Diffraction causes the image of a fluorophore (fluorescent molecule) to be much larger than the fluorophore itself. This behavior is described by the point-spread function (PSF) of the microscope. To accurately position a fluorophore, you must find the centroid of its image by fitting a 2D-Gaussian distribution to the image of the fluorophore. An important input parameter to this fit is the width of the expected PSF, which is directly related to the standard deviation of the 2D-Gaussian model and is known as the PSF size factor. If the width of the PSF changes, the PSF size factor must be adjusted to more precisely locate the fluorophores. The PSF size factor is dependent on the pixel size of the camera, numerical aperture of the objective, the emission wavelength of the fluorophore as well as the optical elements in the microscope.
The Localization Analysis algorithm has default PSF size factor parameters which are used to fit each fluorophore. In some cases, these default values may not provide the best description of the image of the fluorophore. In this case, you may want to measure the PSF size factor for your fluorophore of interest to obtain the most precise data possible.
To measure the PSF size factor value used as input parameter for the Localization Analysis algorithm, use the PSF Width tool.
Calibrating the Camera for Localization
The Localization Results Viewer