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Dear listserv,

24 seconds of fame ... for widefield brightfield and fluorescence 
microscopy (did not need confocal, lattice light sheet, temporal 
focusing or multiphotons). Sorry I cannot provide popcorn to each of you.

See bottom of this web page

http://www.cancerfrontline.org/sleeping-beauty-car-t-cells/

Cells: our favorite "clone4" Activating and Propagating Cells (AaPC's) - large, green fluorescent (most of them anyway).
CD19 CAR T-cells unlabeled (I'll add an NIRF tracking dye next time) (see the p.r. for explanation of CAR.
"Red is dead" (1 uM PI).
The 22 frames spanned 3.5 days of a 6 day experiment.
Leica DMI6000 inverted fluorescence microscope
PECON shroud and stage incubators (37 C).
Lumencor SOLA (v1.0) 6-LED lamp ... operated in single LED mode (see thanks below).
ASI 8 position exciter filter wheel
Chroma and Semrock filters in the wheel (narrow excitation)
Leica and Semrock filter cubes
Hamamatsu FLASH4.0 (v1.0)
Original video was from center 1024x1024 pixels, Microsoft Video 1 codec (75%Q)(right click online to see online format).
MetaMorph Premier 7.8.9, Multi-dimensional acquisition, saved locally.



My thanks to:
Laurence Cooper - PI.
Tiejuan Mi and Harjeet Singh for cells.
Rest of Cooper lab&  Lee lab for putting up with me.
Leica folks in Texas for support.
MetaMorph support for working through some crash issues (solved by DCAM-API 14.12 - Hopefully this driver does not have new bugs).
SOLA single LED through MetaMorph my thanks to Tom DiMatteo for exhibiting at ABRF 2014 and telling me about this v1.0 feature. We now have one of his CaliCubes, http://www.epitechnology.com/contact-1/ and if I was not busy making movies of serial killers and single molecule FISH -- http://works.bepress.com/gmcnamara/61/ and FISH Z-series/ GPU deconvolution pages at http://works.bepress.com/gmcnamara/subject_areas.html -- I might thoroughly test the hardware.
Also my thanks to MetaMorph marketing for their "Amazing Image" program from a couple of years ago that netted me a personal license of MetaMorph - later upgraded with Review Multi-Dimensional Data -- used in the analysis of the project -- from an "amazing software incentive - see https://mdc.custhelp.com/euf/assets/content/MetaMatters%20vol%202_iss6.pdf (also my TAM series in issues 3-6 that year).

enjoy,

George

-- 



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42