ELYRA 7
Hardware Aspects
ZEISS
03/2019 V_02
000000-2262-999
9
2
Hardware Aspects
2.1
Principles of Superresolution Microscopy (ELYRA)
A point object imaged by an objective lens will always be a blurred spot, the so called point spread
function (PSF), due to the diffraction of light.
Therefore two points cannot come closer than a certain limit in order to still be resolved. This minimal
distance in object space corresponds to a maximal cut-off frequency in the frequency space that can be
transmitted through the objective lens.
The transmittable frequencies represent the so-called optical transfer function (OTF), which is the Fourier
transform of the PSF. As a rule of thumb the resolution of a far field light microscope in the lateral
direction is approximately half the wavelength, whereas it is 3 fold worse in the axial direction.
Hence, for a light microscope lateral and axial resolutions are approximately 200 and 600 nm,
respectively. In order to enhance resolution, the PSF has to be narrowed, which is synonymous to the OTF
to be expanded.
This exactly is accomplished by structured illumination microscopy (SIM) and photo activated localization
microscopy (SMLM).
2.1.1
Principle of Structured Illumination Microscopy (SIM)
In SIM a sinusoidal pattern, e. g. a line grid pattern (Stripe SIM), or a dot pattern (Lattice SIM) with a
defined periodicity, is positioned in the excitation path in a plane that is conjugated to the image plane
(Fig. 2).
Hence the pattern is projected onto the image and the excitation intensity is modulated along the
pattern. This results in a modulation of the fluorescence as well.
The highest modulation contrast is obtained in the focal plane. The contrast gets weaker with distance to
the focal plane so that depth discrimination in the axial direction is possible. The grid constant is ideally
the cut off frequency of the system.
The interferences of the diffraction orders generated through the grid are used for the lateral and axial
structuring of the light.
Fig. 2
Principle of structured illumination – diffraction orders from the
modulation grid (line grid pattern) and the sample structures are
indicated