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Native fluorescence, or autofluorescence (AF), consists in the emission of light in the UV-visible, near-IR spectral range when biological substrates are excited with light at suitable wavelength.

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Photobleaching is the chemical alteration of the indicator dye.
it is unable to fluoresce due to the destruction of covalent or non-covalent bonds due to non-specific binding caused by excitation light.

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photo bleaching
auto- fluorescence
non specific fluorescence

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use of two antibodies.
promary recog. secondary Ab*  conjugated to a fluorescent dye.
eg: Bovine IgG

technique : first introduced by beutner and jordan. detection of Ab* to the intracellular spaces of squamous epithelia in sera of patients with pemphigus

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single Ab*
conjugated to a fluorescent dye.
eg: ATTO fluorescent conjugates

technique: first introduced by burnham et al., - lesional skin of lupus erythromatosis patients.

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direct immunofluorescence
indirect immunofluorescence

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distribution of proteins, glycoproteins, antigen targets

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tissue sections, cultured cells and individual cells.

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Fluorescence is the process where a material absorbs light at a high energy, short wavelength and emits light at a lower energy, usually visible, wavelength.

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these are used to visualize the location of bound antibodies

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assess both localization & endogenous expression levels of POI

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first to describe fluorescence

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an optical microscope that fluorescence instead of reflection, scattering, and absorption.