1. SHRIMP – Super High Resolution IMaging with Photobleaching 2a. PALM – Photoactivated Localization Microscopy b. STORM – Stochastic Optical Reconstruction Microscopy How to get super-resolution microscopy. Nanometer-scale instead of micron-scale FIONA & Turn on/off dye (accuracy and resolution)
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1.SHRIMP – Super High Resolution IMaging with Photobleaching 2a. PALM – Photoactivated Localization Microscopy b. STORM – Stochastic Optical Reconstruction.
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1. SHRIMP – Super High Resolution IMaging with Photobleaching
You have PALM spelled out with really tiny molecules separated by a tiny distance—such that each letter is less than a diffraction limit apart.How to see what is written? First you try regular fluorescence, labeling it with some fluorescent dye and shine light to make it fluoresce. What do you see? Each dye emits with a diffraction-limited (i.e., about 250 nm) size. The result is B. It’s not well resolved.However, if you can make each fluorescent molecule emit one at a time, then you can determine where the dye is by doing FIONA—taking the SEM (instead of the Standard Deviation), where you can determine it’s position to within a few nanometers. Then you repeat this measurements many many times, until you get the entire image. See next page.
Activate with weak near UV-light; Once activated, shine visible light to get out fluorescence. Locate each fluorphore to within a few nanometers by taking the center of the emission (rather than the diffraction-limited width). Record the position of these molecules, Then repeat, until you get all of the position of all of the fluorophores.
“Regular” dyes can be made to blink They are off; then can be made to come on.
(Cy3B, Cy5, Alexa 647…)
You have measured kinesin movingYou will measure the width of microtubules.