Why using a monochromator in a microplate reader?

Microplate readers are used in a broad range of methods in many scientific fields. Some methods, such as luminescence, do not require any means of wavelength selection, because all photons are being bundled together to calculate the luminescence of the sample. However, most methods require the ability to limit the measured wavelengths to a narrow bandwidth: e.g. measuring the absorption around the absorption maximum of the substance of interest, exciting only a single fluorophore or separating the emission of different reporter proteins.

Traditionally, filters were used to select the desired wavelength: they are affordable, easy to use and offer high sensitivity thanks to their high transmittance and blocking properties. However, they lack flexibility, since the use of different wavelengths requires the use of different filter sets, and they are not suitable for measuring the spectrum of a substance. Learn more about the principle and types of monochromators.

Monochromators are used in microplate readers mainly to measure absorbance and fluorescence intensity.

The most relevant monochromator specifications for microplate readers

Spectral range (wavelength range)

Many microplate readers on the market cover an absorption range from 230 nm to 1000 nm. With our systems you can use the monochromator up to 200 nm. When measuring fluorescence, many microplate readers can only measure up to 700 or 740 nm. Our instruments can cover the range up to 850 nm.

 

Spectral Bandwidth

The spectral bandwidth (also called spectral bandpass) is defined as the width at the points where the light has reached half the maximum value (full width at half maximum, abbreviated FWHM). The slit width determines the spectral bandwidth: the wider the slit width, the narrower the spectral bandwidth. Many microplate readers on the market have a fixed spectral bandwidth. However, our microplate readers, which are equipped with a monochromator, all have a variable bandwidth: from 4 nm (or 6, depending on the device) to 22 nm. A narrower bandwidth improves resolution and is recommended for fluorescence when excitation and emission peaks are very close together. A wider bandwidth improves the signal to noise ratio. A monochromator with variable spectral bandwidth is very useful for optimizing tricky assays.

Blocking efficiency

Blocking efficiency is the ability of a wavelength selection system to block unwanted wavelengths (other than the range selected in the monochromator), and is critical to achieve a good signal-to-noise ratio. It is expressed as the fraction of unwanted light which exits the monochromator: a blocking efficiency of 10-3 means that 1/1000 of the light of unwanted wavelengths is not blocked and exits the monochromator. Most microplate readers use a double monochromator configuration to achieve a blocking efficiency of 10-6, which is required for most fluorescence intensity assays.

Stray Light

Stray light is the measured amount of light that exits the monochromator at a wavelength different from the selected wavelength. It is radiation that is the result of imperfections in the dispersing element or other optical surfaces, diffraction effects, other optical aberrations or damaged and worn components.

In absorption measurements, scattered light causes deviations from Lambert-Beer's law. At high absorption values, the linear relationship between absorption and concentration is strongly influenced by the factor of scattered light. It introduces a systematic bias towards lower absorptions at increasing concentrations. Stray light is also the primary influencing parameter for the upper limit of the linear dynamic range for an analysis and will also cause problems in fluorescence measurements.

Stray light is expressed as a fraction of the light exiting the monochromator: an index of 10-4 means that 1/10000 of the light exiting the monochromator is stray light.

While most microplate readers in the market have a stray light index between 3 x 10-4 and 5 x 10-4, instruments manufactured by Berthold Technologies feature an excellent index of 10-6, that is, at least 20 times better, providing top reliability in all monochromator-based measurements.

Spectral Resolution

Resolution is the minimum bandpass which can be set in the monochromator; once the monochromator elements and their positions are fixed, the resolution is determined by the minimum slit width. Resolution is critical to accurately determine the spectrum of a sample. Originally sharp spectral peaks broaden when measured at low resolution, and may even disappear using a broad slit width; a narrow slit achieves a spectrum shape closer to the original spectrum. On Figure 1 you can see an example of the impact of resolution in a spectral scan: with a resolution of 4 nm, the 3 peaks around 500 nm look flatter that at 1 nm, but they are still easily distinguishable; with a 15 nm resolution, however, they appear fused as a single peak, resulting in a loss of information.

The monochromators used in our microplate readers have a spectral resolution of 4 or 6 nm (depending on the device), while many competitors only have a fluorescence resolution of 9, 15 or even 20 nm.

Figure 1: Effect of spectral resolution on a spectrum scan

When should you measure with filters and when using a monochromator?

This depends on the particular assay. For some measurements, e.g. Fluorescence Polarization, filters must be used because the polarization in a monochromator cannot be easily adjusted. In other cases, such as BRET, the required sensitivity will be difficult to achieve with monochromators.

In general, measurements made with filters provide higher sensitivity than the same measurements made with a monochromator in a comparable instrument. In many cases, however, the sensitivity offered by the monochromator is good enough, and the greater flexibility and convenience is a welcome plus.

Monochromator-based microplate readers from Berthold Technologies offer the best of both worlds: You are free to choose between filters and monochromator, providing the option that offers the best performance for your assay.

Our Microplate Readers with monochromator

We offer 2 different instruments with monochromator: the TriStar² S LB 942 has one double monochromator for absorbance and fluorescence excitation, and the Mithras² LB 943 has two double monochromators, one for absorbance and fluorescence excitation, and one for emission.