What to consider when choosing a luminescence plate reader?
If you are on the market for a new luminescence plate reader, there are a few factors to take into account, as the performance of the instrument can significantly affect the quality of your assay results.
Sensitivity
Luminescence is typically generated by a bio-chemical or chemical reaction and does not require excitation light. Consequently, it generally offers a higher sensitivity when compared to absorbance or fluorescence. The unspecific background or noise signal is extremely limited when compared to fluorescence intensity, since there is no excitation process that can trigger light scatter or autofluorescence from both the sample and the microplate.
Nevertheless, a luminescence microplate reader with better sensitivity will deliver data with a better statistic, less variability among replicates, and a larger delta (distance) between responding and not-responding samples. In addition, a better sensitivity will allow you to reduce the amount of reagents and/or samples used to achieve good quality data and consequently reduce costs.
Dynamic range
Luminescent reactions are usually based on the addition of a substrate to start an enzymatic reaction. Once the reaction is started and the amount of substrate is metabolised by the enzyme, the reaction is exhausted and cannot be retriggered again, unless new reagents and samples are used. Consequently, it is imperative to acquire the signal of all samples that have to be measured at once with a luminescence plate reader, in order to avoid repeating the assay and consuming more reagents.
If you have samples spanning over a large range of signal intensities, it may be difficult to acquire them all in one measurement with one setting. Very bright samples may saturate the luminescence detector of the microplate reader. Very dim samples may be undiscernible from the blanks.
Having a large dynamic range allows your luminescence plate reader to acquire highly divergent samples in one run, avoiding waste of reagents and time caused by multiple trial-and-error measurements to find the correct dilution-gain-integration time ratio. The Enhanced Dynamic Range feature on the CLARIOstar Plus and VANTAstar offers the largest possible dynamic range on the market (8 concentration decades), and makes it possible to automatically measure very bright and dim signals in the same measurement – no adjustment required.
Wavelength selection
A wavelength selection capability in luminescence plate readers is not mandatory, especially when detecting basic luminescent assays. However, depending on the application and especially for luminescent assays with multiple signals, the possibility to select the emission wavelengths is beneficial. Filters or monochromators can be employed for this purpose. This option is useful when developing new assays or detecting multiple luminescent signals. Moreover, it helps to optimise the assays by reducing background noise.
Additionally, the capability of detecting two emission channels simultaneously on a luminescence plate reader is quite beneficial for BRET and nanoBRET assays. This option halves measurement time and reduces data variability. Simultaneous Dual Emission (SDE) detection is available on the Omega series and on the PHERAstar FSX.
Compatible microplate formats
Commonly, basic luminescence assays are measured in 96-well plates. However, if you have multiple samples and/or wish to save precious reagents or time, 384-well and 1536-well formats can also be used. Just make sure that your luminescence plate reader of choice is also capable of reading the plate format you plan on using. Light cross-talk should also be considered when using high-density microplates.
Flash or glow reactions
When looking for a luminescence plate reader, you should consider the nature of the assays you want to run. According to their kinetic, luminescent assays can be divided in flash and glow reactions. Flash luminescence is a short-lived reaction that gives off a very bright signal for a very short amount of time, usually seconds. Glow luminescence is rather long-lived. The reaction can last for several minutes, but usually emits a less intense signal.
Any luminescence plate reader can detect glow reactions. Flash assays require instead the use of reagent injectors. By manually pipetting reagents into the wells you can run the risk of losing the peak of your reaction as the short-lived signal could already be exhausted before you detect it. Our luminescence plate readers equipped with reagent injectors can automatically inject and detect the signal of a well and then move to the next one.
Reagent injectors are also beneficial for Dual Luciferase Reporter (DLR) assays. Here, the activity of two luciferases is sequentially measured from a single sample by luminescence microplate reader and reports the transcription of a gene of interest and a transfection control. Both assays can be completed in about 4 seconds using luminescence plate readers with reagent injectors.
Light cross-talk
Light cross-talk happens when during the measurement of a well, the light signal from the neighbouring well(s) is also picked up by the detector. This leads to adulterated results. Cross-talk is specifically a factor when working with high-density plates (384-wells and higher).There are two ways by which cross-talk light can be picked up by the detector, either from the top of the well or through the plastic wall of the microplate.
High-end luminescence plate readers such as the CLARIOstar Plus, the VANTAstar and the PHERAstar FSX can compensate this factor by physically blocking the light coming from the neighbouring wells. Specific algorithms can calculate the amount of light transmitted through the plastic wall and subtract it from your samples.
Our luminescence plate readers
Luminescence detection can be performed on BMG LABTECH´s dedicated luminescence plate reader LUMIstar® Omega, and multi-mode microplate readers including the PHERAstar® FSX, CLARIOstar® Plus, VANTAstarTM and FLUOstar® Omega.
All of our luminescence plate readers are certified for Dual-Luciferase Reporter assays utilize luminescence-optimized low-noise PMT (photomultiplier tube) and can be equipped with high-precision reagent injectors.
