Modern examination methods in cell biology
Various modern optical, chemical and biological methods are used or combined in order to understand the function of cells and to resolve structures under the optical resolution of light microscopes . These methods are summarized under the term "Live Cell Imaging". As this name suggests, it is about studies on living cells and cell complexes to entire living organisms such as zebrafish - embryos . Electron microscopic methods that look at fixed tissue are therefore useless.
Methods
- Confocal microscopy : A form of light microscopy that enables optical sections to be made through a specimen. The advantages of confocal microscopy lie in the very sharp and almost background-free acquisition of images. Furthermore, the reconstruction of three-dimensional structures in the computer is made possible.
- 2-photon microscopy:
- YFP / GFP / CFP labeling : labeling using fluorescent proteins synthesized in the cell itself
- Staining using ion-sensitive dyes : Investigating processes on (nerve) cells. Cells are filled with different dyes, the emission spectrum or intensity of which depends on the concentration of certain ions. This makes it possible to measure changes in the concentration of these ions. In certain cases it is also possible to determine absolute ion concentrations.
- FRET (Förster resonance energy transfer): FRET is used to prove the interaction of proteins, to determine the concentration of nucleic acids and as an "optical nanometer scale".
- BRET (bioluminescence resonance energy transfer): Modification of FRET, where the acceptor fluorophore is a bioluminescence source.
- BiFC (Bimolecular fluorescence complementation): The method is used to detect and localize protein-protein interactions. It can be used as a replacement or supplement to FRET and BRET.
- FRAP (fluorescence recovery after photo-bleaching): a fluorescent dye is bleached selectively by strong lighting. The increase in fluorescence in the bleached area depends on the mobility of the dye. In addition to diffusion coefficients of molecules, FRAP can be used to determine a whole range of parameters that influence the mobility of molecules.
- FLIP (fluorescence loss in photo-bleaching): An area of a cell is repeatedly bleached by strong light. At the same time, the fluorescence is observed throughout the cell. Areas that become darker are in a continuum with the bleached area.
- FLIM (fluorescence lifetime imaging microscopy): instead of fluorescence intensity, the fluorescence lifetime (FL) is used as image information. The FL is the mean residence time of a fluorophore in the excited state. While the fluorescence intensity depends on various factors such as the local concentration of a dye, the FL mainly reflects the chemical environment.
- TIRFM (Total Internal Reflection Microscopy): Often referred to as evanescent wave microscopy . Laser light is radiated onto the underside of the specimen at such a flat angle that it comes to total reflection . Along with the reflection, part of the light penetrates approx. 40 nm deep into the specimen (the so-called evanescent wave ). This makes it possible to excite fluorescence in a very thin optical section.
- FCS (Fluorescence Correlation Spectroscopy): Investigation of the Brownian movement of dye molecules, with which one can infer the size distribution of the particles to which they have docked
- DIC (differential interference contrast): method of contrast enhancement, which is mainly used in tissue sections. Often, infrared light is used. One then speaks of IR-DIC .
Individual evidence
- ↑ Neuropathology LMU ( Memento from September 29, 2007 in the Internet Archive )