Mitochondrial calcium measurement with highpass filtering

Parameters:

Name	#	Type	Description
Background Level (percentile)	1	real	Background is calculated as frame-by-frame median of those pixels that are the darker than this percentile of image histogram for the duration of the entire recording. This has no effect if rolling ball is used.
Filter file	2	string	Filter files can be saved by the Setup DFT Filter dialog.

Description:

Measurement of mitochondrial (and cytosolic) and cytosolic calcium concentration. The mitochondria-specific fluorescence of mitochondrial calcium indicators (rhod-2 or x-rhod-1) are determined by high pass filtering, with the same filtering as it was originally published by Gerencser and Adam-Vizi in Cell Calcium 2001 Nov;30(5):311-21. As a surrogate for cytosolic calcium, the fluorescence of the same dye over mitochondria-free areas, e.g. over nuclei is determined.

Input: Time series of rhod-2 or x-rhod-1 fluorescence in cells, captured at high magnification ~0.15 µm/pixel (typically using a 100x lens).
Output: Processed images for mitochondrial and cytosolic
ucleosolic calcium measurement.

The pipeline subtracts background (this affects only the cytosolic
ucleosolic measurement), and aligns (registers) the time series. A copy of the image is high pass filtered with absolute value calculation enabled. This copy is used for mitochondrial calcium measurement. The original image is used to determine cytosolic
ucleosolic calcium by ROI circling mitochondrion-free areas.

Protocol:
1. Adjust background level (this is important only for measuring cytosolic calcium). 5 for confluent, 10 for subconfluent, 20 for sparse cultures.
2. Process time series
3. On the image “Draw mitochondrial ROIs here” use area ROIs to mark perinuclear, mitochondrion-rich areas to measure mitochondrial fluorescence.
4. On the image “Draw nucleus ROIs here” use area ROIs to mark parts of nuclei that is not covered with mitochondria.
5. Use the Plotting/Plot function (or the Plot toolbar button) to plot intensities.
6. Use the context menu of plots to plot DF/F0 values. The number of timepoints averaged for a baseline can be set at Preferences/Misc/Normalization and DF/F0 Calculation.