FLUORESCENCE

Lifetime

(in picoseconds): duration of the excited state of a fluorophore before returning to its ground state. It refers to the time taken for a population of excited fluorophores to decay to 1/e (≈0.368) of the original amount

Extinction Coefficient

(or molar absorption, in Mol−1cm−1): links the quantity of absorbed light, at a given wavelength, to the concentration of fluorophore in solution. The efficiency with which the fluorochrome absorbs the excitation light is known as the extinction coefficient. The greater the extinction coefficient, the greater the possibility of light absorption in a given wavelength region (a prerequisite to ensuing fluorescence emission).

From the lowest excited singlet state, the electrons are then able to "relax" back to the ground state with simultaneous emission of fluorescent light. How does the wavelength of the emitted light compared with the wavelength of excitation light?

The emitted light is always of longer wavelength than the excitation light (Stokes Law) and continues so long as the excitation illumination bathes the fluorescent specimen. If the exciting radiation is halted, the fluorescence ceases.

Who coined the word "flourescence"?

The phenomenon of fluorescence was known by the middle of the nineteenth century. British scientist Sir George G. Stokes first made the observation that the mineral fluorspar exhibits fluorescence when illuminated with ultraviolet light, and he coined the word "fluorescence"

How does one achieve proper separation of excitation and emission wavelengths without overlap of the spectra

The separation of excitation and emission wavelengths is achieved by the proper selection of filters to block or pass specific wavelengths of the spectrum

What diagram depicts fluorescence activity

Fluorescence activity is sometimes depicted diagrammatically (termed a Jablonski energy diagram).

What is fluorescence

Fluorescence, on the other hand, describes light emission that continues only during the absorption of the excitation light. The time interval between absorption of excitation light and emission of re-radiated light in fluorescence is of extraordinarily short duration, usually less than a millionth of a second.

What is phosphorescence?

If the emission of light persists for up to a few seconds after the excitation energy (light) is discontinued, the phenomenon is known as phosphorescence.

What is a triplet state?

In a triplet state the excited electron is no longer paired with the ground state electron; that is, they are parallel (same spin). Since excitation to a triplet state involves an additional "forbidden" spin transition, it is less probable that a triplet state will form when the molecule absorbs radiation.

what is immunofluorescence?

In direct immunofluorescence, a specific antibody is labeled by chemically attaching a fluorochrome to form what is known as a conjugate, which is then spread on a microscope slide containing the suspected presence of a particular antigen known to stimulate production of the antibody. If the antigen is present, the labeled antibody conjugate binds to the antigen and remains bound to the specimen after it is washed. The presence of the chemically attached fluorescent conjugate and antigen is demonstrated when the fluorochrome is excited at its excitation peak, and the subsequent emission intensities at various wavelengths can then be observed visually or captured by a detector system (digital or traditional camera).

What do you do in order to achieve maximum fluorescence intensity

In order to achieve maximum fluorescence intensity, the fluorochrome is usually excited at the wavelength at the peak of the excitation curve, and the emission detection is selected at the peak wavelength (or other wavelengths chosen by the observer) of the emission curve.

What is a doublet state?

In quantum mechanics, a doublet is a mixed quantum state of a system with a spin of 1/2, such that there are two allowed values of the spin component, −1/2 and +1/2

1930's that Austrian investigator Max Haitinger and other scientists developed what technique?

Max Haitinger and other scientists developed the technique of secondary fluorescence, which employs fluorochrome stains to label specific tissue components, bacteria, and other pathogens that do not autofluoresce.

How can the techniques of fluorescence microscopy be applied?

The techniques of fluorescence microscopy can be applied to organic material, formerly living (biological) material, or to living material (with the use of in vitro or in vivo fluorochromes) or to inorganic material (especially in the investigation of contaminants on semiconductor wafers). There are also a burgeoning number of studies using fluorescent probes to monitor rapidly changing physiological ion concentrations, such as calcium and magnesium, and pH values in living cel

What is photobleaching?

There are specific conditions that may affect the re-radiation of light by an excited fluorophore, and thus reduce the intensity of fluorescence. This reduction of emission intensity is generally called fading or photobleaching.

How can you prevent photobleaching from occurring?

To reduce the degree of fading in some specimens, it may be advisable to use a neutral density filter in the light path before the illumination reaches the excitation filter, thus diminishing the excitation light intensity. In other instances, fading effects may be reduced by changing the pH of the mounting medium or by using anti-bleaching agents (several of the more important agents are listed in Table 2). For digital imaging, photomicrography, or simply visual observation, rapidly changing the field of view may also avoid fading effects.

What is photoluminescence?

When specimens, living or non-living, organic or inorganic, absorb and subsequently re-radiate light, the process is described as photoluminescence.

Upon absorbing a photon of excitation light, usually of short wavelengths, electrons may be raised to a higher energy and vibrational excited state. How long does this process take?

a process that may only take a quadrillionth of a second (a time period commonly referred to as a femtosecond, 10E-15 seconds).

How long does it take for the excited electron to lose some vibrational energy to the surrounding environment and return to the lowest excited singlet state?

approximately a trillionth of a second (a picosecond or 10E-12 seconds)

Prior to excitation, the electronic configuration of the molecule is described as being in the what state?

described as being in the ground state.

Stokes shift

difference between the maximum excitation and maximum emission wavelengths

Quantum yield

efficiency of the energy transferred from incident light to emitted fluorescence (= number of emitted photons per absorbed photons). The yield of emitted light is referred to as the quantum yield, the ratio of the number of quanta ("packets" of energy) emitted compared to the number of quanta absorbed (usually the yield is between 0.1 and 0.9).

-Maximum excitation and emission wavelength

expressed in nanometers (nm)): corresponds to the peak in the excitation and emission spectra (usually one peak each),

What is autofluorescence?

fluorescence emitted naturally by a biological substance. Many plant and animal tissues, as well as materials specimens, inherently fluoresce when irradiated with shorter wavelength light (primary or autofluorescence)

The greater the stokes shift, the

the easier it is to separate excitation light from emission light

What are the characteristics of fluorophores?

-Maximum excitation and emission wavelength -Extinction Coefficient -Quantum yield -Lifetime -Stokes shift

What are fluorochromes and why are they highly valuable in biological applications?

A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorochromes are stains which attach themselves to visible or sub-visible organic matter. These fluorochromes, capable of absorbing and then re-radiating light, are often highly specific in their attachment targeting and have significant yield in absorption-emission ratios (a concept termed quantum yield).

What is a fluorophore?

A fluorophore, is a part of a molecule which makes a molecule to be fluorescent. It is similar to a chromophore, the element of a molecule accountable for its color. Flurophore is a functional group (specific groups of atoms within molecules that are responsible for the distinctive chemical reactions of those molecules ) in a molecule which absorbs energy of a particular wavelength and emits energy at a different but specific wavelength. The quantity and wavelength of the emitted energy depend on the fluorophore and the chemical atmosphere of the fluorophore.

What is photon?

A photon is an elementary particle, the quantum of the electromagnetic field including electromagnetic radiation such as light, and the force carrier for the electromagnetic force (

What is a singlet state?

A singlet state is a molecular electronic state such that all electron spins are paired. That is, the spin of the excited electron is still paired with the groundstate electron (a pair of electrons in the same energy level must have opposite spins, per the Pauli exclusion principle).

Phosphorescensce or delayed fluorescence is caused by what state?

Occasionally the excited electrons, instead of relaxing to the lowest singlet state through vibrational interactions, make a forbidden transition to the exited triplet state and then to the ground state in a process where the emission of radiation may be considerably delayed-up to several seconds or more. This phenomenon is characteristic of phosphorescence, as shown in Figure 4(b). In some instances, the excited electrons may go from the triplet state back to the lowest excited singlet state and then return to the ground state, subsequently emitting fluorescent light. This action takes a little longer (about a microsecond or two) than usual fluorescence and is called delayed fluorescence (Figure 4(c)). Under other circumstances (for example, photobleaching or the presence of salts of heavy metals or other chemicals), emitted light may be significantly reduced or halted altogether, as discussed below.

What was it that he observed about the wavelength of fluorescing light compared to the excitation light, and what was this phenomenon called?

Stokes observed that the fluorescing light has longer wavelengths than the excitation light, a phenomenon that has become to be known as the Stokes shift

What is the basic task of the fluorescence microscope?

The basic task of the fluorescence microscope is to permit excitation light to irradiate the specimen and then to separate the much weaker emitted fluorescent light from the brighter excitation light. Thus, only the emission light from the specimen reaches the eye or other detector (usually a digital or conventional film camera).