What happens when a fluorophore is excited?

An excited fluorophore molecule emits lower-energy light than the light it absorbs. Therefore, there is always a shift along the spectrum between the color of the light absorbed by the fluorophore during excitation, and the color emitted.

How do you choose fluorophore?

Select fluorophores with high extinction coefficients ( ε ) One defining factor of a fluorophore’s brightness is its extinction coefficient (a measurement of the probability of absorbing a photon of light); the higher the value of the extinction coefficient the brighter the fluorophore.

Which fluorophore is the brightest?

It is concluded that cyanine 3.18 should be useful for multicolor fluorescence experiments and that it may be the brightest fluorophore available for single-color fluorescence immunocytochemistry.

Does a fluorophore absorb and emit light at the same wavelength?

Fluorophores absorb a range of wavelengths of light energy, and also emit a range of wavelengths. Within these ranges are the excitation maximum and the emission maximum.

Is the emission wavelength of a fluorophore shorter or longer than the excitation wavelength?

The emitted light will always be of lower energy, and hence a longer wavelength than the exciting light. Because the colors vary from the use of different dyes, the exciting and emitting light are different and they can be separated from one another incorporating use of optical filters.

Is fluorophore the same as chromophore?

The main difference between fluorophore and chromophore is that fluorophore is a part of a molecule, re-emitting the absorbed photon at a longer wavelength whereas chromophore is a part of a molecule, absorbing UV or visible light to emit light in the visible region.

What is a good fluorophore?

Fluorescein, via its amine-reactive isothiocyanate derivative fluorescein isothiocyanate (FITC), has been one of the most popular fluorophores.

What are the two main requirements for selecting an ideal fluorophore for fluorescence microscopy?

Ideally, a fluorescent label should be small, bright, and stable, without any perturbation to the biological system. Furthermore, the label should be specific without the tendency to oligomerize and with the possibility to label multiple proteins at the same time.

What determines fluorophore brightness?

A fluorophore’s brightness is defined by two parameters: its extinction coefficient and quantum yield. The extinction coefficient is a measure of the quantity of absorbed light at a given wavelength. Therefore, a high extinction coefficient will lead to a greater amount of light being absorbed.

Is PE Cy7 bright?

PE-Cy7 conjugated reagents are as bright as PE conjugates. However, PE-Cy7 is highly sensitive to photo-induced degradation resulting in loss of fluorescence. Care should be taken to avoid light exposure and prolonged exposure to paraformaldehyde fixative.