howto:calculate_and_fit_fcs_traces_with_the_fcs_script
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howto:calculate_and_fit_fcs_traces_with_the_fcs_script [2014/06/18 09:21] – admin | howto: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 " | + | |
- | 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 is a technique that can be used over a wide range of concentrations, | + | |
===== Step-by-Step Tutorial ===== | ===== Step-by-Step Tutorial ===== | ||
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{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_7.png? | {{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_7.png? | ||
- | **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 | + | **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 |
* If the sample has beenexcited with cw light, we would now save the data by clicking "Save Result" | * If the sample has beenexcited with cw light, we would now save the data by clicking "Save Result" |
howto/calculate_and_fit_fcs_traces_with_the_fcs_script.txt · Last modified: 2015/11/03 14:11 by admin