Fluorescence assays are increasingly being used in areas such as biotechnology, clinical diagnostics, drug development and environmental monitoring, where standards for instrument qualification and method validation are important considerations. Scientists at the National Institute of Standards and Technology (NIST) in Gaithersburg, Md., have developed two new standard reference materials to help correct and validate the performance of analytical instruments, such as fluorescent spectrometers, that detect, measure and identify substances based on fluorescence. According to the lead researcher, Paul DeRose, the materials could be particularly useful for satisfying the method validation requirements of regulatory agencies, such as the FDA and the Environmental Protection Agency, because they provide an easy, reliable and systematic approach to qualifying a fluorescent instrument. The fluorescence spectroscopy technique involves directing a beam of light at a certain wavelength into a sample, exciting electrons in particular analytes or fluorescent labels, which then emit light at longer wavelengths, which can be measured along with corresponding fluorescence intensities by a spectrometer. Fluorescent compounds have distinct spectral signatures, and observed fluorescence intensities are used to determine analyte concentrations. The new standard reference materials, borate glass blocks about the size of a pack of gum, can be used to correct fluorescence emission spectra for relative intensity. SRM 2940 (orange emission) has certified values for emission wavelengths from 500 to 800 nm when excited with light at 412 nm. SRM 2941 (green emission) has certified values for emission wavelengths from 450 to 650 nm when excited at 427 nm.NIST has completed the certification of SRM 2940 and 2941, Relative Intensity Correction Standards for Fluorescence Spectroscopy, with orange emission and green emission, respectively. The cuvette-shaped glass artifacts are calibration tools for validating fluorescent instruments. Courtesy of NIST.To calibrate a fluorescent spectrometer, for example, scientists would excite SRM 2941 with light at 427 nm and collect the fluorescence emission from 450 to 650 nm. They could then determine spectral correction factors for the instrument by comparing the measured intensity value to the certified values. The fluorescence spectrum of an unknown sample taken on that instrument that emits between 450 and 650 nm then could be corrected to determine its true spectral shape. A need for these calibration tools has been expressed at NIST fluorescence workshops over the past several years, according to DeRose. Unlike the dye-based reference materials of this type, he explained, the glass-based materials are portable, photostable and robust, with long shelf lives (estimated at 10 years or more). These attributes make the tools suitable for day-to-day instrument validation, enabling them to measure fluorescence intensity under a fixed set of conditions. If the measurement intensity stays constant over time, he added, the instrument has not changed, thereby validating consistent performance over time. This day-to-day validation is suitable for both steady-state and time-resolved instruments, including spectrometers and filter-based types. The standard reference materials have applications for spectral correction in benchtop and portable steady-state and fluorescent spectrometers. They can be used for method validation for assays that use fluorescence intensity ratios, enabling the instrument to be qualified in one step, thereby ensuring the accuracy of the measured intensity ratios.The tools enable the peak positions and shapes of spectra, along with the fluorescence intensities of analytes, to be compared between instruments, and they can be used to compare fluorescence intensities between steady-state instruments with continuous-excitation sources. The team is in the process of developing similar glass standards that cover other wavelength regions where fluorescence is measured, such as in the UV and the NIR. It also is investigating standards for high-throughput fluorescence instruments, such as microwell plates and readers.