ELYRA 7
Hardware Aspects
ZEISS
03/2019 V_02
000000-2262-999
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SIM image requires a minimum of 3 x 5 = 15 images for a line grid pattern. Finally a back transformation
from the frequency to the real space generates the high resolution image.
In Lattice SIM we have a 7 orders. Hence 13 (2 x 7 + 1) phase shifted images are minimally required. In
ELYRA 7 15 phase images are used to have more information for reconstruction of the SIM image. Since
the Lattice SIM pattern is symmetrical, no rotaion is necessary.
2.1.2
Principle of Photo Activated
Localization Microscopy (SMLM)
In SMLM the sample is illuminated in a regime that
statistically activates only one fluorescent molecule
per PSF.
Hence, the centre of mass of the PSF can be
determined, which can be done more precisely
than the PSF itself. The accuracy of determination
depends solely on the number of photons
collected.
The resolution enhancement is by a factor of 1/
√
N
with N representing the number of photons
(Fig. 5).
To image molecules one at a time in a PSF requires
certain properties of the fluorescence dye.
It has to exist in a bright and dark state, which can
be converted between each other.
Most of organic dyes show such a behavior in
certain reducing environments.
And there are also three classes of fluorescent
proteins (FPs) with the required switching
characteristics.
Photoactivatable FPs can be irreversibly switched from a dark to a bright state by violet light.
The bright state is than bleached by the imaging laser light. Photoconvertable FPs can be irreversible
switched from one spectral state to another by violet light.
The latter one is than bleached by the imaging laser.
Photoswitchable FPs are reversibly switched from a dark state to bright state by violet light and will turn
back to the ark state by illumination with the imaging laser light.
It is the correct balance between the activation light (mostly violet light) and the imaging light (mostly
strong powers needed) to that determines how many molecules are in the on-state and how many
molecules are in the off-state.
Fig. 5
The accuracy of the determination of
the center of mass depend on photon
number N