Two-photon excitation fluorescence microscopy and its application in functional connectomics

Nemoto, Tomomi; Kawakami, Rika; Hibi, Terumasa; Iijima, Ko-Ichiro; Otomo, Kohei

doi:10.1093/jmicro/dfu110

Cited by 15 publications

(9 citation statements)

References 44 publications

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“…For long-term imaging, we combined a PEO-CYTOP nanosheet with a glass cover slip, to prevent the PEO-CYTOP nanosheet from floating and breaking. Finally, a 35 mm disposable dish was fixed onto the head of the mouse, to preserve the immersion solution and suspend the head from the adapter stage, as reported previously (Kawakami et al, 2013(Kawakami et al, , 2015. Moreover, for long-term imaging, we set a glass cover slip (a circle with a diameter of 4.2 mm, as shown in Figures 5 and S6, S7; a circle with a diameter of 2.7 mm, as shown in Figure 6; and a 5 × 6 mm rectangle, as shown in Figure S5; #1S, approximately 0.17 mm in thickness, Matsunami Glass Ind., Ltd, Japan) on top of the PEO-CYTOP nanosheet, followed by similar procedures.…”

Section: Cranial Window Surgerymentioning

confidence: 99%

“…In contrast, in vivo two-photon fluorescence microscopy visualizes neural activities and ensembles at subcellular resolution in living mammalian brains across multiple regions ( Theer et al., 2003 ; Kondo et al., 2017 ; Ebina et al., 2018 ). This microscopic technique is used especially in deeper regions of the living brain because the near-infrared laser light pulses employed for two-photon excitation are less scattered and absorbed than is the excitation laser light in the visible range that is usually employed in confocal microscopy ( Denk et al., 1990 ; Helmchen and Denk, 2005 ; Nemoto et al., 2015 ).…”

Section: Introductionmentioning

confidence: 99%

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PEO-CYTOP Fluoropolymer Nanosheets as a Novel Open-Skull Window for Imaging of the Living Mouse Brain

Takahashi,

Zhang,

Kawakami

et al. 2020

iScience

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Summary In vivo two-photon deep imaging with a broad field of view has revealed functional connectivity among brain regions. Here, we developed a novel observation method that utilizes a polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet with a thickness of ∼130 nm that exhibited a water retention effect and a hydrophilized adhesive surface. PEO-CYTOP nanosheets firmly adhered to brain surfaces, which suppressed bleeding from superficial veins. By taking advantage of the excellent optical properties of PEO-CYTOP nanosheets, we performed in vivo deep imaging in mouse brains at high resolution. Moreover, PEO-CYTOP nanosheets enabled to prepare large cranial windows, achieving in vivo imaging of neural structure and Ca 2+ elevation in a large field of view. Furthermore, the PEO-CYTOP nanosheets functioned as a sealing material, even after the removal of the dura. These results indicate that this method would be suitable for the investigation of neural functions that are composed of interactions among multiple regions.

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Section: Cranial Window Surgerymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

PEO-CYTOP Fluoropolymer Nanosheets as a Novel Open-Skull Window for Imaging of the Living Mouse Brain

Takahashi,

Zhang,

Kawakami

et al. 2020

iScience

Self Cite

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“…, ion transport, membrane potential), and through chemical or environmental parameters ( e.g. , mechanical strains, pH) or even potentially connectomics (Nemoto et al 2015). …”

Section: Resultsmentioning

confidence: 99%

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells

Ricard

Arroyo

et al. 2018

Brain Struct Funct

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Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

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“…A 2-photon microscope (2-P) relies on a nonlinear excitation reaction via a 2-photon excitation process (Nemoto et al 2015). Some advantages include increased penetration depth, lower laser toxicity, and compatibility with a second harmonic generation (SHG) signal.…”

Section: Two-photon Microscopementioning

confidence: 99%

Tissue Clearing and Its Application to Bone and Dental Tissues

Jing

Luo

et al. 2019

J Dent Res

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Opaqueness of animal tissue can be attributed mostly to light absorption and light scattering. In most noncleared tissue samples, confocal images can be acquired at no more than a 100-µm depth. Tissue-clearing techniques have emerged in recent years in the neuroscience field. Many tissue-clearing methods have been developed, and they all follow similar working principles. During the tissueclearing process, chemical or physical treatments are applied to remove components blocking or scattering the light. Finally, samples are immersed in a designated clearing medium to achieve a uniform refractive index and to gain transparency. Once the transparency is reached, images can be acquired even at several millimeters of depth with high resolution. Tissue clearing has become an essential tool for neuroscientists to investigate the neural connectome or to analyze spatial information of various types of brain cells. Other than neural science research, tissue-clearing techniques also have applications for bone research. Several methods have been developed for clearing bones. Clearing treatment enables 3-dimensional imaging of bones without sectioning and provides important new insights that are difficult or impossible to acquire with conventional approaches. Application of tissue-clearing technique on dental research remains limited. This review will provide an overview of the recent literature related to the methods and application of various tissue-clearing methods. The following aspects will be covered: general principles for the tissue-clearing technique, current available methods for clearing bones and teeth, general principles of 3-dimensional imaging acquisition and data processing, applications of tissue clearing on studying biological processes within bones and teeth, and future directions for 3-dimensional imaging.

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Two-photon excitation fluorescence microscopy and its application in functional connectomics

Cited by 15 publications

References 44 publications

PEO-CYTOP Fluoropolymer Nanosheets as a Novel Open-Skull Window for Imaging of the Living Mouse Brain

PEO-CYTOP Fluoropolymer Nanosheets as a Novel Open-Skull Window for Imaging of the Living Mouse Brain

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells

Tissue Clearing and Its Application to Bone and Dental Tissues

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