UC San Diego
Automation in image cytometry : continuous HCS and kinetic image cytometry
- Author(s): Charlot, David J.
- et al.
Image based high content analysis (HCA) and high content screens (HCS) are powerful tools used predominately in the study of cellular and molecular dynamics, function, and structure and for cDNA, RNAi and compound library screens. Analyses and screens based on fully automated image cytometry create a vast wealth of information in a hands free, unbiased manner. Large-scale screens of tens of thousands to millions of compounds and potential clinical diagnostic applications would benefit from increased image acquisition speeds. Section I of this dissertation (Chapters 1-6) discusses the development of an HTHCS instrument that uses TDI image capture and dynamic autofocus. Current instruments typically scan at peak speeds of 20,000 to 50,000 wells/day (1-4 image(s)/well), whereas many define "high throughput" screening as > 100, 000 wells/day. Progress is reported about routine continuous scanning with time-delay-and-integrate (TDI) 3- color fluorescence imaging at ̃70,000 wells/day (20 x 0.75 NA Nikon objective, 384-well plate, 8-10 usable images/ well depending on wall thickness and edge effects). Image cytometry quality (e.g., precision, accuracy and dynamic range of measurements) is directly linked to the contrast and resolution (detail) available in each image, and is thus dependent on the quality of autofocus. The system was validated using a synthetic assay and compared with the Perkin Elmer Opera using an assay screened by the Sanford- Burnham Center for Chemical Genomics (Chapter 4). Section II of this dissertation discusses the development of an HCS instrument capable of automatically stimulating, monitoring, and analyzing kinetic activity in cells. The Kinetic Image Cytometer or KIC will electrically stimulate (0r pace) the cells, record the resulting Ca⁺⁺ transients from cells in microtiter plates (e.g., with 96 wells), and automatically quantify characteristics such as the duration of the Ca⁺⁺ waves on a cell-by-cell basis in a fully automated manner on large scale screens. Chapters 1 and 2 are a summation of research to develop the technology using cardiomyocytes as the control model. Chapters 3 and 4 are a summation of research to use the KIC to perform dose response assays of different sources, including hESC--derived cardiomyocytes (hESC--CMs) and hiPSC--derived cardiomyocytes (hiPSC--CMs)