Example of hyperspectral image analysis using the software HIA2.1

Unzip Data.zip
Data.txt: hyperspectral CARS intensity images of a U2OS cell acquired with the multimodal CARS microscope at School of Biosciences (Cardiff University) using spectral focusing. Software: MultiCARS.
Offset.txt: CARS intensity image of the same cell taken at zero temporal overlap of the Pump and Stokes pulses. Software: MultiCARS.
gwratio.dat: Glass/Water CARS ratio spectrum

1. Import hyperspectral data

File>Load>MultiCARS

select Data.txt

for tiff or ascii import, check type (CARS, SRS, Raman) and scaling (pixel size, spectral axis, intensity scale)

2. Remove offset

Image>Subtract Offset>Image

select Offset.txt

3. SVD denoising

Analysis>SVD denoising
SVD settings:

if negative values are present in the data, use (sign(d)^1)*(d^2+0)^0.25

Press Ok
Denoising settings:

Press Ok
Check SVD denoised data (Data 1: original data, Data 2: denoised data)

4. Calculate CARS ratio

Analysis>CARS ratio calculation
CARS ratio settings:

Press Ok
Load gwratio.dat
Select region corresponding to water

Type>Mask
Selection>Freehand (double click on the selected area to activate)

Press Ok

5. Correction for uneven background

Analysis>Uneven background correction>Background region determination>Manual
Settings:

Press Ok
Select region corresponding to water

Type>Mask
Energy>Range

select full range

Selection>Freehand (double click on the selected area to activate) - select area similar to previous step
Press Ok

Check the results (Data 1: uncorrected data, Data 2: corrected data)

6. Field retrieval

Analysis>Field retrieval (PCKK)
Settings:

Press Ok

7. Save compressed data

It is recommended to save the data at different step of the analysis.
File>Save>HIA file
Data type: 16bit
SVD denoising parameters:

Press Ok

8. FSC³

Limit spectral range (if needed)

Image>Limit spectral range

Limit from 2700 to 3100 1/cm

Analysis>FSC3
FSC³ settings

Press Ok

Save FSC3 results

Recommended settings for improved reproducibility

We noticed improved reproducibility of the factorization of the example data using the FSC3 settings shown below. Settings can be imported in the v2.1 using the 'Load FSC3 parameters' function on the FSC3 panel menu and selecting the file 'FSC3_2p1.par'.

These settings produce some results were the water is factorized in only one component and DNA and lipid are factorized separately from the cytosolic protein.

07/05/2015, Francesco Masia. Updated by FM 26/06/2015