CSU-X1, CSU22, CSU10 Applications

Application: CSU-X1, CSU22, CSU10

• Vesicle trafficking
• Cell Division
• FRET Imaging
• Calcium Imaging
• in vivo imaging
• Multi-color imaging
• Long-term, multi-dimensional live-cell imaging
• Micro PIV

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Vesicle trafficking

*Movies from Indiana Center for Biological Microscopy, taken by Kenneth Dunn, Ph.D. (movies of Vesicle trafficking)

All images were collected using the system described below:
Nikon TE 2000U inverted microscope, using Nikon 60 or 100 NA 1.4, oil-immersion plan apochromatic objectives, with a Hamamatsu Orca-ER CCD system

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Cell Division

Cell Division of an LLCPK1 cell

*Movies from Patricia Wadsworth’s laboratory

Cell Division of an LLCPK1 cell expressing GFP-tagged EB1, a microtubule plus-end binding protein (kind gift of Lynne Cassimeris).

(All movies were taken by Dr. U. Serdar Tulu by using a MicroMax camera on a Nikon TE-300 inverted microscope coupled to a Yokogawa spinning disc confocal unit (Perkin Elmer Life & Analytical Sciences. )and are shown at the speed of 10 frames/sec.)

Cell Division

*Movies by Drs. Jonathan M. Scholey& David J. Sharp

Intravital Molecular Imaging

Movies by Paul Maddox, Ludwig Inst. of Cancer Research Time-lapse images were acquired by using a CSU10 Yokogawa spinning-disk confocal system (Perkin-Elmer Life Sciences Wallac) mounted on a Nikon Eclipse TE300 inverted microscope (Nikon Instrument Group). The specimens were illuminated at 488 nm with an air-cooled Ar/Kr laser (60mW, Melles Griot). Digital images were acquired with an Orca ER, Hamamatsu 16-bit cooled CCD camera (Hamamatsu Photonics), providing the goa-1 and gpa-16 DNA constructs, Stephan Grill, and the acquisition system was controlled by MetaMorph software (Universal Imaging). Fluorescence images were acquired with 350 ms exposure at 2- or 5-s intervals by using a Plan Apochromat 100_/1.4 NA objective and 2 _ 2 binning in the camera. Images were analyzed by using MetaMorph software and Microsoft Excel (Microsoft) and were processed with Adobe Photoshop (Adobe Systems)

C. elegans embryo

*Movies by Fumio Motegi & Asako Sugimoto
(Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology)

Tobacco BY-2 cell

*Movies by Takumi Higaki & Seiichiro Hasezawa
Laboratory of Plant Cell Biology in Totipotency,
Department of Integrated Bioscience, Graduate school of Frontier Science, The University of Tokyo

Time-lapse imaging of neural progenitor cells during cortical development

Tangential time-lapse monitoring of all cell-cell borders, about 5 µm from the apical surface, in a cortical wall prepared from E13 Lyn-Venus transgenic mouse Data : Mayumi Okamoto, Ph.D., Department of Anatomy and Cell Biology,
Nagoya University Graduate School of Medicine

Blue:  Neural progenitor cell(M phase), Magenta:  Daughter cell

Reference:
Okamoto, M., Namba, T., Shinoda, T., Kondo, T., Watanabe, T., Inoue, Y., Takeuchi, K., Enomoto, Y., Ota, K., Oda, K., Wada, Y., Sagou, K., Saito, K., Sakakibara, A., Kawaguchi, A., Nakajima, K., Adachi, T., Fujimori, T., Ueda, M. Hayashi, S., Kaibuchi, K., Miyata, T.
TAG-1–assisted progenitor elongation streamlines nuclear migration to optimize subapical crowding. Nat. Neurosci., 16: 1556-1566 (2013) DOI: 10.1038/nn.3525

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FRET Imaging

Real-time/real-color FRET imaging/Yellow Cameleon

Takeharu Nagai, and Atsushi Miyawaki A real-time movie of confocal real color images and corresponding pseudocolored ratio images showing Ca²⁺ waves inside cells, which were evoked by histamine, in HeLa cells expressing YC3.60(Yellow Cameleon). The images were taken at video rate (30 Hz) using a color 3CCD camera for simultaneous acquisition of CFP (cyan-emitting mutant of GFP) and YFP (yellow-emitting mutant of GFP) images. To improve z-axis resolution, a spinning disk confocal unit was placed in front of the camera. Ten micromolar histamines was added to the recording medium to activate receptor-evoked Ca²⁺ release from the endoplasmic reticulum.

Real-time/real-color FRET imaging/Yellow Cameleon

A real-time movie of confocal images and corresponding pseudocolored ratio images showing Ca2+ waves inside cells, which were evoked by histamine, in HeLa cells expressing YC3.60(Yellow Cameleon). 

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Calcium Imaging

Real-time intracellular calcium dynamics of the Purkinje fibers in the perfused rat heart

*Movies from Tetsuro Takamatsu’s laboratory (In Japanese)
The fluo3-fluorescence (8 bits, pseudo-color image) of the perfused rat heart was visualized by the CSU-based real-time confocal microscopy at 30 frames/s. 
Pixels 205 x 230. The scale bar denotes 50 m.
Microscope: BX50WI, Olympus、Objective lens: LUMIPlan FL x20, Olympus, with CSU-21, 
CCD camera: ES310 Turbo, Roper Scientific & Image intensifier: VS4-1845, Videoscope.

Simultaneous imaging of phosphatidylinositol metabolism and Ca2+ levels in PC12h cells

BBRC 308 (2003) 673-678
Mitsuhiro Morita, Fumito Yoshiki, and Yoshihisa Kudo
Laboratory of Cellular Neurobiology, School of Life Science, Tokyo University of Pharmacy and Life Science (in Japanese)
Relationship between phosphatidylinositol metabolism and Ca 2+ level was analyzed with high spatial resolution at video rate in PC12h cells double-labeled with GFP fused with the pleckstrin homology domain and FuraRed,
CSU10, C6790 CCD camera, and W-View; simultaneous dual-color imaging system (Hamamatsu), Nikon E600FN microscope with Fluor 40X/0.8W.

Functional multineuron calcium imaging

Yuji Ikegaya
Laboratory of Chemical Pharmacology, Graduate school of Pharmaceutical Sciences, The University of Tokyo.
fMCI is a functional imaging technique with multicell loading of calcium fluorophores. fMCI has unique advantages, including: i) recording en masse from hundreds of neurons in a wide area, ii) single-cell resolution, iii) identifiable location of neurons, and iv) detection of non-active neurons during the observation period

High-speed calcium imaging

Data:  Yuji Ikegaya Ph.D., Graduate School of Pharmaceutical Sciences at the University of Tokyo

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in vivo imaging

Intravital Molecular Imaging

Satoshi Nishimura
Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo
Dr. Satoshi Nishimura is a world-renowned expert of intravital molecular imaging; which means in vivo imaging of microcirculation and molecular dynamics in living animals with high spatiotemporal resolution.

Intravital Molecular imaging is a powerful tool to elucidate not only vascular pathological conditions such as arteriosclerosis but also to study molecular mechanisms of pathological conditions caused by complicated and multi-cellular abnormal interactions, such as cancer or metabolic syndrome. 

He has developed an imaging system based on highly spatiotemporal imaging with the CSU and visualized microcirculation, vascular functions, and multi-cellular dynamics of target molecules in real-time and in three dimensions, in vivo.

Using this technique, he successfully elucidated how obese adipose tissue played a bad role in obesity. 

Long-time, 4 colors, and 3D in vivo imaging.

Mikala Egeblad, Andrew Ewald, Zena Werb., UCSF, Dept of Anatomy

Researchers at UCSF developed in vivo imaging system capable of multicolor, 3D imaging at multi-points to observe tumor microenvironment in the same live mouse for a long time, by using the CSU.

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Multi-color imaging

Multi-color imaging of live cells and tissues

Satoshi Nishimura
Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo

Escape of intracellular vesicles induced by mechanical stress

(By courtesy of Dr. Satoshi Nishimura, Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo and Dr. Seiryo Sugiura, Department of Human and Engineered Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo.)

3D reconstruction image of blood vessels in mouse adipose tissues (unfixed)

(By courtesy of Dr. Satoshi Nishimura, Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo and Dr. Seiryo Sugiura, Department of Human and Engineered Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo.)

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Long-term,multi-dimensional livecell imaging

Long-term, Six-dimensional Live-cell Imaging

Kazuo Yamagata, PhD, Wakayama Lab (Laboratory for Genomic Reprogramming), Center for Developmental Biology, RIKEN
(Present post: Research Institute for Microbial Diseases, Osaka University)

IVF embryos were injected with a mixture of mRNAs for EGFP-α -tublin (Tublin, green) and H2B-mRFP1(H2B, red). The embryos were imaged by being exposed to two different wavelengths of excitation(488 and 561 nm) at 7.5min intervals for about 70h and 51 images were acqired in the Z axis at each time point( total of 56,814 fluorescent images). As a result of transplanting the embryos after imaging, all the pups were healthy, reproductively normal, and not transgenic. Thus, this live-cell imaging technology is safe for full-term mouse development.

IVF:  in vitro fretilization

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Micro PIV

Visualization of the warped interface between two fluids at a curve downstream of the Y junction

(By courtesy of Seika Corporation (Japanese content).​ This experiment was done in the collaboration of the Oshima Laboratory, Institute of Industrial Science, The University of Tokyo.)

Instantaneous velocity distribution analysis of fluid flow in a round shape microchannel (width 100um × depth 50um)

(By courtesy of Seika Corporation (Japanese content). This experiment was done in the collaboration of the Oshima Laboratory, Institute of Industrial Science, The University of Tokyo.)