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• Fluorescence/ EEM
Emission Sensitivity Correction
Excitation Wavelength Assignment
Correction of Excitation Intensity
Remove Rayleigh/ Raman Scatter
Subtract Blank EEM dataset
Show 2D Spectrum (EEM)
Calculate Excitation Spectra

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Just released! The brand-new, top-modern successor software for Spekwin32 Spectragryph 1.0

Fluorescence/ EEM menu

1. Menu Item [Emission Sensitivity Correction]

• For correction of the wavelength dependent sensitivity of detectors of fluorescence spectrometers. Yields "true spectra".
  
  
• Load the correction curve with the button [Load correction Curve] (*.fak file, has to consist of XY pairs with sensitivity against wavelength). Select the spectrum to be corrected in the middle drop down list. The corrected spectrum will be added to the list of spectra, the legend title will be appended with "corrected".
  
  
• Options:
  • [delete original ] deletes the original spectrum.
  • [correct all] corrects all loaded spectra and deletes the original spectra.
    
    

2. Menu Item [Excitation Wavelength Assignment]

• To prepare a set of fluorescence spectra as two-dimensional spectrum or EEM (excitation emission matrix), each spectrum needs its excitation wavelength assigned.
  
  
• This can be entered manually for each spectrum in the menu item [General] (4.1), or automated for a complete set of spectra from this menu item.
  
  
• Just enter the excitation wavelength of the first spectrum and the excitation step width.
  
  
• In case your EEM set comprises only a part of the loaded spectra, select the first and last spectrum of the set in the two drop down lists.
  
  
• For naming the spectras' legend texts with the excitation wavelength, activate the "replace spectrum names…" checkbox.
  

3. Menu Item [Correction of Excitation Intensity]

• For correcting the wavelength dependent excitation intensity (lamp spectrum) of fluorescence spectrometers for a series of fluorescence spectra with known excitation wavelength.
  
  
• Load the lamp correction curve with the button [Load Lamp Spectrum]. Select the range of spectra to be corrected with the two drop down lists. The corrected spectra will replace the original spectra, their legend titles will be appended with "Excitation intensity corrected".
  
  
• Hint: The wavelength can be entered manually for each spectrum in the menu item [General].
  
  

4. Menu Item [Remove Rayleigh/ Raman Scatter]

• Often enough, and especially for samples with low intensity fluorescence, EEMs are distorted by scattering peaks. These come from first and second order Rayleigh scattering as well as Raman scattering and can't be fully suppressed even by choosing optimal geometries of sample container and optical pathways. For being further used in chemometrics analysis, scattering has to be removed. Traditionally this has been done by setting the scattering regions to zero, but there are much better ways to reduce the influence of straylight.
  
  
• This menu item allows to remove all scattering from a whole EEM set at once with this options:
  
  
  • [treat all spectra] applies the treatment to all loaded spectra at once.
    
    
  • " [remove original(s)] removes the original spectrum/ spectra from display.
    
    
  • With [replace by] you can replace the values of the scattering region by
    
    
    • zeroes, this is the traditional & most ugly way of doing it
      
      
    • o a straight line, which might be better in some cases
      
      
    • an interpolated curve, which is most useful, because it give the least distortion. The interpolated values have the same noise level as their surrounding
      
      
  • set the excitation bandwith of your spectrometer, this defines the width of the replaced values area
    
    
  • [remove Rayleigh peaks (1st order)] removes the scattering intensity around your excitation wavelengths (visible because your sample does it)
    
    
  • [remove Rayleigh peaks (2nd order)] removes the scattering intensity around twice your excitation wavelengths (visible because your emission monochromator leaks the 1st order scattered light into it's 2nd order wavelength range)
    
    
  • [remove Raman peaks (1st order)] removes the scattering intensity around the Raman-shifted excitation wavelengths. The wavelength difference is solvent dependent, some solvents can be selected from the drop down list. You can include other solvents by editing the file "RamanBands.csv" from the Spekwin32 program folder.
    
    

5. Menu Item [Subtract Blank EEM dataset]

• It might be useful to subtract the fluorescent properties of a "Blank" sample from the EEM you want to analyze. This can be successfully used to remove background fluorescence and also scattering peaks.
  
  
• In the upper part you can see the dimensions of your current EEM set. Load the Blank dataset to be subtracted with [Load Blank dataset…]. This naturally will only work if the dimensions of both datasets are identical.
  
  
• Optionally, "blank subtracted" can be appended to the legend texts.
  
  

6. Menu Item [Show 2D Spectrum (EEM)]

• Two-dimensional display of a series of fluorescence or excitation spectra. EEM means: Excitation Emission Matrix. The spectra should be connected to a wavelength and be corrected for excitation intensity before (see above). Example:2d_beispiel.zip (315KB)
  
  
• The mouse position is shown in the lower left corner as excitation, emission and intensity values. On the right there are some options for display. By default, excitation is oriented horizontally, emission goes vertically.
  
  
• This orientation can be exchanged by using the [switch x <=> y axes] option. The EEM plot can be copied to clipboard with [Copy => Clipboard].
  
  
• You cannot print or save the graph at the moment, there are no axes.
  
  

7. Menu Item [Calculate Excitation Spectra]

• For calculating a series of excitation spectra from a series of fluorescence spectra. The spectra have to be connected to a wavelength and should be corrected for excitation intensity before(see above).
  
  
• Enter the range and stepwidth of detection wavelengths for the excitation spectra. Fluorescence spectra will be removed if [Remove Fluorescence spectra] is checked.