Hybrid Readers Eliminate the Need to Choose
Multi-mode microplate readers, where fluorescence detection is combined with other detection modes such as luminescence or absorbance, have evolved in response to demands for additional functionality without increased footprint or cost when compared to multiple single-mode microplate readers. The term multi-mode is vague, and can include as few as two detection modes, or up to six or more. Fluorescence-based assays are limited to those compatible with either monochromator-based or filter-based detection technologies as very few readers contain both technologies in one unit.
Monochromator-based fluorescent microplate readers use diffraction gratings to split white light into multiple wavelengths, and select the specific wavelength desired. This excitation light is directed towards a sample, which then emits a specific fluorescent signal. The emitted light signal is passed through an emission monochromator to remove stray light, and sent to a detector to quantify the fluorescent signal.
Monochromator-based readers are very flexible and simple to use, and can perform specialized reads such as spectral scans. A wide range of wavelengths may be selected without use of individual filters. Conversely, monochromator-based readers are usually less sensitive and more expensive than filter-based readers, and are often not compatible with advanced techniques such as time-resolved fluorescence (TRF), time-resolved Förster (fluorescence) resonance energy transfer (TR-FRET) and fluorescence polarization.
Filter-based fluorescent microplate readers use optical filters to select a specific wavelength from white light. These filters are wavelength-specific, and may be removed and stored while other filters are in use. Light is passed through a filter on the excitation side into the sample, and emitted light is passed through a second filter before it is sent to a detector to quantify the fluorescent signal.
Filters are highly effective at transmitting a predetermined bandwidth of light while effectively blocking undesired wavelength, providing maximum transmission on both excitation and emission channels. Filter-based fluorescent microplate readers are usually more sensitive and less expensive than monochromator-based readers, although filter purchases add to the overall cost. Ratiometric-based assays and those requiring rapid switching between two wavelengths are easily accomplished on filter-based systems which are an excellent choice for advanced techniques such as TRF, TR-FRET and FP. Disadvantages of filter-based systems are the use of separate filter sets for each wavelength required, and the inability to run spectral scans.
A Hybrid multi-mode microplate reader, such as the Synergy™ H4 and Synergy H1 (BioTek Instruments, Winooski, VT, USA), combines sensitive filter- and flexible monochromator-based fluorescent detection modes in one unit, and also offers additional non-fluorescent detection modes for broad application flexibility. The monochromator-based optics support luminescence, absorbance, and top and bottom fluorescence detection modes, while the filter-based optics, integrated in Synergy H4 and optional in Synergy H1, provide high sensitivity and enable TRF, fluorescence polarization and Bioluminescence Resonance Energy Transfer (BRET). Both systems are modular, so additional read modes may be added as needed.
Additionally, dual-reagent injection automates inject-and-read applications such as ion-channel or flash luminescence assays. Both are compatible with the Take3™ Multi-Volume Plate for low volume 2 μL microspot, BioCell™ or standard cuvette measurements, and both are controlled with Gen5™ Data Analysis Software for advanced reader control, data analysis, graphing and reporting features.
Filter-based and monochromator-based fluorescence detection each have distinct benefits and limitations, and by combining both technologies in one unit, researchers are no longer faced with choosing between the technologies. Hybrid combines both fluorescence technologies, and other detection modes, into one compact and efficient unit for endless versatility in assay choice.