May 21, 2025

Public workspaceImage Intensity Processing and Analysis with ImagePro and Prism

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l2931qv1y/v1
  • Lexi Simar1,
  • Andrew Barlow2,
  • Andrew Molnar2,
  • Eduardo Rosa-Molinar PhD1,3
  • 1The University of Kansas;
  • 2Media Cybernetics;
  • 3The Washington University in Saint Louis School of Medicine, Washington University Center for Cellular Imaging
Lexi Simar
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l2931qv1y/v1
Protocol Citation: Lexi Simar, Andrew Barlow, Andrew Molnar, Eduardo Rosa-Molinar PhD 2025. Image Intensity Processing and Analysis with ImagePro and Prism. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2931qv1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 27, 2024
Last Modified: May 21, 2025
Protocol Integer ID: 117319
Keywords: ImagePro, intensity, pollen, tiff, OME tiff, Prism, ANOVA, image intensity processing, prism image processing, analysis with imagepro, intensity value, imagepro, use of imagepro, deconvoluted image, images through imagepro, image, prism, graphpad, measures anova, repeated measures anova, analysis, processing, evaluation
Abstract
Image processing and analysis completed with the use of ImagePro and GraphPad: Prism. The intensity values are exported through evaluation of the raw and deconvoluted images through ImagePro. The values are then analyzed through Prism via a two-way repeated measures ANOVA.
Guidelines
This analysis can be run on single images as well as z-stacks. A consistent ROI and n value should be used across each group of the analysis if the repeated measures ANOVA is being used.
Materials
Software:
1. ImagePro (Media Cybernetics, current version 11.1, build 10023)
2. Slidebook (Intelligent Imaging Incorporated, version 2024 2.0.45035)
3. Prism 10 (GraphPad, current version 10.4.1 (627))
Troubleshooting
Before start
To run images in ImagePro that were not obtained through this software, the file needs to be an OME tiff format. You can run inidividual channels at a single time through separate files (_C1 vs _C2) or run all channels at the same time selectively (_C0). File naming is explained in the protocol.
Image Processing: ImagePro
Convert image files into 16-bit OME Tiff files.
Note
Z-stack or single plane images can be converted.

Critical
If images were obtatined through Slidebook, export the images as OME Tiff and check the XML document option in the export window.
Open ImagePro software.
Open additional packages if needed; this can include a macro script that will print out the PSF of the image after the deconvolution is run.
Open initial OME tiff image with notation C0 at the end of the file name (if using Slidebook to convert). This notation includes all channels in the image file, unlike C1 or C2.
On the right side of the image, adjust each channel to correspond to the label/fluorophore of your image.
Note
This can also correspond to what your PMTs are labelled for in two-photon imaging, if looking at autofluorescent signals.

In the Process ribbon, open the AutoQuant Deconvolution function.
Figure 1. AutoQuant Deconvolution sidebar window that opens for the user to provide all values and information needed to run a successful deconvolution.


Note
This needs to be done only once per sample type or objective type.

In "2: Confirm Channel Metadata" fill in the boxes for objective lens, immersion RI, and NA values.
Note
This can be populated through the objective drop down menu if the information has already been tied to the objective and saved within the program.

In "2: Confirm Channel Metadata," select each channel or label, choose the instrument modality from the drop-down menu, and confirm the emission wavelength for that channel.
If all channels do not need to be processed for the deconvolution, deselect the box to the left of the channel name for the unwanted channels.

This can also be done below the image in the channels bar (above z plane bar). Use the purple triangle to "crop out" or exclude the channels not wanted in the deconvolution by moving them left or right.
Note
If the channel is unselected, the deconvolution will take longer than necessary as the channel remains visible to the process; thus, excluding it through the channel bar below the image is preferred.

In "4: Deconvolution Settings," select "default" settings (default can be changed through the settings button) and the theoretical PSF option selected in "3: Choose Starting PSF."


Note
If you have a PSF of a bead for the channel or the system this can be used as well.
Double check the iteration value needed; for this pollen experiment 20 iterations were used (default is 10).


Figure 2. Scripting window within ImagePro. The window shows all open and available macros.
If running without the PSF printout, click deconvolve.

If running with the PSF printout, in the scripting explorer (above AutoQuant Deconvolution icon on the right side of side bar window) right click the DeconSavePSF (or macro option with the scroll; macros are denoted by the scroll icon) then click run.
Once the deconvolution is completed, the file will printout to ImagePro in a new tab with an asterisk by the file name and a number followed by an underscore preceding the file name ("10_" or "20_") to correspond with the iteration value.
Note
If using a macro to run the deconvolution, other files will printout the same way but with .psf following the file name that was printed out.

To gather intensity values, open the Intensity Tracking function from the Count/Size tab; on that tab, the Intensity function is the furthest on the right.

Figure 3. Intensity tracking ribbon showcasing the types of values that can be added or removed to the intensity tracking data collection.

Under types, as shown in Figure 3, select those for intensity mean, OD, maximum, minimum, and standard deviation of the channels.
In the select tab of the ribbon, use the square/rectangle option to select the entire image (or to select smaller regions of interest).
After the selection is made, click the collect button.
Either select Data Table in the data Views section of the ribbon, or look at the bottom of the image if it is already open (Figure 4).
Using the Excel function, a worksheet can be made, or the collected data within ImagePro and then can be copied to the computer's clipboard and then pasted into an existing data collection method.

Figure 4. The data table shown provides the location from which collected values can be exported using the Excel sheet function; or saved and exported as text file using the save function.

Repeat for all images and regions of interest.
Calculating SNR
Using the data collected from the steps described above, isolate the minimum and mean intensity values for each image and each channel.
The ImagePro intenisty print out included sums for each column; these can be used for total or overall image SNR values. For subsets, select an n and isolate that n (51 for this experiment) in the values.
Note
The n should match a range from the image that best suits the following:
Constraint i: overall stack must exceed 70 slices (z-planes)
Constraint ii: ROI cannot be within 5 um of the extremes of the stack (5 um x image step size = # of planes to avoid at the top and bottom of the stack)
Constraint iii: must contain a minimum of 1 full pollen grain within ROI of interest
Constraint iv: size of pollen must be between 8-25 um in diameter

To calculate the channel SNR, for the subset or overall image, sum the mean intensity values and divide that by the sum of the minimum intensity values for that channel.
To calculate the overall SNR for the subset or overall image, sum both channel mean intensity values and divide that by the sum of both channel minimum intensity values.

Note
An SNR over 5 is the goal.

Data Analysis: Prism
Open Prism and select Grouped under the Create column on the opening window.

Figure 5. Opening selection window from Prism. This window provides initial selections for data type as well as easy access to previous files.

Insert data from the ImagePro Intensity collections with the values going across the page (across the rows) rather than down the page (within the column). An example is illustrated in Figure 6.

Figure 6. Data is in a grouped table with each cell containing one intensity value (for this experiment the mean of the image) grouped into media types (a, b, c) across the columns and grouped per imaging condition and channel down the rows.

To analyze this data, for this experiment, a two-way ANOVA was used.

Figure 7. Analysis window to input groupings and parameters to run a two-way ANOVA.

Note
This ANOVA requires the same n per condition. This experiment had a subset of n = 51 images or planes from a z-stack.

Fill out the parameters for the model.

The window shown in Figure 7 provides all the options and choices needed to run the ANOVA. Sphericity for this experiment was not assumed. The choices shown were the ones made for that tab.

Note
Repeated measures is explained in 16.2.
Factor names should match experimental groupings.

Fill out the parameters for multiple comparison options.

The repeated measures, factor names, and multiple comparisons tabs depend on the experiment. For this experiment multiple comparisons were manually selected to ensure the PMT channels (different pollen signals) would not cross in comparisons (cross labels), and the full model was fit.
Fill out the parameters for multiple comparisons tests.

The options tab provides corrections for comparisons (Sidak for this experiment), p value adjustments, and additional results that can be chosen.
Fill out the parameters for residuals.

The residuals tab offers extra graphical options to printout based on the residual values.
Run the analysis.

Note
Significance of 0.05 was set using Greenhouse-Geisser's epsilon test and Sidak's multiple comparison test.

After running the analysis, Prism prints out a result page showing the numerical results ,with additional tabs showing the Sidak's and Greenhouse-Geisser's results. Graphs can be created from here.
Protocol references
Simar, L. & Rosa-Molinar, E. (2025). "Benchmarking Assessment and Implementation of an Imaging Phantom in Nonlinear Optical Microscopy." Unpublished manuscript, Current Protocols.
Acknowledgements
The University of Kansas
The University of Washington at St. Louis, Medical Campus
MediaCybernetics (ImagePro)
GraphPad: Prism