Resources

Spectral Image Analysis - Example Code and Image Data

MATLAB Code:

• Linear Unmixing (LU)
• Spectral Angle Mapper (SAM)
• Constrained Energy Minimization (CEM)
• Matched Filter (MF)

Example Image Data:

• Spectral Image
  
  • Spectral Library
    
    • Note - this spectral library assumes that lumps 2 FRET labels together as one component in the library (e.g., it assumes that FRET in the image is at a constant energy transfer efficiency).  This is for simplicity / demonstration purposes only.

Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification - Example Data

Supplemental resources for the Methods in Molecular Biology book chapter: Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification.  Please cite the book chapter when referencing this work:

Silas J. Leavesley, Arie Nakhmani, Yi Gao, Thomas C. Rich. “Automated image analysis of FRET signals for subcellular cAMP quantification” in cAMP Signaling: Methods and Protocols, Manuela Zaccolo (editor). Methods in Molecular Biology, vol. 1294, Ch. 5. ISBN: 978-1-4939-2536-0. Springer Science+Business Media, LLC, (2015)

For more information on Cell Profiler, please see: http://www.cellprofiler.org/

Note - images are 16-bit greyscale.

• Image - Unmixed Hoechst Signal
• Image - Unmixed CFP Signal
• Image - Unmixed YFP Signal
• Image - Unmixed Donor-Acceptor Total Signal
• Image - FRET Signal
• Image - Root Mean Square Error
• Image - Root Mean Square Error (Percentile)
• Cell Profiler Image Processing Pipeline

Spectral Unmixing, Processing, and Visualization of 4D (x,y,z,λ) FRET/cAMP Signals

Supplemental resources for the JoVE article: Measurement of 3-Dimensional cAMP Distributions in Living Cells Using 4-Dimensional (x, y, z, and λ) Hyperspectral FRET Imaging and Analysis. Please cite the JoVE article when referencing this work.  note that all files should be saved into the same folder.

Naga S. Annamdevula, Hayden Gunn, Rachel Sweat, John R. Griswold, Andrea L. Britain, Thomas C. Rich, Silas J. Leavesley. Measurement of 3-Dimensional cAMP Distributions in Living Cells Using 4-Dimensional (x, y, z, and λ) Hyperspectral FRET Imaging and Analysis. Journal of Visualized Experiments (JoVE) 164 e61720 (2020). PMCID: PMC7968043 

• Linear Spectral Unmixing of 4D Confocal Microscopy Spectral Image Data
  • Linear Spectral Unmixing Function
  • Vectorized Form of Non-Negatively Constrained Linear Unmixing Function - we would like to acknowledge David Provencher for the creation of this algorithm which is available on MATLAB Central
  • Example Spectral Library File
  • Example Wavelength File
• 3D Visualization (Reslicing) and FRET Calculations
  • User Interface (GUI) File
  • User Interface (GUI) Figure
  • Supplemental Function
  • Image Search and Load Function
  • FRET Calculation and Reslicing Function
  • Associated Colormap Function
  • File Handling Function for 2D Anisotropic Smoothing
  • 2D Anisotropic Diffusion Algorithm - we would like to acknowledge Daniel Lopes for the creation of this algorithm which is available on MATLAB Central
  • 3D Anisotropic Diffusion Algorithm - we would like to acknowledge Daniel Lopes for the creation of this algorithm which is available on MATLAB Central
  • Fast 3D Anisotropic Diffusion Algorithm - we would like to acknowledge Daniel Lopes for the creation of this algorithm which is available on MATLAB Central
• Mapping FRET to cAMP
  
  • Associated Colormap Function

Analysis of timelapse fluorescence excitation-scanning hyperspectral image data

Dataset for the Bioengineering article listed below. Please cite the article when referencing this work.

Parker, M., Annamdevula, N. S., Pleshinger, D., Ijaz, Z., Jalkh, J., Penn, R., Deshpande, D., Rich, T. C. & Leavesley, S. J. Comparing Performance of Spectral Image Analysis Approaches for Detection of Cellular Signals in Time-Lapse Hyperspectral Imaging Fluorescence Excitation-Scanning Microscopy. Bioengineering 10, 642 (2023).

• Dataset