When choosing an instrument such as a microplate reader that needs to filter a broadband excitation source into monochromatic wavelengths of light, there were always two options: Optical filters that use thin layer interference coatings on glass to separate light into a fixed wavelength, or monochromators that use diffracting grating prisms to separate light into variable wavelengths.
Optical filters have always offered higher performance over monochromators for two main reasons—greater light transmission and wider bandwidths—whereas monochromators offer greater flexibility; no new filters or filter cubes have to be bought or installed.
Still though, users say there are many assays that do not perform well, or at all, with monochromators; as a result, filters are needed.1 For instance, fluorescent proteins2 like GFP, mKate, mOrange, and CFP-YFP are reported not to perform well, or at all, on monochromator-based microplate readers. The same holds true for FRET and BRET assays.
One obvious reason these assays do not work with monochromators is that wider bandwidths greater than 30 nm are needed. In fact, some BRET assays require an emission bandpass of 60 to 100 nm. Current conventional monochromators have only fixed or adjustable bandwidths up to 30 nm.
Another reason these assays do not work with monochromators is because their signal is weak, emitting significantly less light than common dyes like FITC or rhodamine. Using gratings and fiber optics, monochromators transmit much lower amounts of light compared to filters. In addition, since doubling the bandpass quadruples the amount of transmitted light (true for double monochromators), having bandwidths of less than 30 nm further limits conventional monochromators.
Realizing the need for a more sensitive, broader bandwidth monochromator, BMG LABTECH’s German engineers have developed dual monochromators in a microplate reader. LVF Monochromators™, which can be found in the CLARIOstar® multimode microplate reader, consist of linear variable filters (Figure 1).