The Experts below are selected from a list of 493239 Experts worldwide ranked by ideXlab platform
Markita P. Landry - One of the best experts on this subject based on the ideXlab platform.
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dual near infrared two photon Microscopy for deep tissue dopamine nanosensor imaging
Advanced Functional Materials, 2017Co-Authors: Jackson Travis Del Bonisodonnell, Markita P. Landry, Abraham G Beyene, Ian R Mcfarlane, Ralph H Page, Eric G TindallAbstract:A key limitation for achieving deep imaging in biological structures lies in photon absorption and scattering leading to attenuation of fluorescence. In particular, neurotransmitter imaging is challenging in the biologically relevant context of the intact brain for which photons must traverse the cranium, skin, and bone. Thus, fluorescence imaging is limited to the surface cortical layers of the brain, only achievable with craniotomy. Herein, this study describes optimal excitation and emission wavelengths for through-cranium imaging, and demonstrates that near-infrared emissive nanosensors can be photoexcited using a Two-Photon 1560 nm excitation source. Dopamine-sensitive nanosensors can undergo Two-Photon excitation, and provide chirality-dependent responses selective for dopamine with fluorescent turn-on responses varying between 20% and 350%. The Two-Photon absorption cross-section and quantum yield of dopamine nanosensors are further calculated, and a Two-Photon power law relationship for the nanosensor excitation process is confirmed. Finally, the improved image quality of the nanosensors embedded 2-mm-deep into a brain-mimetic tissue phantom is shown, whereby one-photon excitation yields 42% scattering, in contrast to 4% scattering when the same object is imaged under Two-Photon excitation. The approach overcomes traditional limitations in deep-tissue fluorescence Microscopy, and can enable neurotransmitter imaging in the biologically relevant milieu of the intact and living brain.
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dual near infrared two photon Microscopy for deep tissue dopamine nanosensor imaging
bioRxiv, 2017Co-Authors: Jackson Travis Del Bonisodonnell, Markita P. Landry, Abraham G Beyene, Ian R Mcfarlane, Ralph H Page, Eric G TindallAbstract:A key limitation for achieving deep imaging in biological structures lies in photon absorption and scattering leading to attenuation of fluorescence. In particular, neurotransmitter imaging is challenging in the biologically-relevant context of the intact brain, for which photons must traverse the cranium, skin and bone. Thus, fluorescence imaging is limited to the surface cortical layers of the brain, only achievable with craniotomy. Herein, we describe optimal excitation and emission wavelengths for through-cranium imaging, and demonstrate that near-infrared emissive nanosensors can be photoexcited using a Two-Photon 1560 nm excitation source. Dopamine-sensitive nanosensors can undergo Two-Photon excitation, and provide chirality-dependent responses selective for dopamine with fluorescent turn-on responses varying between 20% and 350%. We further calculate the Two-Photon absorption cross-section and quantum yield of dopamine nanosensors, and confirm a Two-Photon power law relationship for the nanosensor excitation process. Finally, we show improved image quality of the nanosensors embedded 2 mm deep into a brain-mimetic tissue phantom, whereby one-photon excitation yields 42% scattering, in contrast to 4% scattering when the same object is imaged under Two-Photon excitation. Our approach overcomes traditional limitations in deep-tissue fluorescence Microscopy, and can enable neurotransmitter imaging in the biologically-relevant milieu of the intact and living brain.
Fritjof Helmchen - One of the best experts on this subject based on the ideXlab platform.
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miniaturization of two photon Microscopy for imaging in freely moving animals
CSH Protocols, 2013Co-Authors: Fritjof Helmchen, Winfried Denk, Jason N D KerrAbstract:This article describes the development and application of miniaturized Two-Photon-excited fluorescence microscopes ("Two-Photon fiberscopes"). Two-Photon fiberscopes have been developed with the aim of enabling high-resolution imaging of neural activity in freely behaving animals. They use fiber optics to deliver laser light for Two-Photon excitation. Their small front piece typically contains a miniature scanning mechanism and imaging optics. Two-Photon fiberscopes can be made sufficiently small and lightweight to be carried by rats and mice and to allow virtually unrestricted movement within a behavioral arena. Typically mounted to the animal's skull above a cranial window, Two-Photon fiberscopes permit imaging of cells down to at least 250 µm below the brain surface (e.g., in rat neocortex). In freely exploring animals, action-potential-evoked calcium transients can be imaged in individual somata of visual cortex neurons bulk-labeled with a calcium indicator. Two-Photon fiberscopes thus enable high-resolution optical recording of neural activity with cellular resolution during natural behaviors.
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deep tissue two photon Microscopy
Nature Methods, 2005Co-Authors: Fritjof Helmchen, Winfried DenkAbstract:With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional⎯including confocal⎯fluorescence Microscopy. Nonlinear optical Microscopy, in particular two photon–excited fluorescence Microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-Photon Microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear Microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.
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nonlinear anisotropic diffusion filtering of three dimensional image data from two photon Microscopy
Journal of Biomedical Optics, 2004Co-Authors: Philip J Broser, Fritjof Helmchen, R Schulte, S Lang, Arnd Roth, Jack Waters, Bert Sakmann, Gabriel WittumAbstract:Two-Photon Microscopy in combination with novel fluorescent labeling techniques enables imaging of three-dimensional neuronal morphologies in intact brain tissue. In principle it is now possible to automatically reconstruct the dendritic branching patterns of neurons from 3-D fluorescence image stacks. In practice however, the signal-to-noise ratio can be low, in particular in the case of thin dendrites or axons imaged relatively deep in the tissue. Here we present a nonlinear anisotropic diffusion filter that enhances the signal-to-noise ratio while preserving the original dimensions of the structural elements. The key idea is to use structural information in the raw data—the local moments of inertia—to locally control the strength and direction of diffusion filtering. A cylindrical dendrite, for example, is effectively smoothed only parallel to its longitudinal axis, not perpendicular to it. This is demonstrated for artificial data as well as for in vivo Two-Photon microscopic data from pyramidal neurons of rat neocortex. In both cases noise is averaged out along the dendrites, leading to bridging of apparent gaps, while dendritic diameters are not affected. The filter is a valuable general tool for smoothing cellular processes and is well suited for preparing data for subsequent image segmentation and neuron reconstruction.
Sei Kwang Hahn - One of the best experts on this subject based on the ideXlab platform.
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two photon Microscopy of a flt1 peptide hyaluronate conjugate
Nanomedicine: Nanotechnology Biology and Medicine, 2015Co-Authors: Songeun Beack, Jun Ho Lee, Ki Hean Kim, Jun-sub Choi, Choun-ki Joo, Hyemin Kim, Sei Kwang HahnAbstract:Aim: Two-Photon Microscopy was performed to visualize ocular distribution of Flt1 peptide–hyaluronate (HA) conjugate micelles for eye drop treatment of corneal neovascularization. Materials & methods: Flt1 peptide–HA conjugate micelles were topically administered to the eye for Two-Photon Microscopy and antiangiogenic effect assessment after silver nitrate cauterization. Results: In vivo Two-Photon Microscopy revealed that Flt1 peptide–HA conjugate micelles were absorbed and remained on the corneal epithelia with an increased residence time, facilitating the corneal delivery of carboxyfluorescein succinimidyl ester (CFSE) as a model drug. Furthermore, repeated eye drops of Flt1 peptide–HA conjugate micelles showed comparable therapeutic effect to the subconjunctival injection on the corneal neovascularization. Discussion & conclusion: We confirmed the feasibility of Flt1 peptide–HA conjugate micelles for eye drop treatment of corneal neovascularization.
David Kleinfeld - One of the best experts on this subject based on the ideXlab platform.
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ultra large field of view two photon Microscopy
Optics Express, 2015Co-Authors: Philbert S Tsai, Chris B Schaffer, Celine Mateo, Jeffrey J Field, Matthew E Anderson, David KleinfeldAbstract:We present a Two-Photon microscope that images the full extent of murine cortex with an objective-limited spatial resolution across an 8 mm by 10 mm field. The lateral resolution is approximately 1 µm and the maximum scan speed is 5 mm/ms. The scan pathway employs large diameter compound lenses to minimize aberrations and performs near theoretical limits. We demonstrate the special utility of the microscope by recording resting-state vasomotion across both hemispheres of the murine brain through a transcranial window and by imaging histological sections without the need to stitch.
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two photon Microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain
Journal of Cerebral Blood Flow and Metabolism, 2012Co-Authors: Andy Y Shih, Chris B Schaffer, Jonathan D Driscoll, Patrick J Drew, Nozomi Nishimura, David KleinfeldAbstract:The cerebral vascular system services the constant demand for energy during neuronal activity in the brain. Attempts to delineate the logic of neurovascular coupling have been greatly aided by the advent of Two-Photon laser scanning Microscopy to image both blood flow and the activity of individual cells below the surface of the brain. Here we provide a technical guide to imaging cerebral blood flow in rodents. We describe in detail the surgical procedures required to generate cranial windows for optical access to the cortex of both rats and mice and the use of Two-Photon Microscopy to accurately measure blood flow in individual cortical vessels concurrent with local cellular activity. We further provide examples on how these techniques can be applied to the study of local blood flow regulation and vascular pathologies such as small-scale stroke.
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two photon Microscopy reveals that cortical blood flow reverses direction at the first branch that lies downstream from a localized photothrombotic clot
Conference on Lasers and Electro-Optics, 2004Co-Authors: Chris B Schaffer, N Nishimura, Beth Friedman, Philbert S Tsai, Lee F Schroeder, Ford F Ebner, Patrick D Lyden, David KleinfeldAbstract:Using Two-Photon Microscopy, we quantify changes in blood flow after photothrombotic occlusion of individual blood vessels in rat neocortex, and find that flow reverses direction at the first branch that lies downstream from localized clots.
Ryosuke Kawakami - One of the best experts on this subject based on the ideXlab platform.
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in vivo two photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high peak power gain switched laser diode
Biomedical Optics Express, 2015Co-Authors: Ryosuke Kawakami, Yuta Kusama, Yuichi Kozawa, Shunichi Sato, Hiroyuki Yokoyama, Kazuaki Sawada, Yicheng Fang, Shinya Kanazawa, Tomomi NemotoAbstract:In vivo Two-Photon Microscopy is an advantageous technique for observing the mouse brain at high resolution. In this study, we developed a Two-Photon Microscopy method that uses a 1064-nm gain-switched laser diode-based light source with average power above 4 W, pulse width of 7.5-picosecond, repetition rate of 10-MHz, and a high-sensitivity photomultiplier tube. Using this newly developed Two-Photon microscope for in vivo imaging, we were able to successfully image hippocampal neurons in the dentate gyrus and obtain panoramic views of CA1 pyramidal neurons and cerebral cortex, regardless of age of the mouse. Fine dendrites in hippocampal CA1 could be imaged with a high peak-signal-to-background ratio that could not be achieved by titanium sapphire laser excitation. Finally, our system achieved multicolor imaging with neurons and blood vessels in the hippocampal region in vivo. These results indicate that our Two-Photon Microscopy system is suitable for investigations of various neural functions, including the morphological changes undergone by neurons during physiological phenomena.
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7-ps optical pulse generation from a 1064-nm gain-switched laser diode and its application for Two-Photon Microscopy.
Optics express, 2014Co-Authors: Yuta Kusama, Yuichiro Tanushi, Masami Yokoyama, Ryosuke Kawakami, Terumasa Hibi, Yuichi Kozawa, Tomomi Nemoto, Shunichi Sato, Hiroyuki YokoyamaAbstract:In this study, we investigated the picosecond optical pulse generation from a 1064-nm distributed feedback laser diode under strong gain switching. The spectrum of the generated optical pulses was manipulated in two different ways: (i) by extracting the short-wavelength components of the optical pulse spectrum and (ii) by compensating for spectral chirping in the extracted mid-spectral region. Both of these methods shortened the optical pulse duration to approximately 7 ps. These optical pulses were amplified to over 20-kW peak power for Two-Photon Microscopy. We obtained clear Two-Photon images of neurons in a fixed brain slice of H-line mouse expressing enhanced yellow fluorescent protein. Furthermore, a successful experiment was also confirmed for in vivo deep region H-line mouse brain neuron imaging.
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Visualizing hippocampal neurons with in vivo Two-Photon Microscopy using a 1030 nm picosecond pulse laser
Scientific Reports, 2013Co-Authors: Ryosuke Kawakami, Shunichi Sato, Hiroyuki Yokoyama, Kazuaki Sawada, Aya Sato, Terumasa Hibi, Yuichi Kozawa, Tomomi NemotoAbstract:In vivo Two-Photon Microscopy has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than several hundred micrometers from the brain surface. We developed a novel semiconductor-laser-based light source with a wavelength of 1030 nm that can generate pulses of 5-picosecond duration with 2-W output power and a 20-MHz repetition rate. We also developed a system to secure the head of the mouse under an upright microscope stage that has a horizontal adjustment mechanism. We examined the penetration depth while imaging the H-Line mouse brain and demonstrated that our newly developed laser successfully images not only cortex pyramidal neurons spreading to all cortex layers at a superior signal-to-background ratio, but also images hippocampal CA1 neurons in a young adult mouse.
