to increase the resolution, d = /(2NA), the specimen must be viewed using either a shorter wavelength () of light or through an imaging medium with a relatively high refractive index or with optical components which have a high NA (or, indeed, a combination of all of these factors).
This means that for a standard fluorescence system, such as a confocal microscope, the lateral resolving power is at best limited to around 200 to 250 nm and axial resolution to around 600 nm.
These interactions occur within a few nm, but localizing protein pairs is diffraction-limited to approximately 200 nm. STED enables imaging with sub-diffraction resolution down to below 50 nm.

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