Browsing by Subject "Multiphoton microscopy"
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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 Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy(2020-06-22) Hassan, Ahmed Moustafa; Dunn, Andrew Kenneth, 1970-; Harris, Kristen; Milner, Thomas; Yeh, Hsin-Chih (Tim)Multiphoton microscopy is a powerful optical imaging modality renowned for its non-invasive nature and relatively affordable characteristics. In particular, it has found its niche in neuroimaging due to its ability to probe in vivo biological processes in scattering brain tissue approaching millimeter depths with cellular resolution. However, the brain is a large and complex organ, and in order to fully understand its heterogeneous architecture and associated functional roles, several distal regions must be imaged simultaneously. Moreover, due to the critical implications of organelle features in various macroscale processes, whole-brain imaging at subcellular resolution scales presents itself as one of the outstanding challenges faced by the neuroscientific community today. Primarily, this research aims to expand the depth, field-of-view, and temporal throughput of multiphoton microscopy to enable large volume imaging of microvasculature at greater acquisition speeds. To accomplish this, we combine multi-faceted efforts focused on the engineering and development of advanced multiphoton microscopy techniques and technologies. This includes the characterization of novel contrast agents, the optimization of scan system optics, and the integration of high-repetition rate lasers with a resonant galvanometer. In addition, we develop a two-color imaging system capable of enhancing excitation efficiency, improving signal-to- background ratio, and further extending imaging depth. Finally, we present a novel application for two-color non-degenerate mode mixing to effectively circumvent the diffraction-limited nature of optical resolution and enable subcellular imaging. Collectively, these efforts advance the state-of-the art of multiphoton microscopy for routine cerebrovascular and neuroimaging.