Monochromator or Filter-Based Plate Reader? How to Choose

Microplate readers can make quick work of sample analysis across multi-well plates by quantifying key signals like fluorescence, absorbance, luminescence, and turbidimetry. Not all plate readers carry the same features, however, and it pays to know how your lab’s needs intersect with current technologies.

One of the most common questions researchers have when buying is whether to choose a monochromator- or filter-based plate reader.

This guide breaks down the optical mechanisms behind monochromators and filters, along with the pros and cons of each option and why you might choose one over the other.

What are monochromators and filters, and how are they used in microplate readers?

Microplate readers work by selectively emitting or detecting ranges of light. To select the relevant wavelength(s) for analysis, today’s plate readers use either monochromators or optical filters.

Monochromator plate readers

A monochromator – with a name rooted in the Greek words for “single color” – is a device that can mechanically isolate individual wavelengths from a beam of light. Microplate readers containing monochromators can be set to emit or detect light at any specific wavelength within their range for absorbance and emission measurements of samples.

Plate reader monochromators function by diffusing light through a diffraction grating, using a series of mirrors to precisely separate the instrument’s light source beam into single-color components. The monochromator then passes only the selected wavelength of light through to the sample. From there, the sample emits its own light back through the system of selective diffraction grating and mirrors so that, again, only a single wavelength reaches the detector for signal reading.

Filter-based plate readers

Filters are optical devices that only allow certain wavelengths of light to pass through them. Filter-based microplate readers have both excitation filters, which filter light going from instrument to sample, and emission filters, which filter light going from sample to detector.

The wavelength range that a filter can transmit is referred to as the filter’s bandwidth. Light outside of a filter’s bandwidth cannot pass through.

Figure 1. Diagrams of the optics in filter-based and monochromator-based plate readers. (A) In a monochromator-based plate reader, light from the instrument’s light source is diffused through excitation monochromators containing a diffraction grating and series of mirrors to isolate a single wavelength for sample illumination. Light emitting from the sample is diffused back through emission monochromators to ensure that only a specific wavelength reaches the detector. (B) In a filter-based plate reader, light from the instrument’s light source passes through an excitation filter so that only a certain wavelength range illuminates the sample. Light emitting from the sample then passes through an emission filter, ensuring that a specific wavelength range reaches the detector.

What is the difference between filter-based and monochromator-based plate readers? Which is better?

There is no one “right” option for plate readers, and the best choice will depend on your lab’s needs.

In general, microplate readers with monochromators offer the flexibility to select any wavelength and are ideal for running diverse assays or full spectral scans. Filter-based microplate readers are an affordable and robust option for customers performing routine assays.

Summary of key benefits and uses of monochromator-based vs. filter-based microplate readers

Pros of monochromator-based plate readers

Increased flexibility—Monochromators enable selection of any wavelength of light within the instrument range without the need to change physical filters. Monochromator-based microplate readers do not require hardware changes or updating to run new assays or detect fluorophores with diverse spectra. This tunable wavelength selection allows flexibility for labs performing a diverse array of assays and experiments that require different wavelengths for detection. With monochromators, labs do not need to purchase additional filters to analyze different fluorophores and assays, making monochromator-based instruments more adaptable to future laboratory needs.
Precise wavelength selection—Monochromators enable fine-tuning of the wavelength to meet the specific requirements of a dye or experiment, which is critical for developing custom assays and optimizing assay performance. This feature is particularly beneficial when working with fluorophores that have nonstandard spectral characteristics such as close excitation and emission peaks or small Stokes shifts (for instance, GFP and other fluorescent proteins, see Figure 2).
Full spectral scanning—Monochromators offer the ability to scan all wavelengths in the instrument’s range, enabling reading of the entire absorbance or fluorescence spectra. Spectral scanning is useful in characterizing and determining absorbance and emission peaks for an assay or fluorophore when the full spectra is not known, or in assays with spectral shifts.
Reduced spectral cross talk—Because they can detect all wavelengths and have a narrow measurement bandwidth, monochromator-based microplate readers allow better spectral resolution between close wavelengths, resulting in reduced cross talk and enhanced sensitivity for assays and dyes with overlapping spectra.

Cons of monochromator-based plate readers

Increased cost and maintenance—Adjustment of the position of the diffraction grating and movement of mirrors that allow for wavelength selection in monochromator-based microplate readers requires more complex mechanics, which increases cost and can make these plate readers more difficult to maintain. Monochromators also require more powerful light sources, which can add to their expense.
Less sensitive with luminescence and time-resolved fluorescence—Microplate readers that use monochromators are not as efficient in detecting light emission and are not optimal for luminescence and time-resolved fluorescence (TRF) assays.

Pros of filter-based plate readers

Lower cost—Because filter-based microplate readers have fewer moving parts, they are less expensive and require less maintenance than microplate readers with monochromators. Filters also transmit light more efficiently and do not need a strong light source, which further reduces their cost.
Excellent for routine assays—Filter-based microplate readers are a cost-effective and simple option for customers performing routine assays with standard dyes and fluorophores.
Higher sensitivity in certain applications—Filters allow a specific wavelength range or bandwidth of light to pass through and thus are more efficient at transmitting light than monochromators, which only transmit a fraction of light to and from the sample. This efficiency in light transmission for filter-based microplate readers increases sensitivity and results in lower detection limits in luminometric and TRF assays. Filter-based plate readers are necessary for assays that require larger bandwidths of light for high sensitivity, such as AlphaScreen assays.

Cons of filter-based plate readers

Less flexibility—Filters have fixed wavelength ranges, so the selected filter must match the wavelength requirements of the fluorophore or assay. This limits the flexibility of filter-based plate readers to run assays that do not match the wavelength range of the existing filters. In this case, labs may need to acquire new filters.
Lack of spectral scanning—Filter-based microplate readers cannot be used to run full spectral scans to determine absorbance and emission spectra and peaks for novel dyes or to characterize new assays.
Lower spectral resolution—Filters have a relatively wide wavelength range and allow a broad spectrum of light to pass through. This makes it difficult to distinguish between different wavelengths within the filter’s bandwidth. Due to their broader wavelength range, filters are less effective at differentiating between fluorophores with close spectral overlap or detecting fluorophores with small Stokes shifts than monochromators, which allow fine tuning of specific wavelengths by selectively isolating a narrow band of light for illumination and detection (see Figure 2).
Less adaptable—Microplate readers with filter-based detection are less adaptable to future laboratory needs since new filters may need to be purchased to run assays with different wavelength requirements.

Figure 2. Comparison of fitting of double monochromator and filter optics onto fluorophore spectra. (A) Double monochromator optics have triangle-shaped transmission spectra with very steep slopes before and after the peak transmission wavelength. This allows the wavelengths for excitation and emission measurement to be set close to the peak fluorophore wavelengths, even for fluorophores with small Stokes shifts (distance between the excitation and emission peaks). (B) Filters typically have bell-shaped transmission spectra that are relatively wide at low transmission range. Therefore, a longer distance is required between excitation and emission filters compared to double monochromators. This often makes it difficult to excite and detect fluorophores at their peak wavelengths, since the excitation or emission filter (or both) must be selected outside of the fluorophore peak values, resulting in suboptimal excitation or emission detection.

Thermo Scientific microplate reader options

Thermo Scientific offers microplate readers with both monochromator- and filter-based wavelength selection capabilities. For plate readers suitable for absorbance assays, the Multiskan FC Microplate Photometer uses filters to select wavelengths for absorbance measurements, while the Multiskan SkyHigh Microplate Spectrophotometer is a UV/Vis microplate spectrophotometer that uses a monochromator for wavelength selection. The Varioskan ALF and Varioskan LUX Multimode Microplate Readers have both filter- and monochromator-based optics for wavelength selection depending on which detection technology is used.

» Find the best Thermo Scientific microplate reader for your lab

Depending on laboratory needs, a filter-based microplate reader may be sufficient or a microplate reader with the added flexibility of a monochromator may be the best choice. Hybrid plate readers such as the Varioskan ALF and Varioskan LUX Multimode Microplate Readers use both filters and monochromators for wavelength selection, offering another option for labs that may need filter-based detection for certain assays or applications, and monochromator-based detection for others.

Learn more about microplate readers»

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