FRAP acquisition and analysis

_{From left to right: Yeast cells expressing GFP before bleaching, during bleaching and after recovery; images obtained with an iMIC, fitted with Yanus Scan Head, Polytrope Imaging-Mode Switch, Dichrotome Dual Emission Extension, 60x Objective, Sensicam QE;
Images courtesy of Dr. Roland Wedlich and Felix Spira, MPI Martinsried;}

General

Live Acquisition enables photo bleaching of multiple, freely definable regions in a specimen. A FRAP experiment can be recorded in widefield or TIRF illumination mode and either can be combined with the FRET mode in an iMIC. This makes it possible to record two emission wavelengths simultaneously, saving time and treating the cells with care.
Switching from FRAP to widefield or TIRF is done within milliseconds thanks to the innovative Polytrope Image Mode switch. This ensures that the first image after the end of the photo bleaching is immediately acquired.

DynamicFRAP experiments

The optional DynamicFRAP feature includes two new FRAP experiments for users. They allow to either set the time point when the cells are bleached anytime or even both the time point and the position where to bleach the sample.

DynamicFRAP

The DynamicFRAP experiment allows the user to bleach a spot, ellipse or rectangle anytime and anywhere in the sample. This is ideal for FRAP experiments that deal with investigations like vesicle transport, where at the beginning of an experiment it is not know where and when the expected event will happen.

FRAP on demand

FRAP on demand is made for experiments like cell-cell communications in cell cultures or tissues. The user can define multiple FRAP regions of any shape and the software allows to set the FRAP event at any time.
The FRAP on demand item can also be used in the LA Protocol Editor for more flexible experiment planning.

Features

• Cost effective solution in TIRF and Widefield applications
• Multiple user defined regions (spot, line, rectangle, ellipse, polygon)
• ultra fast switching between FRAP and image acquisition
• Multi-Channel image acquisition (TIRF or Widefield)
• Usable in the Protocol Editor for even more flexibility
• use multiple FRAP experiments with different FRAP ROIs in the Protocol Editor

FRAP Analysis

With LA 2.1 TILL Photonics introduces an optional FRAP Analysis module for the Offline Analysis application. Get results fast with the FRAP Analysis module as all the meta data created by the LA software during the acquisition of the FRAP experiment is used for the calculations. The analysis is just a click away and delivers the most important values like t_half, the mobile and immobile fractions. The FRAP analysis module also provides more than five fitting functions to calculate the recovery time for a wide range of FRAP experiments. The resulting values and the graph can be exported to various formats.

Features

• Multiple fitting functions
• Background correction
• Bleaching correction
• Display of the original and corrected curve as well as the fitted curve
• Data export to XLS, CSV; Image export to BMP, PNG,...