Structured illumination microscopy

Principle

The resolution limit to define two very near molecules is defined by the Rayleigh distance (0,61λ/NA). With the classic microscopy we cannot distinguish two molecules that are at a distance inferior to that Rayleigh distance. The SIM microscopy allows a resolution gain of  two times in all axes (100 nm x-y, 300 nm z).At SIM microscopy a structured illumination excites the fluorescent sample. The illumination pattern is superposed with the pattern of the structures in the sample and their inteference creates a third characteristic pattern (Moiré franges, figure 1).

The Moiré franges have a spatial frequency lower than the one of the original structures in the sample and thus can be transmitted by the objective lens and a the end visualize them. The 3 patterns are independent and can be used to knowthe super-resolved details of the sample, i.e. detect the higher frequencies. For an isotropic resolution gain in all dimensions, the illumination pattern is rotated 3 times (3 angles), along 5 phases for each angle (i.e. 15 images per z slice).

Available equipment

The facility disposes an ELYRA PS.1 Zeiss microscope.  The ZEN software can reconstruct the images and provide the super-resolved image.
Objectives : immersion Zeiss 100x , NA 1.46
                       Immersion Zeiss 63x, NA 1.40
Excitation Lasers : 405 nm (50mW), 488 nm (300mW), 561 nm (200mW), 641 nm (150mW).
For every laser line corresponds a different grids to create the structured illumination pattern.
Detection :
EMCCD Andor iXon 885 (1004x1002, pixel size 8μm, QE=65%)

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