Editing Microscopy (section)

=== Limitations ===

Limitations of standard optical microscopy ([[bright field microscopy]]) lie in three areas;
* The technique can only image dark or strongly refracting objects effectively.
* There is a [[Diffraction-limited system|diffraction-limited]] resolution depending on incident wavelength; in visible range, the resolution of optical microscopy is limited to approximately 0.2 &nbsp;[[micrometre]]s (''see: [[microscope]]'') and the practical magnification limit to ~1500x.<ref>Abbe, E. (1874) A contribution to the theory of the microscope and the nature of microscopic vision. Proc. Bristol Nat. Soc. 1, 200–261.</ref>
* Out-of-focus light from points outside the focal plane reduces image clarity.<ref>Pawley JB (editor) (2006). Handbook of Biological Confocal Microscopy (3rd ed.). Berlin: Springer. {{ISBN|0-387-25921-X}}.</ref>

Live cells in particular generally lack sufficient contrast to be studied successfully, since the internal structures of the cell are colorless and transparent. The most common way to increase contrast is to [[staining (biology)|stain]] the structures with selective dyes, but this often involves killing and [[Fixation (histology)|fixing]] the sample.<ref>Lakowicz, Joseph R. (1999) Principles Of Fluorescence Spectroscopy. New York: Kluwer Academic/Plenum.</ref> Staining may also introduce [[Artifact (microscopy)|artifacts]], which are apparent structural details that are caused by the processing of the specimen and are thus not features of the specimen. In general, these techniques make use of differences in the refractive index of cell structures. Bright-field microscopy is comparable to looking through a glass window: one sees not the glass but merely the dirt on the glass. There is a difference, as glass is a denser material, and this creates a difference in phase of the light passing through. The human eye is not sensitive to this difference in phase, but clever optical solutions have been devised to change this difference in phase into a difference in amplitude (light intensity).{{cn|date=June 2022}}