Fluorescence plate reader
A fluorescence plate reader, (longform: fluorescence microplate reader; shortform: fluorescence reader) also called spectrofluorometer or fluorometer, is a piece of equipment capable of detecting and quantifying the light photons emitted by a fluorescent sample present in a microplate upon excitation with light at a specific wavelength.
Fluorescence plate readers are usually intended for fluorescence intensity and FRET detection. Other fluorescence-based detection modes such as fluorescence polarisation or time-resolved fluorescence and TR-FRET can only be detected occasionally and with poor sensitivity, or not at all by single mode fluorescence microplate readers. To additionally cover also these detection modes, as well as absorbance and luminescence, multi-mode microplate readers are usually required.
As opposed to absorbance, fluorescence detection is not an absolute measurement. The intensity of the fluorescent signal is usually relative to other measurements or to a refence measurement taken by an instrument. Consequently, fluorescence plate readers measure the light signal emitted by a sample in Relative Fluorescent Units (RFU).
Fluorescence plate readers are used for different applications in various fields, such as academic life science research, drug discovery and screening, clinical laboratories, synthetic biology and food or water quality monitoring.
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What is a fluorescence plate reader?
Fluorescence plate readers are complex instruments that contain a light source, an optical system for the selection of excitation light, a second optical system for the selection of emission light and a detector, usually a photomultiplier tube (PMT). Because of the nature of fluorescence, samples have to be excited by light at a specific wavelength to emit a fluorescent light signal at a larger wavelength (in nanometers, nm). Fluorescence intensity detection is the measurement of this emitted light.
Fluorescence plate readers are available as stand alone, dedicated readers (fluorometers) or, as part of multi-mode microplate readers, when combined to absorbance and/or luminescence detection.
What to consider when choosing a fluorescence plate reader?
If you are on the market for a new fluorescence plate reader, there are a few factors to take into account, as the performance of the instrument can significantly affect the quality of your research data.
For quantification purposes, fluorescence plate readers generally offer greater sensitivity than absorbance readers. Nevertheless, a fluorescence 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.
In fluorescence intensity detection, samples often cover a vast range of concentrations. Signals that have to be measured on a fluorescence plate reader may span over a wide range of signal intensities – from dim to very bright. Moreover, samples of enzymatic or cell based kinetic assays are often hard to predict in their signal intensity, as their maximum signal builds up over a lapse of time during the course of the kinetic.
In these cases, it may be difficult to acquire all signals in one measurement with one setting. Very bright samples may saturate the fluorescence detector of the microplate reader. Very dim samples may be undiscernible from the blanks.
Having a large dynamic range allows your fluorescence 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 ratio. The Enhanced Dynamic Range feature on the CLARIOstar Plus 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.
A wavelength selection capability in fluorescence microplate readers is mandatory both for excitation and emission. This helps to reduce unspecific background or noise that is usually caused by light scatter or autofluorescence from both the sample and the microplate.
Filters or monochromators can be employed for this purpose. Filters are usually more sensitive, whereas monochromators offer more flexibility. An exception to this paradigm is the Linear Variable Filter (LVF) Monochromator system available on the CLARIOstar Plus. LVF monochromators have filter-like performance and make the CLARIOstar Plus the most sensitive monochromator-based microplate reader on the market.
Additionally, the capability of detecting two emission channels simultaneously on a fluorescence plate reader is quite beneficial for FRET assays. This option halves measurement time and reduces data variability. Simultaneous Dual Emission (SDE) detection is available on the POLARstar Omega and on the PHERAstar FSX.
Compatible microplate formats
Commonly in life science research, basic fluorescence assays are measured in 96 well microplates. 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 fluorescence microplate reader of choice is also capable of reading the microplate format you plan on using.
When looking for a fluorescence plate reader, you should consider the nature of the assay you want to run. For enzymatic kinetics or real-time cell based responses, the use of reagent injectors can be beneficial. By manually pipetting reagents into the wells you can run the risk of losing important data of your reaction. Especially for fast responses, the event you are looking for may already be gone before you detect it. Our fluorescence plate readers equipped with reagent injectors can simultaneously inject and detect the signal of a well, and then move to the next one.
If your research requires long-time live cell based assays and you do not want to shuffle your microplate back and forth between fluorescence microplate reader and incubator, make sure that your reader of choice has the capability to control its internal gas environment.
The Omega series and the CLARIOstar Plus can be equipped with our Atmospheric Control Unit (ACU). The ACU provides the ideal environment for any application with living cells. The ACU independently regulates both O2 and CO2 gas levels within the microplate reader chamber. Combined with shaking and temperature incubation, the ACU provides a real “walk-away” solution for any cell based assay from standard cell growth to hypoxic assays.
Our Fluorescence Plate Readers
Fluorescence intensity detection can be performed on the PHERAstar®FSX, CLARIOstar®Plus, and FLUOstar® and POLARstar® Omega plate readers.
All our fluorescence microplate readers are equipped with a high-intensity xenon flashlamp, assay specific filters, optic modules, or LVF monochromators™ (reader-dependent), and low-noise or extended red-shifted PMTs.
Which assays can be measured by a fluorescence plate reader?
Fluorescence intensity is one of the most popular detection modes in life science. Accordingly, there is a multitude of kits and reagents available on the market for the most different assay types that fluorescence microplate readers both as single- or multi-mode instruments can cover.
Common applications for fluorescence intensity are DNA/RNA quantification, calcium flux, enzymatic activities, gene expression, second messenger signalling for GPCRs, protein-nucleic acid interaction, tryptophan quantification, molecular beacon, biosensors for wastewater analysis and more.
Cell based applications include apoptosis, cell proliferation and cytotoxicity.
Protein interaction can be detected via Fluorescence Resonance Energy Transfer (FRET). FRET is a dual dye fluorescence detection assay based on energy transfer between a donor and an acceptor. If donor and acceptor are in close proximity (10-100 Å), excitation of the donor fluorophore transfers energy to the acceptor fluorophore that will emit light without being directly excited.
Fluorescence intensity/FRET measurements
The following are examples of fluorescence intensity and FRET measurements taken on BMG LABTECH fluorescence plate readers:
- AN 301: Simultaneous detection of GPCR second messengers in living cells
- AN 293: Moss cells as expression system for biopharmaceuticals
- AN 253: Real-time calcium flux measurements in iPSC derived 3D heart tissue
See a complete list of fluorescence intensity application notes.