Molecular Quantification Toolkit

Photobleaching and photoactivation experiments require a precise definition of the cellular compartments for organelles or condensates to be manipulated. ZEN Molecular Quantification Toolkit lets you easily configure regions of interest, either by hand or by automated analysis, and separate them into bleaching or control regions. Thanks to laser technology on the confocal system, these regions are then targeted pixel-by-pixel, at the most efficient wavelength and intensity.

Molecular dynamics experiments imply sophisticated analysis requiring the integration of reference regions or images or exponential curve fitting. The toolkit provides guided analyses for photomanipulation applications, such as diffusion constants from 1st or 2nd order kinetics in FRAP, all typical methods for FRET efficiency calculation, and a flexible ratiometric analysis. Moreover, it also provides ways to beautifully visualize the results.

The Fluorescence Recovery After Photobleaching (FRAP) method serves the investigation of a fluorescent molecule's mobility. The fluorophores are irreversibly inactivated in a small region. The rate at which nearby fluorescent molecules exchange with this region allows determining the diffusion rate, and the amount of bound and unbound molecules.

Beyond standard FRAP, various related methods exist. Inverse FRAP (iFRAP) and "Fluorescence Loss In Photobleaching" (FLIP) help determine whether a region (e.g. an organelle) is actively exchanging with its environment or isolated. "Fluorescence Localization After Photobleaching" (FLAP) allows precise spatial determination of the target molecules. The toolkit supports all these methods.

For measurements, FRET uses the physical principles of Förster transfer, an energy transfer between two matching fluorophores in very close proximity (< 10nm). This reveals precise details of protein dynamic association or dissociation.

The toolkit has dedicated workflows for the two main FRET methods, the first being the Acceptor Photobleaching. Here, a region of interest is bleached in the acceptor channel. This leads to an inactivation of the acceptor and precludes any Förster energy transfer from the donor.