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ThermImageJ - Thermal Image Functions and Macros for ImageJ

ThermImageJ is a collection of ImageJ functions and macros to allow for import and conversion of thermal image files and to assist in extracting raw data from infrared thermal images and converting these to temperatures using standard equations in thermography.

These macros will allow you to import most FLIR jpgs and videos and process the images in ImageJ.

ThermImageJ emerged from Thermimage (https://github.com/gtatters/Thermimage), the latter an R package with similar tools but more emphasis on biological heat transfer analysis.

Compatibility

  • ThermImageJ was developed on OSX, and tested using ImageJ v1.52o. Many features require installation of command line tools that may present future challenges on different operating systems. Testing and troubleshooting is ongoing, especially in Windows. Please report issues here: https://github.com/gtatters/ThermImageJ/issues, or consider converting to Mac or Linux.

How to Cite

  • Glenn J. Tattersall. (2019). ThermImageJ: Thermal Image Functions and Macros for ImageJ. doi:10.5281/zenodo.2652896. DOI

Requirements

External Software Downloads

ThermImageJ Downloads from this Github site

Installation Instructions

  • Install FIJI, exiftool, perl, and ffmpeg according to the website instructions above.
  • Troubleshoot or perform installation checks (see next section).
  • Launch FIJI and follow any update instructions.
  • Launch FIJI–>Help–>Update, allow it to update any plug-ins, then while the update window is open, select Manage update websites, and ensure that the FFMPEG box is ticked. Select ok, then click the Apply option, and restart FIJI. This FFMPEG plugin is required for importing avi files created during the conversion process, although it might require that you have FFMPEG installed at the command line.
  • Navigate to where FIJI is installed to find all the subfolders.
  • Download the ThermImageJ.ijm file from this site and copy into the FIJI/macros/toolsets folder.
  • Open the ThermImageJ.ijm file in any text editor, and verify the paths are properly set for your respective operating system. See the comments with the text file for guidance. Most of the default locations are likely fine, although FFMPEG is sometimes installed in different folders depending on what the user might have selected.
  • Download the additional luts files from this site and copy into your FIJI/luts folder. These are palettes that are commonly used in thermal imaging.
  • Download the perl script, split.pl from this site and copy into a FIJI/scripts folder.
  • Download Byte_Swapper.class to the plugins folder.
  • Restart ImageJ.
  • If everything succeeded (see checks below), the toolset should be installed and visible from your plugins menu.

Installation Checks

Verify exiftool is installed by launching a terminal (or cmd prompt) window and typing the following bash commands:

exiftool -ver
which exiftool
## 12.62
## /usr/local/bin/exiftool

If you see a version number (probably > 10) and no error, then exiftool is installed properly. The second line will tell you the path to where it is installed.

Do the same for perl:

perl -ver
which perl
## 
## This is perl 5, version 38, subversion 0 (v5.38.0) built for darwin-thread-multi-2level
## 
## Copyright 1987-2023, Larry Wall
## 
## Perl may be copied only under the terms of either the Artistic License or the
## GNU General Public License, which may be found in the Perl 5 source kit.
## 
## Complete documentation for Perl, including FAQ lists, should be found on
## this system using "man perl" or "perldoc perl".  If you have access to the
## Internet, point your browser at https://www.perl.org/, the Perl Home Page.
## 
## /usr/local/bin/perl

Verify no errors on your system to ensure perl is installed correctly.

Check that the perl script is accessible by perl (be sure to provide the proper path to the split.pl file on your system):

perl /Applications/Fiji.app/scripts/split.pl

You should see the following warning message:

“Error: Please specify input file, output folder, the output filename base, pattern to split, and output file extension.”

This is a good error, and verifies that the perl script is installed where your machine can access it!

If you see:

“Can’t open perl script”/Applications/Fiji.app/scripts/split.pl”: No such file or directory”

you will need to re-check the location of the script or the path information provided at the top of the ThermImageJ.ijm file.

Now, do the same for ffmpeg:

ffmpeg -version
which ffmpeg
## ffmpeg version 6.1.1 Copyright (c) 2000-2023 the FFmpeg developers
## built with Apple clang version 15.0.0 (clang-1500.1.0.2.5)
## configuration: --prefix=/usr/local/Cellar/ffmpeg/6.1.1_2 --enable-shared --enable-pthreads --enable-version3 --cc=clang --host-cflags= --host-ldflags='-Wl,-ld_classic' --enable-ffplay --enable-gnutls --enable-gpl --enable-libaom --enable-libaribb24 --enable-libbluray --enable-libdav1d --enable-libharfbuzz --enable-libjxl --enable-libmp3lame --enable-libopus --enable-librav1e --enable-librist --enable-librubberband --enable-libsnappy --enable-libsrt --enable-libssh --enable-libsvtav1 --enable-libtesseract --enable-libtheora --enable-libvidstab --enable-libvmaf --enable-libvorbis --enable-libvpx --enable-libwebp --enable-libx264 --enable-libx265 --enable-libxml2 --enable-libxvid --enable-lzma --enable-libfontconfig --enable-libfreetype --enable-frei0r --enable-libass --enable-libopencore-amrnb --enable-libopencore-amrwb --enable-libopenjpeg --enable-libopenvino --enable-libspeex --enable-libsoxr --enable-libzmq --enable-libzimg --disable-libjack --disable-indev=jack --enable-videotoolbox --enable-audiotoolbox
## libavutil      58. 29.100 / 58. 29.100
## libavcodec     60. 31.102 / 60. 31.102
## libavformat    60. 16.100 / 60. 16.100
## libavdevice    60.  3.100 / 60.  3.100
## libavfilter     9. 12.100 /  9. 12.100
## libswscale      7.  5.100 /  7.  5.100
## libswresample   4. 12.100 /  4. 12.100
## libpostproc    57.  3.100 / 57.  3.100
## /usr/local/bin/ffmpeg

Setting and verifying paths to command line tools

Once you have installed everything above, and verified no errors, you can check or change the directory paths in FIJI/ImageJ.

The ThermImageJ.ijm toolset file will detect whether you are using Mac OSX, Linux or Windows and attempts to define the appropriate file paths automatically. Thus, you should not need to change parameters, but it is useful to check and become familiar with the process or do any customisation necessary for your FIJI installation.

Navigate to the ThermImageJ.ijm toolset file and open it using a text editor or the built-in ImageJ macro editor:

Depending on your operating system or how system files are installed you may need to edit the specific path locations for your respective system:

This also applies to the location of the split.pl file that should be placed in the scripts folder inside the Fiji folder.

ThermImageJ assumes you have placed the split.pl file into a scripts subfolder where Fiji is installed, so hopefully you will not need to change this:

Setting ThermImageJ Macros Up in FIJI/ImageJ

Once you have installed everything above, and verified no errors, you can set the macros up in FIJI/ImageJ.

Launch FIJI, left click the more-tools menu, which is the >> on the far right side of the menu bar:

Which will reveal any of the toolsets in the folder. Click on ThermImageJ to replace the present icons with ThermImageJ specific icons / macros:

Once installed, the toolbar menu populates with new icons corresponding to the primary ThermImageJ functions:

Once installed, the toolset should also populate the Plugin Dropdown Menu with the same, and some additional macros used less often:

Customising the Toolset

Feel free to edit your version of ThermImageJ.ijm and if you break it, you can always download a new one.

You can edit it with any text editor or with the built-in ImageJ text editor by selecting Plugins–>Macros–>Edit and navigating to the Fiji/macros/toolset folder and selecting the ThermImageJ file. Or from within ImageJ/Fiji, holding the shift key down, select the >> “More Tools” link and still hold the shift key down, click on ThermImageJ to open the file up within the built-in text editor.

If you do make changes and save them, you will either need to restart Fiji, or restore the toolset bar by clicking on the >> “More Tools” link, selecting Restore Start-Up Tools then clicking on the >> “More Tools” link and selecting ThermImageJ again.

Main Functions and Features

File Operations

Direct Import of Raw Data

  • Raw Import Mikron RTV
    • custom macro to import an old Mikron Mikrospec R/T video format
    • these files had simple encoding and are not likely in use any longer, except by the author
    • see SampleFiles.zip for sample data
  • Raw Import FLIR SEQ
    • custom macro to import FLIR SEQ using the Import-Raw command
    • use only if you know the precise offset byte start and the number of bytes between frames (see Frame Start Byte Macro below).
    • this only works for certain SEQ files (usually those captured to computer), and only formats where tiff format underlies the video.
    • see SampleFiles.zip for sample data
  • Frame Start Byte
    • This macro will scan a FLIR video file (SEQ) for the offset byte position ‘0200wwwwhhhh’ where wwww and hhhh are the image width and height in 16-bit little endian hexadecimal.
    • For example, the magicbyte for a 640x480 camera: 02008002e001”, “8002” corresponds to 640 and “e001” corresponds to 480.
    • The user can provide a custom magicbyte, but should leave this blank otherwise.
    • The function is only used in conjunction with the Raw Import FLIR SEQ macro.
    • The function returns best estimates for the offset and gap bytes necessary for use with the Raw Import FLIR SEQ macro, although is not guaranteed to be correct due to variances in SEQ file saving convention.
    • Note: on unix based OS, this macro calls the xxd executable and runs quickly. For Windows OS, Powershell Core 6 needs to be installed with the updated Format-Hex function, and runs slowly.

Import (and Conversion) using Command-Line Programs

  • Convert FLIR JPG (from the Plugins->Macros Menu only)
    • select a candidate JPG or folder of JPGs, and a call to the command line tool, exiftool, is performed to extract the raw-binary 16 bit pixel data, save this to a gray scale tif or png, placed into a ‘converted’ subfolder.
    • subsequently the user can import these 16-bit grayscale images and apply custom transformations or custom Raw2Temp conversions.
    • some images may be converted in reverse byte order due to FLIR conventions. These can be fixed with the Byte Swapper plugin after import.
  • Import FLIR JPG
    • select a candidate JPG, and a call to the command line tool, exiftool, is performed to extract the raw-binary 16 bit pixel data, temporarily save this to a gray scale tif or png, import that file, and calls the Raw2Temp function using the calibration constants derived from the FLIR JPG file.
  • Import/Convert FLIR SEQ
    • Import: select a candidate SEQ file, and a call to the command line tools, exiftool, perl split.pl, and ffmpeg is performed to extract each video frame (.fff) file, extract the subsequent raw-binary 16 bit pixel data, save these as a series of gray scale files, and collate these into an .avi file or a new folder of png or tiff files. Subsequent .avi file is imported to ImageJ using the Import-Movies (FFMPEG) import tool.
    • jpegls as the output video codec is advised for its high compression, lossless quality, and compatibility between different OS versions of FFMPEG.
    • this function may also work on FCF file types but has not been thoroughly tested
    • Convert: this function may also be used to convert the video into a folder of png or tiff files by selecting png or tiff as the output filetype, instead of avi. File codec is ignore if you choose this approach. The folder will be automatically named according ot the video file without extension. Thus, SampleVid.seq will be converted to files in the folder called SampleVid.
  • Import/Convert FLIR CSQ
    • Import: select a candidate CSQ file, and a call to the command line tools, exiftool, perl split.pl, and ffmpeg is performed to extract each video frame (.fff) file, extract the subsequent raw-binary 16 bit pixel data, save these as a series of gray scale files, and collate these into an .avi file or a new folder of png or tiff files. Subsequent .avi file is imported to ImageJ using the Import-Movies (FFMPEG) import tool.
      • jpegls as the output video codec is advised for its high compression, lossless quality, and compatibility between different OS versions of FFMPEG.
    • Convert: this function may also be used to convert the video into a folder of png or tiff files by selecting png or tiff as the output filetype, instead of avi. File codec is ignore if you choose this approach. The folder will be automatically named according ot the video file without extension. Thus, SampleVid.csq will be converted to files in the folder called SampleVid.

Lookup tables and adjusting colour ranges


  • LUT (Thermal Palette Look Up Table) menu
    • for rapidly accessing different pseudocolour palettes
    • Grays, Ironbow, and Rainbow are more commonly used in thermal imaging
    • ImageJ’s built in LUTs can be always be accessed from the Image-Lookup Tables Menu
  • Next LUT
    • select the next LUT in the list of all ImageJ LUTs, including the ones in the Thermal LUT list
  • Previous LUT
    • select the previous LUT in the list all ImageJ LUTs, including the ones in the Thermal LUT list
  • Invert LUT
    • invert the colour scale of the LUT
    • this can be toggled
  • Brightness/Contrast
    • setting the min and max values of the pseudocolour scale
    • set min equal to the lowest temperature desired on the lookup table scale
    • set max equal to the highest temperature desired on the lookup table scale
  • Add Calibration bar
    • short-cut to ImageJ’s built-in Analyze->Tools->Calibration Bar
    • use this after temperature conversion of image
    • the tool attempts to choose an appropriate sized calibration bar by auto-adjusting the zoom factor
    • to save this permanently on an image you need to duplicate and/or convert your image to RGB format (Image–>Type->RGB Color), then flatten the overlay (Image–>Overlay–>Flatten), then save as a tiff or png. Note: the resolution of the text on the calibration bar depends on your image size and be be unsatisfactory with small images. If I find a fix for this, I will implement it.

Bits and Bytes

  • Image Byte swap
    • short-cut call to the Byte Swapper plugin.
    • since FLIR files are sometimes saved using little endian order (tiff) and big endian order (png), a short-cut to a pixel byte swap is a fast way to repair files once they are imported that have byte order mixed up

Utilities

  • FLIR Dates
    • user selects a candidate FLIR file (jpg, seq, csq) to have the Date/Time Original returned. Use this to quickly scan a file for capture times.
  • FLIR Calibration Values
    • select a candidate FLIR file (jpg, seq, csq) to display the calibration constants and built-in object parameters stored at image capture. Typically, the user would then use the Planck constants and Object Paramters in the Raw2Temp macro.
    • use this function on the original FLIR file if you have a 16-bit grayscale image of the raw data in a separate file and need to convert to temperature under specified conditions.
    • the calibration constants and object parameters are stored in memory for subsequent use of the Raw2Temp function, and should be remembered the next time you re-boot ImageJ, so if you are only working with one thermal camera’s files, you should not have to re-type the calibration constants for future uses.

Temperature Conversion

  • Raw2Temp
    • converts a 16-bit grayscale thermal image (or image stack) into estimated temperature using standard equations used in infrared thermography.
    • user must provide the camera calibration constants, atmospheric attenuation constants, and object parameters that can be obtained using the FLIR Calibration Values macro.
    • various custom versions of Raw2Temp are included for different cameras the author has used, since the calibration constants do not change from image to image, and only when the camera is sent back to manufacturer for re-calibration. Edit these macros once calibration constants are known for other cameras.
    • a Fast and Slow calculation have now been implemented (v 1.4.1).
    • The Slow calculation is slow because it converts the file to a 32-bit file and then converts each pixel to its calculated temperature. This can take time on video files and may be too much for large files or computers with little RAM.
    • The Fast calculation implements a built-in ImageJ function that allows us to fit a 4th order polynomial through the relationship between Temperature and the Raw 16 bit data, providing a pseudo-converted file with both raw data and converted data showing up in the imageJ status bar. This may or may not be an accurate depiction of the response, although I provide the user with cautionary advice on the numbers returned. If you restrict the range of temperatures to fit the polynomial only to reasonable ones for most biological applications, the error seems to be quite low since the polynomial accurately fits the data. It is mainly at the extremely low and high ends of the camera’s temperature range that the fit is poor. For single image analysis, I advise you use the Slow (accurate) conversion, and only consider the Fast conversion for large videos where some trade-off between CPU time vs. accuracy is more crucial.

ROI (Region of Interest) Tools

  • ROI 1 to ROI 6 (from the Plugins->Macros menu)
    • macros coded to short-cut keys, such as: 1,2,3,4,5,6 by adding [#] to the name of the macro in the ThermImageJ.ijm file
    • some extra ROI short-cuts (i.e. d, l) might exist that I have in place for my own analyses - you can ignore these line ROIs
    • extracts mean, min, max, sd, and area of the given ROI and saves to results window as well as to a ROI_Results.csv file to user’s desktop
    • location of ROI_Results.csv file can be changed by user by editting the variable desktopdir at the top of the ThermImageJ.ijm file
    • sample results file: https://github.com/gtatters/ThermImageJ/blob/master/ROI_Results.csv
    • edit the ThermImageJ.ijm to change the nature of the results to extract
    • addition ROIs can be added to the toolset file
    • designed to work with single images or image stacks
    • slice label and number are recorded to the results table as:

Other ROI tools

  • Extract ROI Pixel Values (short-cut: p)
  • Add ROI Measurement to Image
    • adds the result of the ROI parameter to the image as an overlay.
    • will work on stacks or single images.

Math on Stacks

  • ROI on Entire Stack
    • performs an ROI analysis across the entire stack.
    • min, max, mean, median, mode, skewness, kurtosis for every slice are exported to the results window and to file to desktop
    • select what summary statistic to perform discrete fourier analysis to extract dominant frequency components.
    • i have tested this on oscillatory data (metronomes set to move at fixed rates) and the fourier extracted frequencies appear to work correctly
  • Cumulative Difference Sum on Stack (in progress)
    • This function works on stacks, first by subtracting the difference in pixel values between frames, creating an absolute value difference stack n-1 frames in length.
    • Then all pixels from each frame are examined for the mean and standard deviation per frame, stored to the results window, after which a cumulative value is calculated.
    • This cumulative absolute difference value is then detrended and zeroed to remove mean value offset prior to a discrete fourier analysis to return freuquency components.
    • The user should provide time interval in seconds for the image stack.

Typical Workflow

Converted JPG to raw 16-bit PNG or TIFF Workflow

  • Determine your FLIR camera’s calibration constants (i.e. use the Calibration Values Tool)
  • Convert Image to a 16-bit Grayscale File (i.e. Convert FLIR JPG)
  • Import converted file to ImageJ using normal ImageJ file recognition. File->Open or File->Import Image Sequence can work on PNG and TIFF files.
  • You may prefer to work with TIFF files, but the filetype created by these macros depends on how the raw data were stored by FLIR (PNG or TIFF). If so, you might use ImageJ’s batch conversion tool to convert your files before importing them.
  • Run the FLIR Calibration values macro on the original FLIR file in order to extract the calibration constants into memory
  • Run Raw2Temp or one of the custom Raw2Temp macros for your particular camera
  • Choose your palette (LUT in ImageJ)
  • Use ImageJ ROI tools and Measurement tools

Single JPG Workflow

  • Use the Import JPG tool which will scan the file for calibration constants, extract the raw thermal image, convert this to a PNG or TIFF file, and automatically open it.
  • Inspect the opened image, calibration constants, and object parameters to ensure that these values are appropriate to your application.
  • Choose your palette (LUT in ImageJ)
  • Use ImageJ ROI tools and Measurement tools

Video Workflow

  • Use the Import SEQ or Import CSQ functions that scan the file to determine calibration constants before import
  • Select the video import option and jpegls as the codec (i.e. the defaults) This will keep file size as small as possible and preserves compatibility with the ImageJ FFMPEG implementation
  • The Import SEQ and Import CSQ macros will automatically attempt to calculate temperature
  • Once the file is converted and imported, double check that the calibration constants and object parameters are appropriate and select ok. If you escape at this stage, you should still have a 16-bit grayscale image stack, and could run the Raw2Temp function later

Custom ROI analysis

  • First set the parameters you are interested in extracting in the Analyze->Set Measurements menu.
  • Typical values are min, max, mean, modal, median, standard deviation, but ImageJ offers so many other values.
  • In ImageJ terminology, “Intensity” or “Gray Value” corresponds to the number stored in each pixel. This might be the 16-bit raw value or it might be the 32-bit decimal converted temperature, depending on when analysis is performed.
  • Take advantage of all the ImageJ ROI tools, or Tools->ROI Manager to draw regions of interest over sites of interest.
  • Or, use the ROI 1-6 macros included as described earlier in the document.

Sample files to test:

https://github.com/gtatters/ThermImageJ/blob/master/SampleFiles.zip

Video Guide: Installation

See this screen capture introducing the basic installation steps. https://youtu.be/8SXhzOlsmKI

Video Guide: Demonstration of Functions

See this screen capture demonstrating basic functions here: https://youtu.be/5XYZw0kqX64

Performance, Speed, File Size Limits, and Caveats

  • The maximum number of video frames (i.e. stacks) will limited by the CPU and RAM, but success with videos and image stacks of up to ~1000 frames has been tested.
  • Due to memory limits in FIJI, delay converting larger video files to temperature until the files have been otherwise processed. The memory required to work with converted files (32-Bit) is double that required to work with the 16-bit grayscale images. Or consider using the Fast option in the Raw2Temp function, which will create a virtual conversion that is usually quite close to the real data for normal biological temperatures.
  • Consider cropping videos, re-sampling fewer stacks if you have oversampled videos, or performing ROI analyses on the 16-bit raw data and then calculate temperature using the raw2temp function also available in an R package (Thermimage).
  • If you have large video files (>2000 frames and high resolution), it is advisable to convert these videos to folders of png files, and use the File –> Import –> Image Sequence tool to skip files during import as a way of down-sampling.
  • Finally, verify that the temperature values obtained with these macros are similar to the ones obtained using official thermal imaging software. See https://github.com/gtatters/ThermimageCalibration for details on performance accuracy (±0.04C), but a healthy skepticism is advised. Please post in the issues if you do suspect the Raw2Temp conversions are not consistent with expectations.

File Types

  • The radiometric file types at present supported are mainly those from FLIR, however certain file types that can be imported into ImageJ could be used in the future, depending on information from users. Deciphering the radiometric data storage approaches takes time and requires sample files.

  • For a discussion about the Babylonian nature of thermal image file types and strategies employed by thermal camera manufacturers see https://www.irinfo.org/04-01-2006-colbert/

References

The following open source programs were crucial to the development of ThermImageJ.

Contributors

ImageJ Macro Development occurred in association with:

Command Line Development occurred in assocation with:

Raw2Temp development occurred in association with:

Suggestions/Issues/Caveats

  • Suggestions for improvements and additions, as well as bugs or issues can be filed here: https://github.com/gtatters/ThermImageJ/issues

  • Please include a sample image to help with solving issues

  • Please star or follow this github site to keep up to date on new releases as I fix errors following further testing.

  • ThermImageJ will still remain a work in progress as I add features that are useful to myself, but might not be readily apparent to other users. Occasional odd short-cuts that are present are likely the result of a project I am currently working on.

  • Note: I have no affiliation with thermal image companies nor do I receive any funding or free equipment despite the plethora of customers I have sent to them. This project emerged as a result of the frustration of needing to use Windows only software that has limited journaling and customisation. I should acknowledge that in July 2019, FLIR released a more affordable cross-platform analysis software that some users may prefer to invest in rather than this open source solution. It would be unfair of me to not recommend that you try their software first, since they are the experts.

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ImageJ functions and macros for working with thermal image files

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ffmpeg imagej fiji perl thermal-imaging heat-transfer temperature-reading exiftool imagej-macro fiji-macro

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