Microplate-based applications tend to fit into two experimental streams. The first involves discrete, end-point measurements of changes in sample parameters (color, brightness, or fluorescence) as surrogates for intrinsic properties of biological materials that activate, quench, or metabolize substrates. These measurements are pillars of laboratory bioscience. One can use them to quickly obtain protein, RNA, and DNA concentrations in finite series of samples and compare them to standard curves.
The second stream fits into a drive to automate those workflows and to incorporate dynamic labels and technologies to run limitless numbers of samples through cutting-edge screening and characterization assays. In this second stream, there is a premium on throughput, miniaturization, reproducibility, and the flexibility to add or develop new assays based on emerging technology.
The types of microplate readers available fall along two lines appropriate to those experimental streams. A single-mode reader only handles one of the following: absorbance, fluorescence, or luminescence. In contrast, a multimode reader, combines at least two, if not all three, platforms in one system. Moreover, additional capabilities can handle dynamic, real-time assays based on variations of them. Multimode instruments usually come with bigger price tags than their single-mode counterparts. However, users should primarily base purchasing decisions on the current and predicted diversity of their workflows and how different applications will drive needs for different read modes.
Single-mode readers: absorbance, fluorescence, luminescence
A single-mode absorbance reader usually uses an internal monochromator to quickly split focused light across a wide spectrum (typically 230–1,000 nm) and select a wavelength particular to the target being measured. This target is often represented by a colorimetric change forced by the binding of a dye or chromogenic reagent. A classic example is measuring protein concentrations via assays such as Bradford or bicinchoninic acid. Other common absorbance assays include ones for the determination of nucleic acid concentrations and the quantification of ELISA-based antibody-ligand interactions. In these capacities, a plate reader serves a similar function to a spectrometer. A spectrometer, however, only handles one sample at a time, rather than a multi-well plate. As such, a single-mode reader can be thought of as a medium-throughput spectrometer.
A single-mode reader only handles one of the following: absorbance, fluorescence, or luminescence.
A fluorescence reader can also use monochromator technology to resolve fluorescent signal intensity, although with a dual system corresponding to excitation and emission wavelengths. There is a higher degree of sensitivity compared to absorbance, allowing one to measure comparatively rare events in a sample rather than an overall change in the whole sample. Therefore, fluorescence readers are particularly suitable for cell-based assays that use reporters to quantify expression of engineered fusion proteins. Fluorescence intensity assays can also be used to examine cell populations for death, survival, and proliferation using dyes and antibodies that can tag fragmented DNA, regulatory proteins, or newly incorporated nucleotides. They can also measure changes in protein signaling based on quantification of fluorescent dyes that bind downstream effectors such as calcium. The availability of newer fluorophores with narrower absorbance and emission ranges can extend the capabilities of fluorescent readers into multiplex analysis of more complicated expression patterns.
A luminescence reader can quantify the glow or flash of a naturally emitting sample or of an engineered reporter with much greater sensitivity than even a fluorescent measurement. It usually achieves this through the use of filters, rather than a monochromator, favoring sensitivity of detection over flexibility in choice of wavelength. For flash-based assays with short half-lives, instruments use xenon lamps with photomultiplier tubes, but they must be modified to include injectors to properly control assay timing. The most famous luminescence assay uses a fragment of luciferase, the protein that makes fireflies glow, as a reporter to measure the activation of gene promoters, or the formation and dissolution of protein complexes. This technology can also be extended to measure how close drug molecules are to their protein targets, such as in NanoBRET (Bioluminescence Resonance Energy Transfer).
Although single-mode microplate readers satisfy many standard laboratory workflows, and even some specialized ones, it is multimode readers that really extend and expand capabilities into new areas of discovery, particularly via high-throughput screening (HTS) applications. At minimum, a multimode reader provides the ability to choose among absorbance, fluorescence, and luminescence with one machine. Because these three platforms use different light-splitting technologies, the user can choose between monochromator and filter modes, with some models employing large and extensive filter wheels, or dual photomultipliers to improve the available wavelength range. This allows a user to optimize assays for sensitivity, speed, and accuracy. Additionally, it imparts the capability to multiplex several different signals to analyze the response or status of multiple proteins, reporters, or interactions.
Where multimode readers distinguish themselves from single-mode readers is their extension of fluorescence and luminescence capabilities, particularly to plug into HTS-oriented workflows. These capabilities include fluorescence polarization (FP), time-resolved fluorescence (TRF), fluorescent resonance energy transfer (FRET), and ALPHAScreen (Amplified Luminescent Proximity Homogeneous Assay):
FP: In FP, a fluorescent reporter hits a target molecule, altering its rotation and the trajectory of plane-polarized light.
TRF: In TRF, specialized lanthanide chelate fluorophores with wider Stokes shifts cause emission to follow excitation after a delay, rather than occurring almost simultaneously. This improves sensitivity and signal-to-noise ratios, which results in better z prime numbers while validating screening strategies, in comparison to end-point fluorescence intensity readings.
FRET: In FRET, quantification of light energy transfer between donor and acceptor fluorophores functions as a surrogate for the distance between substances conjugated to them.
ALPHAScreen: ALPHAScreen operates on analogous principles but uses laser excitation on donor beads to kick ambient oxygen into a higher energy state that decays across space but causes high-intensity acceptor bead emission if the two beads are close enough. Among the wide array of applications for these assay platforms, streamlining identification and validation of therapeutic molecules and antibodies is paramount.
As a final note, the newest multimode models incorporate the most miniaturized (1536-well) plates and can fully integrate with ancillary instruments to create uninterrupted workstations ideal for HTS discovery. One particularly useful option combines temperature and gas controls that gauge oxygen and carbon dioxide percentages, maintaining consistent cell culture conditions. In this way, cell-based screening assays using sensitive lines can proceed seamlessly without risking loss or data inconsistencies. Suppliers can additionally modify or upgrade systems via integration with incubators and cell imaging systems, along with maximizing their throughput via plate stackers and washers.
However, as a note of caution, it is deceptively attractive to rely on technology to develop and authenticate screening strategies. In dynamic assays with precise cycles of excitation, relaxation, and decay, users need to empirically determine the exact timing of sample reads to avoid lag time building up over thousands of samples and dozens of microplates. This can cause systems to miss optimal windows of measurement, undercutting the power and potential of multimode readers.