Integrating sphere setup for the traceable measurement of absolute photoluminescence quantum yields in the near infrared.

There is an increasing interest in chromophores absorbing and emitting in the near-infrared (NIR) spectral region, e.g., for applications as fluorescent reporters for optical imaging techniques and hence, in reliable methods for the characterization of their signal-relevant properties like the fluorescence quantum yield (Φ(f)) and brightness. The lack of well established Φ(f) standards for the NIR region in conjunction with the need for accurate Φ(f) measurements in transparent and scattering media encouraged us to built up an integrating sphere setup for spectrally resolved measurements of absolute fluorescence traceable to radiometric scales. Here, we present the design of this setup and its characterization and validation including an uncertainty budget for the determination of absolute Φ(f) in the visible and NIR. To provide the basis for better measurements of Φ(f) in the spectral window from ca. 600 to 1000 nm used, e.g., for optical imaging, the absolute Φ(f) of a set of NIR chromophores covering this spectral region are measured and compared to relative values obtained using rhodamine 101 as Φ(f) standard. Additionally, the absolute Φ(f) values of some red dyes that are among the most commonly used labels in the life sciences are presented as well as the absolute quantum yield of an optical probe for tumor imaging.

Evaluation of a commercial integrating sphere setup for the determination of absolute photoluminescence quantum yields of dilute dye solutions.

The commercial availability of stand-alone setups for the determination of absolute photoluminescence quantum yields (Phi(f)) in conjunction with the increasing use of integrating sphere accessories for spectrofluorometers is expected to have a considerable influence not only on the characterization of chromophore systems for use in optical and opto-electronic devices, but also on the determination of this key parameter for (bio)analytically relevant dyes and functional luminophores. Despite the huge potential of systems measuring absolute Phi(f) values and the renewed interest in dependable data, evaluated protocols for even the most elementary case, the determination of the fluorescence quantum yield of transparent dilute solutions of small organic dyes with integrating sphere methods, are still missing. This encouraged us to evaluate the performance and sources of uncertainty of a simple commercial integrating sphere setup with dilute solutions of two of the best characterized fluorescence quantum yield standards, quinine sulfate dihydrate and rhodamine 101, strongly differing in spectral overlap between absorption and emission. Special attention is dedicated to illustrate common pitfalls of this approach, thereby deriving simple procedures to minimize measurement uncertainties and improve the comparability of data for the broad community of users of fluorescence techniques.

An oxygen imaging system for medical applications: preliminary results.

The examination of functional processes in tissue is gaining importance in medical research. As a result the imaging and monitoring of biochemical parameters in vivo is the goal of many imaging methods. One key parameter in photodynamic therapy (PDT) is the molecular oxygen concentration. Two-dimensional monitoring of oxygen is demanded for PDT but has not yet been achieved. The use of optical methods provides a possible means of measuring molecular oxygen. The basis of this method is the measurement of the luminescence lifetime of a dye that is quenched by molecular oxygen. The molecular oxygen concentration can be monitored two-dimensionally by pixel-wise determination of the luminescence lifetime with a CCD-camera. A new O(2)-imaging system based on this principle is presented in this article. The dye Ru(bpy)(3)(2+) is quenched by molecular oxygen and was used in the first experiments with the system.