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Toxicity: Aome fluorochromes can be toxic to cells, to tissues, in vivo, or by producing mutations. [10] Limited resolving power: Fluorescence microscopes are limited in their ability to distinguish close objects at the macroscopic level. In comparison, electron microscopes for example, have the capacity to resolve at a much smaller range.
Multi-color images of several types of fluorophores must be composed by combining several single-color images. [1] Most fluorescence microscopes in use are epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e. through the objective).
A simplified Jablonski diagram illustrating the change of energy levels.. The principle behind fluorescence is that the fluorescent moiety contains electrons which can absorb a photon and briefly enter an excited state before either dispersing the energy non-radiatively or emitting it as a photon, but with a lower energy, i.e., at a longer wavelength (wavelength and energy are inversely ...
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds .
The DyLight Fluor family of fluorescent dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. [4] DyLight dyes are typically used in biotechnology and research applications as biomolecule , cell and tissue labels for fluorescence microscopy , cell biology or molecular biology .
Fluorescence-lifetime imaging microscopy or FLIM is an imaging technique based on the differences in the exponential decay rate of the photon emission of a fluorophore from a sample. It can be used as an imaging technique in confocal microscopy , two-photon excitation microscopy , and multiphoton tomography.
IF can additionally be used in combination with other, non-antibody methods of fluorescent staining, e.g., the use of DAPI to label DNA. [10] [11] Examination of immunofluorescence specimens can be conducted utilizing various microscope configurations, including the epifluorescence microscope, confocal microscope, and widefield microscope. [12]
Most light sheet fluorescence microscopes are used to produce 3D images of the sample by moving the sample through the image plane. If the sample is larger than the field of view of the image sensor, the sample also has to be shifted laterally. An alternative approach is to move the image plane through the sample to create the image stack. [32]