Timelapse imaging of T-cells and Nalm6 "target cells" in Trianja (www.Trianja.com) in a glass nanowell chip, face down on a coverglass inside a Mattek glass-bottom-dishes.com 36 mm imaging dish.
T-cells: unlabeled, visible by transmitted light (often change shape and crawl around).
Nalm6 cells: green fluorescence (GFP expressed throughout cell).
"Red is dead": Propidium iodide is included in the media to identify cell death by loss of plasma membrane integrity. The Nalm6 cells that die also have a decrease in green fluorescence (easier to tell in the single channel data than in the Overlay Images color merge).
Scale: small wells (on right) are 60x60 um; large wells on left are 120x120 um (not shown in this field are 86x86 um wells). Well depth (height, since chip is face down) is 60 um for all wells. Original data was 2048x2048 piel images. The data was zoomed to 25% before making the movie.
Entire chip is 6x6 mm and has several sections of these nanowells. Our lab had Trianja design and manufacture the chips. You are welcome to buy them from Trianja (caveat emptor with respect to your application).
Imaging system: Leica DMI6000B automated microscope, PECON live cell environmental chamber and scope shroud, Lumencor Sola LED fluorescence illuminator, ASI excitation filter wheel, Hamamatsu FLASH4.0 sCMOS camera (great camera!), MetaMorph (currently using 7.8.6). The MetaMorph Graphics-->Overlay Images command was used to generate this color merge. MetaMorph "Make Movie" (to AVI) was used to generate the original AVI movie (Microsoft Video 1 codec) which I then converted to "uncompressed" (no codec) with Apple QuickTime Pro (I could have saved uncompressed in MetaMorph but was at another computer when I decided to go with uncompressed).
Project: Prof. Laurence J.N. Cooper lab, M.D. Anderson Cancer Center. Cells from our colleague Natalya. We've also worked with Ana in our lab on her cells (and added more fluorescent colors to positively identify the T-cells). My thanks to Natalya, Ana, LC, and lab mates.
Related work has been published in collaboration with Prof. Navin Varadarajan's lab, University of Houston - see
I like the Trianja chip because it is glass (and thin and hydrophilic and 'practically sterile') providing excellent optical quality. Navin's chips are PDMS molds with 50x50x50 um nanowells, but ~1 mm thick PDMA "base' (may be possible to reduce this).
Note: I am deliberately not providing much detail of this experiment, so it is not going to be of much use to you (unless you talk or exchange emails with me).
Uncompressed AVI movie ~580 Megabytes (bepress upload limit is currently 800 Mb). Three channels
- Nalm6 cells,
- nanowell chip,
Available at: http://works.bepress.com/gmcnamara/51/