How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards

Abstract

The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro-and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards.

Calibration, standardization, and quantitative analysis of multidimensional fluorescence (MDF) measurements on complex mixtures (IUPAC Technical Report)

“…To date, no such multi-fluorophore standards exist, and consequently both excitation and emission should be calibrated independently using conventional procedures. The reader is directed elsewhere for more detailed discussion of the aspects of standard spectral correction [12,[16][17][18][19][20][21][22][23].…”

The Calibration Kit Spectral Fluorescence Standards—A Simple and Certified Tool for the Standardization of the Spectral Characteristics of Fluorescence Instruments

“…Luminescence techniques always yield signals I u (λ ex ,λ em ) that contain sample-and instrument-specific contributions [6], see Eq. (1).…”

“…Determination of the spectral responsivity s(λ em ) s(λ em ) can be obtained with a source that emits a known and broad spectrum in the UV/-vis/NIR region [6,8,12,13,14]. Its reliable determination as the basis for the spectral correction of emission spectra requires not only control and if necessary consideration of the wavelength accuracy of its emission channel [15] and the operation of the detection system within its linear range, but also consideration of the samples to be corrected and thus measurement conditions commonly employed [8].…”

“…This circumvents errors due to the operation of the detection system within its nonlinear range [8]. 4 In contrast, chromophore-based spectral fluorescence standards [6,8,12,13,14,16] are easy to operate. Especially liquid standards such as BAM-F001-BAM-F005 offer an unique flexibility with respect to measurement geometry, format or type of container, and type of instrument to be calibrated.…”

“…To rule out instrumentation as a major source of variability and to improve the comparability of fluorescence measurements across instruments and over time, fit-for-purpose standards in combination with tested protocols for instrument characterization and performance validation (IPV) are required [6]. As a first step towards simple and multifunctional calibration tools for fluorescence instruments that link luminescence measurements to radiometric units [7], we developed the Calibration Kit Spectral Fluorescence Standards BAM-F001-BAM-F005 including the software LINKCORR [8].…”