This tutorial shows step-by-step, how the Anisotropy script of SymPhoTime 64 is used to calculate a 2D image of the static anisotropy. The anisotropy script is applied to an image of single molecules to calculate the static anisotropy and a histogram with the anisotropy distribution.
Note: The “Samples” workspace is delivered with the SymPhoTime 64 and on the CD-ROM and contains example data to show the function of the SymPhoTime data analysis. If you haven't installed it on your computer, copy it from the DVD onto a local drive before going through this tutorial.
Response: The Anisotropy Image script is applied to the file
Cy5_immo_FLIM+Pol-Imaging.ptu. Thereby, a new window opens which contains the anisotropy image and a histogram with the anisotropy distribution over the different pixels of the image.
Note: The window contains four different regions:
Note: The anisotropy values can theoretically spread between -0.5 and 1 for perpendicular and parallel excitation and emission dipoles respectively. The anisotropy value of 0 is expected in case of very fast rotation or extremely efficient energy transfer processes (e.g. efficient HOMO-FRET). When measuring a randomly aligned ensemble due to photoselection the anisotropy varies with in the limits of -0.2 and 0.4 instead of -0.5 and 1.
Note: At the current setting, the anisotropy is only calculated, if the number of detected photons in one pixel is higher than 25. The calculation only becomes effective after clicking “Calc. Anisotropy”.
Clicking on the help button next to the “Calc. Aniso” button opens the help page, where the equation and calculation of the parameters are explained.
Note: The parameters vary for each system, but are usually constant under identical imaging conditions, i.e. excitation wavelength, filter configuration, objective and detectors. The correction factors L1 and L2 deviate from 0 especially when objectives with a high numerical aperture are used. The factors can be determined with a method described in: Kioshioka M et al., Appl. Spectr. 49, 224-228 (1995).
Note: Scripts can be adapted to perform always in the same way by saving it in a specific user profile. Whenever the user profile is loaded later on, the script is applied with the user specific changes. This is advantageous, if many images acquired under identical conditions (e.g. detector configuration and G-factor) should be analyzed.
Cy5_immo_FLIM+Pol-Imaging.ptuas described before.
Response: A window opens aking where and under which name to save the User Configuration. Select e.g. the name
anisotropy_Cy5.pus and store the new user profile.
Note: Usually, when a system is delivered, a folder “C:\User Configurations” is already present on the hard disc. If not, create it and save your profile there.
Response: The new user settings are saved under the given name as a file.
Response: Software restarts, but applies the user profile