Fluorescence microscopy image an

Interactive analysis protocols

Cell Counting and Colocalization

Count SA-BGAL positive cells using classical staining

This protocol describes how to count senescent cells based on fluorescence labeling of nuclei and senescence associated beta-galactosidase (SA-BGAL) staining absorbance. The provided pipeline segments an image of nuclear stains into single nuclei, and optical density (OD) images are calculated using blank and (optional) dark current images. The outputs are total and positive counts (above threshold OD), histogram, and overlay image showing staining in artificial coloring and nuclei.

Whole well (Seahorse) cell counting

Counts cells based on nuclear staining in single and tiled images. Typical uses are following cell growth and normalization of Seahorse respirometry data to total cell numbers in each the microplate well. For normalization of Seahorse OCR and ECAR data cell numbers can be directly copied and pasted into the Wave software from the Plate Ch1 worksheet of the Excel Data Window.

Intensity and Ratio Measurements

Measure fluorescence intensity or ratio in single frames or time courses

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

Basic fluorescence histometry using nuclear marker (1-3 labels)

Measurement of fluorescence intensities over nuclei or perinuclear areas using a nuclear marker and 1-3 label channels. The intensities of the label channels will be measured, corresponding to the area of the nucleus or a perinuclear ring. The pipeline can run in single or tiled images and in whole wells with automatic suppression of outside of the well areas and debris (see “Suppress debris and outside of well areas”). Different measurements may be done in the same label channel. Multiple background subtraction options are uniformly applied to all channels. This protocol describes the basic version of the histometry pipeline with limited options for configuration for simplicity. See advanced versions in the same Pipelines sub-folder (sub-menu) for more options.

Mitochondrial membrane potential assay - worked example

Mitochondrial membrane potential (ΔψM) is calculated as absolute millivolt values, unbiased by plasma membrane potential (ΔψP), cell size and geometry, autofluorescence and binding. This is based on time lapse fluorescence imaging of adherent cell cultures with two common fluorescence probes. Here the analysis of recorded fluorescence traces is described. This analysis performs all image and data processing tasks required for the calibration of a TMRM / FLIPR (tetramethylrhodamine methyl ester and FLIPR plasma membrane potential assay kit from Molecular Devices; aka PMPI, Plasma Membrane Potential Indicator) to millivolts. The pipeline corrects for misalignment between channels on the frame-by-frame basis, subtracts background, performs spectral unmixing, 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. Then ROIs are generated automatically using the nuclear stain as seed, following the shape of the maximum intensity projection of the summed FLIPR+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. Prepared TMRM and FLIPR images with ROIs are passed to the Membrane Potential Calibration Wizard. For TMRM the complete calibration is described, thus all calibration parameters are calculated. For FLIPR the rate constant kP is assumed, therefore this protocol is more suitable for following slower changes (e.g. using above 10s acquisition intervals).

Calculation of the apparent activity coefficient ratio for the ΔψM assay

This assay measures the apparent affinity coefficient ratio of tetramethylrhodamine methyl ester (TMRM) that is a required parameter for the calculation of the absolute magnitude and time course of mitochondrial membrane potential. The apparent affinity coefficient ratio describes the binding of TMRM to mitochondrial membranes and the dilution of fluorescence by otherwise light microscopically invisible ultrastructural details of mitochondria (e.g. crista density). This is a confocal microscopic assay. To calculate the apparent affinity coefficient ratio, confocal microscopic fluorescence time lapses are recorded to follow the leakage of TMRM from the cells as mitochondria are being completely depolarized immediately before the start of the recording.

Calculation of spectral unmixing coefficients for the ΔψM assay

Recordings with tetramethylrhodamine methyl ester (TMRM) and FLIPR (aka PMPI, plasma membrane potential indicator; FLIPR Membrane Potential Probe from Molecular Devices) will be contaminated with fluorescence crossbleed, more or less, depending on the actual optical setup of the microscope. Mostly FLIPR fluorescence contaminates the TMRM signal. Spectral crossbleed can be calculated most precisely using single-fluorophore stained samples. This means two samples, one incubated with TMRM in basal condition, and an other incubated with FLIPR and then with CDC (or PM-PFA; see the ΔψM assay).

Morphological Measurements

Mitochondria:cell volume fractionator

This assay quantifies mitochondria:cell volume fractions using a confocal microscope and image analysis in Image Analyst MKII. The assay was tuned to result similar volume fractions to electron microscopic stereological measurements when the specified optical configuration is used for image acquisition. The technology is based on segmentation and binarization of high-resolution confocal micrographs of a cytosolic and a mitochondrial stain.