CC BY
6ALT'22 _B-l-22
/|\ BIOMEDICAL PHOTONICS
Imaging of Molecular Oxygen Using Time-resolved Phosphorescence
V. Shcheslavskiy1'2. P. Morozov3, M. Shirmanova2, G. Goltzman3 and W. Becker1
1- Becker&Hickl GmbH, Nunsdorder Ring 7-9, 12277 Berlin, Germany 2- Privolzhsky Research Medical University, Minin and Pozharsky Sq. 10/1, 603005 Nizhny Novgorod, Russia 3- Moscow State Pedagogical University, Ul. Malaya Pirogovskaya 1/1, 119435 Moscow, Russia Main author email address: vis@becker-hickl.de
Oxygen molecules both in the ground state and in the first excited state (1O2) play an important role in photochemical, photophysical and photobiological processes. [1] For this reason, detection and study of oxygen in solutions, cells and whole living organisms is essential for better understanding of the above processes. This presentation describes the development of the technique that allows to map oxygen both in solutions and in biological samples. The technique is based on time-correlated single photon counting and the detection of the photons during the «on» and «off» phases of the high repetition rate laser operation. This way both fluorescence and phosphorescence may be registered simultaneously from the same pixel of an object. We present the results on using this technique for monitoring oxygen content in cells and tissues. Furthermore, this approach can be effectively used to study also singlet oxygen. The most reliable and informative method for 1O2 investigation is the direct measurement of its phosphorescence with a peak around 1270 nm.
[2] Unfortunately, most of the detectors have a low quantum efficiency (typically less than 25%) at these wavelengths.
[3] In addition, singlet oxygen phosphorescence is very weak with efficiency less than 10-6 in aqueous media and has short lifetimes that can be less than a microsecond due to its high reactivity with biomolecules.[4] Therefore, direct 1O2 detection is not in general a trivial task. We show how to do this using a highly efficient superconducting detector [5] in combination with the technique for simultaneous fluorescence and phosphorescence lifetime imaging. The performance of the developed system is verified by measurement of phosphorescence from singlet oxygen generated by the photo-sensitizers that are commonly used in photodynamic therapy, methylene blue and chlorin e6.
Acknowledgements: The authors acknowledge the support of the studies related to singlet oxygen detection from the Russian Science Foundation (Grant # 22-69-00034).
[1] A.A. Krasnovsky, "Photodynamic Action and Singlet Oxygen", Biophysics ,49, pp. 305- 321(2004).
[2] S.Y. Egorov, S.V. Zinukov, V.F. Kamalov, N.I. Koroteev, A.A. Krasnovskii, and B. N. Toleutaev, "Measurement of photosensitized luminescence of singlet molecular oxygen with nanosecond resolution", Optics and Spectroscopy, 65, pp. 530-533 (1988).
[3] www.idquantique.com/single-photon-systems/products/id230
[4] M. Niedre, M. Patterson, and B. Wilson, "Direct Near-infrared Luminescence Detection of Singlet Oxygen Generated by Photo-dynamic Therapy in Cells in Vitro and Tissues In Vivo", Photochem. Photobiol,, 75, pp.382-391 (2002).
[5] G. Gol'tsman, O. Okunev, G. Chulkova, A. Lipatov, A. Semenov, K. Smirnov, B. Voronov, A. Dzardanov, C. Williams, R. Sobolewski, "Picosecond superconducting single-photon optical detector" Appl. Phys. Lett. 79, 705 (2001).
