LVF Monochromators in Microplate Readers
Monochromators are optical devices that are used to select a certain wavelength of light that can then be used for a specific analytical purpose. Monochromators appear in a many types of optical instrumentation related to spectroscopy such as spectrofluorometers and absorbance spectrometers. They’ve been sent into the ocean depths, to the outer reaches of the solar system, and, they have a very important role in the microplate reader on your laboratory bench.
If you’ve ever observed the rainbow pattern of light being diffracted by a piece of glass or a prism then you have observed a rudimentary component of a monochromator in action. Storm Thorgerson famously illustrated this phenomenon on Pink Floyd’s The Dark Side of the Moon, but couple that prism with a mask, or slit, to block out other portions of the spectrum and you have in essence the first monochromator ever used for basic scientific studies (Figure 1).
A more advanced monochromator design replaces the prism with a diffraction grating (D), adds focusing mirrors to the light path (C,E), and adjustable slits (B,F) so that the output light efficiency (G) is improved (image from DrBob, Wiki Commons, Figure 2). This type of monochromator is the familiar Czerny-Turner design and has been in use for many years in various configurations in all matter of optical equipment. It is the most common monochromator employed in microplate readers and generally must be used in pairs, two monochromators for excitation and two for emission, in order achieve acceptable performance with respect to sensitivity.
Monochromators are characterized in performance by a variety of metrics including wavelength range, spectral bandwidth, stray light level, and transfer efficiency. In a microplate reader the goal of the monochromator is to get as much light of a particular bandwidth to and from the sample, while rejecting other wavelengths, thereby improving the sensitivity of the instrument.
However, a lot of light is lost in classical monochromator designs, particularly so in microplate readers where four grating monochromators need to be employed and each monochromator reduces the total amount of light available for detection. That’s the reason the industry standard quadruple monochromator microplate reader designs are not as sensitive as filter-based microplate readers.
At BMG LABTECH we don’t use traditional monochromator designs in our CLARIOstar microplate reader because they cannot be made to perform to our standards. Instead, we use our own patented Linear Variable Filter (LVF) Monochromator (US Patent 9,733,124 B2). The LVF monochromator has superior characteristics when compared to grating based monochromators and provide higher light transmission, superior efficiency, adjustable bandwidths, and these advantages translate into a monochromator based microplate reader that is more sensitive than most filter based microplate readers.
The LVF monochromator is a conceptually simple and elegant design. At the core it is composed of two linear variable filters, a very special filter designed such that the transmission wavelength varies along the length of the filter. One of the LVFs is a long pass filter design, so that it passes light above a certain wavelength, and the other LVF is a short pass design that passes light below a certain wavelength. Each LVF forms one half of the transmitted light peak shape and by sliding the two LVFs back and forth in relation to one another the peak transmission wavelength and bandwidth can be adjusted to sub nanometer accuracy (Figure 3).
The LVF monochromator discriminates light very differently than traditional grating based monochromators found in other instruments. The transmission through the monochromator is based on filters, not light dispersion, and that leads to a high level of light throughput in the monochromator, meaning higher intensities of the light needed for a particular experiment. Since light is not dispersed in the monochromator by a grating the LVF monochromator does not suffer from stray light effects, can more accurately discriminate wavelengths, and therefore can produce a superior signal to noise ratio for higher sensitivity. The performance of the LVF monochromator allows the CLARIOstar to only need two monochromators, one for excitation and one for emission, further improving light throughput over quad monochromator grating designs.
The LVF monochromator has another significant advantage, adjustable bandwidths. Since the selected light output is determined by two LVFs these can be used to modify the transmitted bandpass of the light. Grating based monochromators can only vary the bandwidth by using different sized exit slits, or a variable slit, but the slit is far less efficient than a linear variable filter. The LVF monochromator has much better efficiency and can adjust bandwidths to anything between 8nm to 100nm.
Adjustable bandwidths with high light throughput is very important for microplate readers because it translates into higher sensitivity and much greater flexibility for assay development. An LVF monochromator is essentially like having your own filter design tool because you can select the exact wavelength and bandwidth needed to optimize the signal to noise ratio or Z’ in your particular assay. In the application below the bandwidths used for naphthofluorescein were adjusted to precise values of 42nm and 57nm that produced the best signal to blank ratio available (Figure 4). Wide bandpasses, for example bandpasses greater than 75nm, can be extremely useful for color based luminescence assays such as BRET making LVF monochromators uniquely suited for this demanding application.
The last, and very important component of the LVF monochromator is the LVF dichroic mirror. All high performance microplate readers use a dichroic to help discriminate between excitation and emission light inside the microplate reader. However, dichroic mirrors are for specific applications, like the 505nm long pass dichroic commonly used for fluorescein, therefore dozens of dichroics would be needed to handle all the applications used in a laboratory. The LVF monochromator system has a linear variable filter dichroic that is automatically tuned to be between the excitation and emission wavelengths you select, so it’s as if you had 100s of dichroics inside your microplate reader (Figure 5). The LVF dichroic further reduces background signals in the monochromator providing enhanced sensitivity.
BMG LABTECH’s patented LVF monochromators provide the CLARIOstar microplate reader with superior performance when compared to grating based double or quadruple monochromator systems commonly found in microplate readers. The LVF monochromators emphasize our commitment to innovation and performance in the microplate reader market and form one of our technology backbones that will continue to be incorporated into new microplate reader designs for the future.