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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).
Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy. Fluorescence itself, is a form of luminescence that results from matter emitting light of a certain wavelength after absorbing electromagnetic radiation. Molecules that re-emit light upon absorption of light are called fluorophores. [1] [2]
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]
A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide.
Light sheet microscopy (LSM) was developed to allow for fine optical sectioning of thick biological samples without the need for physical sectioning or clearing, which are both time consuming and detrimental to in-vivo imaging. [10] While most fluorescent imaging techniques use aligned illumination and detection axes, LSM utilizes orthogonal axes.
Köhler illumination is a method of specimen illumination used for transmitted and reflected light (trans- and epi-illuminated) optical microscopy.Köhler illumination acts to generate an even illumination of the sample and ensures that an image of the illumination source (for example a halogen lamp filament) is not visible in the resulting image.
Fluorescence lifetimes can be determined in the frequency domain by a phase-modulation method. The method uses a light source that is pulsed or modulated at high frequency (up to 500 MHz) such as an LED, diode laser or a continuous wave source combined with an electro-optic modulator or an acousto-optic modulator. The fluorescence is (a ...
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]
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