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Plant Proteomics and Metabolomics

Plant Cell Biology

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Homepage > Core Facilities > Plant Cell Biology

 Introduction  Equipment  Service & Fees  Technical resources  Workshops / Seminars  Publications  Contact
Introduction Top

Development of a multicellular organism is a complicated process that requires both developmental/environmental signal perception and proper spatiotemporal regulation of many cellular processes involved in the differentiation of diverse cell types. Some of these key cellular processes include endomembrane trafficking and the cytoskeleton. However, our knowledge of the regulation of these cellular processes and their linkage to developmental patterns is scarce. Recent advances in cell biological technique such as advanced imaging technologies enable us to assess those fundamental questions in the field of biology. “Seeing is believing” is a common proverb in biological sciences, and advances in innovative imaging tools such as novel molecular markers along with the acquisition of the state of the art microscopes will generate unprecedented research opportunities for biologists.

In the Cell Biology Core facility, we provide advanced cell biological equipments as well as technical support to facilitate research activity of researchers in the stress center.

The major functions of the core are as follows:

1. “Provide access to imaging technique.” We obtain/update our instruments to meet with researchers demand. We will provide frequent training sessions and consultation service on research plans to enable researchers.
2. “Maintenance and operation.” We provide reservation/log system to enable efficient use of instruments in the core facility. We will keep our machines in best condition for researchers.
3. “Imaging technology development” We work on imaging technology development to enable customized experiments for research activities in the stress center.

Equipment Top

Intavis In situ pro VSi

Cell biological experiments such as in situ mRNA hybridization or protein immunohistochemistry require multiple liquid changing processes such as wash steps and blocking/antibody incubation steps. Those processes are not only time consuming but also have great potential to cause human errors. In situ pro VSi is an automated liquid handling machine for both in situ mRNA hybridization and protein immunohistochemistry. The machine can handle multiple probes in parallel for high throughput experiments. It equipped with modules for whole-mount samples or sections for adapting broad range of experiments.


Hitachi tabletop SEM TM-3000

Scanning electron microscopy (SEM) detects surface morphology in stereoscopic detail with images in contrast due to different average atomic number composition within the sample. TM-3000 is a user-friendly SEM machine without need of sample preparation steps usually require for SEM imaging.



        

BD FACS ARIA III

With the advancement of “Omics -based studies” such as transcriptome analysis and proteome analysis, it is particularly important to collect groups of biological samples based on their characteristics. Fluorescence activated cell sorting (FACS) is a methodology based on flow-cytometry which enables researchers to selectively collect individual cells into several groups based on their light scattering and fluorescent characteristics. In a modern flow cytometers with multiple lasers and detector combinations, more than several thousands of cells par seconds are sorted inside flow seeds and individually assessed for their characteristics. With the addition of cell sorting capabilities, FACS can separate heterogeneous mixture of biological samples into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. In the plant research field, FACS is used for number of studies to collect tissue specific samples by sorting samples from transgenic lines with differential expression pattern of fluorescent protein such as individual cell layer specific GFP expressing lines in root or shoot.
Our FACS machine is equipped with 3 lines of lasers (375 nm, 488 nm and 633 nm) for flexible choices of combination of fluorescent molecules in differential sorting and high-speed main sorting unit with gel-coupled Cuvette flow cell to ensure optimal resolution and sensitivity. The machine is also equipped with sort collection chamber holder for up to 4-way sorting. The maximum analysis speed is up to 100,000 cells par second, while the maximum sorting speed up to 70,000 cells par second. The ability to selectively collect group of cells with its gene expression patterns is particularly important for plant stress researches, as there are number of stress responsive genes already known and can be used as basis for future omics-based analysis.


Hitachi HT7700 Transmission Electron Microscope

The model HT7700 TEM has been designed so that a wide visual field can be obtined in the low and medium magnification range frequently used for research in medical science and biology, and for research and development of pharmaceuticals, polymer materials, etc., and so that biological spciments can be oberserved with a high contrast. Accelerating voltage is 120kV at maximum. Focal distance is selectively usable between high-resolution and high-contrast applications by means of a double-gap objective lens. Both the TEM main unit and the standard-equipped digital camera system (AMT-made) are integrally contolled by one personal computer. On the liquid crystal display, magnification and other observation conditions are displayed and controlled. In addition, TEM images recorded with the digital camera are displayed on the same LCD. The auto focus function is incorporated as standard. The mounted digital camera is capable of displaying a maximum of 18 frames per second, so you can carry out focu adjustment and astigmatism correction while watching the LCD and save an image as is. Thanks to the high sensitivity of the camera, image observation can be started from an electron beam dose as low as 10-12 A/cm2 orders where nothing can be seen on the fluorescence screen.Specimen damage due to electron beam irradiation can thus bw minimized. 


Leica TCS SP8 SMD

Single molecule detection(SMD) and analysis is an elegant way to examine dynamics and interactions inside cellular systems. The advancement in detector sensitivity as well as innovation in pulsed laser light source enable researchers to perdorm SMD experiments, which detect cellular events in aprecise quantitative manner. SMD experimentd can be mainly divided into 2 categories, FCS(fluorescence correlation spectroscopy) and FLIM (fluerescence lifetime imaging). FCS measures fluctuations of fluerescence intensity in a sub femto litre volume to detect such parameters as the diffusion time, number of molecules or dark states of fluoregation states, binding and biochemical kinetics, both in vitro as well as in vivo. The fluorescence lifetime is a measure of how long a fluorophore remains on average in its excited state before returning to the ground atate by emitting a fluorescence photon. A fluorescence lifetime of a molecule is its intrinsic nature, and slao depends on its microenvitonment such as pH. FLIM combines lifetime measurements with imaging: lifetimes obtaied for each image pixle are color coded topreduce additional image contrast. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information about its biochemical status or nano environment, and used as a method to quantitatively detect changes in environment or biding of flurescent preoteins through FLIM-FRET measurement.
The SMD system requires a confocal microscopy setting along with highly sensitive detector, pulsed laser light source and atrigger unit to synchronize all the components. Our system combines the latest conforcal system from Leica, white light laser and 440 nm laser as pulsed laser light source and detector trigger unit from Pico Quant, which is the leading company of SMD equipments. The system has 2 normal PMT detectors and 1 HyD detector, which enables gated imaging to distinguish true signal from autofluorescence. It has additional 405 nm laser for DAPI near UV imaging. The tunable WLL laser and prism based detector enables free combination of both excitation wavelength and emission range for highly flexible setup for multi-color imaging. This system will be useful for researchers who will test in vivo protein interactions, and quite useful to determine dynamic regulation of protein interaction during plant stress responses as well as plant growth and development. It will also be an important tool to test protein iteractions in nucleus, which is important for research on epigenetic regulations.


Roper VersArray1300B-Plant in vivo imaging

Bioluminescence is the process of light emission in living organisms through the enzymatic activity of luciferase protein towards its substrate. Through thedevelopment of the ultra-sensitive CCD camera to enable the detetion of in vivo bioluminesecence, now researchers can utilize bioluminescence to monitor biological processes in a noninvasive manner. In the plant research field, in vivo bioluminescence imaging is utilized to detect various promoter activities on gene expression in the living plant.The plant in vivo imaging instrument combines high-sensitive Liquid N2 cooled CCD camera from Princeton Instruments with Lumazone dark box system for long-term dark imaging. Versarray 1300 has followig technucal specifications; imaging array of 1340*1300, cooling to -110 C, largr 20 um CCD pixels wij dark current of 1 e-/p/hr. The Dark Box configuration includes a manual stage and provides the anility to automatically acquire bright field images. This instrument will be useful for detecting plant stress included gene expression by monitoring stress induced promoter activity. Scince it has large imaging area, the machine can also be useful for searching mutant plant as a screening experiment.


Leica EMUC7 ultramicrotomes

Leica EMUC7 has several advanced feature necessary for modern ultramicrotomes. It has eucentric movement of the stereomicroscope observation system with patented designated positions for specimen approach, for glass and diamond knives, so that operators can easily observe their sample for sectioning. In addition to the standard LED illumination for top light and backlight and transmitted light, the LED spot illumination offers a focused light beam to enhance observation for cleaning the knife edge and during cryo-sectioning. It also has user-friendly operation design such as Easy-to-learn, fast operation via touch-screen control and displayed hints and data transfer for reporting user, specimen, knife and storage parameters which provides an electronic, paper-free log file. Technical specification for the instruments are; thickness of slice = 1nm - 15um, sectioning speed = 0.05~100mm/s, cutting window = 0.2~14mm.

Zeiss Light Sheet Z.1

Fluorescent proteins allows us to detect cellular dynamics in live cells. However, conventional fluorescent microscopy system always faced limitation in terms of observation angle, which is fixed to a single direction, as well as sample viabilities under observation condition. Also, illumination light often cause photo-damage to the living samples, which makes it difficult to perform long time-lapse experiment. The lightsheet microscopy is intended to overcome those limitations in long time-lapse imaging experiments. In light sheet fluorescence imaging, fluorescence excitation and detection are split into two separate light paths, with the axis of illumination perpendicular to the detection axis. This special design allows researchers to illuminate a single thin section of the sample at one time, generating an inherent optical section by exciting only fluorescence from the in-focus plane, without in need for pinhole or image processing.

Light sheet imaging is the gentlest of all known optical sectioning techniques. Live samples can grow without being adversely affected by the excitation light. The rotating sample holder with cover-slip free optics allows user to rotate their sample to get best imaging direction.Our system is equipped with 405, 488 and 561 nm lasers and 2 detection sCMOS camera unit, which enable flexible multi-color imaging. Max frame rate of the machine is 30 fps at 1000 x 1000 pixel. The sample chamber has a peltier temperature controlling unit for heating or cooling to 10 to 42 degree. The lightsheet microscopy is already used for studies in the plant research field focus on lateral root development or studies focusing on dynamics of proteins involved in vesicle trafficking in plants. Since the instrument has capabilities to add solution into its sample chamber, it will be useful to directly observe salt or other chemical induced stress responses in plants. Also, it will be useful for detecting long-term cellular effects after mechanical damage or other environmental stress conditions.

Leica LMD 7000

Laser micro-dissection (LMD) is an experimental method to isolate specific cell types or tissues. The individual cells or cell clusters are isolated by cutting region of interest through illuminating strong laser, and subsequently be utilized for downstream applications such as transcriptome analysis. The Leica LMD7000 system utilizes simple gravity as a means of collecting isolated cells, which enables researchers to collect cells in a contact-free and contamination-free manner. Various sample preparation method such as paraffin sectioning, cryo-sectioning or live cell cultures can be utilized for experiments.




Spinning Disk Confocal Microscope Andor Revolution WD

Through the advancement of techniques using fluorescent proteins as a marker to detect protein dynamics inside live cell, there is a growing demand for imaging instruments with high sensitivity, accuracy, and speed that are required to capture three-dimensional images showing detailed structures inside live cells, including those in a short time period. By utilizing rotating disc with multiple pinholes (Nipkow disc) to detect signal without out-of-focus light, spinning disc confocal scanner units offer the excellent sensitivity and high-speed scanning to meet those needs, and can observe samples for longer periods of time with little of the damage normally caused by laser beams. In the plant research field, spinning disc confocal systems are used for detecting cytoskeletal dynamics as well as fast moving compartments during endomembrane vesicle trafficking.
The system is equipped with 405, 488, 561 and 640 nm laser lines for flexible combination of fluorescent molecule imaging applications. The main confocal unit for our sytem is CSU-W1, which is the latest spinning disc confocal unit from Yokogawa electronics, the leading company in the field of spinning disc unit. The new CSU-W1 confocal scanner unit has a larger, newly designed Nipkow disk that allows imaging of areas up to four times the size possible with previous models and increases the likelihood that images of phenomena will be captured even when it is difficult to predict where they will occur.
The distance between the pinholes on the disk has also been widened, reducing the flare caused by the leakage of light from adjacent pinholes (crosstalk). As a result, images are clearer. The highest acquisition speed for the instruments is 100 fps, and the imaging field is 17 x 16 mm. The system is also equipped with an active illumination unit with switchable dye based laser/ for bleaching experiment or un-caging of caged compounds. Furthermore, our system has TIRF illumination units to detect membrane dynamics. Our system is useful for detecting cytoskeletal dynamics during stress responses as well as observing dynamic protein movement in epigenetic regulation. By utilizing its active illumination unit, it will also be useful for cell ablation studies to identify key cells/tissues for plant stress responses and other developmental studies.

Microplate Reader Thermo Varioskan Flash

With the advancement of experimental tools utilizing optical signals such as fluorescence or luminescence as an output, not only in vivo assays but also large scale in vitro assays can be performed to detect biologically significant events such as protein interaction or induction of transcriptional activation. Microplate reader is a device that can handle such an in vitro assay in parallel with multiwall plate, and becomes a powerful tool to detect biological, chemical or physical events of samples in solution. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes.

Our instrument, Thermo Varioskan Flash is a spectral scanning multimode reader, including fluorescence intensity, time-resolved fluorescence (TRF), photometric, and luminometric detection technologies. It provides unlimited wavelength selection, and thereby allows both spectral analysis and measurement at any single wavelength. This gives ultimate flexibility for identifying the optimal measurement wavelength. Varioskan Flash is equipped with dual onboard dispensers for easy and accurate reagent additions. Specifications for our instrument are; fluorescence excitation: 200 - 1000nm, fluorescence emission: 270 -840nm, Luminometry sensitivity/dynamic range = < 7 amol ATP/well, > 7 decades, flash ATP reaction. This instrument will be useful for large scale assays to detect protein interaction as well as gene expression/repression during the stress responses and other developmental processes.

Service & Fees Top

(1) Services being offered:
We will offer basic training session for first time users as well as customized training sessions for accommodate specific samples prepared by users. We will also provide consultation service to help users for setting up experimental plans.


(2) Service prices:

(3) Reservations:
Websites under construction

 

(4) Policies:
- All usage for equipments should be logged.
- Cancellations of reserved equipments should be reported as soon as possible. Frequent cancellations of major equipments (confocal microscopies etc) will lead to penalty.

Technical resources Top

(1) Tutorials
Under construction


(2) Web links
http://www.microscopyu.com/
http://zeiss-campus.magnet.fsu.edu/
http://www.leica-microsystems.com/science-lab/

Workshops / Seminars Top

 

 

 

 

 

Publications Top

 

 

 

 

 

Contact Top
Mailing address:
Room 337
Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences
Chenshan Botanical Garden, Research Building
3888 Chenhua Road, Songjiang, Shanghai 201602, P. R. China
Facility manager: Shingo Nagawa
Phone:
E-mail: snagawa@gmail.com
 
 
 
 
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Address: No. 3888 Chenhua Road, Shanghai 201602, P. R. China
Shanghai ICP #05033115