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howto:calculate_and_fit_fcs_traces_with_the_fcs_script [2014/06/18 09:21] adminhowto:calculate_and_fit_fcs_traces_with_the_fcs_script [2015/11/03 14:11] (current) admin
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 ===== Background Information ===== ===== Background Information =====
  
-ATTO488 (ATTO-Tec, Germany) is a suited calibration dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection ~500 – 540 nm.\\ +  * ATTO488 (ATTO-Tec, Germany) is a suited calibration dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection ~500 – 540 nm. 
-The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf ]. This list is extensive, but does not claim to be complete.\\ +  The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf ]. This list is extensive, but does not claim to be complete. 
-Note that the diffusion coefficients of all dyes are temperature dependent.\\ +  * **Note** that the diffusion coefficients of all dyes are temperature dependent. 
-FCS measurements are usually point measurements acquired with very sensitive detectors (typically SPAD or Hybrid PMT detectors).\\ +  FCS measurements are usually point measurements acquired with very sensitive detectors (typically [[glossary:SPAD]] or [[glossary:Hybrid PMT]] detectors). 
-FCS is a technique that can be used over a wide range of concentrations, but ideal FCS concentrations are usually between 1 nM and 20 nM.\\+  FCS is a technique that can be used over a wide range of concentrations, but ideal FCS concentrations are usually between 1 nM and 20 nM.
  
 ===== Step-by-Step Tutorial ===== ===== Step-by-Step Tutorial =====
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 {{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_7.png?600 }} {{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_7.png?600 }}
  
-**Note:** A possibility to remove the afterpulsing when working with cw excitation is splitting the light onto two detectors and calculate a cross correlation. Some detectors (mainly Hybrid PMTs) also have so low afterpulsing that the problem does not occur.+**Note:** A possibility to remove the afterpulsing when working with cw excitation is splitting the light onto two detectors and calculate a cross correlation. Some detectors (mainly [[glossary:Hybrid PMT]]s) also have so low afterpulsing that the problem does not occur.
  
   * If the sample has beenexcited with cw light, we would now save the data by clicking "Save Result" and press afterwards "Transfer to Fit" (both in the "File" dropdown panel). As our sample has been excited with a pulsed laser, we can apply one additional step to increase data quality.   * If the sample has beenexcited with cw light, we would now save the data by clicking "Save Result" and press afterwards "Transfer to Fit" (both in the "File" dropdown panel). As our sample has been excited with a pulsed laser, we can apply one additional step to increase data quality.
howto/calculate_and_fit_fcs_traces_with_the_fcs_script.txt · Last modified: 2015/11/03 14:11 by admin