title: A fluorescence digital image microscopy system for quantifying relative cell numbers in tissue culture plates.
abstract: 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 intensifier, and an 80386 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 (from 264 cells/well to 1.35 x 10(5) cells/well). DIM-SCAN is a versatile system for quantifying total or viable cell numbers in tissue culture plates over a wide dynamic range.
authors.affiliation: Department of Pediatrics, Childrens Hospital of Los Angeles, CA 90027, USA. cpreynol@hsc.usc.edu
authors.forename: R T
authors.initials: RT
authors.lastname: Proffitt
authors.forename: J V
authors.initials: JV
authors.lastname: Tran
authors.forename: C P
authors.initials: CP
authors.lastname: Reynolds
mesh_terms: D000465:Algorithms; D002452:Cell Count; D003201:Computers; D016208:Databases, Factual; D005453:Fluorescence; D006801:Humans; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D018715:Microscopy, Video; D012815:Signal Processing, Computer-Assisted / Q000295:instrumentation; D014407:Tumor Cells, Cultured