HySP: Hyper Spectral Phasor
This software is designed and scripted using Python 3.4 and PyQt5. The nature of the software inspires its multi-platform portability. We currently have Windows and MacOSX versions.
|MacOSX (tested 10.11+)||HySP-0.6.6.dmg||61.6MB|
|Windows 64bit (tested Windows 7 64bit)||HySP-0.6.6-amd64.msi||185.2MB|
Windows: download .msi installer, double click and follow wizard instructions.
Mac: download .dmg image, drag app to Application folder.
Here are 2 sample datasets for testing the software. Datasets were acquired using Zeiss LSM 710 and LSM 780 with Meta/Quasar mode.
If you use HySP for your work or publication, please cite it as:
Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging
Francesco Cutrale, Vikas Trivedi, Le A Trinh, Chi-Li Chiu, John M Choi, Marcela S Artiga & Scott E Fraser
Nature Methods 14, 149–152 (2017) doi:10.1038/nmeth.4134 Link
HySP follows the University of Southern California and Translational Imaging Center Software License available here .
These instructions were written by Wen Shi and Francesco Cutrale at University of Southern California on 2017-01-11
HySP is a multi-platform software for analysing multi-dimensional data. The application here uses hyper- and multi-spectral datasets. The software calculates a 2D phasor plot which can be analyzed interactively with ROI selectors, both phasor-to-image and image-to-phasor. The denoising algorithm improves the quality of data by reducing the poissonian noise.
After opening HySP software, the command window shows as below.
Set up HySP software
In the command window, under Phasor Settings the harmonic number can be typed. (i.e. harmonic:1; default configuration is 2)
As of now HySP supports .lsm and .tiff files. Tiff files should be in a single tiff sequence or multiple multi-layer tiff files. NOT all layers in a single tiff file. Example: if dataset has shape [time, z-stack, channel, x, y] then it should be saved as one multi-layer tiff for each channel or each z-stack [t, :, ch, :,:] , or [t, z, :, :,:]
1. Open .lsm file
Click Show Image > choose the file (file in .lsm format) > click Open .
File > Open LSM >choose the file (file in .lsm format) > click Open .
2. Open generic .tiff file
File > Open Tiff Series > set up Tiff Series Details window.
Detailed info on dataset shape can be set in the window as below, parameters can be edited according to user's data.
Open Spectral Phasor window
On to the commend window > Click Calculate Spectral Phasor.
The phasor window shows as below.
Working with Phasor
Click Filter Data/SDenoise to reduce noise in phasor plot. This step can be repeated up to 10 times to improve phasor quality.
Type in Minimum Value, Maximum Value > Click Update Plot to view fluorescent pixels in interested range.
Here is a comparison of filtering and range controlling
Select Log-Phasor checkbox to visualize the phasor in log-scale. Click Update Plot to update window.
Select Use Alpha-Intensity checkbox to represent ROI phasor selection as an alpha-overlay on top of the image in Image Window. This visualization mode creates a colormap black-to-[color of ROI] and maintains the intensity of the underlying pixel. Generally useful for visual representation/rendering. Use Alpha-Intensity applies to all the ROIs added after the checkbox selection. Default mode (uncheckedg Use Alpha-Intensity) is a solid color represenation.
Rectangle ROI selector can acquire the coordinates in Cursor Info window;
Click Add ROI selector > Choose the options, i.e. Rectangle > Click OK > Choose the color > Click OK > Move the rectangle on phasor.
Note: The phasor window and the image window can be viewed the same time to see the phasor chosen by rectangle is in which part of specimen in image. The specimen in image will be marked the same color user has chosen for rectangle in previous step.
Step1. Add ROI Selector
Step2. Selector color
Click Add ROI selector > Choose the options, i.e. Polygon > Click OK > Choose color > Click OK > Right click to move the polygon; Left click inside polygon close to vertices to change the shape.
Load Saved Polygons
Click Load Saved Polygons and load previous polygons as template.
Navigating Phasor plot
Click Zoom, frame the region surrounding the area of interest and zoom into it.
Click Zoom again to lock the region in case the phasor plot moves.
Click Home to get the original zoom factor.
Click Pan to traslate S and G direction on the phasors.
3D datasets [z,lambda,x,y] and 4D [t,z,lambda,x,y]
Scroll Z-position in Image Window to visualize the chosen volume slice in dataset.
Scroll Timepoint in Image Window to change time position in dataset.
For rectangle ROI and iROI, center coordinates are reported in >Cursor Window.
This option enables an ROI selector on the Image (rather than on the phasor). The (S,G) values of each pixel selected on the image are then reported on the phasor according to the modality selected. E.g. average (S,G) with std for G and S axis.
In Image Window Choose enable iROI > Click iROI options and choose (i.e. iROI avg on top) > Click on iROI >Choose cursor options (i.e. Polygon) > Select color (red as default) > Click OK > Move polygon on interested area > Statistics are reported on Cursor Info tab and in Phasor plot in Phasor window.
Note: Multiple polygons can be added on image with different colors, the area selected is labelled the same color in Phasor plot.
The iROI selector is represented with the same color on top of the phasor plot
Note: To load a new image and acquire the phasor of that image, you need to close all the windows and reopen HySP
Saving Data, ROIs and Images
Processed data can be saved as .TIFF multilayer files by pressing File > Save Volume.. > Save Image Volume..
Each channel will be saved as a separate file. Each file will contain an entire z-stack if present. Multiple timepoint will contain t__ in filename
ROIs selections on phasor can be saved by pressing File > Save i/ROI Phasoe.. > Save ROI Phasor as..
The file saved can be then reloaded on a different dataset by clicking on Add ROI in Phasor Window and selecting Load ROI.
Each image can be saved as a color picture (*.png). On Phasor Window > Save Image.. will save the phasor canvas with ROIs as currently visualized.
On Image Window > Save Image.. will save the image currently represented in the image canvas.