Total Internal Reflection Fluorescence Microscope epi- (TIRFM) diagram
1. Specimen
2. Evanescent wave range
3. Cover slip
4. Immersion oil
5. Objective
6. Emission beam (signal)
7. Excitation beam

Total Internal Reflection Fluorescence Microscope trans- (TIRFM) diagram
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. Image File history File links Tirfm. ... Image File history File links Tirfm. ... Image File history File links TIRFM2. ... Image File history File links TIRFM2. ... The larger the angle to the normal, the smaller is the fraction of light transmitted, until the angle when total internal reflection occurs. ... Robert Hookes microscope (1665) - an engineered device used to study living systems. ... A nanometre (American spelling: nanometer) is 1. ... For other uses, see Observation (disambiguation). ...

Background

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. Cell biology (also called cellular biology or formerly cytology, from the Greek kytos, container) is an academic discipline that studies cells. ... Molecular biology is the study of biology at a molecular level. ... 3D (left and center) and 2D (right) representations of the terpenoid molecule atisane. ... Schematic of cell adhesion The study of cell adhesion is part of cell biology. ... Norepinephrine A hormone (from Greek όρμή - to set in motion) is a chemical messenger from one cell (or group of cells) to another. ... Secretion is the process of segregating, elaborating, and releasing chemicals from a cell, or a secreted chemical substance or amount of substance. ... Chemical structure of D-aspartic acid, a common amino acid neurotransmitter. ... A Fluorescence Microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption. ... A fluorophore is a component of a molecule which causes a molecule to be fluorescent. ... A dynamic equilibrium occurs when two reversible processes occur at the same rate. ...

Solution

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 waves to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface. Evanescent waves are 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. The selective visualization of the plasma membrane renders the features and events on the plasma membrane in living cells with high axial resolution. An evanescent wave is an electromagnetic wave that decays exponentially with distance. ... The larger the angle to the normal, the smaller is the fraction of light transmitted, until the angle when total internal reflection occurs. ... The electromagnetic field is a physical field that is produced by electrically charged objects and which affects the behaviour of charged objects in the vicinity of the field. ... A quantity is said to be subject to exponential decay if it decreases at a rate proportional to its value. ... Drawing of a cell membrane A component of every biological cell, the cell membrane (or plasma membrane) is a thin and structured bilayer of phospholipid and protein molecules that envelopes the cell. ... It has been suggested that Interactive visualization be merged into this article or section. ... Resolving power is the ability of a microscope or telescope to measure the angular separation of images that are close together. ...

TIRF can also be used to observe the fluorescence of a single molecule, making it an important tool of biophysics and quantitative biology. Biophysics (also biological physics) is an interdisciplinary science that applies the theories and methods of physics, to questions of biology. ...

External links

• Glowing molecules can be distinguished one at a time - Nature.com
• Leica Microsystems commercial TIRF microscope systems