Total internal reflection fluorescence microscope

Total internal reflection fluorescence microscope

(TIRFM) diagram
1. Specimen
2. Evanescent wave range
3. Cover slip
4. Immersion oil
5. Objective
6. Emission beam (signal)
7. Excitation beam

1. Objective
2. Emission beam (signal)
3. Immersion oil
4. Cover slip
5. Specimen
6. Evanescent wave range
7. Excitation beam
8. Quartz prism

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nm, can be observed.


In cell and molecular biology, a large number of molecular events in cellular surfaces such as cell adhesion, binding of cells by hormones, secretion of neurotransmitters, and membrane dynamics have been studied with conventional fluorescence microscopes. However, fluorophores that are bound to the specimen surface and those in the surrounding medium exist in an equilibrium state. When these molecules are excited and detected with a conventional fluorescence microscope, the resulting fluorescence from those fluorophores bound to the surface is often overwhelmed by the background fluorescence due to the much larger population of non-bound molecules.


To solve this problem, the TIRFM was developed by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s. A TIRFM uses evanescent wave to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface. The evanescent wave is generated only when the incident light is totally reflected at the glass-water interface. The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium. Thus the TIRFM enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells as shown in the figure above. Note, however, that the region visualized is at least a few hundred nanometers wide, so the cytoplasmic zone immediately beneath the plasma membrane is necessarily visualized in addition to the plasma membrane during TIRF microscopy. The selective visualization of the plasma membrane renders the features and events on the plasma membrane in living cells with high axial resolution.

TIRF can also be used to observe the fluorescence of a single molecule, making it an important tool of biophysics and quantitative biology.

External links

* [ Interactive Fluorescence Dye and Filter Database] Carl Zeiss Interactive Fluorescence Dye and Filter Database.
* [ Glowing molecules can be distinguished one at a time] -
* [ Leica Microsystems] "commercial TIRF microscope systems"
* [ TIRF Microscopy: Introduction and Applications] "TIRF Tutorial from Microscopy U"
* [ Olympus TIRFM Microscopes] "commercial TIRF microscope systems"

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