Super resolution (I) based on Array detectors with

Elyra PS.1 + AiryScan/LSM880

Microtubules imaged with Super resolution

SIM vs. confocal

Since 2018 IRB offers access to three modalities of Super resolution combined in a single System: the Elyra PS.1+ Airyscan.

Airyscan: The implementation by Carl Zeiss of technique "Image Scanning Microscopy" that collects multiple-pixel fluorescence intensity images with point scanning and a 32-channels array detector (as opposed to e.g. a PMT or point detector in confocal microscopy). All 32-pixels images are processed together in a pipeline called "pixel reassignment" to enable a final "up-to" 1.7x factor resolution improvement in scanning mode with deconvolution. The technique is compatible with live imaging and conventional fluorescence. Because the detector collects and uses all the light emitted by the sample around the excitation beam, as opposed to rejecting light with a pinhole in confocal microscopy, Airyscan is much more sensitive hence -much faster or -more gentle than confocal microscopy, or both.

The resolution improvement is achieved in 2D and 3D.

SIM: Structured Illumination Microscopy. A factor 2x resolution improvement achieved by reconstructing various images with a grid illumination pattern. Suitable for live imaging although relatively slow (in the order of 1 reconstructed image per second), the main strength of SIM stands in the easy of use as it is compatible with most conventional fluorophores and does not require specific or advanced sample preparation.

The resolution improvement is achieved in 2D and 3D.

Single Molecule Localization Microscopy: aka PALM/STORM, dSTORM etc...

Using the blinking properties of certain fluorophores, individual fluorophores are images sparsely in time and space, hence enabling to determine their localization with very high precision (20-30nm laterally, 50-60nm axially). The final image is obtained by analyzing thousands of images containing dozens/hundreds of localizations, which are then displayed in a single 2D map.

Resolution improvement can go up to 10x, as compared to conventional microscopy, with suitably selected fluorophores. 3D localization is possible by introducing a diffractive element that will apply a optical pattern to the intensity response of single fluorophores, and such pattern will change orientation according to the relative position of the fluorophore with respect to the image plane, hence enabling to assign an axial position to the fluorophore's localization. 3D SMLM generally induces a slight resolution decrease compared to 2D SMLM.

SMLM is in general not suitable for live imaging, and special sample preparation is required.

For info, please contact Nikolaos Giakoumakis  nikolaos(dot)giakoumakis(at)irbbarcelona(dot)org