A fluorescence digital image microscopy system for quantifying relative cell numbers in tissue culture plates
✍ Scribed by Robert T. Proffitt; James V. Tran; Patrick Reynolds
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 916 KB
- Volume
- 24
- Category
- Article
- ISSN
- 0196-4763
No coin nor oath required. For personal study only.
✦ Synopsis
Microwell tissue culture plates provide a convenient format for conducting cell growth and cellular cytotoxicity assays, but such assays often have a limited dynamic range. We have developed the digital imaging microscopy scanning system (DIM-SCAN), which is a semiautomated fluorescence digital imaging system for quantifying relative cell numbers in situ in a variety of tissue culture plate formats, especially 96-well plates. The system consists of an epifluorescence inverted microscope with a motorized stage, video camera, image inten-sitZer, and an 80% microcomputer with a PC-Vision digitizer. Turbo Pascal software controls the stage and scans the plate taking multiple images per well, calculates total fluorescence per well, provides for daily calibration, and configures easily for a variety of tissue culture plate formats. Thresholding of digital images is used to remove background fluorescence without rinsing. Using cells pretreated with fluorescein diacetate (FDA) and loaded into 96-well plates, linearity was seen over 3.0 logs of cell density (r = 0.996), with similar results in 6-well (r = 0.972), 24-well (r = 0.970), and 48-well plates (r = 0.981). For cells stained directly in plates with Hoechst 33342, linearity was observed over 2.7 logs of cell density (r = 0.975). In low-viability cultures stained with FDA in 96-well plates, excessive background fluorescence completely masked viable cells, but digital thresholding dramatically reduced background fluorescence, producing a linear response (r = 9.333) over 2.7 logs of cell density @om 264 celldwell to 1.35 x lo5 celldwell). DIM-SCAN is a versatile system for quantiQiig total or viable cell numbers in tissue culture plates over a wide dynamic range. o 1996 wiley-Liss, ILK.