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- Calibration tool (1)
- Fluorescence (1)
- Fluorescence spectroscopy (1)
- Fluorescence standard (1)
- Glass (1)
- Instrument performance validation (1)
- Intensity standard (1)
- Liquid standard (1)
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The potential of spectrofluorometry and fluorescence microscopy for the characterization and quantification of different functionalities like OH and NH2 groups at plasma-chemically modified polymer surfaces is assessed using traditional reactive dyes such as dansyl derivatives and a sophisticated VIS-excitable chromogenic and fluorogenic pyrylium label showing binding-induced spectral and intensity changes in absorption and emission. Aiming at an improved fluorometric surface analysis, based upon these measurements, several sources of uncertainty inherent to fluorescence measurements are illustrated ranging from environment-dependent dye absorption and emission features over spectral correction and nonspecific adsorption to the critical influence of label choice on the measured background. Solutions to these drawbacks are given thereby underlining the potential of fluorometry for surface analysis.
To improve the reliability of fluorescence data in the life and material sciences and to enable accreditation of fluorescence techniques, standardization concepts are required that guarantee and improve the comparability of fluorescence measurements. At the core of such concepts are simple and evaluated fluorescence standards for the consideration of instrument-specific spectral and intensity distortions of measured signals and for instrument performance validation (IPV). Similarly in need are fluorescence intensity standards for the quantification from measured intensities and for signal referencing, thereby accounting for excitation light-induced intensity fluctuations. These standards should be preferably certified, especially for use in regulated areas like medical diagnostics. This encouraged us to develop liquid and solid standards for different fluorescence parameters and techniques for use under routine measurement conditions in different formates. Special emphasis was dedicated to the determination and control of the spectral responsivity of detection systems, wavelength accuracy, homogeneity of illumination, and intensity referencing for e.g. spectrofluorometers, fluorescence sensors and confocal laser scanning fluorescence microscopes. Here, we will present design concepts and examples for mono- and multifunctional fluorescence standards that provide traceability to radiometric units and present a first step towards a toolbox of standards.