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ZEISS
Hardware Aspects
ELYRA 7
12
000000-2262-999
03/2019 V_02
Since only a few molecules are active at a time the procedure of activating to convert molecules to the
on-state followed by deactivation to convert them to the off-stateh has to be repeated many times in
order to detect all labeled molecules (Fig. 6).
So the aim in SMLM is to activate a few molecules, calculate their center of mass, plot them in a new
coordinate system and deactivate them quickly (preferentially after 1 to 3 frames) and repeat the process
until statistically all molecules have been activated.
So a SMLM experiment easily needs 10 000 frames and more.
The resulting image is a plot of all molecules localizations with a certain precision depending on the
photon numbers obtained from the molecule.
Fig. 6
Principle of SMLM – Whereas in normal widefield images the PSF of
closely juxtaposed point sources can overlap heavily (upper panel)
their sequential illumination and plotting of their center of mass will
result in their clear separation (lower panel)