High-content analysis (HCA) describes the use of automated fluorescence microscopy and a wide array of fluorescent probes for the study of fixed or live cells, tissues, or even whole organisms, where quantitative phenotypic data is obtained from images. The cellular images provide rich multiparametric information concerning biological processes and their modulation by diseases, hence the use of the term high content.
When HCA applications call for screening chemical libraries in an effort to find compounds that induce or inhibit a desired phenotypic change, the process is typically more automated and uses microplates and automated liquid-handling devices to increase sample throughput, allowing libraries to be screened in a reasonable time frame.
While this additional level of automation is comparable to that used in high-throughput screening (HTS), the need to capture and process images for the multivariate data ensures that a greater time is required to execute a screening campaign. Typically HCA imagers require almost 10 minutes to process one 96-well microplate for one fluorophore generating a phenotypic response. Compared to HTS microplate readers that quantify light emission from the whole microplate well using PMT-based detection, these read times are at least 10x slower.
This problem is compounded for each additional fluorophore quantified. This attribute has typically limited the use of HCA to secondary screening of hits from a primary HTS screen, target discovery (i.e., RNAi screening) or lead optimization (i.e., toxicity) applications where the compound number screened is relatively small.
In addition, the data-storage requirements are arduous as the screening of a full 96-well plate for a single fluorophore will require about 200 MB storage space. The hard drives of typical computers used to drive these imagers are not compatible for HCS data storage, and dedicated external data storage devices in the 1–10 terabyte range are required.
Lastly, HCS instruments are typically cost prohibitive for many laboratories, especially for academic labs relying on government funding agencies. HCS instruments are typically reserved for industry or core facilities in universities.
BioTek Instruments’ new Cytation™3 Cell Imaging Multi-Mode Reader aims to remedy some of these issues in addition to providing a highly flexible microplate reader. The Cytation3 is built from BioTek’s Hybrid Technology™ consisting of dual optical paths, each using PMT-based detection, but using separate methods for wavelength selection. One optical path uses quadruple grating monochromator-based light dispersion for spectral scanning applications and wavelength selection flexibility; the other, filter cubes consisting of a dichroic mirror and both excitation and emission spectral filters providing rapid analysis and high sensitivity.
In addition to this established architecture, the Cytation3 incorporates a microscopy module that provides automated digital widefield fluorescence and brightfield microscopy (Figure 1). This microscopy module uses high-quality optical components including a 16-bit scientific grade CCD camera, LED, and spectral filter cubes that allow high sensitivity analysis using a wide selection of fluorescent probes including the family of fluorescent proteins.