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howto:separation_of_2_species_with [2014/05/23 07:45] – adminhowto:separation_of_2_species_with [2014/06/18 12:22] (current) – admin
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 {{tag> howto tutorial FCS FLCS analysis SymPhoTime}} {{tag> howto tutorial FCS FLCS analysis SymPhoTime}}
  
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-====== Separation of 2 Species with Different Lifetimes Using the FLCS Analysis ======+====== Separation of 2 Species with Different Lifetimes Using FLCS ======
  
  
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 This tutorial shows step-by-step, how the FLCS analysis can be used to calculate separate autocorrelation curves for the two components of a mixture of ATTO655 and Cy5 based on their different lifetimes. This tutorial shows step-by-step, how the FLCS analysis can be used to calculate separate autocorrelation curves for the two components of a mixture of ATTO655 and Cy5 based on their different lifetimes.
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 ===== Background Information ===== ===== Background Information =====
  
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-**Note:**\\ 
 The two fluorophores ATTO655 (ATTO-Tec, Germany) and Cy5 (GE Healthcare) have almost identical spectral properties and are thus excited with the same wavelength (e.g. 635 nm) and detected at the same spectral range. As also their molecular weight is comparable, the diffusion constant is too similar to distinguish between both dyes in a mixture by means of standard FCS.\\ The two fluorophores ATTO655 (ATTO-Tec, Germany) and Cy5 (GE Healthcare) have almost identical spectral properties and are thus excited with the same wavelength (e.g. 635 nm) and detected at the same spectral range. As also their molecular weight is comparable, the diffusion constant is too similar to distinguish between both dyes in a mixture by means of standard FCS.\\
 Nevertheless, the autocorrelation curves of both dyes differ in the microsecond range. ATTO655 shows practically no photophysical fluctuations. Therefore, FCS curves of ATTO655 can be fitted with the simplest 3D diffusion model, making it very suited for calibration. Cy5 on the contrary, exhibits a distinct bunching term on its FCS trace originating from photophysical processes, i.e. cis/trans isomerization.\\ Nevertheless, the autocorrelation curves of both dyes differ in the microsecond range. ATTO655 shows practically no photophysical fluctuations. Therefore, FCS curves of ATTO655 can be fitted with the simplest 3D diffusion model, making it very suited for calibration. Cy5 on the contrary, exhibits a distinct bunching term on its FCS trace originating from photophysical processes, i.e. cis/trans isomerization.\\
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   * Click on "Save result" to save this curve. Rename the generated file as "Cy5-FLCS".   * Click on "Save result" to save this curve. Rename the generated file as "Cy5-FLCS".
   * These curves can now be fitted by selecting "Transfer to fit". The process of how to fit a FCS curve is explained in the tutorial [[howto:Calculate and Fit FCS Traces with the FCS Script]].   * These curves can now be fitted by selecting "Transfer to fit". The process of how to fit a FCS curve is explained in the tutorial [[howto:Calculate and Fit FCS Traces with the FCS Script]].
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howto/separation_of_2_species_with.txt · Last modified: 2014/06/18 12:22 by admin