These instructions were written by Wen Shi and Francesco Cutrale at University of Southern California on 2019-03-30

Introduction

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 poissonian noise.

SEER is a pre-processing visualization approach for hyper- and multi-spectral fluorescent images. The technique is incorporated in the HySP software platform, a multi-platform software for analysis of multi-dimensional spectral data. SEER provide rapid enhanced visualization of spectral datasets, starting from fluorescent data.

Starter Guide / README

This is a Starter Guide including updates on SEER (Spectrally Encoded Enhanced Representations) [Starter Guide SEER]

This is a Starter Guide including updates on Hybrid Unmixing (HyU) [Starter Guide HyU]

Detailed Guide PDF

This is a Detailed Guide of HySP including updates on SEER and HyU in pdf format [Detailed Guide PDF]

Command window

After opening HySP software, the command window shows as below.

Menu

Image window

Phasor window

Set up HySP software

Harmonic

In the command window, under Phasor Settings the harmonic number can be typed. (i.e. harmonic:1; default configuration is 2)

Loading images

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

Filter

Click Filter Data/SDenoise to reduce noise in phasor plot. This step can be repeated up to 10 times to improve phasor quality.

Range controls

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

Scale controls

Select Log-Phasor checkbox to visualize the phasor in log-scale. Click Update Plot to update window.

ROI controls

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.

ROI selector

Rectangle
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

Step3.

Polygon
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

Zoom
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.

Home
Click Home to get the original zoom factor.

Pan
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.

Cursor info

For rectangle ROI and iROI, center coordinates are reported in >Cursor Window.

iROI selector

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

Save Data
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

Save ROIs
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.

Save Images
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.

SEER Visualization

SEER Maps

After loading data and applying denoising, return to the Image window and on the bottom left click on the dropdown menu labeled “Grayscale” and the different SEER options of Gradient ascent, descent, radius, angle, and contour will appear.

SEER Reference Maps

Toggle the Map Reference checkbox, corresponding reference map will be updated on phasor window.

SEER Modes

The “Mode” dropdown menu is right below the SEER map menu. The initial value is set as “Original Maps” and there are “Scaled Mode” and “Morphed Mode” available.

Scale Mode

Under Scaled mode there are auto-scale and manual scale mode.

Manual Scaled Mode

Selecting “Scaled Maps” will enable the sub-modes for this mode.

One can type in the G/S values to specify the reference map position on phasor plot, click Update SEER to scale and relocate the map reference on phasor plot. See figure below.

Morph Mode

Under Morph mode, there are Max Morph mode, Mass Morph mode, and Manual morph mode. As an example, selecting Morphed Maps in the “Mode” dropdown menu will activate the different Morph modes in the drop down right below.

Manual Scaled Mode

Select Morphed Maps,Manual Morph Maps to activate manual morph mode. Click Start.

Mouse double click on phasor plot to morph the map reference to a new center, (G, S) coordinate of this new center is also updated when mouse released.

To explore the dataset, one can always switch to another map under any mode and sub-mode.

Save Volume Image

Click Save Seer Volume will yield a series of Tiff files in a name required folder for future processing, i.e. 3D rendering.