Spinning Disk Confocal

Dual Spinning Disk Confocal Technology

As the pioneer in dual spinning disk confocal technology, Yokogawa has revolutionized live cell imaging in optical microscopy. The multi-beam scanning method offers not only high speed imaging but significantly reduced phototoxicity and photo bleaching, making our confocal scanner units the de facto standard tool for live cell imaging.

High-Speed, High Resolution Imaging

Yokogawa’s confocal scanners employ advanced imaging technologies to help researchers achieve high-speed and high-resolution live cell imaging.

  • Fast time-lapse confocal images of living cells
  • Minimal phototoxicity and less photobleaching
  • Live-cell confocal fluorescence imaging capabilities
  • Stability during long-term and high-speed imaging
  • Facilitates quantitative analysis of huge amounts of data
  • Super Resolution via Optical Re-assignment.

  • The CSU-W1 is our answer to researchers’ requests for “Wider FOV” and “Clearer Images”.

  • The CSU-X1 is widely recognized as the leading tool for live cell imaging with 2,000 fps capability.

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Comparison of CSU series

Model CSU-W1 CSU-X1
High-end Basic
Imaging Speed
(Max. fps)
200 2,000 360
Scanner Motor
Rotation Speed
(rpm)
1,500-4,000 1,500-10,000
(Variable)
1,800
(Fixed)*2
Recommended
camera
exposure time
5msec 0.5msec 33msec
Effective FOV 17x16mm 10x7mm
Disk unit Selectable up to
2 disks
Pinhole size :50µm, 25µm
1 disk
Pinhole size :50µm
Rotation
position trigger
signal
External signal output possible None*2
Filters EX Option
DM Option (up to 3 filters) Option
(1 filter)
EM Option
(up to 10 filters with
filter wheel)
Option
(up to 12 filters with
filter wheel)
Option
(1 filter)
Addition or
exchange of
filters
At user site :DM block and filters (EX, EM.)
At Yokogawa factory :DM
*1 option

Comparison between CSU and other confocal systems

Model CSU Conventional
Point-scan Confocal
Conventional
Slit-scan Confocal
Other spinning
disk Confocal
Epi- fluorescence
(Wide field)
Scan Type Microlens-enhanced
multi beam scan
Single beam scan Line Scan Disk scan (Multi beam or slit) None
Light Source Lasers Hg or Xenon arc lamp
Detector CCD, EMCCD PMT Line CCD CCD, EMCCD
Microscope Flexible Specific Flexible / Specific Flexible
Scan speed of
full-size image
2000fps ~1fps ~120fps (512X512) <200fps Any
Photo bleaching/
photo toxicity
Low Severe Low
Confocality High (X-Y-Z confocal) Modest
(Compromise
y-resolution)
Modest
(X-Y-Z confocal with pinhole,
Compromise y-resolution
with slit-scan)
None
Image Quality
(Background)
High Good S/N
(Low background)
High
(multiple averaging
necessary)
Modest Modest
(High background
with dim samples)
Low
(High background)

User labs

  • Ted Salmon Lab., Dept. of Biology, University of North Carolina, Chapel Hill
  • Waterman-Storer Lab., Laboratory of Cell and Tissue Morphodynamics (LCTM),NHLBI(Bethesda Campus)
  • Tim Mitchison Lab., Dept. of Systems Biology, Harvard Medical School
  • Scholey Lab., Dept. of Cell and Computational Biology, University of California, Davis
  • The Vale Lab., Dept. of Cellular and Molecular Pharmacology, University of California, San Francisco
  • The Wadsworth Lab., Biology Dept., University of Massachusetts, Amherst
  • The Kiehart Lab., Dept. of Biology, Duke University
  • HSC Core Facilities School of Medicine, University of Utah
  • Indiana Center for Biological Microscopy, Indiana University Medical Center
  • Ehlers Laboratory - Department of Neurobiology, Duke University
  • Bob Goldstein Lab., University of North Carolina Chapel Hill
  • Andrew Matus Lab., at Friedrich Miescher Institute for Biomedical Research
  • Laboratory of Developmental Dynamics,Graduate School of Life Sciences, Tohoku University
  • Zena Werb Lab., Anatomy, University of California San Francisco
  • Satoshi Nishimura Lab., Dept. of Cardiovascular Medicine, the University of Tokyo
  • Oshima Lab., Graduate School of Interdisciplinary Information Studies,The Univ. of Tokyo
  • The Huser research group at UC Davis
  • Nakano Lab., Graduate School of Science, University of Tokyo

Useful Sites

1) Microscopy & Imaging Resources on the WWW

Complete list of all aspects of microscopy and imaging, by Douglas W. Cromey From Cellular Imaging Core of Southwest Environmental Health Sciences Center, University of Arizona College of Pharmacy, University of Arizona

 

2) The Centro de Biologia Molecular “Severo Ochoa” (CBMSO)

Contains links to general information, microscopy laboratories, publications, courses and meetings, societies, images. Especially useful for finding microscopy workshops.

 

3) The Cell Imaging Facility, a part of the University of Utah Health Science Center's Core Research Facilities Department

Good explanation of CSU-Disk scanning confocal system by Chris Rodesch

 

4) Molecular Expressions Website

Run by National High Magnetic Field Laboratory, Florida State University.
One of Web's largest collections of excellent optical microscopy images, and quite thorough information on all types of microscopy.
Interactive Java Tutorials sponsored by Nikon (Nikon MicroscopyU) and Olympus(Olympus Microscopy Resource Center) are extremely useful to learn not only confocal but all kinds of microscopies, and related technologies.

 

Life Science Textbooks

Microscopy Techniques, Advances in Biochemical Engineering/Biotechnology Vol.95
Serial Editor T. Scheper, Volume Editor J. Rietdorf, Springer(2005)
ISBN-10 3-540-23698-8

Confocal Microscopy for Biologists
Edited by Alan R.Hibbs,Kluwer Academic / Plenum Publishers(2004)
ISBN:0-306-48468-4(hardback) 0-306-48565-6(e-Book)

Live Cell Imaging, A Laboratory Manual
Edited by Robert D. Goldman & David L. Spector. 2nd Edition,Cold Spring Harbor Laboratory Press (2010)
ISBN:0-87969-893-4(pbk), ISBN 0-87969-892-6 (hardcover)

Handbook of Biological Confocal Microscopy, 3rd Edition
Edited by James B. Pawley, Springer(2006)

VideoMicroscopy, The Fundamentals
Shinya Inoue, Kenneth Spring, Second Edition Plenum Press. New York,(1997)

ISBN: 0-306-45531-5

Direct-View High-Speed Confocal Scanner: The CSU-10, Chapter 2: Cell Biological Applications of Confocal Microscopy (Methods in Cell Biology)
Shinya Inoue and Ted Inoue
Edited by Brian Matsumoto Academic Press
ISBN:0-12-580445-8 ; 2nd Rev (2002/12)

Articles:  CSU Technology and its applications

Quantification and clustering of actin cytoskeletal structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsis guard cells.
Higaki T, Kutsuna N, Sano T, Kondo N, Hasezawa S
submitted.
Current Application and Technology of Functional Multineuron Calcium Imaging.
Shigehiro Namiki and Yuji Ikegaya
Biological and Pharmaceutical Bulletin Vol.32(2009) , No.11
Live imaging of yeast Golgi cisternal maturation.
Kumi Matsuura-Tokita, Masaki Takeuchi, Akira Ichihara, Kenta Mikuriya and Akihiko Nakano
Nature 441, 1007-1010 (22 June 2006)
Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems.
E. Wang, C. M. Babbey & K. W. Dunn
Journal of Microscopy, Vol. 218, Pt 2 May 2005, pp. 148 ?159
Optically sectioned fluorescence lifetime imaging using a Nipkow disk microscope and a tunable ultrafast continuum excitation source.
D.M.Grant, D.S. Elson, D.Schimpf, C.Dunsby, J.Requejo-Isidro, E.Auksorius, I.Munro, M.A. A. Neil, P. M. W. French ,E. Nye G. Stamp, P.Courtney
Optics Letters Vol. 30, No. 24 (2005 ) 3353
Optimization of Spinning Disk Confocal Microscopy: Synchronization with the Ultra-Sensitive EMCCD.
F.K.Chong, C.G.Coates, D.J.Denvir, N.McHale, K.Thornbury & M.Hollywood
Proceedings of SPIE 2004
Spinning disk confocal microscope system for rapid high-resolution, multimode, fluorescence speckle microscopy and green fluorescent protein imaging in living cells.
Maddox PS, Moree B, Canman JC, Salmon ED
Methods Enzymol. 360:597-617 (2003)
A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells.
Adams, M.C., Salmon, W.C., Gupton, S.L., Cohan, C.S., Wittmann, T., Prigozhina, N. & Waterman-Storer, C.M
Methods, 29, 29?41 (2003)
Spinning-disk confocal microscopy ? a cutting-edge tool for imaging of membrane traffic.
Nakano, A.
Cell Structure Function, 27, 349?355.(2002)
High Speed 1-frame/ms scanning confocal microscope with a miclolens and Nipkow disks.
T.Tanaami, S.Otsuki,N.Tomosada, Y.Kosugi. M.Shimizu & H.Ishida
Applied Optics, Vol.41,No.22(2002)
New imaging modes for lenslet-array tandem scanning microscopes.
T. F. Watson, R. Juskaitis & T. Wilson
Journal of Microscopy, Vol. 205, Pt 2 February (2002) 209?212
High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3-D imaging of single fluorescence molecule in real time.
A.Ichihara, T.Tanaami, K.Isozaki, Y.Sugiyama, Y.Kosugi, K.Mikuriya, M.Abe and I.Uemura
Bioimages 4, 57-62(1996)

Articles:  Cell Biology

(Vesicular transport, actin dynamics, microtubule dynamics, cell division)

Long-term, Six-dimensional Live-cell Imaging for the Mouse Preimplantation Embryo That Does Not Affect Full-term Development.
Yamagata, K., Suetsugu, R. and Wakayama, T., J. Reprod. Dev., 55: 328-331(2009) ”
The Caenorhabditis elegans DDX-23, a homolog of yeast splicing factor PRP28, is required for the sperm-oocyte switch and differentiation of various cell types.
Konishi, T., Uodome, N., and Sugimoto, A.,Developmental Dynamics 237, 2367-2377(2008)
Determining the position of the cell division plane.
J. C. Canman, L. A. Cameron, P.S. Maddox, A. Straight, J, S. Tirnauer, T. J. Mitchison, G. Fang., T. M. Kapoor & E. D. Salmon, Nature 424, 1074-1078 (28 August 2003) “Cover”
Taxol-stabilized Microtubules Can Position the Cytokinetic Furrow in Mammalian Cells.
Katie B. Shannon, Julie C. Canman,C. Ben Moree,Jennifer S. Tirnauer, and E. D. Salmon, Mol Biol Cell. September; 16(9): 4423?4436 (2005)
Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase.
G. C. Rogers, S. L. Rogers, T. A. Schwimmer,S. C. Ems-McClung, .C E. Walczak, R. D. Vale, J. M. Scholey & D. J. Sharp, Nature 427(6972):, 364-370 (2004)
Crm1 is a Mitotic Effector of Ran-GTP in Somatic Cells
Arnaoutov, A., Azuma, Y., Ribbeck, K., Joseph, J., Boyarchuk, Y. and Dasso, M, Nat Cell Biol. Jun;7(6):626-32 (2005).
Nuclear congression is driven by cytoplasmic microtubule plus end interactions in S. cerevisiae.
J.N. Molk, E.D. Salmon, and K. Bloom, JCB, Vol. 172, Number 1, 27-39 (2006)
Centrosome fragments and microtubules are transported asymmetrically away from division plane in anaphase
Nasser M. Rusan and Patricia Wadsworth ,JCB 168 (1) 21?28 (2005)
The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line.
G. Goshima and R.D. Vale,JCB 162(6) 1003?1016 (2003)
Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables.
Hwang, E., Kusch, J., Barral, Y. & Huffaker, T.C, J. Cell Biol. 161, 483?488 (2003)
Cell migration without a lamellipodium : translation of actin dynamics into cell movement mediated by tropomyosin
S.L. Gupton, K.L. Anderson, T. P. Kole, R.S. Fischer, A. Ponti, S.E. Hitchcock-DeGregori, G. Danuser, V.M. Fowler, D.Wirtz, D. Hanein, and C.M. Waterman-Storer ,JCB 168(4) 619-631(2005)
Actin dynamics in the contractile ring during cytokinesis in fission yeast.
Pelham, R.J. & Chang, F, Nature, 419, 82?86. (2002)
CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity.
Nishimura S, Manabe I, Nagasaki M, Eto K, Yamashita H, Ohsugi M, Otsu M, Hara K, Sugiura S,Yoshimura K, Kadowaki T, Nagai R, Nature Medicine 8, 914- 920 (15 August 2009)
T-cell engagement of dendritic cells rapidly rearranges MHC class II transport.
M. Boes, J. Cerny, R. Masso, M. Op den Brouw, T. Kirchhausen, J. Chenk & H. L. Ploegh, Nature 418, 983- 988 (29 August 2002) “Cover”
Functional coordination of intraflagellar transport motors.
G. Ou, O.E. Blacque, J.J.Snow, M.R. Leroux & J.M. Scholey,Nature 436(7050):583-7. (2005).
Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells
Kreitzer, G., Schmoranzer, J., Low, S.H., Li, X., Gan, Y., Weimbs, T., Simon, S.M. & Rodriguez-Boulan, E, Nature Cell Biol. 5, 126?136. (2003)
Dynamics of Membrane Clathrin-Coated Structures During Cytokinesis.
James H. & Wang, Yu-Li Warner, Anne K., Keen, Traffic 7 (2), 205-215. (2006 ) 

Articles:  Neuroscience

Activity-induced targeting of profilin and stabilization of dendritic spine morphology.
Ackermann, M., and A. Matus, Nat Neurosci. Nov;6(11):1194-200 (2003)
Dynamics and Regulation of Clathrin Coats at Specialized Endocytic Zones of Dendrites and Spines.
T.A. Blanpied, D.B. Scott, M.D. Ehlers, Neuron, Vol. 36, 435?449, October 24(2002)
Neurabin/Protein Phosphatase-1 Complex Regulates Dendritic Spine Morphogenesis and Maturation.
R.T.Terry-Lorenzo, D.W. Roadcap, T. Otsuka , T.A. Blanpied, P.L. Zamorano, C.C. Garner, S. Shenolikar, and M.D. Ehlers, MBC Vol. 16, Issue 5, 2349-2362, May (2005)
Phosphatidylinositol phosphate kinase type I regulates dynamics of large dense-core vesicle fusion.
L.W..Gong , G. D.Paolo, E. Diaz ., G.Cestra , M-E. Diaz, M. Lindau , P.De Camilli , and D. Toomre , PNAS, vol. 102 no. 14 5204-5209 (2005)

Calcium Dynamics

Formation of planar and spiral Ca2+ waves in isolated cardiac myocytes.
Ishida, H., Genka, C., Hirota, Y., Nakazawa, H. & Barry, W.H, Biophys. J. 77, 2114?2122 (1999)
Simultaneous imaging of phosphatidyl inositol metabolism and Ca2+ levels in PC12h cells.
Morita,M,Yoshiki, F,and ,Kudo,Y, BBRC 308, 673-678 (2003)
Calcium oscillations in interstitial cells of the rabbit urethra.
Johnston, L., Sergeant, G. P., Hollywood, M. A., Thornbury, K. D. & McHale, N. G,The Journal of Physiology 565 (2), 449-461 (2005).

Microvascular Blood Flow

Real-time observation of hemodynamic changes in glomerular aneurysms induced by anti-Thy-1 antibody.
Oyanagi-Tanaka, Y., Yao, J., Wada, Y., Morioka, T., Suzuki, Y., Gejyo, F., Arakawa, M. & Oite, T, Kidney Int. 59, 252?259. (2001)
Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.
Shahrokh Falati, Prter Gross, Glenn Merrill-Skoloff, Barbara C. Furie & Bruce Furie, Nat Med 8 (10) 1175-1180(2002)

Other Applications

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage
Hiroko P. Indo,Mercy Davidson,Hsiu-Chuan Yen,Shigeaki Suenaga,Kazuo Tomita,Takeshi Nishii,Masahiro Higuchi,Yasutoshi Koga,Toshihiko Ozawa,Hideyuki J. Majima,Mitochondrion No.7 106?118(2007)
Application Note
Overview:

Wide and Clear
Confocal Scanner Unit

Overview:

Real time live cell imaging of mitochondria

Image provided by Dr. Kaoru Kato, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST)


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Overview:

In the fertilization and early embryonic development process, various events are spatiotemporally controlled, and many events are connected in the cause-effect relations toward the final goal of ontogenesis. To understand the mechanism of this process, conventional experimental techniques by fixing and destruction of the cells have limitations. If this process can be observed over time and the development process can be continued after the observation, it will open a new era in the Genetics research. A mammalian developmental biology researcher, Dr. Kazuo Yamagata, established such technique by using the CSU system.
He successfully imaged mouse embryos over a long period of time, from the post-fertilization through to the blastocyst stage, to acquire approximately 60,000 of 3D confocal images. Thereafter, the embryos were transferred to a recipient mouse, and the pups were born all normally, grew healthy, and were capable of reproduction; a firm evidence that this early embryo imaging technique does not adversely affect the process of full-term development. The high speed image acquisition and extremely low excitation light unique for the CSU system enabled greatly reduced phototoxicity and realized intensive but damage-free long time observation. Only by using this technology which does no harm on the embryonic development, it is possible to “utilize the same embryo after intensive analysis by imaging” , and thus to investigate cause- and-effect relationship of various early stage phenomena and their influence on the development.

Experimental flow
Movie example

Figure : The long-time, multi-dimensional live cell imaging on early stage embryos does not affect the process of ontogenesis.
(a) Experimental flow
(b) Movie example: Images were acquired at 7.5-minute intervals over approximately 70 hours.
      This figure shows extracted images at 2-hour intervals.
    Each image is the maximum intensity projection of a total of 51 images in the Z-axis direction.
    Green:Spindle (EGFP-α‒tubulin), Red:Nucleus (H2B-mRFP1)
 

Experimental conditions
Total time 70 hours
Interval 7.5 min/stack
Z-axis slices 51 sections (at 2μm intervals)
Channel 3(DIC, EGFP, mRFP1)
Position 6(Total 72 embryos)
Laser power(Measured at objective lens) 488nm (0.281 mW), 561nm (0.225 mW)


Data: Kazuo Yamagata, PhD., Laboratory for Genomic Reprogramming,Center for Developmental Biology, Riken


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Overview:

In recent years, obese adipose tissue is attracting attention as an “active metabolic organ” that causes various diseases. Especially, visceral obesity and inflammation play a central role in metabolic syndrome. It was found that visceral obesity caused remodeling of adipose tissues based on chronic inflammation, and insulin resistance was occurred, which eventually leads to development of arteriosclerosis lesion, and cause new blood vessel events.
To elucidate the molecular mechanisms of pathological conditions consisted by the complicated and multi-cellular abnormal interactions in remodeling tissues, an “in vivo molecular imaging” based on the CSU system was developed.
By using this technique, it becomes possible to precisely evaluate the three-dimensional changes in the structures in living tissue, and the multi-cellular dynamics in vivo with high time and spatial resolutions.

 

Images of the remodeling of adipose tissue in live animals

Figure 1: Images of the remodeling of adipose tissue in live animals
a: Conventional adipose tissue specimen (lean, db/+ mouse)
b & c: Images of a white adipose tissue of an 8-week-old thin mouse (lean, db/+)
d: Adipose tissue of an 8-week-old obese animal (obese, db/db)
 

 

An example of real-time multi-color movie of microcirculation in mouse

Figure 2: An example of real-time multi-color movie of microcirculation in mouse, which clearly shows dynamic movement and interactions among leucocytes, platelets, macrophages and endothelium.

 

Application of “ in vivo molecular imaging” on various organs

Figure 3: Application of “ in vivo molecular imaging” on various organs
(Blood flow images of a: Skeletal muscle, b: Liver, c & d: Kidney glomeruli)

Data: Satoshi Nishimura M.D., Ph.D www.invivoimaging.net
Dept. of Cardiovascular Medicine, Translational Systems Biology and Medicine Initiative,
The University of Tokyo & PRESTO, Japan Science and Technology Agency


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Overview:

To investigate interactive dynamics of the intracellular structures and organelles in the stomatal movement through live imaging technique, a CSU system was used to capture 3-dimensional images (XYZN) and time-laps images (XYT) of guard cells.

Overview:

List of Selected Publications : CSU-X1

Overview:

The neuronal network is a computing system that transforms input to output. This computation involves complex nonlinear processes through polysynaptic feedforward and feedback microcircuitry, and thus cannot be addressed either with isolated neuron responses or averaged multineuronal responses. Functional multineuron calcium imaging (fMCI) is promising to solve this problem.
The fMCI is a large-scale recording technique that simultaneously monitors the firing activity of more than a thousand neurons through their somatic Ca2+ signals.
Because of several advantages, including i) simultaneous recording from numerous neurons, ii) single-cell resolution, iii) identifiable location of recorded neurons, and iv) detection of non-active neurons during the observation period, fMCI attracts attention as a new-generation large-scale recording method.
In vitro fMCI is made more sophisticated by using multipoint ilumination and scanning with the CSU in combination with low-intensity lasers and an EM-CCD (electron-multiplying charge-coupled device) camera.
This CSU system allows to achieve ultra-high-speed and high-resolution fMCI in hippocampal slices; the Ca2+ fluorescent intensity of a large number of neurons can be monitored at the speed of up to 2,000 frames per second. This is one of the applications that make best use of the high-speed performance of the CSU Confocal Scanner Unit.

fMCI

Data: Yuji Ikegaya, PhD, Associate Professor at University of Tokyo Graduate School of Pharmaceutical Sciences.

 


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Overview:

List of Selected Publications : CSU-W1

Overview:

Time-lapse imaging : Early stage mouse embryo

Following the injection of mouse embryos with mRNA, nearly 25,000 multicolor and multilayer confocal images of the embryos were acquired over 48 hour period as they developed to the blastocysts stage.Thereafter, they were transferred to a recipient mouse that gave birth to healthy pups, each of which developed normally and had full reproductive capability.This is firm evidence that long-term, multi-dimensional confocal imaging with CSU causes no harm to a delicate specimen such as an early stage embryo.

Early stage mouse embryo

Time lapse (MIP)  |  Full size movie Play

Measurement condition
Z-sections/
stack
100um
(1um/101slices)
Fluorescent probe 488nm:
H2B-EGFP
561nm:
mCherry-MBD-NLS
Pinhole 50um
Objective lens 60x silicone
Total time 48 hours
(Interval:10mins)
 Early stage mouse embryo

Excerpts from Time lapse (MIP of chromosome)

Data:  Kazuo Yamagata, Ph.D., Center for Genetic Analysis of Biological Responses, The Research Institute for Microbial Diseases, Osaka University

Overview:

Faster, Brighter, and More Versatile
Confocal Scanner Unit

Yokogawa Technical Report
The World as Seen from Cells
(rd-te-r06002-001)
2.2 MB
Yokogawa Technical Report
6.1 MB

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