Fluorescence microscopy image analysis
automation - time series - physiology

Image Analyst MKII provides tools both for routine manual and robust automated analysis of fluorescence time-lapse microscopic recordings. Most of the functionalities highlighted below are also incorporated into ready-to-use pipelines.

Time lapse graphing, fluorescence ratio graphing, potentiometric calibration, and specific cell and mitochondrial physiology assays distinguish Image Analyst MKII from available free software. Compare to ImageJ and Cellprofiler.

How to analyze your recordings

• Image Analyst MKII Workflow
• Quick How To
• Protocols
• Video tutorials

• Time lapse (time series) analysis of intensities, morphology, cell numbers, motility.
• Frame-to-frame background subtraction algorithms, using histogram interpolation allowing bit noise-free handling of low light level imaging data.
• Local background subtraction using Fourier-domain (high pass) spatial filtering or median filtering. Tile pattern removal. Fast 2D median calculation allows median-based rolling ball-style background subtraction.
• Automatic ROI drawing using magic wand or image segmentation-based techniques.
• Intensity measurements using ROIs or segments and shape measurements of segments with data output directly to Microsoft Excel.
• Image registration routines (image stabilizer and channel alignment) and lens distortion correction routines can correct a variety of imaging artifacts, also working with tiled imaging, fixing each tile individually.
• Fastest available template matching algorithm (compare to ImageJ): use template matching routinely to match live recordings with upfront or post-hoc stainings.
• Image masking: hide unwanted details (background, debris) from quantification to improve signal to noise and make analysis more robust.
• Image filtering spatially and in time, using nonlinear (min, max, median) or linear (mean, differentiation, kernel convolution and Fourier domain filtering (spatial only; e.g. high pass filtering)). Measurement of mitochondrion-specific fluorescence using optimized high pass or band pass spatial filter.
• Temporal and spatial differentiation: work with rates of fluorescence increase and Ca2+ waves.
• Local thresholding, and watershed-based segmentation for detecting objects with very different intensities or on uneven background. 
• Automatic threshold level calculation - use positive control images but no manually set levels to provide an unbiased and automated analysis.
• Spectral unmixing, determination of spectral crossbleed from cross-correlations or with nonnegative matrix factorization.
• Optical density image calculation for analysis of histological stainings
• Colocalization analysis using Pearson's or Spearman's correlations or with image segmentation.
• All functions work with floating point data and handle time lapses.
• Real time axis in seconds from image metadata, if available.
• Automation of analysis of multi-position or microplate recordings.

• Mitochondrial swelling assay using optimized spatial filtering (described: 6, used: 11, 22)
• Organelle motion assay using optical flow (7)
• Absolute millivolts calibration of mitochondrial membrane potential (described: 17, used: 10, 14, 26, protocol: 61)
• Mitochondria:cell volume fraction assay with confocal microscopy (described: 17 , used: 13, 14, 21, 24, 26, protocol: 61)

Algorithms developed to evaluate mitochondrial physiology by Dr. Gerencser and colleagues have been implemented to Image Analyst MKII: