This tutorial shows step-by-step, how to determine the Point Spread Function (PSF) of a microscopic system using the Focal Width Script. It requires the image of a subresolution bead, in which the center is marked in the image. The script applies then a Gaussian fit of the bead and calculates the Full Width Half Maximum (FWHM) in both scanning directions.
Note: The “Samples” workspace is delivered with the SymPhoTime 64 and on the DVD-ROM and contains example data to show the function of the SymPhoTime 64 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, e.g. to a folder “C:\SPT_User”.
TS-Bead_immo_xy-scan.ptuby a single mouse click.
Note: This file contains the image of a TetraSpeck Bead with 100 nm diameter. As this is smaller than the optical resolution, beads of this size can be used to measure the focal width in a microscope system.
Note: The window contains two different regions:
Optional: The following parameters can be adapted:
Note: The data in the file is a bead that was excited via pulsed interleaved excitation (PIE) using two perpendicularly polarized lasers, which were guided through a DIC prism. This causes a displacement of the beams, which is used in dual focus FCS (2fFCS) measurements.
If data are acquired with a scanner controlled by the SymPhoTime 64 software (i.e. the piezo scanner in the MicroTime200), the image dimensions are automatically correct. If the image is acquired using an external scanner, the image size has to be defined or transferred before the measurement to make sure that the measured image dimensions are correct. Otherwise, one pixel is considered to a size of 1 µm.
.pqres)-file, which is assigned to the corresponding raw data (
TS-bead_immo_xz-scan.ptuto get an idea about the ratio of the focal width in xy vs. z.