Christoph Florian Schaller - FU Berlin, FB MI

Christoph Schaller - STORMicroscopy 5
1.3 STORM - the basic idea
The basic idea is presented in principle in the
adjacent scheme in Figure 1.5. At rst photoswitchable
uorophores are attached to specic
molecules, e.g. nucleic acids or proteins, in an
immobilized sample. Then one (optically resolvable)
subset is activated by laser excitation of
a specic wavelength. Thereafter the activated
uorophores emit photons while imaging occurs.
That way one obtains a coarse matrix for every
frame, which contains the number of collected
photons within every pixel. Using these one is
now able to reconstruct the spot centers utilizing
a so called tting algorithm. After waiting for
the activated uorophores to go back into a dark
state, one can repeat the described process several
times to obtain a STORM image until most
of the uorophores were excited at least once.
As the cautious reader might have observed
there are several preconditions to be satised.
On the one hand we would like to have wellseparated
objects to easily distinguish them from
each other, on the other hand we need to be able
to label only specic subsets in a discriminable
Figure 1.5: The STORM imaging process. [8]
way. Furthermore if our objects are too large
themselves, reducing them to one point is not very meaningful, thus we assume them small enough to
be considered punctate.
The following Figure 1.6 depicts the improved resolution due to STORM compared to immunouorescence
microscopy.
Figure 1.6: STORM imaging of microtubules
in a mammalian cell. [9] (A)
Conventional immunouorescence image
in a large area. (B) STORM image of the
same area. (C and E) Conventional and
(D and F) STORM images corresponding
to the boxed regions in (A).