Light Microscopy: Exploring Cell Structure and Visualizing Proteins Within Cells
The resolution of the light microscope, about 0.2 µm, is limited by the wavelength of light.
Differences in refractive index can be used to observe parts of single cells by employing phase-
Tissues generally have to be fixed, sectioned, and stained for cells and subcellular structures to be observed.
Fluorescence microscopy makes use of compounds that absorb light at one wavelength and emit it at a longer wavelength.
Ion-
Immunofluorescence microscopy makes use of antibodies to localize specific components in fixed and permeabilized cells.
Indirect immunofluorescence microscopy uses an unlabeled primary antibody, followed by a fluorescently labeled secondary antibody that recognizes the primary one and allows it to be localized.
Short sequences encoding epitope tags can be appended to protein-
Green fluorescence protein (GFP) and its derivatives are naturally occurring fluorescent proteins.
Fusing GFP to a protein of interest allows its localization and dynamics to be explored in a live cell.
Deconvolution and confocal microscopy provide greatly improved clarity in fluorescent images by removing out-
Total internal reflection fluorescence (TIRF) microscopy allows fluorescent samples adjacent to a coverslip to be seen with great clarity.
Fluorescence recovery after photobleaching (FRAP) allows the dynamics of a population of molecules to be analyzed.
Förster resonance energy transfer (FRET) is a technique in which light energy is transferred from one fluorescent protein to another when the proteins are very close, thereby revealing when two molecules are close in the cell.
Super-
Light-