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January 15, 2014 (Vol. 34, No. 2)

Improving upon Monochromator Technology

System Measures Fluorescent Proteins as well as FRET and BRET Assays

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    Figure 1. Simplified schematic of one of the LVF Monochromators found in the CLARIOstar microplate reader. Linear variable filters allow white light to be filtered into definable wavelengths and bandwidths up to100 nm.

    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).

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    Figure 2. mTFP1-YFP tandem FRET signal in HEK293 cells as measured on a confocal microscope and on the CLARIOstar microplate reader using LVF Monochromators. When similar wavelengths to ones optimized on the confocal microscope are used, a similar percent change is seen in the donor-to-acceptor ratio when using LVF Monochromators.

    In addition, a linear variable dichroic mirror (not shown) improves the spectral separation between excitation and emission light. When properly aligned, linear variable filters separate light not only into definable wavelengths, but also into bandwidths up to 100 nm wide. Having adjustable bandwidths up to 100 nm, LVF Monochromators can easily perform assays that used to require filters.

    Besides having wider bandwidths, LVF Monochromators attain greater light transmission for two additional reasons. First, the design of the LVF Monochromator is based on transmission and interference, not on diffraction, like grating-based monochromators. Second the transmission-based design allows an optical system free of inefficient fiber optic guides, which typically lose a significant amount of light.

    With the LVF Monochromator, the signal is guided to and from the microplate by a direct optical path through an adjustable dichroic mirror. For these two reasons alone, LVF Monochromators transmit several times more light than conventional, grating-based monochromators.

    One of the most challenging needs for researchers is to adapt their microscope-based fluorescent protein assay to a microplate format. Now with LVF Monochromators, similar wavelengths and bandwidths used on the microscope can be used on a CLARIOstar microplate reader, making it a seamless transition from one instrument to the other.

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    Figure 3. BRET assay measuring the arginine vasopressin receptor response. The BRET ratio is determined from dividing the two emission wavelengths (460–60 and 660–100 nm). One reason LVF Monochromators outperform conventional monochromators is that they have adjustable bandpasses up to 100 nm.

    As an example, Figure 2 shows a FRET response in HEK293 cells between mTFP1 and YFP fluorescent proteins as measured on a confocal microscope and on the CLARIOstar. FRET assays require two different emission signals to be measured after excitation. For the microscope, bandwidths of 30 nm and 45 nm were used for the two emission wavelengths3, which were the starting points for CLARIOstar’s LVF Monochromators.

    Further optimization shows that the same percent change can be seen in the FRET ratios on the confocal microscope and on the plate reader, making it an almost seamless transition to a higher-throughput method.

    Another example of how LVF Monochromators have filter-like performance is with bioluminescence resonance energy transfer or BRET assays. Like FRET, BRET applications require the measurement of two emission signals, but usually with even wider bandwidths (60 to 100 nm). Therefore BRET applications rarely work, if at all, on instruments with conventional monochromators that have fixed or narrow bandwidths. However, the CLARIOstar’s LVF Monochromators measure BRET assays with filter-like performance (Figure 3).

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