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howto:determination_of_the_focal_width_with_the_focal [2014/05/07 16:13] – adminhowto:determination_of_the_focal_width_with_the_focal [2015/01/22 14:48] (current) – admin
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 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. 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.
  
- \\ +{{ youtube>WkSbWoVdw_g?large }} 
 ===== Step-by-Step Tutorial ===== ===== Step-by-Step Tutorial =====
  
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 **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.\\ **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 MicroTime 200), 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.+If data are acquired with a scanner controlled by the SymPhoTime 64 software (i.e. the piezo scanner in the [[products:MicroTime#MicroTime 200|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.
  
 {{ determination_of_the_focal_width_with_the_focal_Image_6.png }} {{ determination_of_the_focal_width_with_the_focal_Image_6.png }}
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   * Optional: Apply the same script to the file ''TS-bead_immo_xz-scan.ptu'' to get an idea about the ratio of the focal width in xy vs. z.   * Optional: Apply the same script to the file ''TS-bead_immo_xz-scan.ptu'' to get an idea about the ratio of the focal width in xy vs. z.
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howto/determination_of_the_focal_width_with_the_focal.txt · Last modified: 2015/01/22 14:48 by admin