Fluorescence microscopy image analysis
automation - time series - physiology

Sample interactive analysis protocol

Control Image Analyst MKII from interactive protocols to speed up routine work or to learn into image analysis. Click any section below to open. Buttons and links marked by callouts operate the application. You can analyze single time courses or entire multi-dimensional data sets simply by button-clicking, when using this protocol in the Primer Window of Image Analyst MKII.

Measure fluorescence intensity or ratio in time courses
Abstract

This interactive assay protocol helps selection and operation of an appropriate pipeline to analyze intensity or ratiometric fluorescence time lapse recordings. Pipelines are located in the Pipelines/Intensity Measurements main menu item. 

Assay Version 1.0 10/30/2017

Input

Single or multi-channel time lapse recordings in single or multiple positions.

Analysis protocol
  • Open the recording.  Use multi-select if time points or segments of time courses were recorded in individual files. If the recording is already open, click refresh instead.
Protocol
  • Measure intensity of whole view field with background masking

    The pipeline calculates average fluorescence intensity for the whole view field after stabilization of the image time series, background subtraction and masking of background regions. The mask is calculated from maximum intensity projection of the time series therefore accounts for cellular movement during the timelapse. This pipeline is ideal for obtaining fluorescence intensities from low-light level single-channel recordings, where cellular movement occurs. Applicable to Ca2+-imaging (e.g. Fluo-3, GCaMP), reactive oxygen species imaging (e.g. DCFDA), GFP-based sensor imaging.

  • Measure intensities in single-cells using ROIs drawn by segmentation

    This pipeline stabilizes the time lapse (registers frames in time) and subtracts background. To eliminate background the “Median of pixels below percentile of max projection” with the percentile given at “Background level” is used. Then segments the maximum intensity projection of a time lapse, creates ROIs from the segments and copies ROIs to the original image. Finally ROI means are plotted. Use: generic pre-processing for single-channel recordings, before single-cell analysis. Applicable to Ca2+-imaging (e.g. Fluo-3, GCaMP), reactive oxygen species imaging (e.g. DCFDA), GFP-based sensor imaging.

  • Measure intensities corresponding to segments

    Two pipelines are provided here to segment an image of cytosolic or nuclear stain into single cells or nuclei, and then measures intensities in the same or in an other channel, after performing background subtraction. .

  • Measure fluorescence ratio of whole view field with background masking

    This pipeline calculates average fluorescence ratio of all cells in the view field after stabilization of images, background subtraction and masking of background regions. The mask is calculated from maximum intensity projection images therefore accounts for cellular movement during the timelapse. The fluorescence ratio is calculated as the ratio of mean fluorescence intensities allowing working at low signal to noise ratio. This pipeline is ideal for obtaining fluorescence ratio from low-light level recordings, where cellular movement occurs. Applicable to Ca2+-imaging (e.g. Fura-2, yellow cameleons), other ratiometric ion probes imaging (e.g. PBFI, SPFI, SNARF-1, BCECF), reactive oxygen species imaging (e.g. HyPer, roGFP), and intramolecular FRET sensor imaging.

    • Enter the channel numbers for the numerator and denominator channels for the ratio calculation, e.g. Fura340 and Fura380, respectively.
  • Measure fluorescence ratio in single-cells using segmentation-based ROIs

    This pipeline computes ratio image and shows it in Intensity Gated Pseudocolor, draws ROIs and plots ratio values as ratio of the average fluorescence intensities measured in the two source images for each individual cell. This pipeline stabilizes the time lapse (registers frames in time) and subtracts background. To eliminate background the “Median of pixels below percentile of max projection” with the percentile given at “Background level” is used. Then segments the maximum intensity projection of a time lapse, creates ROIs from the segments and copies ROIs to the original image. The fluorescence ratio is calculated as the ratio of mean fluorescence intensities allowing working at low signal to noise ratio. This pipeline is applicable to Ca2+-imaging (e.g. Fura-2, yellow cameleons), other ratiometric ion probes imaging (e.g. PBFI, SPFI, SNARF-1, BCECF), reactive oxygen species imaging (e.g. HyPer, roGFP), and intramolecular FRET sensor imaging.

    • Enter the channel numbers for the numerator and denominator channels for the ratio calculation, e.g. Fura340 and Fura380, respectively.
  • Measure intensities in single-cells in two channels with spectral unmixing

    This pipeline corrects for misalignment between channels on the frame-by-frame basis, subtracts background, performs spectral unmixing with pre-defined coefficients, and stabilizes the time lapse (registers frames in time). To eliminate background the “Median of pixels below percentile of max projection” with the percentile given at “Background level” is used.
    To determine spectral crossbleed coefficients use the Tools/Calculate Crossbleed Correction Factor main menu point, or the Math/Blind Spectral Unmix with NMF function. See more on the layout of the Spectral Unmix Coefficient Matrix in the description of Math/Spectral Unmix.
    Then ROIs are generated automatically using the nuclear stain as seeds, following the shape of the maximum intensity projection of the summed PMPI+TMRM image. Therefore if a cell moves during the experiment, the ROI will contain the cell for the whole duration of the recording. The calibration is not affected by the amount of background recorded in the ROI.

Adjustments
Output

The output is average ROI fluorescence intensity or ratio values for each time point. Average ratio is calculated as the ratio of average pixel intensities. Data can be recorded into Microsoft Excel, Graphpad Prism or simple tab separated text files: