January 15, 2008 (Vol. 28, No. 2)
Paul Held Ph.D. Lab Manager BioTek Instruments
Synergy 4 Combines the Features of Filter- and Monochromator-based Systems
As assays and detection technologies have advanced in microplate format, space-saving and user-friendly multimode (or multidetection) readers have developed that allow researchers to perform multiple assay types in one instrument.
These readers are typically built with a focus on fluorescent detection as it is the most diverse, complex, and common read mode used in biotechnology applications. In fact, it is estimated that over one-half of multimode readers are used for some form of fluorescent detection, including fluorescent intensity, fluorescence resonance energy transfer, fluorescence polarization, and more.
Scientists incorporating multimode microplate readers for fluorescent detection have found themselves choosing between two types of detection systems: filter-based readers and monochromator-based readers.
It is estimated that in the 2007 global market, up to 67% of multimode microplate readers in laboratories depend mainly on filter-based technology, while 19% use monochromator-based technology, and 13% use both types. Looking forward, these ratios are expected to shift as researchers incorporate more assay platforms into their work, with the combined use of both reader types more than doubling from 13% to 32% in the next two years.
Filter-based Multimode Microplate Reader Technology
Filter-based multimode microplate readers (Figure 1a) usually incorporate several sets of optical filters. White light is passed through a filter on the excitation side, which typically transmits at least 60% of the desired wavelength in the visible range to the sample well.
The light excites the sample, which in return emits a specific fluorescent signal according to its unique properties. An emission filter, coupled with a dichroic mirror in some cases, cleans the sample’s signal and typically transmits at least 60% of the desired wavelength to the detector. The high transmission efficiency on both the excitation and emission channels provides high sensitivity.
There are several advantages of filter-based readers when compared to monochromator-based microplate readers. First, filter-based readers are typically less expensive than monochromator-based microplate readers. Filter wheels or slides are less expensive components than monochromators, and the light source required to produce the same level of sensitivity does not need to be as powerful.
The second important advantage is that of sensitivity; a filter-based reader is more effective at delivering light to the sample and light blocking between the excitation and emission channels for superior sensitivity. Bandwidth selection is an advantage as a filter can be dedicated to a specific assay for maximum sensitivity and can have a bandwidth from a few nanometers to greater than 100 nm, which is necessary for low-level fluorescence assays.
Finally, filter-based microplate readers can rapidly switch between two wavelengths, or be designed with two measurement channels, for ratiometric-based assays while monochromator-based systems are typically much slower.
A disadvantage of filter-based microplate readers is that separate filter sets must be purchased and maintained for separate applications. Accordingly, the fixed wavelength of a filter also disallows the use of spectral scanning applications.
Monochromator-based Multimode Microplate Reader Technology
Monochromator-based microplate readers (Figure 1b) use diffraction gratings instead of filters to deliver a wavelength to the sample. White light is reflected by one or more diffraction gratings on the excitation side. Each grating has an average efficiency of about 40% at the desired wavelength, so the total efficiency of the two gratings is about 16%.
Similar to the process in filter-based readers, the light excites the sample in a microplate well, which in turn emits a specific fluorescent signal. An emission monochromator cleans the signal through another diffraction grating or set of diffraction gratings, with a combined efficiency of approximately 16%, before sending the signal to a detector. The combined excitation/emission efficiency of this system is much lower compared to the efficiency of a filter-based design.
A major advantage of monochromator-based readers is their high degree of flexibility. Wavelengths can be conveniently selected through the software, so manipulation of the system and storage of additional accessories are not required. This also means that new applications can be accommodated with relative ease and low additional cost. Additionally, these systems can run spectral scans that are used to study spectral shifts and profiles, or to characterize new fluorophores, although they exhibit higher background noise than comparable cuvette-based readers.
The disadvantages of monochromator-based microplate readers include sensitivity limitations and overall cost. The inherent design of the diffraction gratings causes significant loss of light both to and from the sample, which decreases overall sensitivity. Another disadvantage is the higher cost associated with the units, especially when compared to the performance in common applications.
Detecting a Market Need
Although the aforementioned designs have specific advantages and disadvantages, a gap has existed for those requiring the benefits of both technologies (Figure 2) without the related expense of two separate microplate readers.
In fact, high sensitivity and limits of detection on one hand, flexibility, and multiple read modes on the other hand are estimated as the most critical technical requirements influencing the purchase of a multimode microplate reader. These represent, not coincidentally, a combination of the best features in each separate technology.
Further strengthening an emergent need, up to 70% of researchers indicated an interest in a multimode microplate reader that incorporated both a filter-based and monochromator-based mode of operation.
The Hybrid Solution
The Synergy™ 4 Multi-Detection Microplate Reader from BioTek Instruments (www.biotek.com) was designed to bridge the gap between flexible monochromator-based readers and sensitive filter-based readers.
Both detection technologies (Figure 3) are combined into one compact unit, each able to compensate for the weaknesses of the other so that true multidetection is achieved. Users benefit from high sensitivity and increased versatility, with the option to choose the detection system, including the use of both systems concurrently, that is best suited for their preferences.
In addition to the monochromator- and filter-based technologies, modular detection modes are available for UV-visible absorbance, luminescence, fluorescence intensity, time-resolved fluorescence, and fluorescence polarization. Users can choose the detection modes applicable for their needs and add onto the instrument as necessary. This combination of detection modes and features ensures that Synergy 4 can satisfy current microplate assay detection needs, and as user assay requirements change, this unit will seamlessly support their evolution.