Fluorescence microscopy image analysis
automation - time series - physiology

New functions: Template Matching to retrieve regions of images based on similarity; Correct Intensity Jumps to fix illumination artifacts; Z-drift Correction; Weighted Z-projection. Excel window automation in pipelines. Calculation, copying and saving error bars for mean traces. Scalar arithmetic can be constrained to specified frames of a time lapse.
Improved functions: Blind spectral unmixing: a lot improved convergence, and additional features e.g. fixed coefficients for known crossbleed. Image registration functions run now even faster than before. Improved multi-dimensional file opening with bio-formats. The internal lsm reader was re-optimized for loading large files. Membrane potential calibration wizard.

• New functions:
  • High DPI and 4K display support, support of differing scaling between displays
  • TIFF folder open: flexible open method for folders with many TIFF files or multi-dimensional data sets organized into folders of many TIFF files
  • Opening of BD Pathway recordings
  • Measurement of distance of segments from ROI (Plot type:'Each') or from other segments (Distance from Segments function)
• Improved functions:
  • Improved Excel Data Window behavior
  • Improvements in opening AVI files
  • Opening Metafluor files with numbers only extensions
  • File creation time is used to establish time axis if TIFF metadata is missing
  • Smarter detection of well names in position names (for all file types)
• Other changes:
  • 23 other minor improvements or fixes

• New functions (V3.5.x):
  • Windows 64-bit version for working with image series up to the size of physical memory and ca. 22k x 22k pixel image size
  • Organize technical and experimental replicates of time courses directly in Graphpad Prism files
  • Interactive primer including analysis protocols
  • Conditionals in pipelines ( If, IfValue, Select and Endif)
  • Segment welding
  • Add scale bar to images
  • Multiple select in Pipeline window, copy-paste entire pipelines with pipeline parameters and related evaluated expressions.
  • Compatibility with regional settings using comma as decimal separator
  • Blinded stage positions
  • Additional parameters for plotting intensities, tracking parameters; spherical mean intensity classifier
  • Convenience features
    • File naming helper buttons: add measurement, position or pipeline names from save dialogs to file names
    • Switch between recent pipelines and functions from context menu
    • Improvements to the Membrane Potential Calibration Wizard user interface
    • Send to Excel or to Prism from Plot Window context menu
• Pipelines
  • Improved segmentation of cells or nuclei
  • Various options for local, image or frame-by-frame  background subtraction in fluorescence intensity and ratio measurement pipelines 
• Improved functions:
  • Improved Excel handling
  • Improved multi-display multi-DPI behavior
  • Altogether over 100 improvements
• 3.5.1 hotfix
  • Improved Primer Window and Excel Data Window behavior
  • Fixed sizing of property bar items
  • Improved file date and time reading
  • The occasional  "kernel32.dll" or "nt.dll" crash has been fixed
  • Altogether 26 smaller improvements
• 3.5.3 update
  • About 20 improvements

• New ΔψP calibration methods: "Goldman" and "Iterative"
• Other enhancements to the Membrane Potential Calibration Wizard:
  • Estimation of residual spectral crossbleed
  • Flipping y-axis on potential graphs
  • White background plots
  • Showing calculated parameters in the Wizard tab
• DPI aware plot trace thickness
• Batch processing of multiple image data sets with different pipelines and pipeline parameters (see Tools menu)
• 2 new pipelines (ΔψM calibration variants)

• Batch processing:
  • The batch processor (find it in the Tools main menu) is a simple, table-based automation using Excel spreadsheets.
  • The batch processor also provides a novel way to organize recordings, and speeds up repeated opening of complex data sets.
• Pipeline optimizer:
  • Find the optimal pipeline parameters to achieve an arbitrary goal on a test data set (find the Pipeline Optimizer in the Tools main menu).
• New Import and export formats
  • Most common video formats, including mp4, mov, avi and others; including mpeg4 and h.264 compressions
  • Faster jpg export
• New functions:
  • 7 New functions: ROI Ratiometric Classifiers, Shift Time Scale, Plot ROI Dimensions, Invert, Clear Segmentation Classifiers,  Mirror or Rotate in Place, Mirror or Rotate (new image)
  • A variety of tracking parameters can be calculated by the Plot Tracking Parameters function: 'average-path velocity, average-path acceleration, curvilinear velocity, straight-line velocity, mean average-path velocity, mean-square displacement, diffusion coefficient, track length, duty cycle, linearity, straightness, wobble, tracks
• Pipelines
  • 17 new pipelines, updates to existing pipelines
• Improved functions:
  • Plots are bar graphs for single frame recordings.
  • Segments can be selected by clicking plots
  • Black or white plot background
  • Improved multi-display and high/mixed DPI support
  • Improved locality handling (, as decimal)
  • Improved Prism export (transpose for single-frame intensity or morphology data)
  • More robust Excel export
  • Improved Membrane Potential Calibration Wizard
  • Altogether over 100 improvements
• Changes that may affect results obtained by this compared to older versions of Image Analyst MKII:
  • Membrane Potential Calibration Wizard: an error in r^2 calculations was fixed. This may affect data when using r^2 values for quality controlling calibration by affecting which traces are passed or rejected.
  • The conversion of segments to ROIs has been improved resulting slight differences in which edge pixels are contained in the resultant ROIs.
  • An error was fixed in all cell and nucleus segmenting and counting pipelines that prevented proper adjustment minimum fluorescence classifier. After updating Image Analyst MKII you may keep on using the old pipelines - find them at Pipelines/Legacy Pipelines.
  • Number of neighbors object classifier has been fixed - segmentation using this classifier may run differently

• New features
  • Histogram and sorted bar graphs for ROI and segmentation-based analysis
  • Highlight segments corresponding to graph or histogram bars
  • Image intensity histogram with data export
  • More information in the image property box, some editable, e.g. numerically adjustable image window zoom, size and color
  • Copy/paste pipeline and function parameters as text
  • Ordinal range definitions can take 'All'
  • Rate calculation and markers on plots can be toggled

• Core improvements
  • Faster pipeline processing of larger images
  • Improved high DPI support
  • Improved stability of pipeline processing and Prism output

• Changes to visuals
  • Grayscale images are shown now colored based on metadata. RGB images are shown in color. To load grayscale images and use no automatic overlaying disable this feature in the Preferences/LUT page.
• New interactive assays (see Image Analyst MKII Primer window)
  • Basic Histometry
  • Senescence-associated beta galactosidase analysis
• 21 New pipelines including:
  • Histometric pipelines
  • Aggregate / foci counting pipelines
  • Microplate whole-well counting with automatic well positioning and debris rejection
  • Optical density measurement (micro densitometry) and succinate dehydrogenase assay pipelines
  • Quantitation of fat droplets
  • Count HE and DAB-positive nuclei in brightfield RGB images
  • Pipeline snippets for common pipeline coding examples
• Improved pipelines
  • Single pipeline application folders were replaced with multiple thematic  ones.
  • Show color composite image
  • Tracking and velocity measurement pipelines
  • Measure fluorescence ratio in single cells using segmentation
  • Seahorse well cell count pipelines
  • Mitochondrial membrane potential pipelines
  • SA-BGAL pipeline
• New function
  • Crop in place
• Functions with added parameters
  • Crop - more linkage and ROI options
  • Plotting functions - Histogram option
  • Set scaling - "As recorded or custom" color option and custom color picker
• Improvements to Membrane Potential Calibration Wizard
  • Menu-loadable dilutions worksheets for MMP calibration (see Tools menu)
  • Improved stability and verbose error messaging
  • Shorter MDC (kernel size +1) segments can be used now
  • Showing standard errors can be toggled
  • Improved graph scaling with error bars
• Bug fixes
  • Range markers in plots
  • Export function from menu hanging fixed
  • Template matching hanging fixed
  • Fixes to run pipeline on partial microplate
  • Fixed interactive assay buttons that did not work
  • Fixed per frame intensity scaling during exporting ovrlayed images
  • Batch processing can take now single (not Multi-Dimensional Open) images
  • Multiple fixes to the internal tif reader.
  • ND2 file timing, when longer than 24h
  • Recent pipelines list cycles around properly
  • Improved Excel output of resampled time courses with gaps
  • Fixes to selecting segments and highlighting corresponding graph
• Changes that may affect analysis results when compared to analysis performed with earlier versions of Image Analyst MKII.
  • The calculation of shape factor was changed resulting in lower values and changed gating. The updated pipelines were adjusted for this. If keeping on using old pipelines with Set Segment Classifier and shape factor set other than 0 (disabled), then decrease this value. E.g. from 0.5 to 0.3.
  • The order of RGB channel loading was changed from BGR to RGB for Windows AVI and FFMPEG (MP4, MOV, JPG, TIF…) 
  • A small change in percentile calculation is expected to cause negligible effects
  • Changes to Excel output
  • Segments deselected using ROI masking are not outputted to the table as masked columns.
  • Some header items were renamed for consistency

• Over 184 improvements, including:
• New functions:
  • Run EXE – command line execution with image export and import, supporting passing arguments and launching multiple instances of external programs. This function is used for Cellpose and Elastix integration.
  • Create Segments from ROIs
  • Fix Vignetting (for confocal bidirectional scanning errors)
  • Set ROI Annotation
• New pipelines:
  • “AI” flavors for histometry and select automatic ROI-based pipelines, using Cellpose.
  • Snippets
  • Updates and improvements to several pipelines.
  • Pipelines to fix hot pixels in digital camera images
• New features:
  • ROI Annotations – named ROIs, support of PASCAL VOC annotation format
  • Frame labels – carrying original image names when multiple images are merged
  • Variables and Expression Evaluation dialog
  • New math evaluator variables added: roicount, segmentcount, imageentropy, imagemean, imageaccutance
  • String expression evaluation in pipelines can use the %param% variable now
  • Text file with a file list can be used to open recordings
  • Removal of segments close to edges
  • Faster temporal median filter
  • Time-averaged Z-stacks
  • Additional crop functions when loading large images with Bio-Formats.
  • Manual Channel Alignment
  • ROI transformation for affine, rescale, rotate functions.
  • Copy/paste of time point values, ROI annotations and Frame Labels
  • Updated histometry pipelines with new features (see descriptions)
• Improvements to:
  •  Multi-Dimensional Open Dialog, including fixing a critical memory leak in Bio-Formats loading, fixed loading of Keyence TIF files, improvements in Z-stabilizer, improved well label handling
  •  Membrane Potential Calibration Dialog: improved constraints during ΔψM calibration resulting fewer cells rejected. Output can be baseline potentials (a bar graph) only, or ranges can be averaged, baseline can be subtracted to calculate relative changes.
  •  Prism Export, fixed scrambling of subcolumn titles when more subcolumns are added, fixed occasional failure of time course synchronization
  • Pipeline Optimizer and in Batch Pipeline Processor
  • Improved stability of video and jpg export using FFMPEG.
• Changed behavior:
  • Baseline normalization for plots does now df/f0 and there is a separate df/f0+1 normalization.
  • Cross-correlation image now centers the origin

• Over 30 improvements, including:
• New functions:
  • Brightfield Color Deconvolution with PCA – Work with bright filed RGB images, white balance, stain normalization, stain to concentration, concentration to stain. Stain concentration images allow using standard pipelines designed for fluorescence on bright field images such as H&E staining.
  • AI Create Training Set - crop and augment annotated images
• New features:
  • Bundled Java, no more Oracle Java needed
  • GitHub support
  • Reading labels for Keyence microplate-based images
  • Finding ROIs based on annotations and overlaps
  • Cycle around ROIs in zoomed-in images with < > keys
  • More automations supporting annotations
  • Support of multiple annotation formats, *.geojson, *.yaml+txt
  • Plot functions Histogram parameter supports cell or object counting in a variety of ways
• Improvements
  • Speed optimizations for geometric transformations and segmentation
  • Excel data output
  • Histogram plot

• Over 20 improvements, including:
  • Fixes to internal nd2 loader to correctly load files that were also problematic with the Bio-Formats reader.
  • Improved large tif and Bio-Formats loading of large images
  • New variables (see Tools/Variables and Expression Evaluation)
  • Improved z-stabilizer (tracked plane optimization option)
  • Improved Github repository handling
• New function:
  • Replace or modify ROI/Annotation - Use the ROI tools toolbar button for additional manual ROI operations.