Photobleaching is the photochemical destruction of a 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...

. In microscopy

Microscopy

Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...

, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing. This is especially problematic in time-lapse microscopy.

However, photobleaching may also be used prior to applying the (primarily antibody

Antibody

An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...

-linked) fluorescent molecules, in an attempt to quench autofluorescence

Autofluorescence

Autofluorescence is the natural emission of light by biological entities such as mitochondria and lysosomes, and is used to distinguish the light originating from artificially added fluorescent markers...

. This can help to improve signal-to-noise ratio

Signal-to-noise ratio

Signal-to-noise ratio is a measure used in science and engineering that compares the level of a desired signal to the level of background noise. It is defined as the ratio of signal power to the noise power. A ratio higher than 1:1 indicates more signal than noise...

.

Photobleaching may also be exploited to study the motion and/or diffusion of molecules, for example via the FRAP

Fluorescence recovery after photobleaching

Fluorescence recovery after photobleaching denotes an optical technique capable of quantifying the two dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells. This technique is very useful in biological studies of cell membrane...

 or FLIP

Fluorescence loss in photobleaching

Fluorescence Loss in Photobleaching, or FLIP, is a technique in fluorescence microscopy which can be used to examine the movement or diffusion of molecules inside cells or membranes. Typically a cell membrane is labelled with a fluorescent dye, and a specific area of the labeled membrane is...

 techniques.

Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-span of light exposure, by increasing the concentration of fluorophores, by reducing the frequency and thus the photon energy of the input light, or by employing more robust fluorophores that are less prone to bleaching (e.g. Alexa Fluor

Alexa (fluor)

The Alexa Fluor family of fluorescent dyes is produced by Molecular Probes, a subsidiary of Invitrogen. Alexa Fluor dyes are typically used as cell and tissue labels in fluorescence microscopy and cell biology....

s or DyLight Fluor

DyLight Fluor

The DyLight Fluor family of fluorescent dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. DyLight dyes are typically used in biotechnology and research applications as biomolecule, cell and tissue labels for fluorescence microscopy, cell biology or molecular...

s). To a reasonable approximation, a given molecule will be destroyed after a constant exposure (intensity of emission X emission time X number of cycles) because, in a constant environment, each absorption-emission cycle has an equal probability of causing photobleaching.

Lifetime

Depending on the material, dyes can produce different photon numbers and therefore have different lifetimes (at e.g. 10⁵ photons/s):

• Green fluorescent protein
  Green fluorescent protein
  The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
  
  : 10⁴-10⁵; 0.1-1 s
• Typical organic dye: 10⁵-10⁶; 1-10 s
• CdSe/ZnS Quantum dot
  Quantum dot
  A quantum dot is a portion of matter whose excitons are confined in all three spatial dimensions. Consequently, such materials have electronic properties intermediate between those of bulk semiconductors and those of discrete molecules. They were discovered at the beginning of the 1980s by Alexei...
  
  : 10⁸; > 1000 s

This use of the term "lifetime" is not to be confused with the "lifetime" measured by fluorescence lifetime imaging

Fluorescence lifetime imaging

Fluorescence-lifetime imaging microscopy or FLIM is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample...

.

External links

• Introduction to Optical Microscopy an article about photobleaching