Editing Microscopy (section)

==== Fluorescence ====
[[File:Anther of thale cress (Arabidopsis thaliana), an artefact.jpg|thumb|Images may also contain [[Artifact (error)|artifacts]]. This is a [[Confocal laser scanning microscopy|confocal laser scanning]] [[Fluorescence microscopy|fluorescence]] [[micrograph]] of [[Arabidopsis thaliana|thale cress]] anther (part of [[stamen]]). The picture shows among other things a nice red flowing collar-like structure just below the anther. However, an intact thale cress stamen does not have such collar, this is a fixation artifact: the stamen has been cut below the picture frame, and [[Epidermis (botany)|epidermis]] (upper layer of cells) of stamen stalk has peeled off, forming a non-characteristic structure. Photo: Heiti Paves from [[Tallinn University of Technology]].]]
{{Main|Fluorescence microscopy}}

When certain compounds are illuminated with high energy light, they emit light of a lower frequency. This effect is known as [[fluorescence]]. Often specimens show their characteristic [[autofluorescence]] image, based on their chemical makeup.

This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules. Many fluorescent [[dye]]s can be used to stain structures or chemical compounds. One powerful method is the combination of [[antibody|antibodies]] coupled to a fluorophore as in [[immunostaining]]. Examples of commonly used fluorophores are [[fluorescein]] or [[rhodamine]].

The antibodies can be tailor-made for a chemical compound. For example, one strategy often in use is the artificial production of proteins, based on the genetic code (DNA). These proteins can then be used to immunize rabbits, forming antibodies which bind to the protein. The antibodies are then coupled chemically to a fluorophore and used to trace the proteins in the cells under study.

Highly efficient fluorescent [[protein]]s such as the [[green fluorescent protein]] (GFP) have been developed using the [[molecular biology]] technique of [[fusion gene|gene fusion]], a process that links the [[gene expression|expression]] of the fluorescent compound to that of the target protein. This combined fluorescent protein is, in general, non-toxic to the organism and rarely interferes with the function of the protein under study. Genetically modified cells or organisms directly express the fluorescently tagged proteins, which enables the study of the function of the original protein [[in vivo]].

Growth of [[Protein crystallization|protein crystals]] results in both protein and salt crystals. Both are colorless and microscopic. Recovery of the protein crystals requires imaging which can be done by the intrinsic fluorescence of the protein or by using transmission microscopy. Both methods require an ultraviolet microscope as proteins absorbs light at 280&nbsp;nm. Protein will also fluorescence at approximately 353&nbsp;nm when excited with 280&nbsp;nm light.<ref name="Laboratory Communications">{{cite journal|last=Gill|first=Harindarpal|title=Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals|journal=Acta Crystallographica|date=January 2010|volume=F66|issue=Pt 3|pages=364–372 |pmid=20208182|pmc=2833058|doi=10.1107/S1744309110002022|bibcode=2010AcCrF..66..364G }}</ref>

Since [[fluorescence|fluorescence emission]] differs in [[wavelength]] (color) from the excitation light, an ideal fluorescent image shows only the structure of interest that was labeled with the fluorescent dye. This high specificity led to the widespread use of fluorescence light microscopy in biomedical research. Different fluorescent dyes can be used to stain different biological structures, which can then be detected simultaneously, while still being specific due to the individual color of the dye.

To block the excitation light from reaching the observer or the detector, [[filter (optics)|filter sets]] of high quality are needed. These typically consist of an [[excitation filter]] selecting the range of excitation [[wavelength]]s, a [[dichroism|dichroic]] mirror, and an [[Emission (electromagnetic radiation)|emission]] filter blocking the excitation light. Most fluorescence [[microscope]]s are operated in the Epi-illumination mode (illumination and detection from one side of the sample) to further decrease the amount of excitation light entering the detector.

See also:
[[total internal reflection fluorescence microscope]]
[[Neuroscience]]