The goal of this research program is to optimize three-photon fluorescence microscopy (3PM) for large scale, noninvasive, volumetric imaging of neuronal activity. To leverage the superb performance of green-fluorescent protein based genetically engineered Ca-probes (e.g., GCaMPs), 3PM at the 1300-nm spectral window will be developed, which not only preserves the tissue penetration capability of 3PM at the longer excitation wavelength but also enables a wide variety of blue and green fluorophores, including a number of fluorescent proteins and Ca-indicators, to be excitable via three-photon excitation. To improve the signal-to-noise ratio (SNR) so that a practical frame rate can be achieved for imaging dynamic brain activity even at a penetration depth of 1.1 mm or beyond, new objective lenses will be designed and fabricated that will collect the signal efficiently at depth. In additin, the lens design will also support convenient integration with adaptive optics (AO), with the goal of making AO a routine imaging tool in a neuroscience lab. To improve both SNR and spatial resolution, AO in 3PM at 1300 nm will be employed. The impact of AO for increasing signal generation is significantly higher for 3PM than 2PM because of the higher order nonlinear process. The impact of AO is also expected to increase with increasing imaging depth.
The aim i s to achieve close to diffraction limited spatial resolution for 3PM at 1300 nm, which will be sufficient to resolve individual dendrite. A strong interdisciplinary research team has been assembled, including participants from both industry and academia, to perform the research and development. The successful completion of this program will have a broad impact on neuroscience where high-resolution, high speed imaging deep within an intact mouse brain is required.
The proposed program, if successfully completed, will lead to a practical tool for large scale, noninvasive, volumetric recording of brain activity. The successful completion of this program will have a broad impact on a wide variety of biological and biomedical research fields where high-resolution imaging deep within intact tissue is required.
|Sinefeld, David; Paudel, Hari P; Ouzounov, Dimitre G et al. (2015) Adaptive optics in multiphoton microscopy: comparison of two, three and four photon fluorescence. Opt Express 23:31472-83|