The luminescence lifetime as a beneficial analytical parameter is known for many years and is well described by a large variety of publications. Many instruments including 2D measuring systems with cameras have been developed and applied in the past years. However, since the current instrumentation to perform either time- or frequency-domain lifetime measurements is rather complex, new developments in CMOS image sensor technology have achieved to create new image sensors, which can efficiently be integrated into easier-to-handle luminescence lifetime measuring systems. The principle of these modulatable CMOS image sensors, while initially being designed for distance measurements, shows a clear analogy to frequency-domain FLIM measurements, which was proven by researchers [1, 2]. Based on this principle a new CMOS image sensor has been developed, integrated into a camera system and has been investigated within a research project. The image sensor has a resolution of 1024 × 1024 pixels with a 5.6 μm pitch and can be modulated up to 50 MHz. First measurements show an effective dynamic range of larger than 1:1024 (corresponding to 10 bit dynamic). The maximum frame rate is in the range of 90 frames/s in dual-tap mode, resulting in an effective lifetime image frame rate for realistic measurements of approximately 22 frames/s. The camera system pco.flim, featuring that image sensor, generates all required modulation signals from 5 kHz to 50 MHz (sinusoidal and rectangular). It performs advanced pixel correction to generate linear and high-quality images, while the basic lifetime image processing is done in the computer. The modulation frequency can be freely adjusted within the specified range. The characteristics of the camera systems are presented, and first results are discussed using different representations of the data like for example the phasor approach [3], which has been established to provide a more global view to pixelwise fluorescence lifetime data and compare time- and frequency-domain results. Based on these results and the experiences of the on-going tests, it can be expected, that the pco.flim will significantly ease the introduction of luminescence lifetime imaging systems to broader applications.

Date Published: 2 March 2015
PDF: 19 pages
Proc. SPIE 9328, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XIII, 93281K (2 March 2015); doi: 10.1117/12.2087589

Published in SPIE Proceedings Vol. 9328:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XIII
Daniel L. Farkas; Dan V. Nicolau; Robert C. Leif, Editor(s)