Optical microscopy has developed rapidly in recent years and has thus become an indispensable tool in cellular and molecular biology research. The focus of the optical microscope unit of the core facility BioSupraMol is on fluorescence-based microscopy, in particular on confocal laser scanning microscopy (CLSM) and total internal reflection fluorescence microscopy (TIRF microscopy).
Confocal laser scanning microscopy enables a significantly higher image contrast and inherent depth discrimination compared to conventional microscopy and is used mainly for fluorescence microscopy tasks in biology and medicine. In addition, conventional contrast methods such as the differential interference contrast (DIC) can be imaged and for the analysis of e.g. particles or surfaces, the reflection mode can be used.
TIRF microscopy can be used to investigate structures close to surfaces. The fluorescence of the specimen is excited by an evanescent field, which is generated by total internal reflection of the incident light in a restricted region of the specimen immediately adjacent to the cover glass. This limits the fluorescence emission to the region near the glass-specimen interface (approximately 100 nm) and leads to a markedly enhanced resolution compared to CLSM.
Optical super-resolution microscopy allows the imaging of structures in cells below the diffraction limit of visible light. Single-molecule-based high-resolution microscopy is one of the methods that was awarded the Nobel Prize in 2014. The method bypasses the diffraction limit by sequential imaging and localization of single molecules. These techniques (for example (d)STORM, DNA-PAINT and qPAINT) are available by using the Vutara 532 microscope and software.
Users of the core facility BioSupraMol have access to two confocal laser scanning microscopes from Leica Micro Systems (SP8), an inverse fluorescence microscope from Zeiss (Axiovert 200M) with Visitron Laser TIRF expansion and CoolSNAP CCD camera and an inverse single-molecule microscope Vutara 532 from Bruker with a large-area Hamamatsu sCMOS camera and 1W 640 nm laser excitation.