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Applications

Photoactivation and Photoswitching

Many cellular particles move quickly and unpre-dictably—for example, vesicles or intracellular pathogens such as viruses. For this reason, fluorescent molecules exhibiting photoactivation and photoconversion properties are used as markers to track the dynamic behavior. For example, fluorophores are available that can be changed-reversible or irreversible-in their spectral characteristics upon illumination with specific wavelengths—for example PA-GFP, KFP1, and Kaede. In particular, following the fate of, for example, a protein by an activated tagged molecule without the need to constantly providing addional fluorophores provides clear signals and minimal background conditions.

‘Click & Bleach’

The ‘Click & Bleach’ function in the cellSens software enables the user to react to sample dynamics and to photomanipulate a target using direct mouse interaction. Multiple points can be bleached or activated and the resultant images can be captured during, or immediately after, the bleaching procedure.

FRAP

Fluorescence recovery after photobleaching (FRAP) provides an ideal method for calculating the coefficient of diffusion of a particular molecule within a certain target area of the cell. This is achieved by photobleaching the fluorophore attached to a specific molecule within the target area and then assessing the dynamics of the return of fluorescence (recovery) to that area.

FLIP

Fluorescence loss in photobleaching (FLIP) is useful for studying molecular movement along cell membranes (lateral membrane fluidity) and membrane continuity, especially for membranous organelles. A defined area of the membrane or cell is continuously bleached, and the dynamic loss of fluorescence from the remaining area is measured based on the replacement of the bleached molecules in the defined area by unbleached ones from the remainder.

FLAP

Fluorescence localization after photobleaching (FLAP) is a clever adaptation of FRAP where the dynamics of a particular molecular species are of interest. The target molecule is coexpressed with two different fluorophores such that distribution and localization overlap. One of the fluorophores is then bleached at a defined location, and the movement of the molecule can be followed by comparing the relative and absolute distribution of the two fluorophores.

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