Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo
TL;DR: A small, lightweight two-photon fiberscope is presented and functional imaging of calcium signals in Purkinje cell dendrites in the cerebellum of anesthetized rats is demonstrated.
Abstract: We present a small, lightweight two-photon fiberscope and demonstrate its suitability for functional imaging in the intact brain. Our device consists of a hollow-core photonic crystal fiber for efficient delivery of near-IR femtosecond laser pulses, a spiral fiber-scanner for resonant beam steering, and a gradient-index lens system for fluorescence excitation, dichroic beam splitting, and signal collection. Fluorescence light is remotely detected using a standard photomultiplier tube. All optical components have 1 mm dimensions and the microscope’s headpiece weighs only 0.6 grams. The instrument achieves micrometer resolution at frame rates of typically 25 Hz with a field-of-view of up to 200 microns. We demonstrate functional imaging of calcium signals in Purkinje cell dendrites in the cerebellum of anesthetized rats. The microscope will be easily portable by a rat or mouse and thus should enable functional imaging in freely behaving animals.
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TL;DR: This Primer briefly reviews the general mechanisms of neuronal calcium signaling, and introduces the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices used in freely moving animals.
1,113 citations
Cites background from "Ultra-compact fiber-optic two-photo..."
...Finally, there are increasing efforts directed toward recordings in freely moving animals, involving the development of miniaturized head-mounted imaging devices (Engelbrecht et al., 2008; Flusberg et al., 2008; Helmchen et al., 2001; Sawinski et al., 2009)....
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TL;DR: A miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor enables high-speed cellular imaging across ∼0.5 mm2 areas in active mice and allows concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones.
Abstract: The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals for relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor. This device enables high-speed cellular imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens.
868 citations
Cites methods from "Ultra-compact fiber-optic two-photo..."
...Prior approaches to fluorescence imaging in miniaturized format have generally required accessory, tabletop optical instrumentatio...
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TL;DR: A novel approach is presented by utilizing disordered light within a standard multimode optical fibre for lensless microscopy and optical mode conversion and showing how such control can realize a new form of mode converter and generate various types of advanced light fields such as propagation-invariant beams and optical vortices.
Abstract: technology. This has been necessitated by the need to access hostile or difficult environments in situ and in vivo. strategies to date have included the use of specialist fibres and miniaturized scanning systems accompanied by ingenious microfabricated lenses. Here we present a novel approach for this field by utilizing disordered light within a standard multimode optical fibre for lensless microscopy and optical mode conversion. We demonstrate the modalities of bright- and dark-field imaging and scanning fluorescence microscopy at acquisition rates that allow observation of dynamic processes such as Brownian motion of mesoscopic particles. Furthermore, we show how such control can realize a new form of mode converter and generate various types of advanced light fields such as propagation-invariant beams and optical vortices. These may be useful for future fibre-based implementations of super-resolution or light-sheet microscopy.
575 citations
TL;DR: A new and versatile scanning fiber‐imaging technology is reviewed and its implementation for ultrathin and flexible endoscopy is described, providing new options for in vivo biological research of subsurface tissue and high resolution fluorescence imaging.
Abstract: In modern endoscopy, wide field of view and full color are considered necessary for navigating inside the body, inspecting tissue for disease and guiding interventions such as biopsy or surgery. Current flexible endoscope technologies suffer from reduced resolution when device diameter shrinks. Endoscopic procedures today, using coherent fiber-bundle technology on the scale of 1 mm, are performed with such poor image quality that the clinician's vision meets the criteria for legal blindness. Here, we review a new and versatile scanning fiber-imaging technology and describe its implementation for ultrathin and flexible endoscopy. This scanning fiber endoscope (SFE) or catheterscope enables high-quality, laser-based, video imaging for ultrathin clinical applications, while also providing new options for in vivo biological research of subsurface tissue and high resolution fluorescence imaging.
341 citations
TL;DR: Progress in Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers.
Abstract: Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists.
303 citations
Cites background or methods from "Ultra-compact fiber-optic two-photo..."
...…under exploration have included microelectromechanical systems (MEMS) scanning mirrors (Piyawattanametha et al. 2007, Piyawattanametha et al. 2006) as well as nonresonant (Sawinski & Denk 2007) or resonant vibration of the illumination optical fiber (Engelbrecht et al. 2008, Flusberg et al. 2005b)....
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...F/F (vertical) (panels d–g based on Engelbrecht et al. 2008)....
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...An alternative approach to laser-scanning drives the illumination fiber in a resonant, spiral pattern at frame rates up to 25 Hz (Figure 8d,e) (Engelbrecht et al. 2008)....
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...The small size of GRIN microlenses also permits their incorporation into miniaturized, fiber-optic two-photon microscopes (Engelbrecht et al. 2008, Flusberg et al. 2005b, Göbel et al. 2004, Hoy et al. 2008, Jung et al. 2008, Le Harzic et al. 2008) (see section below on Fiber-Optic Microscopy)....
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...More recent portable two-photon microscopes for brain imaging have had masses as small as ∼1–4 g, in part by employing GRIN microlenses similar to those used for microendoscopy (Engelbrecht et al. 2008, Flusberg et al. 2005b, Göbel et al. 2004, Piyawattanametha et al. 2006)....
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References
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TL;DR: The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation.
Abstract: Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.
8,905 citations
TL;DR: Fundamental concepts of nonlinear microscopy are reviewed and conditions relevant for achieving large imaging depths in intact tissue are discussed.
Abstract: 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.
3,781 citations
TL;DR: In vivo two-photon calcium imaging recordings indicated that whisker deflection-evoked Ca2+ transients occur in a subset of layer 2/3 neurons of the barrel cortex, demonstrating the suitability of this technique for real-time analyses of intact neuronal circuits with the resolution of individual cells.
Abstract: Two-photon calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo. In the mammalian brain, such imaging studies have been restricted largely to calcium recordings from neurons that were individually dye-loaded through microelectrodes. Previous attempts to use membrane-permeant forms of fluorometric calcium indicators to load populations of neurons have yielded satisfactory results only in cell cultures or in slices of immature brain tissue. Here we introduce a versatile approach for loading membrane-permeant fluorescent indicator dyes in large populations of cells. We established a pressure ejection-based local dye delivery protocol that can be used for a large spectrum of membrane-permeant indicator dyes, including calcium green-1 acetoxymethyl (AM) ester, Fura-2 AM, Fluo-4 AM, and Indo-1 AM. We applied this dye-loading protocol successfully in mouse brain tissue at any developmental stage from newborn to adult in vivo and in vitro. In vivo two-photon Ca2+ recordings, obtained by imaging through the intact skull, indicated that whisker deflection-evoked Ca2+ transients occur in a subset of layer 2/3 neurons of the barrel cortex. Thus, our results demonstrate the suitability of this technique for real-time analyses of intact neuronal circuits with the resolution of individual cells.
1,366 citations
TL;DR: A technique for two-photon fluorescence imaging with cellular resolution in awake, behaving mice with minimal motion artifact is reported, demonstrating that running-associated brain motion is limited to approximately 2-5 microm.
1,071 citations
TL;DR: The unique niche that light microscopy occupies in biology is based on the ability to perform observations on living tissue at relatively high spatial resolution, but this resolution is limited by the wavelength of light and does not rival that of electron microscopy.
734 citations