Browsing by Subject "Optical parametric amplifier"
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Item Extending the depth limit of multiphoton microscopy for in vivo brain imaging(2016-08) Miller, David Roger; Dunn, Andrew Kenneth, 1970-; Yeh, Hsin-Chih "Tim"The benefit of high-resolution imaging provided by optical microscopy has resulted in many discoveries in both biology and neuroscience. Two-photon fluorescence microscopy (2PM) is widely used for in vivo brain imaging to visualize cerebral vasculature and neuronal physiology. Conventional 2PM using titanium-doped sapphire oscillators is typically limited to imaging depths less than 600 um due to their short excitation wavelengths (700 -1,000 nm) and low pulse energy (~10 nJ). The ideal approach for deep imaging is to use both longer wavelengths to reduce the effects of scattering by heterogeneous brain tissue and higher energy pulses such that more photons reach the excitation volume at deeper tissue depths. I perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier (OPA). The OPA outputs 400 nJ pulse energies and is tunable from 1,100 to 1,400 nm. The tunability of the OPA is an advantage over other high-pulse-energy lasers because the OPA wavelength can be matched to the peak absorption of the target fluorophore, enabling the excitation of numerous different fluorophores. I demonstrate an imaging depth of 1,200 um in vasculature labeled with Texas Red and 1,160 um in neurons labeled with tdTomato, and perform line scans as deep as 1200 um to measure the blood flow speed in a single capillary. I also demonstrate deep-tissue imaging using Indocyanine Green (ICG), which is FDA approved and a promising route to translate multiphoton microscopy to human applications.Item In vivo optical imaging to investigate neurovascular structure and cerebral hemodynamics(2018-05-01) Miller, David Roger; Dunn, Andrew Kenneth, 1970-; Milner, Thomas E; Yeh, Hsin-Chih "Tim"; Jones, Theresa AThe ability to visualize structural features of the brain and associated functional information has fueled a revolution in our understanding of the brain. The optical technique two-photon microscopy (2PM) is widely used to study individual neural circuits and blood vessel networks in vivo because it is minimally invasive and provides three-dimensional images with cellular resolution. There is rising interest from neuroscientists for the ability to extend the traditional imaging depth of 2PM, which is typically limited to ∼500 μm below the surface of the brain. In this dissertation, I detail the development of a novel laser source that enables deep-tissue in vivo multiphoton microscopy imaging of blood vessel networks and neurons. Using an excitation wavelength near 1,300 nm at which scattering in tissue is minimized, I demonstrate the ability to chronically study vascular morphology and dynamics as well as neuron morphology at imaging depths of 1 mm and beyond.