Timelapse data - GM imaging cytometer needs stitching part 2 of 2
Imaging challenge: align multiple time series over time to overcome "stage slop" in the imaging cytometer used in this experiment.
http://works.bepress.com/gmcnamara/53 (previous) and http://works.bepress.com/gmcnamara/54 (this)
are zip files containing 8-bit stacks (MetaMorph multiplane TIFF files) acquired on an imaging cytometer.
"53" is the brightfield data, raw and automatic aligned stacks in MetaMorph (Apps menu, MM7.8.6). "54" is GFP fluorescence, raw and automatic aligned stacks in MetaMorph (Apps menu, MM7.8.6). The GFP fluorescence decreases over time due to photobleaching. GFP disappears from cells when they die (soluble GFP diffusing into the media).
The MetaMorph Align Stack command only operates on one image stack. While I could use "Stacks menu - Montage Stacks" to get two or more image series into a single stack, this is time consuming and tedious (and would not be very effective if there were rotation issues - fortunately, not expected here).
I have posted these files in the hopes that someone - whether MetaMorph, Fiji ImageJ, Bitplane Imaris, CellProfiler, FARSIGHT Toolkit, or other developers, take interest in this imaging challenge and come up with a fast, efficient way to register multiple stacks are closely as possible.
The best stack to work from is likely the brightfield data, since the GFP (in this case) photobleached and some cells died and went dim.
With respect to the imaging cytometer, it is not a bad instrument, just not designed for 'precision alignment' 20x objective lens timelapse imaging.
20x lens, 4 minute interval, mouse EL-4 cells, some expressing GFP (not shown: others expressing mCherry RFP). Acquired by Dr. George McNamara, Prof. L.J.N. Cooper lab, UT M.D. Anderson Cancer Center, Houston, TX.
My thanks to the vendor for loaning us their imaging cytometer. Ideally in the future, image alignment will be dealt with "up front".
My thanks also to Greiner Bio-One for half area 96-well plates.
- timelapse data,
- live cells movies,
- imaging cytometer,
- image registration
Available at: http://works.bepress.com/gmcnamara/54/