Microscopy - L6

• TIRF

• STED

• PALM

• dSTORM/STORM

• SIM

Total Internal Reflection Fluorescence Microscopy

• based on total internal reflection of sample -> creates evanescent field at glass-water surface

• fluorescence on surface of sample -> does not penetrate deeply -> no background fluorescnce

• highly depends on alignment/angle

• prism method: produces cleaner evanescent field with less scatter

• through-the-object-method: angle of excitation can be easily changed

Stimulated Emission Depletion

• problem: distinguishability between two points

• selective suppression of fluorescence

• after normal excitation, donut-shaped STED beam forces fluorophores back to ground state via stimulated emission

• imaging beyond diffraction limit

PhotoActivated Localization Microscopy

• different fluorophores seperated by time

• only random set is activated at a time, then irreversible bleached

• each single spot can be precisely localized within nm by Gaussian fit

• all spots combined in one image

Stochastic Optical Reconstruction Microscopy (STORM)

• d -> only one dye

• fluorescence signals described mathematically by point spread function (-> Gaussian function as approximation)

• center of PSF can be calculated -> more photons (brightness) = higher accuracy

Structured Illumination Microscopy

• illuminate sample with fine spatial pattern

• creates moiré effect at points, where structure is finer than diffraction

• multiple images rotating the light pattern

• compute structure off all these images

• fast record of large field-of-view, twice the resolution of widefield microscopy