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C1si Spectral Imaging Confocal Laser Scanning Microscope System
True Spectral Imaging Confocal Laser Scanning Microscope System
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Bringing the world of spectral imaginginto your laboratory
The C1si is a revolutionary true spectral imaging confocal laser microscope system with the amazing
capability to acquire 32 channels of fluorescence spectra over a 320 nm wide wavelength range in a
single pass. Easy switching between the spectral detector and a standard fluorescence detector makes
the C1si useful for a wide range of applications.
By cleanly unmixing overlapping spectra of different fluorescent labels, the C1si dramatically improves
dynamic observations of live cells and facilitates the acquisition of detailed data.
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"One-shot" acquisition of a 320 nm wavelength rangeA single pass of the laser is sufficient to obtainspectra over a broad 320 nm range, minimizingdamage to live cells. Also, simultaneous acquisitionof 32 channels facilitates the creation of highlydetailed spectral time-lapse images.
True spectral imagingImaging of fluorescence spectra in real colors hasbeen realized thanks to a host of new innovationsfor accurately correcting spectral data andwavelength resolution independent of pinholediameter.
Spectral imaging focusing on brightnessSignal loss has been minimized by use of proprietaryoptics that very efficiently transmit fluorescenceemission photons to the detector and the signal-processing circuitry that digitizes for the full pixelperiod.
Eliminating spectral crosstalkOverlapping wavelengths of fluorescent labels arecleanly separated for images with no spectralcrosstalk.
Highly versatileWavelength resolution can be set to 2.5 nm, 5 nm, or10 nm. Compatibility with a wide variety of lasersand easy switching to the standard fluorescencedetector makes this microscope system perfect for awide range of applications.
C1si–True Spectral Imaging Confocal Laser Scanning Microscope System
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Simultaneous acquisition of 32 channel spectral images*
The C1si boasts a multi-anode PMT with 32 channels, the most of
any confocal microscope. Innovations such as multiple high-speed
digital conversion circuits and LVDS (Low Voltage Differential
Signal) high-speed serial transmission technology allow a full 32
channels of spectral images to be obtained from a single scan. This
allows for dramatically reduced imaging time and real-time
observation.
One-step acquisition of a broad 320 nm range
Three wavelength resolution settings of 2.5, 5, and 10 nm are
available. At 10 nm, spectra over a full range of 320 nm can be
obtained in a single pass, a capability unmatched by previous
spectral imaging systems.
Gentle on living cells
Spectral images over a broad wavelength range can be obtained
with only a single laser excitation. Therefore, adjustment of laser
intensity and PMT gain is simple and quick, and there is no need to
make multiple scans to acquire a broad spectrum, keeping
fluorescence fading and specimen damage at a minimum. The C1si
spectral imaging system is gentle on living cells and tissue.
* Nikon original
Overlay image of all 32 simultaneouschannels (True Color)
Rat olfactory bulbFITC: Anti-calbindin-antibodyCy3: Anti-calretinin-antibody
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SpeedSignificant reduction in image acquisition time
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Expression of GFP and YFP in the nucleus ofHeLa cells, with actin stained by Alexa 488.1:GFP, 2:YFP, 3:Alexa488
Acquiring real fluorescence colors
Fluorescence spectra peak wavelengths and differences in spectral
shapes can be detected by spectral acquisition with a high degree of
reliability and accuracy. Whereas previously false colors were used to
portray detail, the C1si allows observation of specimens in True Color.
High wavelength resolution*
High performance wavelength resolution at a minimum of 2.5 nm has been achieved, with three
resolution levels (2.5, 5, 10 nm) independent of confocal pinhole diameter.
Superb error and deviation correction*
Accuracy of spectra is maintained with highly precise correction technologies, including
wavelength correction using emission lines and luminosity correction utilizing a NIST (National
Institute of Standards and Technology) traceable light source.
Also, multi-anode PMT sensitivity correction technology* allows correction of sensitivity error and
wavelength transmittance properties on a per-channel basis, allowing researchers to minimize
measurement errors and deviations among different equipment. * Nikon original
(Brightness)
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Spectral data from C1si Spectral data from the probe manufacturer
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Peak wavelengths and spectral shapes obtained in the C1si image above closely match those
obtained by the probe manufacturer.
AccuracyTrue spectral imaging
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Multi-anode PMTThe spectral imaging detector utilizes a newly developed laser shielding
mechanism. Coupled with the wavelength resolution independent of
pinhole diameter, this mechanism prevents the reflected laser beam from
contaminating data. The blocking mechanism can be moved freely with
software, allowing users to block any laser wavelength, making the C1si
compatible with virtually any laser selection.
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Diffraction ratio (%)
Wavelength (nm)
Spectral detector with polarization control technology*
Nikon’s proprietary DEES (Diffraction Efficiency Enhancement
System) for polarization control has been adopted in the spectral
detector of the C1si to maximize brightness. By co-aligning the
direction of polarization, the efficiency of the diffraction grating is
optimized, resulting in exceptionally bright images.
In particular, increasing the diffraction efficiency in the long
wavelength range leads to improved brightness and linearity of
spectral data over the whole visible range from blue to red.
*Nikon original
Grating properties
Standard fluorescence detector
Optical fiber
Unpolarized light
Polarized beam splitter
S1
P
S1S2
S2
Polarization rotator Multiple gratings
(2.5/5/10nm)
Spectral detector
Laser unit
Scanning head
Overview of DEES
BrightnessOptics and signal processing designed to efficiently capture fluorescence photons
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High-efficiency fluorescence transmission technology*
The ends of the fluorescence fibers and detector surfaces use a
proprietary anti-reflective coating to reduce signal loss to a
minimum, achieving a high optical transmission.
Dual integration signal processing*
Newly developed DISP (Dual Integration Signal Processing)
technology has been implemented in the image processing circuitry
to improve electrical efficiency, preventing signal loss while the
digitizer processes pixel data and resets. The signal is monitored for
the entire pixel time resulting in an extremely high S/N ratio.
*Nikon original
Integrator (1)
Integration Hold Reset
Pixel time
Integrator (2)
Separation of GFP and Alexa 488 spectra
Fluorescence spectra of GFP and Alexa 488 It has always been difficult to cleanly separate fluorescence proteins
in multi-stained specimens with greatly overlapping wavelengths
with a confocal microscope. By mathematically processing the
spectral data of closely overlapping probes such as CFP, YFP, RFP,
and Alexa 488, the C1si cleanly separates emissions from each,
yielding clear images with no cross-talk. This is particularly useful in
observations of multi-stained specimens with localized protein
molecules, and in FRET experiments. Spectral separation of probe
signals from autofluorescence is also possible.
Unmixing of images with no crosstalk
GFP expressed in HeLa cell nuclei and actin stained with Alexa 488. Excitationwavelength 488 nm.
Combined 32 channel True Color imageobtained with 2.5 nm wavelength resolutionin 493-570.5 nm range
Image with separated spectra after usingunmixing software
Sepctra displayed at 2.5 nm wavelength resolutionper channel of the fluorescence detector in the 493-570.5 nm range
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Fluorescence
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DISP
Two integrators work in parallel as the optical signal is read to ensurethere are no gaps.
ons
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Quick detector mode change
Switching from standard confocal imaging to spectral confocal
imaging is a matter of turning the switch on the scanning head.
The imaging mode of the EZ-C1 software is automatically switched.
Mouse cerebellum observed with Z series images acquiredwith spectral imaging, then unmixed and projected.Green (FITC):Inositol 1,4,5-trisphosphate receptor (IP3R)Red (Rhodamine):Glial fibrillary acidic protein (GFAP)Blue (Hoechst):DNA
Spectral confocal image acquisition mode
Standard confocal image acquisition mode
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SimplicityEffortless acquisition of spectral images
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Quick parameter setting
Each parameter of the spectral detector, e.g. excitation laser
wavelength, wavelength resolution, or acquisition wavelength
range, can be easily set from the menu with the mouse. When it is
set, spectral imaging can be performed with common imaging
procedures. Parameter profiles may be saved for later use. A
binning function that combines signal from adjacent channels to
increase brightness is provided. Therefore, when determining the
target area, it is possible for users to lower excitation laser intensity
and reduce damage to the specimen.
One-click acquisition of spectral images
Once spectral detector settings have been set, spectral confocal
images can be acquired with a single click of the Start button.
Parameter selection screen
Parameter setting screen
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Unmixing red fluorochromes
Red flourochromes, which had previously posed a challenge, are
now simple to unmix as well.
Rhodamine’s fluorescence spectral peak is at approximately 579 nm,while that for RFP is approximately 600 nm. RFP’s fluorescence isweaker than Rhodamine’s, but their spectra are cleanly unmixed.
True Color image overlay of 32 images in the 495-575 nm range at 2.5 nm wavelength resolution.HeLa cell nuclei (YFP)HeLa cell actin (Alexa 488)
After fluorescence unmixing
After fluorescence unmixing
RFP expressed in HeLa cell nuclei and actin stained withRhodamine. True Color image overlay of 32 images in the550-630 nm range at 2.5 nm wavelength resolution.
Spectra for ROI 1 and 2
ROI spectra
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Effortless fluorescence unmixing
One-touch fluorescence unmixing
Even without specifying a reference spectrum, simply drawing an
ROI (Region of Interest) within the image and clicking the Simple
Unmixing button allows separation of fluorescence spectra. Use the
Unmixing button when you wish to specify the color each
fluorescence probe will be displayed in after separation.
The C1si contains a built-in database of spectral data provided from
manufacturers of fluorescence labels, which can be specified as
reference spectra for fluorescence unmixing. Users may also add
spectral information for new labels to the database.
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Wavelength information of the entire range can be obtained in a single spectral
imaging operation, so you no longer have to acquire only the limited wavelength
range at the beginning or re-shoot other ranges after the imaging session.
After spectral imaging, you can easily display images that are filtered with any
desired wavelength range.
A variable-color-filtered image with green allocated to filtered GFP spectrum(10 nm band pass filter used to capture 507.5-517.5 nm wavelength), blueallocated to filtered YFP spectrum (10 nm band pass filter used to capture527.5-537.5 nm wavelength), and red allocated to filtered RFP spectrum (10 nm band pass filter used to capture 612.5-622.5 nm wavelength). The GFP, YFP, and RFP spectra are all unmixed for clear observation.
True Color display of an overlaid spectral image combining colorsallocated for all wavelengths acquired
Variable-color-filtering setting screen
Variable-color-filtered image
This natural color image is close to what you see through eyepiece lenses(including fluorescence cross-talk). GFP, YFP, and RFP expressed in HeLa cellnuclei.
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Simple variable-color-filtering
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Fluorescence intensity
Time (sec)
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“Variable delay” to adjust acquisition frame rate to the temporal dynamics of the experiment
C1si’s variable delay function allows you to freely set acquisition speed based on different
reaction speeds of the specimen such as before, during, and after stimulation of the
specimen, allowing efficient image capturing.
Real-time observation of spectral changes with synchronized graph
The spectral graph is generated synchronously with imaging, so changes in
spectra are observable in real time. This is especially useful in time-lapse
observation of living cells or tissue.
The graph at right is a graph display of spectral data with third-party software.
Simple time-lapse recording of spectral images
Calcium ion concentration changes in HeLa cells expressing Yellow Cameleon.CFP/YFP fluorescence spectra captured at approximately 1 second intervals. ATPstimulation on the 10th frame (11.58 sec). Excitation wavelength 408 nm, wavelengthresolution 10 nm, acquired wavelength range 451-751 nm.
Spectral images (True Color)
Spectral analysis of time-lapse imaging
Spectral FRET observation
t=13.90 (sec) t=15.17 (sec) t=16.31 (sec) t=17.46 (sec) t=18.62 (sec) t=19.79 (sec)
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13.90 15.17 16.31 17.46 18.62 19.79
After fluorescence unmixing CFP: Green YFP: Red
13.90 15.17 16.31 17.46 18.62 19.79
Spectra
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Initial state After 2 sec. After 8 sec. After 17 sec. After 30 sec. After 60 sec.
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Fluorescence intensity
Bleach
FRAP observation
FRAP (Fluorescence Recovery After
Photobleaching) experiments are also
possible on the macro program. Since the
laser can be precisely pointed to ROI to
photobleach only a specific area of cells, it is
possible to observe the fluorescence recovery
process as the molecules translocate over
time.
Diascopic observation
The spectral detector can acquire both diascopic and confocal
images simultaneously, like a standard fluorescence detector that
acquires confocal images. This is especially effective in locating
fluorescent labels.
Simple, flexible microscope settings
Switching between eyepiece observation and laser scanning modes
is accomplished with a simple click on an icon. When the motorized
TE2000-E Inverted Microscope or the ECLIPSE 90i Upright
Microscope is used, the microscope can be controlled via the C1si
system software, which frees users from the burden of changing
optical paths and allows them to concentrate on data collection.
Superbly versatile functions
Part of a specimen in which H1 Histon-GFP inthe nuclei of HeLa cells is expressed, and therecovery of fluorescence intensity is observed intime-laspse recording.
FRAP experiment (HeLa cell histone GFP)
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Modules
Standard fluorescencedetector
AOM controller
Spectral detector
Z focus module
Scanning head
Laser unit
Diascopic detector
Diffraction efficiency increased withDEES. Three wavelength resolutionsavailable, with 32-channel PMT.Moveable laser blocking mechanismto expand laser compatibility.
Super-precise focusing is possiblewith a minimum focal adjustment of50 nm. You can easily accomplish ahost of image acquisition settingsfrom the software, includingcombinations of space and time axes(XYZ, XYZt, etc.).
High quality DIC images can beobtained simultaneously withconfocal fluorescence images. Bothimages can be superimposed to aid inimage analysis such as locatingfluorescence labels. Compact andretrofittable to microscopes.
Easy upgrades to new computersComputers controlling the C1si system are highly independentof the system itself. They can, for example, analyze existingimage data while obtaining new images. This makes upgradingof computers a simple task. Because spectral imaging with the C1si system generates vastlymore data than previous systems, ease of computer upgrades isan important factor.
Has the flexibility to handle a varietyof modes, including simultaneous 3-channel fluorescence observation orsimultaneous 3-channel + diascopicDIC observation. Filters are allexchangeable, so new probes anddyes can be used with no hassle.
Scan rotation ability allows scanningof long, thin specimens such asneurons without rotating the stage.Bi-directional scanning increasesscanning speed and captures rapidchanges in the specimen.
AOM (Acousto-Optical Modulator)regulates laser power within aspecific ROI. Laser power can be finetuned easily. This allows for finetuning of brightness for individualfluorescent labels in multi-stainedspecimens or critical regulation ofpower for photobleaching orphotoactivation.
Now more laser lines than ever can beused, with a greater degree of freedomin selecting laser frequency. A MultiArgon laser (488/514 nm) is availablefor YFP, while a 408 nm laser isavailable for DAPI and CFP. Laserillumination can be restricted to theROI, so FRAP is possible as well.
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Objectives
Superb selection of CFI60 series of objectives
CFI Plan Apo VC 60 x WI/1.20
CFI Plan Apo TIRF 60 x /1.45 oil (Left)CFI Plan Apo TIRF 100 x /1.45 oil (Right)
37˚C (with correction)
Correction ring effects (severity distribution)
23˚C 37˚C (no correction)
Perfectly suited for digital imaging
These top-of-the-line objectives achieve both full correction ofchromatic aberration in the visible range and high peripheralresolution. They are perfect for digital imaging, which requiresuniform resolution from the image center to the periphery. Theseobjectives remove aberrations in the peripheral visual field and alsoeliminate shading, resulting in images that are sharp all the way tothe edges, a feature absolutely necessary when stitching imagestogether.
Fluorescence observation of organic tissue
These lenses boast exceptional optical performance in brightfield,DIC, and multi-stained fluorescence observations. In addition to thechromatic aberration correction range (435-660 nm) of the previousPlan Apo series, axial chromatic aberration has been corrected up to405 nm (h line), making this series appropriate for confocalobservations. The 60x WI lens achieves high spectral transmittancein the UV range, making it optimal for fluorescence observation oforganic tissue.
Highest NA
These lenses boast the highest NA ever within the Nikon objectivelens line, with nearly complete aberrational correction. Theirpowerful optical capabilities are perfectly suited to multi-wavelength observations, and they can be used with normalcoverglasses and immersion oils.
World’s first temperature correction ring
The 60x oil immersion lens utilizes the world’s first temperaturecorrection mechanism. Changes in the refraction index of theimmersion oils resulting from changes in temperature affect imagequality. With a 60x lens, this change can be easily corrected with acorrection ring in the range of 23˚C (room temperature) to 37˚C(incubation temperature). The correction ring is also effective in improving visualization offine structures in DIC and epi-fluorescence microscopy, making thislens optimal for laser tweezers microscopy as well. As this lensallows for correction of the slight optical degradations that arisefrom temperature and coverglass thickness changes, improvingobservation quality on a consistent basis is possible.
CFI Plan Apochromat TIRF series
CFI Plan Apochromat VC series
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System diagram
Epi-fl Filter Cube
Standard Epi-fl Detector (2-PMT or 3-PMT)
Laser Fiber
Laser Unit
ESCAPE
COARSE FOCUS
Dichroic Mirror 1
Scanning Head
Ring Adapter
Software
Computer
Diascopic Detector*2
Diascopic Detector*2
ECLIPSE 90i/80i*1
TE2000-E
Spect
BD Laser (408nm) Y-HeNe Laser (594nm) R-HeNe Laser (633nm)
Ar Laser (488 nm) Ar Laser (488/514 nm)
G-HeNe Laser (543 nm)
L1
L2
L3
L1L2
L3
C1 Adapter
Optical Fiber
* When using laser wavelengths other than the above, consult Nikon or its distributors
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r*2
etector*2
TE2000-U
Controller AOM Controller
Optical Fiber
Spectral Detector
Z-focus Module
Diascopic Detector*2
*1: Z-focus module necessary for the ECLIPSE 80i.*2: Diascopic detector or motorized diascopic detector
ber
Detector
Pin-hole
Laser Dichroic Mirror
Objective
Scanning Mirror
Specimen
Focal Plane
YX
Principle of confocal imaging
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Recommended layout
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650
(820
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W=1500mm (two 19-inch monitors)W=1000mm (24-inch monitor)
Note 1) Computer table size is for reference only.
Controller
Laser UnitScanning Head
Spectral Detector
Standard Epi-fl Detector
(142
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700
AOM Controller
(2400 or 2900)
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(149
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Controller
AOM Controller
Laser Unit
Scanning Head
Spectral detector
Note 1) Computer table size is for reference only.
StandardEpi-fl Detector
Combination with the Inverted Microscope TE2000-E/TE2000-U
Combination with the Upright Microscope ECLIPSE 80i/90i
Item Specifications
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Major specifications
Laser light source Laser wavelength BD laser (408 nm, 17mW variable)BD laser (405 nm, 25mW)Ar laser (488 nm/514nm, 40mW)Ar laser (488 nm, 10mW)G-HeNe laser (543 nm, 1mW)Y-HeNe laser (594 nm, 2mW)R-HeNe laser (633 nm, 5mW)* When using laser wavelengths other than the above, consult Nikon or its distributors.
Maximum loading number 3Laser control AOM (continuous variable)Laser shutter Motorized mechanical shutter (each laser)
Confocal pin-hole Variable Motorized switching
Standard fluorescence Number of channels 2 channels or 3 channelsdetector Dichroic mirror 1 20/80 Beam Splitter, 408/514, 488/543, 488/594, 408/488/543,
488/543/633, 408/488/561, Q-DOT440Filter cube 488/543, 488/543/633, 408/488/543, 488/594, 408/514,
405/488, 408/488/561
Scanning specifications for Display mode 160x160 to 2048x2048 pixelsa standard fluorescence Scanning speed Standard: 1.68µs/pixel (512x512 pixels)detector Bidirectional scanning: 0.7 sec@512x512 pixels
Scanning mode 2 D: X-Y, X-t3 D: X-Y-Z, X-Y-t4 D: X-Y-Z-tArbitrary ROI scanPoint scan
Spectral detector Number of channels 32 channelsCorresponding wavelength 400-750 nmWavelength resolution 2.5/5/10 nm (Switchable)Minimum wavelength step 0.2 nmDichroic mirror 1 20/80 beam splitter
Scanning specifications for Display mode 160x160 or 512x512 pixelsa spectral detector Scanning speed Standard: 2.4µs/pixel (512x512 pixels, 32-channel simultaneous recording)
Scanning mode 3 D: X-Y-λ4 D: X-Y-Z-λ, X-Y-t-λ5 D: X-Y-Z-t-λPoint scan
Diascopic detector 1 channel (motorized or manual)
Optical zoom 1X-1000X (continuous variable)
FOV Square inscribed in a φ18mm circle
Image bit depth 12 bit
Compatible microscopes Upright type ECLIPSE 80i/90iECLIPSE E600 (Motorized diascopic detector cannot be attached)ECLIPSE E800/E1000 (Motorized diascopic detector cannot be attached)
Inverted type ECLIPSE TE2000-E/UFixed stage type ECLIPSE E600FN (Motorized diascopic detector cannot be attached)
Z-axis control Built-in microscope monitor ECLIPSE 90i, ECLIPSE E1000, ECLIPSE TE2000-EExternal motor Stepping motor, 50 nm step
Control computer OS Windows® XP Professional (Hard disk capacity 4TB)Interface Ethernet
Scan rotation 360˚ (1˚ step)
Analysis software 2 D analysis, 3 D structure, 4 D analysisSpectral analysis, time lapse
Consumption power BD laser 15WAr laser 2000W max.Solid laser (488nm, 25mW) 140WSolid laser (561nm, 10mW) 25WHeNe laser 25W (G-HeNe, Y-HeNe, R-HeNe)C1si system 830W (PC, monitor, C1si controller, AOM controller)Epi-fl microscope 530W
Installation condition Temperature 5 to 35˚C, Humidity 65% (RH) or less (non-condensing)
En
Images and specimens courtesy of:
1 Specimen provided by Dr. Kazunori Toida, Department of Anatomy and Cell Biology,Institute of Health Bio Sciecnces, The University of Tokushima Graduate School
23567 Cells provided by Drs. Takuya Saiwaki and Yoshihiro Yoneda, Department of Medicine,Osaka University
4 Image provided by University of California, San Diego National Center for Microscopy &Imaging Research. Specimen provided by Dr. Tom Deerinck
8 Image provided by Dr. Takashi Sakurai, Photon Medical Research Center, HamamatsuUniversity School of Medicine
9 Specimen provided by Dr. Hiroshi Kimura, Horizontal Medical Research Organization,Faculty of Medicine, Kyoto University
! Shooting cooperation: Dr. Chieko Nakada, Kusumi Office, Institute for Frontier MedicalSciences, Kyoto University
This brochure is printed on recycled paper made from 40% used material.
Specifications and equipment are subject to change without any notice or obligationon the part of the manufacturer. June 2005. ©2005 NIKON CORPORATION
Printed in Japan (0506-15)T Code No. 2CE-SAPH-1
www.nikon.com/
Fuji Bldg., 2-3, Marunouchi 3-chome, Chiyoda-ku, Tokyo 100-8331, Japan
NIKON CORPORATION
Please contact Nikon for a handy pamphlet listing compatibleaccessories, including objectives and epi-fluorescence filters.Some models are not available in certain areas. Please checkwith your local Nikon representative for details.
This product is controlled byEAR (Export AdministrationRegulations). It should not beexported without authorizationfrom the appropriategovernment authorities.
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WARNINGTO ENSURE CORRECT USAGE, READ THE CORRESPONDINGMANUALS CAREFULLY BEFORE USING YOUR EQUIPMENT.
* Monitor images are simulated.CompactFlash is a trademark of SanDisk Corporation, Sunnyvale, CA, U.S.A. Company names and product names appearing in this brochure are their registered trademarks or trademarks.
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