Critical Considerations for Choosing the Right Microplate Reader

Do I need a single-mode reader or a multi-mode reader?

If you are only doing one read type (just absorbance, fluorescence, or luminescence), then a single-mode reader we be more appropriate for you. However, if you think your needs may change or you’re running a variety of applications with various read types, then a multi-mode reader would be best for you.

Which light sources are available and which should I choose?

There are three light sources available: flash lamp, LEDs, and laser.

A xenon flash lamp will provide substantial intensity over a very broad range of light wavelengths (200-1000 nm). Generally, it will be more efficient in the visible range, but it can also be used in the ultraviolet (UV) and near-infrared ranges.

LEDs can be used as your light source in the spectral range of 375 to 1000 nm. Most LEDs have a narrow bandwidth, between 20 and 50 nm, and they are dedicated to a specific range. They generate a higher light intensity than most xenon flash lamps and can therefore provide greater sensitivity for certain assays. LEDs also have a very long lifetime.

Lasers are much higher intensity and are used for specific applications, such as AlphaScreen, where you need a lot of energy at a specific wavelength.

In general, LEDs will give you better performance than flash lamps. However, newer generations of instruments, such as the SpectraMax® iD5 Multi-Mode Microplate Reader, which has a high performance (high intensity) flash lamp will give you similar performance to that of an LED-based reader. If you do not want to have to choose between light sources, you can go with a system like our SpectraMax i3x Multi-Mode Microplate Reader, which offers all three, so you can choose the best one for your application.

Do I need filters or monochromators?

Monochromators and filters are two different wavelength selection technologies integral to microplate reader design.

In a filter-based instrument, optical filters with specific wavelengths are incorporated into the excitation and emission optics. In addition, a beam splitter, such as a dichroic mirror (or semi-transparent filter) will pass the excitation light into the sample and will allow the emission light to go through the emission filter so the signal can be detected. This design provides minimal signal loss, as well as effective separation of excitation and emission wavelengths.

Monochromator-based instruments have a more complex design that can vary between instruments. A monochromator generally consists of an entrance slit, a dispersive element, such as a prism or holographic grating, and an exit slit. Monochromators are used for filtering excitation and emission light. A diffraction grating will separate white light into a spectrum so that the exit slit isolates a specific wavelength of light to excite the sample. Within the exit slit, a rotating dispersive element allows the selection of the desired wavelength. Filter- and monochromator-based microplate readers each have their advantages.

Filter-based instruments use less expensive components due to a simpler design (compared to a monochromator). Dedicated filters allow for minimal signal loss and effective separation of the excitation and emission wavelengths, making them more sensitive for some applications. You will probably need an initial inventory of common-use filters, you will likely need to purchase additional filters over time to accommodate changing needs. Applications such as BRET, fluorescence polarization, and TR-FRET typically require filters to achieve the necessary sensitivity. On the other hand, monochromator-based instruments are very convenient and flexible, meet the sensitivity requirements of many applications, and they do not require you to maintain an inventory of application-specific filters. Monochromators also offer a spectral scanning function for the characterization of new dyes and the study of spectral shifts.

Which one should you choose? If you are always working with the same application or the same wavelengths, the filter-based instrument may be cheaper for you. In general, filters provide higher sensitivity than a monochromator in a comparable instrument. However, the sensitivity provided by monochromators is sufficient for most assays, and they have more flexibility and convenience compared to filter-based instruments. Fortunately, Molecular Devices’ SpectraMax i3x and iD5 readers offer the best of both worlds. These systems are hybrids that allow you to choose between filters or monochromators to get the best performance for your assay.

Do I need a wide or narrow bandwidth?

A narrow bandwidth improves resolution and is recommended for making fluorescence measurements when a fluorophore’s excitation and emission peaks are close together (narrow Stokes shift). It minimizes the amount of unwanted light measurement so that background levels are diminished, and signal is measured more specifically.

Increasing the bandwidth allows more light to enter the instrument’s optical path, which can improve the signal-to-noise ratio and sensitivity. A wider bandwidth for excitation increases the amount of light exciting the sample, while for emission it captures a wider range of the emission signal.

Which spectral bandwidth you choose for your microplate reader will depend on multiple things. Which applications are you running? Are you doing dual reads, such as FRET or TR-FRET (e.g., HTRF®)? Which fluorophores are you using, and what are the shapes of the excitation and emission peaks? How much background is there with your assay? As a general rule, narrow bandwidths are best for fluorophores with a small Stokes shift, and for making multiple fluorescence measurements at wavelengths that are close together. Wider bandwidths are best for wide excitation and emission peaks because they will increase an assay’s sensitivity. However, you must be careful to monitor your assay background, as wider bandwidths can allow increased background fluorescence to be detected, lowering the signal-to-noise ratio.

Fortunately, Molecular Devices offers a system with variable bandwidth that can be specific and sensitive at the same time and is suitable for assay optimization, so you do not have to choose. The SpectraMax i3x reader has variable bandwidth for fluorescence and luminescence, and you are able to choose either 9nm or 15 nm for excitation and either 15 nm or 25 nm for emission. In luminescence mode, if you are specifying a wavelength for the read, you can select an emission bandwidth of either 15 nm or 25 nm.

Do I need Injectors?

You will need injectors if you are doing a fast kinetic assay like a dual-luciferase assay or fluorescence-based calcium (GPCR) assay. Fast kinetic assays are characterized by a very fast response upon addition of a compound or other reagent to a well. The speed of the response, as well as a rapid decline in the resulting signal, means that the instrument used to run these assays needs to read a well while reagent is being added, or very shortly afterward, so that all of the signal is captured.

Do I need a top-read or bottom-read instrument?

If you’re doing only experiments where the assay material is distributed evenly throughout the well (homogeneous), top read will be enough for you. However, if you are looking to run cell-based experiments where the relevant signal is located at the bottom of the wells, and may even be masked from above by quenchers or masking dyes, then you will need a bottom-reading microplate reader.