Fluorescence measurements with fluorometers, microtiter plate readers, fluorescence microscopes, and other fluorescence instruments are widely performed in life and materials sciences. This is related to the inherently multiparametric nature of fluorescence, providing spectral, intensity, polarization, and lifetime information with a high speed and sensitivity, suitability for online and in situ measurements. As fluorescence data always contain sample- and instrument-specific contributions, instrument-independent fluorescence data, comparable across instruments and laboratories, require reliable and validated instrument calibration strategies and reference materials (RMs) with well characterized fluorescence properties. Such RMs can act as calibration and instrument performance validation tools and provide benchmark values.

Spectral fluorescence standards and corrected emission spectra

Comparable and reliable fluorescence spectra as well as fluorescence intensity and quantum yield measurements require the determination of the wavelength-dependent spectral responsivity of fluorescence instruments, also termed emission or spectral correction curve, for subsequently correcting measured instrument-specific data. This is essential for comparing different fluorescent labels and reporters, quantitative fluorescence measurements, and the determination of the fluorescence quantum yield, the spectroscopic measure for the fluorescence efficiency of a fluorophore. To implement such correction procedures in the fluorescence community, simple calibrations tools are needed such as chromophore based RMs, referred to as fluorescence standards. Emission correction curves can be obtained with spectral fluorescence standards with precisely known, preferably certified instrument-independent fluorescence spectra. Meanwhile, spectral fluorescence standards for use as solids or solutions are available for the ultraviolet (UV) and visible (vis), but for the increasingly used near infrared (NIR) wavelength region > 700 nm, presently no spectral fluorescence standards exist. This gap has been now closed with spectral fluorescence standards BAM F007 and BAM-F009.

Traceable fluorescence measurements

For applications, which require traceable fluorescence measurements and hence, a traceable instrument calibration or performance validation, e.g., for laboratories accredited according to ISO/IEC 17025, a traceability statement is required. Metrological traceability is defined as a property of a measuring result whereby the result can be related to a reference through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty. Typically, traceability to SI units (Système international d'unités = International System of Units) is established during the certification procedure of the RM, commonly via the calibration of the instruments used for determination of the certified property, i.e., the corrected fluorescence emission spectra of spectral fluorescence standards. Traceable fluorescence measurements require high-quality RMs and the measurement of the standard´s application-relevant fluorescence properties with a traceably calibrated spectrofluorometer with a well-documented calibration including uncertainty statements, that are wavelength-dependent for fluorescence spectra. For spectral and intensity fluorescence standards, traceability either to the spectral radiance or spectral photon radiance scale is established, in the latter case considering the photonic nature of the emitted light. All BAM-certified fluorescence standards are characterized with traceably calibrated fluorescence instruments with a known uncertainty budget and provide traceability to the spectral photon radiance scale.

BAM spectral fluorescence standards – Design criteria

The design of spectral fluorescence standards for the determination of the spectral responsivity s(λ) of fluorescence instruments requires emitter-matrix combinations, that are excitable with common light sources and measurable with typical instrument settings, photostable, and have broad and unstructured fluorescence emission spectra independent of excitation wavelength. Thereby, a dependence on the spectral bandpass of the fluorescence instrument to be calibrated is minimized. To cover a spectral window of several hundred nanometers, standard sets are needed such as the BAM Calibration Kit F001-F005, consisting of five dyes with overlapping emission spectra to ease the generation of an overall spectral correction curve for the UV/vis/NIR (Figure 1). Such an emission correction curve equals the inverse relative spectral responsivity of the fluorescence instrument to be calibrated. This correction curve is obtained by calculating the quotients of the certified normalized corrected fluorescence emission spectra and the instrument-specific, i.e., uncorrected (non-normalized) emission spectra of the BAM standards measured with the fluorescence instrument to be calibrated for each set component, followed by the weighted merging of the individual correction curves of the Kit dyes (Figure 1). As this merging procedure can be challenging, BAM developed the user-friendly software LINKCORR, which contains the certified emission spectra of the BAM dyes including their wavelength-dependent uncertainties and performs this data evaluation in a standardized way previously validated by us. Multiplication of measured fluorescence emission spectra with this emission correction curve yields instrument-independent corrected emission spectra.

The BAM spectral fluorescence standards have been designed for use as liquid fluorescence standards, i.e., as ethanolic dye solutions. Advantages of liquid fluorescence standards are a homogeneous chromophore distribution, flexibility with respect to emitter concentration and measurement geometry, and ease of format adaptation. Such liquid standards can be utilized for calibrating and characterizing different fluorescence instruments such as fluorescence spectrometers, microtiter plate readers, and fluorescence microscopes. Also, dye solutions closely resemble typically measured liquid samples and are not prone to local photobleaching effects as well as to polarization effects, contrary to solid fluorescence standards. As dye solutions have a limited shelf life of a few months, the fluorescence standards are provided as solid dyes. BAM provides a SOP for RM handling and usage, including the preparation of the dye solutions using ethanol of high purity and documentation on the storage conditions of the standards and their shelf life.

Extending the BAM Calibration Kit to the NIR with two novel NIR fluorophores

To expand the wavelength range of the BAM Calibration Kit from 300-730 nm, we developed the novel fluorescence standards BAM-F007 and BAM-F009 and certified their emission spectra in ethanol, extending from 580 nm to 940 nm (Figure 1). Certification involved the calculation of the respective wavelength-dependent uncertainty budgets including contributions from homogeneity and stability studies. Thereby, fluorescence instruments can now be traceably calibrated from 300 nm to 940 nm, enabling comparable fluorescence measurements in the UV/vis/NIR.

Future Work 

In the future, the applicability of the BAM Calibration Kit for calibrating fluorescence instruments such as microplate readers and fluorescence microscopes is explored and the assessment of aging-induced changes in spectral sensitivity. This also includes the development of fluorescence standards for the emerging short-wave infrared (SWIR) to improve the comparability of fluorescence measurements > 1000 nm.

Figure 1. Working principle of the BAM Calibration Kit F001-F005 and the BAM certified instrument independent emission spectra of the seven kit components.