Perinuclear optical density measurement (SDH activity)

Parameters:

Name	#	Type	Description
Brightfield channel to calculate OD (formazan)	1	integer	The first linked image window with matching channel number will be invoked.
Fluorescence channel for nuclear markers	2	integer	The first linked image window with matching channel number will be invoked.
Number of tiles in x	3	integer	The image consists of this number of equal sized tiles in x dimension.
Number of tiles in y	4	integer	The image consists of this number of equal sized tiles in y dimension.
Top OD threshold	5	real	Pixel value, percentile, or factor times Otsu optimal threshold value (typically 1)
Dark current intensity (if no reference image used)	6	real	Average pixel value in an unilluminated imaged. If a dark current reference image is present, pixel-by-pixel dark current values will be used instead of this uniform value to calculate OD.
Approximate nucleus diameter	7	integer	Volume in 3D, area in 2D. 0 for not checking
Debris cutoff (percentile)	8	real	This percentile of the image histogram sets the intensity value where the maximum of the Look Up Table (LUT) is scaled. Use -1 to override this with fixed value set below at "Max value".
Minimum cell fluorescence (%)	9	real	Pixel value, percentile, or factor times Otsu optimal threshold value (typically 1)
Ring width (pixels)	10	integer	Diameter of the structuring element in pixels

Description:

Measurement of average optical density (OD) in a bright field channel in rings around nuclei marked in a fluorescence channel. This pipeline has been used for end-point measurement of succinate dehydrogenase (SDH) activity by measuring the amount of developed formazan product. The average OD is calculated only around nuclei, specified by “Ring width”. This mitigates effect of varying cell density. Debris and bubbles may show much higher OD than the actual signal, this can be eliminated by the “Top OD threshold” value.

Input: 2 channels recording: a brightfield channel showing a light absorbing staining such as formazan or DAB, and a nuclei marker fluorescence image such as Hoechst or DAPI. Imaging is performed at low resolution, using 10-20x magnification. In addition, OD calculation requires a blank and a dark current image. These are cell-free areas of the sample, and an image with no light reaching the camera, respectively, using the same camera settings, in particular image size. Please set blank (and optionally dark current) reference images by loading blank and dark recordings and using the context menu of the image window. The blank may be recreated using the “Create BLANK reference image for multiwell plate using median” pipeline in microplate data with low density cultures without recording actual blank images. An average intensity value may be used instead of dark current image provided in “Dark current intensity”.

Output: Excel table showing an OD value and a cell count for each position (well).
Adjustments of pipeline parameters:
Set channel associations and number of tiles matching how images were recorded.
Set “Approximate nucleus diameter” and “Minimum cell fluorescence” to match the appearance of the nucleus marker image.

This pipeline was used in:
Brand MD, Goncalves RL, Orr AL, Vargas L, Gerencser AA, Borch Jensen M, Wang YT, Melov S, Turk CN, Matzen JT, Dardov VJ, Petrassi HM, Meeusen SL, Perevoshchikova IV, Jasper H, Brookes PS, Ainscow EK. Suppressors of Superoxide-H2O2 Production at Site IQ of Mitochondrial Complex I Protect against Stem Cell Hyperplasia and Ischemia-Reperfusion Injury. Cell Metab. 2016 Oct 11;24(4):582-592.