Super Resolution (SR) Microscopy is a name given to a family of fluorescence-based microscopies that break the resolution limit (determined by diffraction in perfect optical system). There are a handful of different SR technologies available in the imaging facility.
With high-NA lenses the diffraction limit for standard fluorescence microscopy is ~200 nm. Our Zeiss Elyra Super Resolution Microscope supports several SR technologies: localization-based (e.g. PALM or STORM), structured illumination (SR-SIM) and total internal reflection fluorescence (TIRF). Our inverted Zeiss LSM880 supports Airyscan technology.
Super Resolution technologies available in the Imaging Facility.
A) Localization methods stem ultimately from fluorophore blinking. In A1 individual fluorophore positions cannot be resolved (yellow dot in the center of the diffraction-limited red glow) However, if the fluorophores can be put into a non-fluorescent state from which a very limited number turn on during individual frames (A2), they can each be resolved to the centroid of the diffraction-limited spot (A3). By reconstructing an image from thousands of localizations taken from many frames, a synthesized image can be produced with up to 10 nm resolution. The two most common localization methods are referred to as STORM (STochastic Optical Reconstruction Microscopy), which uses photoswitching organic dyes, and PALM (PhotoActivated Localization Microscopy), which employs photo-activatable genetically-encoded proteins. Localization methods require specialized fluorophores and often specialized buffers. They are relatively slow and generally not appropriate for living cells, but localization methods can yield images with tenfold enhanced resolution (~20nm). Figures A1-A3 are adapted from Huang et al, Ann Rev Biochem 17, 993-1016 (2009).
B) Super Resolution Structured Illumination Microscopy (SR-SIM) works by illuminating the sample with a series of patterns that mix with sub-resolution sample frequencies to become resolvable “beat” information. SR-SIM is faster, does not require specialized fluorophores or sample preparations and is usually the method of choice for live cells. This method typically results in a two-fold increase in both the lateral and axial resolution.
C) TIRF illumination only excites fluorescence in the evanescent field directly at the coverslip surface (<100 nm away), typically five-fold smaller than the axial resolution in a standard microscope. On our Elyra Microscope, TIRF illumination can be used separately or in conjunction with PALM or STORM.
D) Airyscan technology, supported by our inverted Zeiss LSM880 confocal microscope, uses a PMT array rather than a pinhole-PMT arrangement. Any imaged point of light results in an Airy Disk or Point Spread Function (PSF, D1), the size of which determines the resolution in any microscope. In a standard confocal microscope, the PSF is focused to the pinhole and collected by a single element PMT. In Airyscan mode, the PSF is instead focused to a 2-D PMT array (D2). Because the PSF is detected by multiple elements rather than a single element, information about its shape rather than just its overall intensity is collected, enabling a resolution that is smaller than the size of the PSF. Airyscan, like SR-SIM, is relatively fast, does not require specialized fluorophores, can be used in live cell applications and results in a 1.8-fold resolution increase. Airyscan is more appropriate to thicker preparations than the other technologies.