Calibration of systems for quantitative fluorescence analysis of thin layers

Imaging fluorescence analysis is a powerful tool for the characterization of thin functional layers. Due to the development of new components such as cost-efficient and long life diode lasers and LEDs as well as sensitive cameras, the number of industrial in situ sensors based on fluorescence analysis technology increased rapidly in recent years. Of crucial importance for all these new sensors are efficient and robust methods for calibration. Although there are many examples for the calibration of laboratory setups for single specialized applications, there is no standardized method for the traceable device independent calibration of imaging fluorescence systems. This paper presents the evaluation of five different methods for the calibration of systems for quantitative fluorescence analysis. Each method is applied for the calibration of an imaging fluorescence laser scanner. In addition to characterizing the precision of the methods, the work analyzes the usability of the methods for different applications. The results show for the first time that a calibrated IR point sensor can be used for the auto calibration of high resolution imaging inline fluorescence sensors. In addition, we present a novel method for the transfer of calibration data between analysis systems with different optical setups by using a solid material fluorescence standard.