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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)

(Channel)

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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.

0

400 750

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S-polarizing

P-polarizing

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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493.

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Fluorescence

Fluorescence wavelength (nm)

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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5

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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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Wavelength (nm)

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

380

700

650

(820

)43

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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

0)

700

AOM Controller

(2400 or 2900)

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(820

)43

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(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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