Analysis for Single Molecule Localization Microscopy
Fluorescence microscopy is currently the most important tool for visualizing biological structures at the sub?cellular scale. The combination of fluorescence, which enables a high imaging contrast, and the possibility to apply molecular labeling, which allows for a high imagingspecificity, makes it a powerful imaging modality. The use of fluorescence microscopy has risen tremendously, in particular since the introduction of the green fluorescent protein (GFP) in the mid-1990s and the possibility to genetically engineer cells to express these proteins.
It shows the basic layout of a fluorescence microscope. Excitation light of a certain wavelength is reflected via a dichroic beamsplitter and projected onto the specimen via the objective lens of the microscope. The light is absorbed by the fluorescent labels and re-emitted, slightly Stokes-shifted by ?100 nm, at a larger wavelength, typically a few nanoseconds later. The emission light is captured by the objective lens and directed toward the camera via the dichroic beamsplitter.
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