In this project we address the need in probes for two-photon microscopy - the leading imaging technique for dynamic visualization and quantification of biological processes in vivo in 3D with micron-scale spatial resolution. We propose to develop a new class of multiphoton probes, termed dendritic UCNPs, which comprise lanthanide-based up converting nanoparticles (UCNPs) and dendritic ligands. The key advantage of UCNPs is their enormously high multiphoton absorption cross-sections, which exceed those of the most efficient multiphoton probes available today by several orders of magnitude. Recently, we demonstrated that due to this remarkable property, in vivo two-photon depth-resolved microscopic imaging with UCNPs can be accomplished using simple low-power continuous-wave (CW) infrared light sources, which is in contrast to conventional two-photon experiments requiring very expensive pulsed femtosecond lasers. This remarkable advantage comes on top of other benefits of UCNPs, which include record-high photo stability, zero background fluorescence (due to CW infrared excitation) and greatly diminished risk of photo damage. However, lack of robust methods of UCNP solubilization and functionalization has been a major obstacle preventing their inclusion into the toolkit of modern imaging methods. We propose to solve this problem by using dendritic macromolecules. Our key proposition is that modification of UCNP surfaces with hydrophilic shape-persistent dendrimers will make up an efficient and general route to soluble bio-compatible UCNPs, whose luminescence will be coupled to analyte detection via UCNP-to-dendrimer excitation energy transfer (EET). Our approach capitalizes on unique structural features of dendritic architecture, i.e. intrinsic polyvalency and pseudo- globular shape. Both "colorless" probes for morphologic angiographic two-photon imaging and dedicated probes for imaging of specific analytes (pH and Ca2+) will be developed. To test the probes we will perform: (a) angiographic imaging in vivo in rodent brain, determining changes in blood rheology upon functional stimulation;(b) simultaneous in vivo multiphoton imaging of alterations in tissue pH and partial pressure of oxygen (pO2) in stroke rodent models;d) imaging of extracellular Ca2+ flux in mouse neurohypophysis upon electrical stimulation. All these experiments will differ from conventional multiphoton imaging in that the cost of the excitation sources will be lower by ca 1000 fold. These applications will demonstrate the ability of the new probes to a) replace currently used expensive multiphoton setups;and b) go beyond and address questions and hypotheses in neuroscience for which no alternative solutions are currently available.
In this project we propose to develop a new class of optical probes for two-photon microscopy. The new probes will be excitable in the infrared by low-power continuous wave (CW) sources and emit visible light, permitting depth-resolved high-resolution microscopic imaging. The probes will have very high photo stability and generate zero-background luminescence. These probes will comprise lanthanide upconverting nanoparticles (UCNPs) and shape-persistent dendrimers. We will develop dendritic UCNPs for morphologic angiographic imaging as well for multiphoton pH and Ca2+ sensing. The probes will be validated in: (i) measurements of changes in cerebral blood rheology upon functional activation;(ii) recording changes in pH, blood flow and oxygenation in stroke-related conditions;and (iii) quantification of extracellular Ca2+ upon electrical stimulation of mouse neurohypophysis. These experiments will validate the new probe technology and provide valuable physiological information, not currently obtainable by any other method.