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Super-resolution fluorescence microscopy

Super-resolution in fluorescence microscopy can be defined as all techniques with a resolution higher than the classical diffraction limit of light.

The Centre for Cellular Imaging has an ELYRA 7, which can perform Lattice Structured Illumination Microscopy (SIM) and Single Molecule Localization Microscopy (SMLM). The ELYRA 7 microscope is a widefield inverted microscope designed for high-speed super-resolution imaging of biological specimens with minimal light exposure.

Structured Illumination Microscopy

Linear SIM

Structured Illumination Microscopy (SIM) is a technique, which overcomes the diffraction limit by using a patterned illumination and hence creating a moaré effect in the image. An image with twice the resolution in all three spatial dimensions can then be calculated from the known illumination pattern and the recorded image.

SIM was pioneered by Gustaffson, and in its first implementation a line grid pattern was used to produce the SIM illumination pattern. This example is shown in the image below [Vangindertael]

The principle of SIM: The sample is illuminated by a striped pattern. Different illumination rotations creates different Moaré patterns in the image.

This line structuring element allows to obtain higher resolution along one spatial direction. Therefore, the pattern must be rotated to improve the resolution in other directions and obtain an isotropic image resolution. A common approach was to rotate the pattern on 3 different orientation and translate it on 5 different steps, generating a total of 15 images. These 15 images are then combined to obtain the final super-resolved image. Such rotation is time consuming and thus limits the imaging speed.

Lattice SIM

Three-dimensional point patterns, referred to as Lattice SIM,  for SIM have been described previously by [Betzig] and [Heintzmann]. This is the technology used by the ELYRA 7. In Lattice SIM, the spot pattern is shifted laterally, without rotation steps, which leads to faster imaging and allows live cell imaging of dynamic processes. The image bellow shows simulations of Linear, Quadratic Lattice and Hexagonal Lattice light patterns. The Elyra 7 uses a quadratic  lattice pattern for structured illumination microscopy.

Simulated Lattice SIM illumination patterns
Illumination pattern at 488 nm excitation (NA 1.4).
Foto: ZEISS
Example of lattice SIM light pattern
Example of lattice SIM light pattern while imaging mitochondria. Sample by Syam Bhuvanachandran Nair
Foto: Rafael Camacho CCI
Example of SIM reconstruction on Mitochondria, samples by Syam Bhuvanachandran Nair
Example of SIM reconstruction on Mitochondria. The image changes from Widefield (resolution ~300 nm), to SIM (resolution ~150 nm), to SIM² (resolution <100 nm) samples by Syam Bhuvanachandran Nair
Foto: Rafael Camacho CCI

Single Molecule Localization Microscopy

Single Molecule Localization Microscopy (SMLM) is the common name of several techniques, which utilizes the blinking of fluorophores to surpass the diffraction limit, such as PALM, dSTORM etc. In all of these techniques, a small subset of fluorophores are imaged in each frame of a long time series. The localization of each detected flourophore is found and the final superresolution image is reconstructed from all localization positions.

The principle of SMLM: Instead of collecting the signal from all the labeled molecules in the sample at the same time, like in the widefield (WF) technique, only a few molecules are imaged in each frame of a time series.
Example of blinking for SMLM, NPC
Blinking of fluorescent molecules attached to the Nuclear Pore Complex of a cell. Processing of these "blinking" movies allows for the reconstruction of super resolved images with resolution < 100 nm
Foto: Rafael Camacho CCI
Example of SMLM processing, NPC
Output image after SMLM reconstruction. Top left WF image. Bottom right super resolved image.
Foto: Rafael Camacho CCI