This report summarizes problems associated with the comparability of measurements of photoluminescence and procedures for the characterization of relevant instruments, focusing on physical and chemical fluorescence standards. To provide recommendations on selecting and using such standards, we derive general and scope-specific requirements and quality criteria for suitable devices and materials and briefly address metrological requirements linked to the realization of comparable measurements. Special emphasis is dedicated to liquid and solid chromophore-based fluorescence standards developed or currently tested by us.
Molecular engineering of polymer surfaces that includes the attachment of functional molecules to existing or previously generated reactive chemical groups like e.g., - OH, - NH2, or - CHO requires simple strategies and tools for the controlled generation of surface functionalities and their derivatization as well as for their identification and eventually quantification. Here, we systematically investigate the plasma-chemical surface modification of polypropylene films in combination with dansyl labeling chemistry and conventional, yet costly, XPS and highly sensitive fluorescence spectroscopy for the detection of surface groups. Based on these results, the potential of and requirements on the fluorometric characterization and quantification of surfaces functionalities are discussed.
A straightforward strategy toward the sensitive fluorometric detection of primary amino groups on plasma-chemically modified polypropylene supports is presented, exploiting the transformation of the sterically nonhindered pyrylium dye Py-1 into its pyridinium counterpart. The reaction-induced blue-shifted absorption and emission bands and an increased fluorescence quantum yield provide the basis for the spectroscopic distinction between covalently bound and free, that is, nonspecifically adsorbed label molecules. With this label, for the first time, plasma-chemically introduced amino functionalities could be monitored on the surface of a polymer film employing fluorescence spectroscopy and confocal laser scanning microscopy.
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.
The aim of this article is to illustrate the need for an improved quality assurance in fluorescence microscopy. From the instrument-side, this can be achieved by a better understanding, consideration, and regular control of the instrument-specific parameters and quantities affecting measured fluorescence signals. Particularly, the need for requirements on physical- and chemical-type instrument standards for the characterization and performance validation of spectral fluorescence microscopes (SFMs) is discussed and suitable systems are presented. Special emphasis is given to spectral fluorescence standards and to day-to-day intensity standards for SFMs. Fluorescence standards and well-characterized fluorescence microscopes are the first and essential steps towards the comparability and the understanding of the variability in fluorescence microscopy data in medical and life sciences. In addition, standards enable the distinction between instrument-specific variations and fluorescent label- or probe-related uncertainties as well as generally sample-related effects.
Fluorescence microscopic and spectroscopic monitoring of degradation processes upon polymer ageing
(2017)
The majority of all routinely used methods to assess polymer aging are based on destructive tests and methods. Early indicators for the deterioration of polymer materials are e.g., physical or mechanical properties like tensile strength, adhesion, brittleness, and color. It is well-known, however, that predominantly chemical changes are the underlying process of the physical changes that occur in organic materials upon aging over time. Typical initial steps during polymer degradation are crosslinking or chain breaking, alteration of autofluorescence, “yellowing” or bleaching caused by the formation of new functional groups. A straightforward strategy towards the sensitive detection and monitoring of chemical changes in the course of polymer aging is based on non-destructive optical measurements. Luminescence techniques, one of the most sensitive spectroscopic methods are the method of choice. Here, we present first results of luminescence-based monitoring of polymer degradation induced by different environmentally relevant weathering factors (e.g. humidity and UV exposure). Our studies include fluorescence spectroscopy as well as spectral scanning confocal fluorescence microscopy and clearly demonstrate the possibility to follow accelerate-aging processes by luminescence detection.