Molecular Quantification Toolkit
Study Life in Motion
ZEISS imaging systems enable experiments involving photobleaching, photoactivation or ratiometric imaging. This toolkit provides advanced functionality for acquiring and analyzing such experiments, including dedicated user interfaces for Fluorescence Recovery after Photobleaching (FRAP), Förster resonance energy transfer (FRET) and ratiometric analysis.
Pixel-Precise Photomanipulation
Your Prerequisite for Successful Photobleaching Experiments
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.
Guided Analysis and Visualization
Complex Analysis Made Simple and Visually Appealing
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.
Workflow Customization and Automation
Maximize the Potential of Your Dynamics Experiments
The toolkit further offers customization and automation on different levels. You can modify acquisition parameters such as frame rate in real-time to capture rapid kinetics. Bleaching protocols can be optimized in terms of intensity, wavelength or dwell time to find the optimal conditions for each fluorophore. And having established your optimal parameters, you may go on and perform bleaching experiments fully automatically over arbitrary numbers of regions and image scenes.
What is FRAP?
FRAP Principle
Unveil Molecular Dynamics with Photobleaching
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.
FRAP Variants
Photobleaching Methods and When to Use Them
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.
What is FRET?
FRET in a Nutshell
Physical Principle and Molecular Design
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 Acceptor Photobleaching Method
Förster Energy Transfer Made Easy
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.
The Sensitized Emission Method
FRET with Full Spatial and Temporal Resolution
Sensitized Emission allows measurements in live imaging mode and precise localization of FRET events. This requires recording of reference samples and separate donor and acceptor spectra, but the image correction can conveniently be done with the help of an analysis wizard. Three different algorithms optimized for different molecule densities are available.