Principle Investigator	Masanobu Kano Visiting Professor at Center for Multidisciplinary Brain Research, National Institute for Physiological Sciences
Keyword for Support Function	Support for using techniques for light microscopes, electron microscopes, magnetic resonance imaging, image analysis and training.
Grant period	FY 2016 - 2021
URL	http://www.nibb.ac.jp/abis/

Light Microscopy

Intravital deep imaging	We support intravital deep tissue imaging using multi-photon excitation microscopy. We can help real-time imaging or 3D imaging of several tissues and diseases in living mouse. For this purpose, we prepared three multi-photon excitation microscopes with original long wavelength excitation and/or adaptive optics systems. In addition, a biomedical confocal laser microscopy, a confocal laser macro zoom integrated microscope, a super-resolution microscopy (SIM) and a light sheet microscope are also available.
Digital light sheet fluorescence microscopy (DLSM)	We support three-dimensional observations using digital light sheet fluorescence microscopy (DLSM). With DLSM, we can observe less faded and less phototoxic biospecimen in a wide field of vision. Our support ranges from selecting the observation method, prepare and capture the images of the samples to visualize the obtained image data for observation of tissues, embryo, live observation of animals, and large samples.
4D Microscopy	Multidimensional timelapse observations over several days will be supported using spinning disc confocal microscopes and multi photon microscopes equipped with incubators. In addition to observation of cells, organs and tissues, observation of living animals will be supported. To realize these observations, we will help with preparation of samples and arrangement of mice expressing fluorescent markers maintained in our institute. We also offer technical support on searching for culture conditions of tissues and imaging conditions.
IR-LEGO microscope	A heat shock response is often used for inducible gene expression of target gene. The heat shock promoter system can control temporal gene expression, while our technology, IR-LEGO (InfraRed Laser-Evoked Gene Operator), enables additionally spatial gene induction in vivo at single-cell level by focusing infrared laser to a target cell. We support your use of this microscope technology from an experimental planning to observation and analysis.
Fluorescence live imaging	We will support fluorescence live imaging of various samples ranging from tissue culture cells to small animals. Transgenic mice expressing fluorescence proteins or biosensors and three-dimensional tissue culture cells are particularly fit to our instruments. The multiphoton excitation microscopes (MPEM) are equipped with vacuum organ stabilizers, auto-tracking system, gas anesthetizer, and electrocardiograph, which enable users to observe mice more than several hours. The incubator-type MPEM multiphoton excitation microscope is versatile to observe three-dimensional tissue culture cells or tissues for days. The MPEM equipped with time-correlated single-photon counting is for the fluorescence lifetime imaging microscopy (FLIM), which can be used to visualize fluorescence resonance energy transfer (FRET) and thereby protein-protein interaction.
In vivo two-photon imaging	Two-photon microscope enables us to observe not only neurons and glial cells in the brain, but also various types of cells and structure, such as bone cells, blood vessels, and immune cells. 1. Visualization of cells in living animals and in vitro systems 2. Visualization of various cells such as neurons, glial cells, blood vessels, intestinal cells and immune cells 3. Long-term imaging of same structures in same animals (e.g. Long-term spines imaging for more than 2 months)
Two-photon Fluorescence Lifetime Imaging Microscopy (2pFLIM)	This facility provides access to a state-of-the-art microscopy (2pFLIM), which enables to detect Fluorescence Resonance Energy Transfer (FRET). The facility also provides image acquisition and analysis services. Cultured cell or tissue are compatible to our microscope. Using 2pFLIM, users can visualize spatiotemporal information of protein-protein interaction and protein activity.
Imaging marine organisms	Marine organisms accompany several difficulties in collection and culturing of marine organisms. We support the imaging of cellular activity in marine organisms, including reproduction, development, behavior and ecology. In particular, high-speed ciliary movement and locomotion of zoospore, spermatozoa, protists and ciliary planktonic larvae are visualized with the dynamics of intracellular calcium. In response to researchers’ request, we handle to visualize other signaling molecules using several types of marine organisms.
Plant imaging analysis	ITbM Live Imaging Center (http://www.itbm.nagoya-u.ac.jp/liveimagingcenter/en/) is an imaging and processing site that provides access to researchers worldwide. We possess a number of leading-edge laser microscopes which allow the following advanced imaging. The imaging subjects cover a wide variety of biological samples. Users will have supports ranging from experimental design to data acquisition by coordinators.
Support using 2-photon-CSU microscopy and advanced laser microscope considering regional support	Nikon Imaging Center (NIC@HU) has continued to support researchers with its equipment such as novel laser-scanning microscopy systems (including multi-point scanning two-photon microscopy system we developed), fluorescence microscopy systems and several analytical software.
Super-resolution microscopy	We will support super-resolution microscope applications including STED (Stimulated Emission Depletion) microscope, SDSRM (Spinning Disk Super-Resolution Microscope), as well as localization microscopy (also known as PALM, STORM or GSDIM).
Chemical probe synthesis	Synthesis, photochemical and physical properties of chemical probes including caged compounds of neurotransmitters, signaling molecules, small molecular weight agonists and inhibitors, quantitative measurement of two-photon action cross-section of caged compounds using a femtosecond pulsed near-IR laser.

Electron Microscopy

High-voltage & Cryo-microscopy	Detailed structures of cultured cells and so on with the sample thickness of several microns can be determined by cryo-microscopy using a high-voltage electron microscope. The native structures of biological specimen can be observed by rapid freezing to trap them in amorphous ice at millisecond order. For reconstruction of the structures by electron tomography and single particle analysis, we support the data collection by cryo-electron microscopy. By electron tomography, the cell structures at nano-meter resolution can be determined. By single particle analysis, the atomic structures of biological complexes with high molecular weight can be determined.
Immunoelectron microscopy (GFP/double labeling)	Using the transmission electron microscopy, we are mainly able to support the green fluorescent protein (GFP)-labeling and double immunohistochemistry (both pre- and post-embedding methods). Most vertebrates are possibly used. We are going to consult about the other fluorescent proteins/variants or model plants.
FIB/SEM	The aim of this support is to assist morphological research using next generation electron microscope, FIB/SEM, FIB/SEM tomography, and also related techniques. The high-resolution images of single sections over wide range area on Epon block surface are obtained in the fundamental methods, and serial images enabling three-dimensional ultrastructure reconstructions, finally analyzing cell and tissue fine structures. Depending on the size of the samples, etc., we may recommend the use of the SBF-SEM method in this support platform in order to make the best use of the advantage of the FIB/SEM.
Volume-CLEM for cultured cell	Correlative light electron microscopy (CLEM) is a powerful tool to observe an identical target using different modalities. Cellular dynamics are able to be observed by various light microscopic technics. Additionally, electron microscopic observation of a same target provides important insights into biological processes and cellular architecture. We are supporting a volume-CLEM observation combined with light microscopic live imaging and latest electron microscopic 3D reconstruction method using focused ion-beam scanning electron microscopy.
Immunoelectron microscopy using Tokuyasu cryosectioning technique	The advantages of immunoelectron microscopy using ultrathin cryosections (Tokuyasu technique) are easy sample preparation, double/multiple labeling, and good preservation of membranes and membranous organelles. Combining colloidal gold- and fluorescent secondary antibodies enables correlative light- and immunoelectron microscopy on cryosections. Furthermore, preservation of fluorescence in the specimens themselves would be another advantage. Novel flat-embedding procedure for ultrathin cryosections can be applied to cultured neurons in situ. For supporting researches using this technique, the information on the specificity of antibodies is essential.
SBF-SEM	In this support, resin-embedded specimens are prepared from biological samples, and serial section images are obtained from large areas (ranging from tens to hundreds of ㎛ 2), using serial block-face scanning electron microscopy (SBF-SEM). Structures of interest will be segmented from the obtained data and complex structures such as cellular processes and 3D parameters such as volume and surface areas can be obtained from the segmented data. The throughput of SBF-SEM is relatively high, and special conductive resin will be used in this support to facilitate acquisition of high quality images from small and thin samples.
ZPC cryoEM	With Zernike phase contrast cryo-electron microscopy, we will support all imaging processes: sample preparation, data collection, image processing, 3D reconstruction, segmentation, movie making, etc. We at first discuss about the project with applicant and decide what kinds of support it needs.
SDS-digested freeze-fracture replica labeling (SDS-FRL)	We would provide researchers the assistance in distribution analysis of biomolecules and for three-dimensional ultrastructural analysis of organelle both at nano-scale spatial resolution by employing SDS-digested Freeze-fracture Replica Labeling (SDS-FRL), preembedding immunoelectron microscopy and reconstruction of target structures from serial electron micrographs. These assistance include advice on experimental plans for ultrastructural analysis, access to special experimental equipment and practical training of researchers for experimental procedures. It is notable that SDS-FRL is unique in its uniform and high-sensitivity detection of membrane molecules over the biological membranes and in two-dimensional visualization of their distribution.
ImmunoEM & Antibody production	①Support for Immunoelectron Microscopy This supports immunoelectron microscopic analyses using pre-embedding and post-embedding methods. Applicants are required to provide specificity-guaranteed antibody and materials to test the specificity. ②Support for High-Quality Antibody Production This supports production of specific antibodies that can not be purchased or obtained by commercial outsourcing. Applicants are required to possess materials to test the specificity and to perform specificity test.
Cryo-EM work flow	This facility provides a precious transmission electron microscope (TEM) incorporating 300kV FEG, Ω-type energy filter, and liquid helium cooled specimen stage. The cryo-TEM can observe the cryo-section of fragile biological samples with extremely reduced beam irradiation damage. And a modified scanning electron microscope (SEM) incorporating liquid nitrogen cooled specimen stage with VCT100 vacuum cryo transfer system, is also provided for the observation of the surface of frozen specimen. This facility can fully support the preparation of cryo-TEM and cryo-SEM samples of biological and/or organic materials.

MRI

Diffusion MRI	For groups of researchers who studies the clinical state of the disease using diffusion MRI adopted from public applicants, we provide research environment for their data analysis or instruct them to analyze their own data. According to their own highly original hypothesis, we will support applicant who analyze their diffusion MRI data. We also presented tutorials with the general participants having applied for subscription in order to expand the range of research activities. We guide participants through a hands-on experience of MRI analysis by demonstration how to analyze practically in the tutorial targeting researchers who do not have sufficient ability of statistical analysis method gathered from across Japan.
Functional MRI	We will support establishing the experimental design of an fMRI study, implementing the experiment and analyzing obtained data. Our facility supports the brain research on 7T, MRS research and technical development for ultra-high field MRI. We respond to a call for support for research using dual fMRI which could simultaneously acquire brain activation from two brains during real-time social interaction.
sMRI/rs-fMRI	We will provide support for researchers working on human MRI studies using T1-weighted structural MRI (sMRI) and resting state fMRI (rs-fMRI). Specific themes of support include: 1) to provide standard protocols for sMRI/rs-fMRI and support image acquisition/quality control; 2) to instruct analysis techniques including voxel-based morphometry (VBM) and automated ROI analysis using FreeSurfer; and 3) to instruct analysis regarding combination of imaging and neurobehavioral and molecular biomarkers. We will also provide advice for ethical issues on conducting MRI studies and managing data, and hold tutorials on MRI analysis in collaboration with Dr. Sadato’s and Dr. Aoki’s groups.

Image Analysis

Development of image processing/analysis algorithms for biological data	We develop image analysis algorithms especially for bio-medical images for extracting morphological features and building mathematical theory underlying that shape. By applying these approaches, we will provide supports for feature extraction and quantification, and image enhancement techniques for bio-medical imaging data. We will also support batch processing of large data set such as images obtained by multi-dimensional time-lapse microscopy.
Software and algorithm for bioimage analysis	We can support image recognition, such as quantification of targets, by pattern recognition and image processing. We will provide advice for appropriate parameters to conduct multiple methods. In response to user’s request, we develop appropriate methods for individual purpose and implement them for analyses. We also can support machine learning and feature extraction.
Electron Microscopic Images	We support image processing, analysis, mainly, for electron microscopic images (TEM, SEM). We have been developing an image analysis system, Eos, which has more than 500 commands and can perform image processing of 2D and 3D images (noise reduction, correction of EM-specific contrast transfer function, atomic modeling), with including 3D reconstruction from 2D images, such as ’single particle analysis’ and ‘electron tomography’. We also support the other useful programs such as RELION and imod, which have been developed by the other researchers in the field of structural biology. We can also develop a new algorithm to solve what you hope.
Consultations, developments and introduction supports of image analysis	We have established consistent research system for bioimage analysis including the development of visualizing techniques (immunoflouro-staining and fluorescent-proteins expressing lines), the capturing of microscopic images and the implementing of original software for image analysis. In this support, we suggest optimum solutions of image analysis to a researcher’s problem and properties of bioimages. In addition, we provide integrated supports considering imaging conditions, working hypothesis and statistical testing.

Training

Light Microscopy	Course Objectives Lectures and practical courses of fluorescence microscopy techniques to analyze localization and mobility of intracellular molecules in living cells. Principles and practices of fluorescence microscopy will be provided. Course Topics/Modules Principles of microscopy, measurements of the point-spread function, introduction of fluorescent reagents into the cell, observation of living cells, time-lapse imaging using wide-field and confocal microscopes, measurements of protein mobility by FRAP, FLIP, photoactivation in living cells.
Light Microscopy	Course Objectives: Contribute to the spread of basic knowledge and skills on light microscopy, especially fluorescence microscopy. Promote knowledge and skills on high-end microscopes with advanced technology, including wide-field deconvolution system, confocal laser scanning microscopes, super resolution microscopes. Course Topics/Modules: Seminars and lectures on basics of light microscopy. Hands on training course of advanced microscopes.
Electron Microscopy	Course Objectives　 Imaging structures of biological samples by electron microscopy remains some difficulties how to use special expensive machines. And there are several steps on the sample preparation, especially on cryo-techniques, such as high pressure freezing, rapid freezing, freeze substitution, cryo-sectioning, and freeze fracture. This facility provides access to the observation with both transmission and scanning electron microscopes, as well as practical training for the preparation of biological samples for electron microscopy. Course Topics/Modules EM techniques from sample preparation to data collection Basic techniques: Conventional EM (Chemical fixation, Embedding, Sectioning, TEM/SEM) Applied techniques: Cryo-EM (High pressure freezing, Freeze fracture, Cryo-sectioning, Cryo-TEM/SEM)
Image Analysis	Course Objectives: Image analysis consists of many steps, such as acquisition of image data by microscopy and output of the processed results. Especially, image processing, which includes image digitization, image enhancement, binarization, image transforms and filtering, is crucial step. In order to obtain an optimal analysis results, it is required to design an effective combination of these techniques based on understanding of image processing theories. Course Topics/Modules This course will cover the basic principles of image processing algorithms and it will give biologists an opportunity to learn how to analyze a wide variety of image data derived from the biological and medical imaging modalities, such as optical and electron micrographs of molecules and cells, radiographic images of tissues and MRI images of brains. Through this course, the participants can learn computational methods for biological image analysis to apply workflows of the analysis techniques to image data in their research.