A total internal reflection

Total internal reflection

Total internal reflection is an optical phenomenon that happens when a ray of light strikes a medium boundary at an angle larger than a particular critical angle with respect to the normal to the surface. If the refractive index is lower on the other side of the boundary and the incident angle is...

 fluorescence microscope (TIRFM) is a type of microscope

Microscope

A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

 with which a thin region of a specimen, usually less than 200 nm, can be observed

Observation

Observation is either an activity of a living being, such as a human, consisting of receiving knowledge of the outside world through the senses, or the recording of data using scientific instruments. The term may also refer to any data collected during this activity...

.

Background

In cell

Cell biology

Cell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...

 and molecular biology

Molecular biology

Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

, a large number of molecular

Molecule

A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...

 events in cellular surfaces such as cell adhesion

Cell adhesion

Cellular adhesion is the binding of a cell to a surface, extracellular matrix or another cell using cell adhesion molecules such as selectins, integrins, and cadherins. Correct cellular adhesion is essential in maintaining multicellular structure...

, binding of cells by hormone

Hormone

A hormone is a chemical released by a cell or a gland in one part of the body that sends out messages that affect cells in other parts of the organism. Only a small amount of hormone is required to alter cell metabolism. In essence, it is a chemical messenger that transports a signal from one...

s, secretion

Secretion

Secretion is the process of elaborating, releasing, and oozing chemicals, or a secreted chemical substance from a cell or gland. In contrast to excretion, the substance may have a certain function, rather than being a waste product...

 of neurotransmitter

Neurotransmitter

Neurotransmitters are endogenous chemicals that transmit signals from a neuron to a target cell across a synapse. Neurotransmitters are packaged into synaptic vesicles clustered beneath the membrane on the presynaptic side of a synapse, and are released into the synaptic cleft, where they bind to...

s, and membrane dynamics have been studied with conventional fluorescence microscope

Fluorescence microscope

A fluorescence microscope is an optical 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...

s. However, fluorophore

Fluorophore

A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...

s that are bound to the specimen surface and those in the surrounding medium exist in an equilibrium

Dynamic equilibrium

A dynamic equilibrium exists once a reversible reaction ceases to change its ratio of reactants/products, but substances move between the chemicals at an equal rate, meaning there is no net change. It is a particular example of a system in a steady state...

 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.

Overview

To solve this problem, TIRFM was developed by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s. A TIRFM uses an evanescent wave

Evanescent wave

An evanescent wave is a nearfield standing wave with an intensity that exhibits exponential decay with distance from the boundary at which the wave was formed. Evanescent waves are a general property of wave-equations, and can in principle occur in any context to which a wave-equation applies...

 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 internally reflected

Total internal reflection

Total internal reflection is an optical phenomenon that happens when a ray of light strikes a medium boundary at an angle larger than a particular critical angle with respect to the normal to the surface. If the refractive index is lower on the other side of the boundary and the incident angle is...

 at the glass-water interface. The evanescent electromagnetic field

Electromagnetic field

An electromagnetic field is a physical field produced by moving electrically charged objects. It affects the behavior of charged objects in the vicinity of the field. The electromagnetic field extends indefinitely throughout space and describes the electromagnetic interaction...

 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 visualised is at least a few hundred nanometers wide, so the cytoplasmic zone immediately beneath the plasma membrane is necessarily visualised in addition to the plasma membrane during TIRF microscopy. The selective visualisation

Visualization (graphic)

Visualization is any technique for creating images, diagrams, or animations to communicate a message. Visualization through visual imagery has been an effective way to communicate both abstract and concrete ideas since the dawn of man...

 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

Biophysics

Biophysics is an interdisciplinary science that uses the methods of physical science to study biological systems. Studies included under the branches of biophysics span all levels of biological organization, from the molecular scale to whole organisms and ecosystems...

and quantitative biology.

External links

• Interactive Fluorescence Dye and Filter Database Carl Zeiss Interactive Fluorescence Dye and Filter Database.
• Cells come into focus: Glowing molecules can be distinguished one at a time - cmbi.bjmu.edu.cn
• cell^TIRF - Multicolor TIRF - Olympus commercial TIRF microscope systems
• Leica Microsystems commercial TIRF microscope systems
• TIRF Microscopy: Introduction and Applications TIRF Tutorial from Microscopy U
• TIRF Microscopy: Overview TIRF Tutorial from Olympus Microscopy Resource Center
• Olympus TIRFM Microscopes commercial TIRF microscope systems
• Carl Zeiss Laser TIRF 3 commercial TIRF microscope systems
• Lightguide- and prism-based TIRF microscopy Commercial TIRF Microscopy, TIRF Spectroscopy, TIRF ElectroChemistry, and TIRF Dielectrophoresis systems
• TIRF FLIM microscopy Lambert Instruments TIRF - FLIM microscopy