Fluorescence microscopy image analysis
automation - time series - physiology

Fluorescence ratio measurement

Complete analysis of a fura-2 calcium recording in rat cortical neurons.

Key features:

  • Image registration to fix movement of the specimen during pipetting
  • Automatic background subtraction
  • Automatic single-cell ROI drawing
  • True ratio image and ratio calculation from ROI means
  • Ratio values are directly transferred to Excel

Run time: 4 min 00 s.


H2O2 assay using normalized DCFDA rates

Complete analysis of intracellular H2O2 levels using normalized rate measurement of DCFDA fluorescence in C2C2 cells.

Key features:

  • Image registration to fix movement of the specimen due to stage drift
  • Automatic background subtraction and masking
  • Normalization of intensities in plot windows
  • Rate calculation
  • Automation of analysis for microplates
  • Rates are directly transferred to Excel

Run time: 3 min 35 s.

Protocol

Whole well cell counting

This tutorial video demonstrates how to perform automated cell counting in images recorded in microplates showing a nuclear stain. This particular recording was used for normalizing Seahorse cell respirometry data to cell counts.

Key features:

  • Inhomogeneous background related to debris or tiling pattern is removed
  • No constraint on maximal number of cells
  • Results are directly transferred to Excel

Run time: 3 min 36 s.

Pipeline

Unbiased and absolute calibration of ΔψM in millivolts

This tutorial video demonstrates how to work out millivolt values of the mitochondrial and plasma membrane potentials in intact adherent cells.  Automated image and data analysis of recordings using TMRM and the FLIPR membrane potential assay kit* (aka PMPI) is demonstrated using pipelines and the Membrane Potential Calibration Wizard. The example data relates to our recent publication (26), and the image files are available for download here. See corresponding protocol here.
Key features:

  • The menu-activated pipeline performs all required image processing: background subtraction, image registration, spectral unmixing and automatic ROI drawing.
  • Basic usage of the Membrane Potential Calibration Wizard allows point-and-click entering the frame ranges of calibrant additions, and converts each fluorescence trace into a millivolt trace.
  • Advanced usage of the Membrane Potential Calibration Wizard allows setting up quality control based on estimated error of calibration.

Run time: 11 min 40 s.

 *The FLIPR membrane potential assay kit is from Molecular Devices.

Protocol