As drug discovery and development efforts continue to seek balance between throughput and translatability of in vitro studies to in vivo results, understanding the advantages and disadvantages of several optics-based endpoints- from plate reading to flow cytometry, to imaging- is ever more critical to striking that balance.

Traditionally, in vitro screening assays, particularly those aimed at high throughput screens have levered plate-reader based endpoints. Since a number of reagents have been built out into assay kits, containing detection reagents, buffers, standards, and sometimes even microwell plates, these assays are quick, relatively cheap, and easy to execute. For example, kits as well as well validated protocols are available for assays like Promega’s Cell TiterGlo (for the quantification of cellular ATP content), antibody-based assays such as ELISA (for the quantification of protein analytes from cell supernatants or lysed cell samples), or assays that rely on enzymatic functionality. Most platereaders can read a 96-well plate in a matter of seconds and offer the flexibility of being able to quickly and simply analyze absorbance, fluorescence, and luminescence.

While plate-reader based assays may be amenable to high throughput screening, where costs and assay time must be kept to a minimum, these assays most often cannot be easily multiplexed to assess several endpoints from the same sample and moreover do not offer single cell resolution. When an assay stipulates a need for these parameters (i.e., when determining cytotoxicity in cell subpopulations present within a sample), other approaches must be leveraged. While limited to fluorescence endpoints, flow cytometry can provide single cell resolution of any endpoint that can be indicated with a fluorescent dye, probe, or antibody. Able to acquire tens to hundreds of data points (i.e. single cells) per second, amenable to multiplexing (limited only by the laser/filter capacity of the flow cytometer), and in some cases pairable with 96-well microplate samplers, flow cytometry can accommodate assays of moderate throughput where single cell resolution is required.

However, larger samples such as spheroids or organoids are more challenging to assess via flow cytometry; not only can their large size clog the fluidics systems of many cytometers, but spatial distribution of markers of interest is lost. These samples may be best analyzed via more traditional microscopy approaches. In the interest of increasing throughput of these more traditional microscopy approaches, a number of platforms are currently available that offer the ability to image cells, spheroids, and even larger samples in a multi-well format (i.e. the Thermo Scientific CellInsight to the Molecular Devices ImageXpress, GE IN Cell, and PerkinElmer Opera Phenix). Moreover, most models offer the flexibility of imaging in either widefield or confocal imaging mode, such that throughput and image quality can be optimized. For example, many endpoints in thicker samples, such as 3D cell culture models may require confocal imaging to eliminate out-of-plate light, while simple monolayer cell cultures can be imaged using widefield mode. While widefield scanning enables faster scan times and thus facilitates more high throughput assays, combining tissue clearing (i.e. with Visikol® HISTO-M™) with confocal imaging of 3D tissues enables for unmatched resolution to facilitate single cell quantification and localization.

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