Many fluorescent protein cell-based assays (like GFP, CFP-YFP FRET, mCherry, mOrange, etc.) that work well on a confocal microscope, do not translate well to other instrumentation because there is a loss in sensitivity when moving away from the microscope. Until recently, microplate readers were in this category of instruments. When measuring a live, real-time cell-based assay on a microplate reader, it is necessary to read from the plate bottom since a microplate lid is used on top to prevent cell contamination and liquid evaporation. Most microplate readers use longer, more flexible fiber optics to reach the microplate bottom. Since fiber optics are inefficient in capturing all of the light emitted from cell-based assays, sensitivity is lost when measuring fluorescent proteins from the microplate bottom. Hence, assays that work on a microscope do not always work on a microplate reader.
To overcome this problem and to improve sensitivity in fluorescent protein cell-based assays, the PHERAstar FS microplate reader was designed with a unique, proprietary technology that is analogous to the microscope. This technology, Direct Optic Bottom Reading, uses a series of software controlled, motor-drive mirrors to focus light on the bottom of the microplate. Since the light travels in a free-air optical path to the plate bottom, and not through inefficient fiber optics, the bottom reading sensitivity on the PHERAstar FS is unmatched by any other microplate reader that uses fiber optics.
Thus, Direct Optic Bottom Reading will enable GFP and other fluorescent protein cellular assays to be measured on the PHERAstar FS that cannot be measured on other microplate readers. As an example, when GFP tagged BAE cells are read on the PHERAstar FS, there is a 300% higher overall signal-to-blank ratio when compared to a fiber optic based microplate reader (see figure). This 3-fold improvement in the assay window will allow experiments to be performed on the PHERAstar FS that are only possible on a microscope.
Click here to find out more about BMG LABTECH’s Direct Optic Bottom Reading.