James Netterwald, PhD, is a freelance science and medical writer based in New Jersey. His writing serves every life science industry.
Imagine a world in which scientists had to read fluorescence assays manually, one tube at a time, in a spectrophotometer dialed up to measure light emission in the visible range. Well, that world has transformed into a modern one in which there are automated fluorescence readers. These instruments are capable of reading fluorescence emitted from the sample wells of assay plates that contain anywhere from six wells to 384 wells per plate. These instruments are used for a wide variety of applications including, but not limited to, enzyme kinetics, gene reporter assays, cell viability assays, cytotoxicity assays, ELISAs and other immunoassays, nucleic acid or protein quantification and fluorescence resonance energy transfer (FRET). The clinical applications include measurement of thyroid hormones, amino acids and galactose. However, before you even put a plate into the reader, you want to make sure you have the right instrument. Consider the following features prior to purchasing a fluorescence plate reader.
What should I look for in a fluorescence detector?
Of course, the most important consideration to make before purchasing a fluorescence plate reader is choosing a detection system that is compatible with your workflow. The detection system consists of several components. One, the filter, is optional. With its role in filtering out specific wavelengths while allowing others to shine through, the filter, whether used in the excitation of a sample or to measure its fluorescence emission, may or may not be a part of your detection system. The light source of the detection system is also important. This is often a xenon lamp with a wavelength range in the visible part of the spectrum (typically 300 nm to 1,000 nm).
Another feature to consider in a plate reader is the type of monochromator its detection system uses. Plate readers that contain a single monochromator enable the user to select a single wavelength for both excitation and emission. In contrast, instruments that contain dual monochromators, which is the case for most fluorimeters, have one monochromator to select the excitation wavelength and another to select the emission wavelength, creating an optimized wavelength pair that eliminates the need to change filters prior to a reading. Having dual monochromators also enables the instrument to scan a wider range of wavelengths in the visible spectrum, vs. single monochromators.
What about dynamic range and data analysis?
It’s also important to consider the amount of fluorescence accurately detected, which is expressed in relative fluorescence units (RFUs) and is determined by the instrument’s dynamic range of signal intensity. This feature is a function of the detector’s photomultiplier tube. Some instruments have a narrow dynamic range—approximately three orders of magnitude—and therefore encounter fluorescence saturation. Others have signal optimization systems that increase the dynamic range by adjusting the fluorescence detector to each sample well’s concentration and normalizing raw data. These optimizers enable the instrument to capture the lowest and highest signals from the same plate. In addition, calibrating against an internal standard enables the instrument to make accurate comparisons of RFU levels between samples.
Finally, most fluorescence plate reader packages include software that enable users to analyze raw fluorescence data, make comparisons, run statistics and generate reports.
Summary
Fluorescence plate readers have definitely made an impact on the way in which fluorescence-generating reactions are detected. In fact, they have made the process easier. With monochromators, photomultiplier tubes, filters and analytical software, fluorescence plate readers are necessary instruments in clinical, research and pharmaceutical laboratories worldwide
The image at the top of the page is from BioTek.