Champaign, Illinois - November 30, 2021 - Phosphorescence has a number of promising applications in biomedical research. Phosphorescent probes can be engineered to respond to environmental parameters, such as tissue O₂ concentration. These probes typically have decay lifetimes in the range of micro- to milliseconds, as compared to fluorescence probes that have lifetimes on the order of nanoseconds. Therefore, measurements of phosphoresce lifetimes require excitation light sources operating at low repetition rates.

Previously, ISS introduced a phosphorescence lifetime imaging (PLIM) module compatible with excitation sources having repetition rates as low as 1 Hz. Recently, ISS developed a new module that enables PLIM using Ti-Sapphire femtosecond oscillators operating at repetition rates of 80-100 MHz, which are typically used in two-photon (2P) microscopy. This new module has been combined with Nikon A1 2-photon microscope and shown capable of lifetime measurements in the range of 100 ns to 100 ms.

Oxygen quenches phosphorescence of probe Oxyphor 2P [1] (kindly provided by the center for Oxygen Imaging by Phosphorescence Quenching, NIH grant U24-EB028941, PI: Dr. Sergei Vinogradov, University of Pennsylvania). When this probe is injected into tissue, oxygen concentration can be imaged with micron-scale resolution using the method of two-photon-excited phosphorescence [2-4] at depths reaching 600µm below the tissue surface. As a demonstration of principle, the probe was injected systemically in a mouse under anesthesia, and the oxygen concentration in the inhaled gas mixture was varied between 15% and 100%. Two-photon PLIM in the brain cortex was performed through a cranial window using a laser emitting at 960 nm. The phosphorescence was detected using a GaAs PMT and a FastFLIM card. The decay times of Oxyphor 2P vary in the range of ~9-38 µs, quantitatively reflecting changes in the brain tissue oxygenation. All institutional IACUC guidelines were followed during the procedure. [Funded by the grant NS083858 from the NIH (PI: Dr. Sergei Kirov, Medical College of Georgia at Augusta University).]

Figures courtesy of Dr. J. Sword, Medical College of Georgia at Augusta University Medical Center, Augusta, GA

ISS Nikon A1 FLIM & PLIM Upgrades Kits provide solutions for:

• FFS (FCS, PCH, RICS, and N&B)
• Scanning FCS
• FLIM/FRET
• PLIM
• Orbital Particle Tracking

References

1. Esipova, T. V.; Barrett, M. J. P.; Erlebach, E.; Masunov, A. E.; Weber, B.; Vinogradov, S. A.;
   Oxyphor 2P: A High-Performance Probe for Deep-Tissue Longitudinal Oxygen Imaging.
   Cell Metabolism 2019, 29 (3), 736-744.
2. Finikova, O. S.; Lebedev, A. Y.; Aprelev, A.; Troxler, T.; Gao, F.; Garnacho, C.; Muro, S.; Hochstrasser, R. M.; Vinogradov, S. A.;
   Oxygen Microscopy by Two-Photon-Excited Phosphorescence.
   ChemPhysChem 2008, 9 (12), 1673-1679.
3. Sakadzic, S.; Roussakis, E.; Yaseen, M. A.; Mandeville, E. T.; Srinivasan, V. J.; Arai, K.; Ruvinskaya, S.; Devor, A.; Lo, E. H.; Vinogradov, S. A., Boas D. A.;
   Two-Photon High-Resolution Measurement of Partial Pressure of Oxygen in Cerebral Vasculature and Tissue.
   Nature Methods 2010, 7 (9), 755-U125.
4. Lecoq, J.; Parpaleix, A.; Roussakis, E.; Ducros, M.; Houssen, Y. G.; Vinogradov, S. A.; Charpak, S.;
   Flow Simultaneous Two-Photon Imaging of Oxygen and Blood in Deep Cerebral Vessels.
   Nature Medicine 2011, 17 (7), 893-U262.