Ryosuke Kawakami

Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser

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.

A rapid optical clearing protocol using 2,2'-thiodiethanol for microscopic observation of fixed mouse brain.

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2′-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.

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

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.

7-ps optical pulse generation from a 1064-nm gain-switched laser diode and its application for two-photon microscopy

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.

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

Two-photon excitation fluorescence microscopy has become widely used in various life science fields in this decade. In the field of neuroscience in particular, in vivo two-photon microscopy has provided vital information on neural activity and brain function. In the current era of connectomics, visualization of the morphology and activity of numerous neurons in ever larger regions of the living brain are required within short periods. Based on this viewpoint, we discuss the fundamentals, advantages and potential of two-photon excitation fluorescence microscopy for the investigation of neural circuit functions.

Opposing roles for SNAP23 in secretion in exocrine and endocrine pancreatic cells

Kunii et al. reveal that the SNARE protein SNAP23 plays distinct roles in the secretion of amylase in exocrine cells and of insulin in endocrine cells the pancreas and show that MF286, a novel inhibitor of SNAP23, may be a new drug candidate for diabetes.

Multi-point Scanning Two-photon Excitation Microscopy by Utilizing a High-peak-power 1042-nm Laser

The temporal resolution of a two-photon excitation laser scanning microscopy (TPLSM) system is limited by the excitation laser beams scanning speed. To improve the temporal resolution, the TPLSM system is equipped with a spinning-disk confocal scanning unit. However, the insufficient energy of a conventional Ti:sapphire laser source restricts the field of view (FOV) for TPLSM images to a narrow region. Therefore, we introduced a high-peak-power Yb-based laser in order to enlarge the FOV. This system provided three-dimensional imaging of a sufficiently deep and wide region of fixed mouse brain slices, clear four-dimensional imaging of actin dynamics in live mammalian cells and microtubule dynamics during mitosis and cytokinesis in live plant cells.

Fluoropolymer Nanosheet as a Wrapping Mount for High-Quality Tissue Imaging

In the field of biological microscopy technology, it is still a practical challenge to obtain high-quality tissue images, due to the tissue desiccation that occurs during observations without an effective sample mounting. Inspired by the use of plastic food wrap, this study proposes the use of polymer thin films (also known as nanosheets) to fix the tissue samples. Water-repellent nanosheets composed of the amorphous fluoropolymer CYTOP are prepared with adjustable thicknesses and their hydrophobicity, transparency, and adhesion strength are evaluated. They show excellent water-retention effect and work well for sample fixation. By wrapping cleared mouse brain slices with a 133 nm thick CYTOP nanosheet, this study achieves high spatial resolution neuron images while scanning over a large area for a long period of time. No visible artifacts arising from sample shrinkage can be detected. This study also expects that nanosheet wrapping could be effective over a longer time span by combination with conventional agarose embedding.

Visualizing in vivo brain neural structures using volume rendered feature spaces

BACKGROUND Dendrites of cortical neurons are widely spread across several layers of the cortex. Recently developed two-photon microscopy systems are capable of visualizing the morphology of neurons within deeper layers of the brain and generate large amounts of volumetric imaging data from living tissue. METHOD For visual exploration of the three-dimensional (3D) structure of dendrites and the connectivity among neurons in the brain, we propose a visualization software and interface for 3D images based on a new transfer function design using volume rendered feature spaces. This software enables the visualization of multidimensional descriptors of shape and texture extracted from imaging data to characterize tissue. It also allows the efficient analysis and visualization of large data sets. RESULTS We apply and demonstrate the software to two-photon microscopy images of a living mouse brain. By applying the developed visualization software and algorithms to two-photon microscope images of the mouse brain, we identified a set of feature values that distinguish characteristic structures such as soma, dendrites and apical dendrites in mouse brain. Also, the visualization interface was compared to conventional 1D/2D transfer function system. CONCLUSIONS We have developed a visualization tool and interface that can represent 3D feature values as textures and shapes. This visualization system allows the analysis and characterization of the higher-dimensional feature values of living tissues at the micron level and will contribute to new discoveries in basic biology and clinical medicine.

In Vivo Imaging of All Cortical Layers and Hippocampal CA1 Pyramidal Cells by Two-Photon Excitation Microscopy

Conventional in vivo two-photon microscopy has revealed vital information about neural activity in relation to brain function, despite its limitations in imaging events at depths greater than several hundred micrometers from the surface of the brain. Here, we developed a novel two-photon microscope consisting of a 1064-nm gain-switched laser diode-based light source with average power above 4 W, pulse width of 7.5 ps, repetition rate of 10 MHz, and a high-sensitivity photomultiplier tube for efficient detection of fluorescence. By applying this newly developed two-photon microscope to in vivo imaging, we were able to successfully visualize hippocampal neurons in dentate gyrus and panoramic views of CA1 pyramidal neurons and cerebral cortex, in both young adult and adult mice. Fine structures of dendrites in CA1 neurons could be visualized with a high peak signal-to-background ratio that could not be achieved by titanium sapphire laser excitation. We hope that our two-photon microscopy system will be applicable to investigations of various neural functions, including the morphological changes undergone by neurons during physiological phenomena.