Example of hyperspectral image analysis of Raman data using the software HIA2.3

1. Import hyperspectral data

• File>Load>Ascii
• select RamanData.asc
  
• alternatively drag and drop the file on the HIA panel
  

2. Select the column with data

• For data acquired with the Andor Newton Camera, Andor Solis software, the Raman intensity is stored in the second column of the ascii file.
• Press OK

3. Set import parameters

• Insert the acquisition settings
• For the Andor Solis
• see Galileo_Raman webpage for grating calibration and sensitivity
• Notice that in the import panel the polynomial is in (pixel-1), so that the constant term for the import panel is the sum of the constant, first and second order terms in the webpage where the polynominal is in (pizel).
  
• If you have saved the parameters previously you can import them with Load Ascii Parameters
• It is recommended to save the parameters as they are common to the data taken with identical settings
• For the RamanData.asc the parameters are saved in the ImportSettings.par file
• Press OK

4. Select pixel range for offset

• For the 300l/mm: range 1525-1585
• Press OK
• If you are not sure, first import the data without offset and without conversion, and look at the spectra to identify the range which is blocked be the filters
  

5. Set interpolation setting

• Set the interpolation settings for the wavelength to wavenumber correction
• Press OK
• To verify the laser position, which should be at zero wavenumbers, use a negative minimum wavenumber. After verification, it sufficent to keep the relevant range which is transmitted by the filter.
  

6. SVD filtering

• Analysis>SVD denoising
  
• SVD settings:  

• the offset term is the read noise squared.
  

• Press Ok
• Denoising settings:

• Press Ok
• Do not overwrite Data 1
• You can also use a lower threshold (e.g. 1.2) not to remove possibly relevant components.
  
• Check SVD denoised data (Data 1: original data, Data 2: denoised data)

7. Background fit

• Select the SVD filtered data (Data2)
  
• Analysis>Background subtraction (robust fit)
  
• Background subtraction (robust fit) settings

• For the RamanData.asc the parameters are saved in the BGfit.par file
• Press Ok
• Two new datasets are generated
• Data 3 is the residual of the fit (Raman resonances)
• Data 4 is the background fit (Auto-fluorescence)

8. Save compressed data

• You can save the data after certain steps of the analysis, Data compression uses SVD filtering
  
• Select Data3
  
• File>Save>HIA file
• Data type: 16bit
• SVD denoising parameters:

• Press Ok
  

• Press Ok
• Repeat for Data4
  

8. Limit Spectra Range 

• Select Data3
  
• Only if needed
  
• Image>Limit spectral range
• Limit up to 3050.25 1/cm
• Do not overwrite Data3 (creates Data5)
  

9. Exclude regions

• Only if needed
• Select Data5
  
• Image>Exclude Regions
• Type>Crop
• Energy>Range
• Select full range
• Selection->Polygonal

• Double-click inside the selected region

• Press Ok

10. FSC3

• Analysis>FSC3
• FSC³ settings
  

• Press Ok
• Save FSC3 results