Bandwidth of the wavelength selector (filters or monochromators) is an important consideration for researchers when purchasing a microplate reader. Described as the range of wavelengths from the monochromator that the microplate detects, bandwidth significantly impacts assay sensitivity and linearity. More specifically, spectral bandwidth—the width of the band when the light is at half the peak maximum—determines the amount of light that can pass through the filter. A narrow bandwidth correlates with high resolution, allowing researchers to specify and distinguish signals between fluorophores with very close wavelengths maximum. Alternatively, a wide bandwidth improves signal-to-noise ratio by allowing more light to excite a sample and reach the detector. This can be beneficial for experiments with limited sample amounts and weak signals.

Why variable bandwidth might be of preference

Essentially, researchers must work with a microplate reader that balances precision with sensitivity. While some microplate readers feature a fixed bandwidth monochromator, others offer a variable bandwidth option that grants researchers the versatility to fine-tune their instrument settings. With this technology, users can adjust the bandwidth to achieve both high resolution and high sensitivity for the assay. This is particularly beneficial for researchers who need flexibility with their R&D workflows. For instance, a variable bandwidth allows multiplexing such that researchers can simultaneously detect a multi-peak dye or multiple fluorophores in the same solution. It also offers a clear advantage for narrow peaks or small stock shifts fluorophores to achieve optimal sensitivity and increase the signal-to-noise ratio for experiments. For these reasons, there is a considerable shift toward variable bandwidth for specific applications such as fluorescence resonance energy transfer (FRET), time-resolved fluorescence TRF / TR-FRET (HTRF), and bioluminescence resonance energy transfer (BRET).

While a variable bandwidth monochromator may sound like a must-have microplate reader feature, it’s important to note that it comes with considerable challenges. Users switching to variable bandwidth must have the expertise to handle proper user input, adjustments, and calibration to ensure accurate wavelength selection. When used improperly, variable bandwidth can significantly disrupt quantitative outputs and jeopardize the results and validity of assays, especially for drug discovery studies.

Variable bandwidth can compromise signal, data analysis, and time

For researchers not fully aware of the correlation between monochromator bandwidth and signal, bandwidth adjustment can lead to inaccurate measurements. Imagine a bright red flower in a field full of vibrant-colored flowers. A narrow bandwidth is similar to focusing on the flower with a magnifying glass to minimize interference from the other colors. However, a sudden adjustment of the focus—while capturing all the other flowers in the field—overshadows the signal coming from the red flower. By the same logic, changing the monochromator bandwidth while running the same assay can alter results. To account for this change, users should optimize the instrument settings each and every time they change the monochromator bandwidth. Otherwise, they will keep generating highly variable signals from the same assay without understanding the reason behind it. Such optimizations are time-consuming and challenging if you don’t have the correct guidelines and experience.

Along with signal changes come downstream changes in microplate reader data, which heavily depends on the monochromator bandwidth and the specific fluorophore used. While varying the instrument bandwidth, the user is altering the amount of light reaching the sample or the detector. These have a direct impact on the assay limit of detection and linearity. Consequently, the signal detected can differ even for identical samples, making it difficult to compare quantitative data between different plates, and even between different readouts of the same plate. Furthermore, when working with samples with low concentration, even a slight decrease in bandwidth causes a loss of sensitivity and linearity, generating inaccurate plots. Similarly, widening the bandwidth during fluorescent assays might cause the instrument to detect unwanted fluorescence signal and reduce the signal-to-noise ratio, therefore increasing the detection limit.

The versatility of variable bandwidth is also overshadowed by the complexity of optimization. In particular, monochromators with variable bandwidth offer more than 200 excitation/emission (ex/em) combinations, and finding the ideal ex/em pair can be time-consuming. Researchers using multiple dyes would need to optimize for each dye, not to mention that even different concentrations of the same dye can have different ex/em pairs. If the instrument is used by multiple users, one also must verify that their original assay settings have not been modified by the previous user. Overall, researchers might find themselves spending hours just optimizing instrument settings. Impractical optimization eventually sacrifices standardized quantification, which is crucial for drug development and assessment pipelines.

Reconsidering fixed-bandwidth monochromator microplate readers with filter options

Are variable bandwidth monochromators a revolution in the microplate reader world? It’s important to not disregard the versatility gained in R&D labs where assay optimization is part of daily work and users fully understand the impact of variable bandwidth on their data. Yet, it’s critical to consider that variable bandwidth can become a nuisance when used improperly and is arguably unnecessary for most researchers. That said, what solutions are available today that achieve a balance between resolution and signal-to-noise ratio while minimizing the risks we’ve covered?

Hybrid optics microplate readers offer the option to choose between a fixed monochromator bandwidth for day-to-day experiments and dedicated filters with specific bandwidth to meet certain application requirements. These systems are often set up with a fixed medium bandwidth monochromator ranging from 10 to 25 nm, which allows them to perform seamlessly with most classic fluorophores. For small stock shift fluorophores, researchers can cover the dynamic range by shifting the excitation and emission wavelengths to 85–90% of the signal maximum. For assays requiring a very small or a large bandwidth, dedicated filters will ensure that assay specifications can be met without compromise. These instrument designs allow the user to be future-ready without the trouble of intense optimization.

When it comes down to variable bandwidth for the microplate reader monochromator, “to be, or not to be” is the ultimate question. And the answer is for the end user to decide. Each system has its advantages and drawbacks. It is important to choose a microplate reader design that will meet a lab’s unique needs and with which all users will feel comfortable in their daily research. The key to choosing wisely is to know which applications will run on the instrument and how much versatility that research can afford.

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