Modular Confocal Laser Microscope System C1 Series
Modular Confocal Laser Microscope System
C1si / C1plus
State-of-the-Art Resolution, Contrast, and Image Brightness
Nikon has fused its first-class optics and electronics technology to develop the Eclipse C1 series
Modular confocal laser microscope system
modular confocal microscope systems that provide the highest-quality digital imaging with an
DIGITAL ECLIPSE C1plus
ultra-compact and lightweight design. With basic C1plus and spectral imaging C1si, Nikon now
Employs high resolving power to capture images for spatial information—3D data, for
example—and high-speed scanning for time-lapse information. Simultaneous 3-channel
fluorescence, 3-channel plus DIC, and other advanced imaging is possible.
has a confocal solution to suit a broad range of advanced research needs.
High-resolution image acquisition
Molecule dynamics with optical stimulation
Incorporating Nikon’s renowned optical technologies—including VC
objectives—in a confocal mechanism to eliminate flare from fluorescence
images, the sophisticated C1 series allows high-resolution image capture. The
system also supports multi-dimensional acquisition including XYZ imaging,
time-lapse imaging, spectral imaging, and multi-point imaging.
The C1 series can handle a variety of the latest optical stimulation
applications, including observation of photoconversion fluorescence proteins
that change fluorescence characteristics when exposed to laser light, and
observation of FRAP (fluorescence recovery after photobleaching) in the ROI
(region of interest).
Spectral image acquisition (C1si)
Powerful image analysis function
The C1si true spectral imaging confocal laser scanning microscope system is
the powerful solution to autofluorescence, which is a problem when
observing live specimens as it is difficult to distinguish autofluorescence from
the target fluorescence. The spectral detector can acquire a broad
wavelength range up to 320nm with a single scan to provide high-resolution
images. The dedicated software allows unmixing of target fluorescence
signals and autofluorescence. Also, the short scan time reduces damage to
cells and improves time resolution.
Nikon appreciates that the nalysis of captured images is an important task.
The latest analysis functions that the C1 series offers include analysis over
time of intensity and spectra changes in ROI (region of interest), and
colocalization analysis for studying intensity data between channels.
True spectral imaging confocal laser scanning microscope system
DIGITAL ECLIPSE C1si
Supports the latest optical stimulation applications through the acquisition of spatial,
time-lapse and spectral information. Enables the simultaneous acquisition of 32-channels
of fluorescence spectra, and the wavelength resolution can be switched between 2.5, 5,
and 10nm. Changeover of the spectral detector and the 3-channel PMT detector is a snap.
Colocalization analysis
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The highest quality optical performance
Unprecedented brightness and contrast
Compact modular design
The Eclipse C1 series—the culmination of Nikon’s many years of dedication
as an optical equipment manufacturer—delivers optical performance of the
highest level in this class of confocal systems. With the Eclipse C1 series,
fluorescence images are rendered with unprecedented brightness, while DIC
images are pin-sharp and of the highest possible contrast.
• Modular Components: Expansion and
maintenance are easy.
• Pre-calibrated Modules: No need for
calibration during set-up.
• Compact Design: Does not fill up bench
space. The C1plus features the world’s
smallest and lightest scanning head.
DIC image
Overlay of DIC and fluorescence images
Actin/Mitochondria/DAPI
Precision objectives for aberration-free confocal microscopy
CFI Plan Apochromat VC series
CFI Apochromat TIRF series
These objectives correct axial chromatic aberration on the whole visible light
spectrum including 405nm (h line), making this series perfect for multistained confocal observations
Among Nikon objectives, this series has the highest resolution. It boasts an
unprecedented NA of 1.49 even when a standard coverslip and immersion oil
are used.
C1si true spectral imaging confocal laser scanning microscope
system configured with a 4-laser unit
Advanced scanning features
• Up to 1000x optical zoom
The desired area can be optically zoomed. The GUI can be
used to specify the area to be zoomed and rotate it.
Specimen: Cerebellum Purkinje cell with mouse monoclonal anti-calbindin
antibody stained with FITC
Specimen courtesy of Assistant Prof. Kazunori Toida, Department of Anatomy and
Cell Biology, Institute of Health Biosciences, the University of Tokushima Graduate
School
4x
•Corrected shift in Z-axis direction with VC objective lens
With the conventional objective, DAPI fluorescence (blue) may
shift in the Z-axis direction due to axial chromatic aberration.
However, because the VC objective lens’ axial chromatic
aberration has been corrected up to the violet range, the DAPI
fluorescence (blue) shift in the Z-axis direction is also corrected.
The VC objective lens sample image on the right clearly shows
that the DAPI-stained nucleus is positioned within the cell.
XY
Fluorescence image of actin (green: Alexa 488, excitation: 488nm), mitochondria
(red: Mito Tracker Organe, excitation: 543nm) and nucleus (blue: DAPI, excitation:
408nm) of HeLa cell. Consecutive cross-sectional XY and XZ images acquired with
a confocal laser microscope and CFI Plan Apo VC 100x oil objective lens.
XZ
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Conventional objective lens
8x
2x
CFI Apo TIRF 60x oil/1.49 (left)
CFI Apo TIRF 100x oil/1.49 (right)
CFI Plan Apo VC 100x oil/1.40 (left)
CFI Plan Apo VC 60x oil/1.40 (middle)
CFI Plan Apo VC 60x WI/1.20 (right)
• ROI scanning with AOM/AOTF
Free shape scanning is possible with AOTF*(Acousto Optical
Tunable Filter) and AOM** (Acousto Optical Modulator). It
is effective for bleaching specific areas in FRAP/FLIP
experiments or optical stimulation with a 405nm laser.
VC objective lens
*Comes standard with the 4-laser unit
**Optional with the 3-laser unit
Excitation of all areas
XY
Excitation of all but specific areas
Excitation of specific areas only
Broad selection of laser options
Leveraging the latest PC functions
The C1 series accommodates a greater variety of lasers with wavelengths
ranging from 405 to 640nm. It also supports solid-state lasers.
Because image processing is conducted with the controller instead of the PC,
the user has greater freedom in selecting a PC. This makes it easy to upgrade
the PC depending on requirements. Also, handling large amounts of data of
up to four terabytes in size is possible, allowing large-size data acquisition,
such as 3D time-lapse imaging.
XZ
5
Easy to use software for seamless image acquisition
Versatile multi-dimensional imaging functions
All settings and procedures required for live image capture—fundamentals in
confocal microscopy—can be viewed in a single window, eliminating the need
for the operator to switch between many windows. The operation panel gives
you an at-a-glance picture of all important settings including scan speed, pixel
XYZ image
Time-lapse (XYT) image
With precise Z-axis information of a specimen—a feat not possible with
ordinary fluorescence microscopy—clear cross section images are possible.
Changes over time can be analyzed using images with high spatial resolution.
Intensity change analysis, ratio analysis and real time graph display during
image acquisition is possible.
size, zoom/pan, PMT settings, pinhole, shutter, and color image look-up
table. Scanning modes range from 2D (XY, XT), to 3D (XYZ, XYT), and even
further to 4-dimensional (XYZT) scans.
XZ (cross section)
3D orthogonal display
At-a-glance setting panel
1Simple GUI 2Microscope setting 3Thumbnail display
Scan start/stop
Image capture area
Scan speeds
Pixel size
Initial state
Digital zoom
9 sec
86 sec
Specimen: visualized changes of calcium in cultured cells loaded with Fluo-4 AM
Pan
16 sec
110 sec
YZ (cross section)
Time-lapse (XYZT) image
Volume rendering
Two-dimensional (XY) or three-dimensional (XYZ) time-lapse images can be
captured. The C1 series allows flexible interval time settings.
Angles of 3D images can be freely changed.
PMT amplification
Color display setting
1Simple GUI
Simple tool bar
Available for easy image capture.
3D tiled image
Images with different depths can be viewed simultaneously on a single screen.
XYZT images of HeLa cells stained with Hoechst33342 are captured at 15-minute intervals.
Multi-point imaging within the field of view
Time-lapse, Z-stack images of multiple points within the objective’s field of
view can be captured.
Configuration window
(basic)
Selecting laser and
objective lens types.
Brightness window
Adjusting the brightness of each laser.
Navigation window
Adjusting zoom magnification and Z position in preview image.
Specimen: mouse’s whole brain stained with mCB (mouse monoclonal anti-calbindin: green), rPV (rabbit
polyclonal anti-parvalbumin: red) and nucleus (Hoechst: blue)
Specimen courtesy of Assistant Prof. Kazunori Toida, Department of Anatomy and Cell Biology, Institute
of Health Biosciences, the University of Tokushima Graduate School
Optical path
Displaying information such as laser wavelength in use, 1st
dichroic mirror, 2nd dichroic mirror and detector.
C1plus
C1si
2Microscope setting
Acquisition window
Image acquisition settings.
Spectral (XYλ) image (C1si)
By using a spectral detector, spectral images with high spatial resolution can
be captured with a single scan.
3Thumbnail display/Parameter resetting
Thumbnails and parameters of captured images can be displayed. It is easy to reset the
same parameters while confirming the images.
Nikon motorized microscopes can be operated with the intuitive GUI.
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2
3
Specimen: respective expression of GFP, YFP and RFP in the nucleus of HeLa cell
Cells courtesy of Dr. Yoshihiro Yoneda and Dr. Takuya Saiwaki, Faculty of Medicine, Osaka University
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Observation of molecular dynamics
FRET(Fluorescence Resonant Energy Transfer)
FRAP(Fluorescence Recovery after Photobleaching)
FRET is a physical phenomenon that occurs when there are at least two
fluorescent molecules within 10nm distance and it can be observed when
energy transfers from one fluorescent molecule to another. The C1 series
enables imaging of the changes in three-dimensional protein structures as
well as the interaction of two different proteins with high spatial resolution.
After bleaching ROI with strong laser exposure, the recovery process of
fluorescence is observed to analyze movement of molecules. With the C1
series, only a specific area can be bleached by controlling the laser intensity
with AOTF (comes standard with the 4-laser unit) and AOM (optional with
the 3-laser unit).
Donor
Acceptor
Donor
Acceptor
FRAP setting panel
Before induction of apoptosis
FRAP image
After induction of apoptosis
Specimen: after bleaching a part of a HeLa cell in which H1-GFP is expressed, the recovery of
fluorescence intensity is observed in time-lapse recording. One image is displayed for every 11
images captured.
Specimen courtesy of Dr. Tokuko Haraguchi, Kobe Advanced ICT Research Center, National Institute
of Information and Communications Technology
Specimen: HeLa-S3 cell in which SCAT (sensor for activated caspase based on FRET) is expressed. CFP fluorescence
(480nm: green) is detected within the cell undergoing apoptosis, and YFP fluorescence (545nm: red) is detected within
the cell prior to apoptosis by FRET.
Specimen courtesy of Dr. Tokuko Haraguchi, Kobe Advanced ICT Research Center, National Institute of Information and
Communications Technology
FRET analysis setting panel
Kaede Photoconversion fluorescence protein
i-FRAP (inverted FRAP) / FLIP(Fluorescence Loss in Photobleaching)
This photoconversion fluorescence protein originally emits green fluorescence
that changes to red when exposed to ultraviolet light (408nm). By controlling
the laser exposure position, area, intensity, timing and number of times,
imaging the changing Kaede protein color in the cell is possible.
With i-FRAP, the area outside ROI is bleached to analyze movement of
fluorescence molecules that leave ROI.
With FLIP, the movement of fluorescence molecules that enter the ROI from
outside is analyzed by continuously bleaching ROI.
Bleached
10 sec
20 sec
Kaede image (Overlay of DIC and Kaede fluorescence images)
30 sec
40 sec
50 sec
1600
1400
Initial state
1200
1000
spots 1
spots 2
800
spots 3
spots 4
600
400
200
405nm optical stimulation
405nm optical stimulation
Specimen: fluorescent protein vector “CoralHue® Kaede” of Amalgaam Co., Ltd. expressed in HeLa cell. Fluorescence changes after optical stimulation by 405nm laser are captured over time. Diascopic DIC and
fluorescence images of Kaede (green/red) are simultaneously acquired.
Cells courtesy of Amalgaam Co., Ltd.
8
0
0.0
405nm optical stimulation
30.6 61.1
91.5 122.0 152.5 182.9 213.4 243.9 274.3
Specimen: BDV (Borna disease virus) P protein fused with GFP expressed in human-derived glia cells infected by BDV.
Images courtesy of Dr. Keizo Asanaga, Dept. of Virology, Research Institute for Microbial Diseases, Osaka University
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True spectral imaging
In addition to the conventional fluorescence detector, the C1si true spectral imaging confocal
laser scanning microscope is equipped with a dedicated spectral detector. By switching
between these detectors, accurate spectral data of fluorescence signals can be obtained. The
C1si captures minute changes of wavelength in true color and unmixes even overlapping
spectra. Moreover, it has the capability to acquire spectra over a 320nm-wide wavelength
range in a single scan, minimizing damage to living cells.
Fluorescence labels with closely overlapping spectra can be unmixed cleanly with no crosstalk.
Spectral detector with polarization control technology
Effortless fluorescence unmixing
Nikon original
Nikon’s proprietary DEES (Diffraction Efficiency Enhancement System) for polarization
correction is employed in the C1si’s spectral detector to maximize brightness. By coaligning the direction of polarization of fluorescent light, 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 over the whole visible range from blue to red.
Even without a given reference spectrum, simply specifying a ROI within the
image and clicking the Simple Unmixing button allows separation of
fluorescence spectra. The C1si contains a built-in database of given spectral
Grating properties
100
Diffraction ratio (%)
90
Overview of DEES
(internal structure of spectral detector)
80
S-polarizing
70
60
50
40
30
P-polarizing
20
10
S-polarizing
P-polarizing
0
400
S1
S2
S2
S1
Unpolarized light
Polarization rotator
P
Multiple gratings (2.5/5/10nm)
Polarized beam splitter
Fluorescence
unmixing
750
Wavelength (nm)
Multi-anode PMT
The spectral imaging detector
utilizes a newly developed laser
shielding mechanism. Coupled with
a wavelength resolution
independent of pinhole diameter,
this mechanism successfully shuts
out the reflected laser beam. 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.
Specimen: HeLa cell in which GFP (Tubulin) and YFP (Golgi) are expressed. Spectral image captured with a 488nm laser (left). After fluorescence unmixing,
GFP is indicated in green and YFP is indicated in red (right). The graph at left shows the spectral curve in the ROI.
Specimen courtesy of Dr. Sheng-Chung Lee, Dr. Han-Yi E. Chou, National Taiwan University College of Medicine, Institute of Molecular Medicine
What is spectral unmixing?
Optical fiber
Nikon original
Nikon original
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 (Nation Institutes 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.
Multi-anode PMT sensitivity correction
(Brightness)
4000
3500
3500
3000
3000
2500
2500
2000
2000
1500
1500
1000
1000
500
500
1
4
7
10
13
16
19
(Channel)
10
(Brightness)
Pre-correction
4000
0
data provided by manufacturers of fluorescence dyes that can be specified as
reference spectra for fluorescence unmixing. Users may also add spectral
information for new labels to the database.
22
25
28
31
0
Post-correction
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 high
optical transmission.
Nikon original
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.
The spectrum obtained by actual measurement is a mix of spectral elements with a certain proportion. An imaging
algorithm is used to compare the spectra of each pixel with reference curves for each spectral element. Each fluorescent
probe in the specimen is displayed in a unique color in the final unmixed image.
fn = Sn*P
Intensity f
fn
fn =Wave pattern of spectrum obtained by actual measurement
Sn=Wave pattern of individual reference spectrum
P =Ratio of elements for each wave pattern
P1 Sn 1
P2 Sn 2
Wavelength λ
DISP
Reference wave pattern (S) is selected from the following three depending on the experiment.
1 Spectrum obtained by actual measurement of the zone with less crosstalk in the captured image
2 Data obtained by another actual measurement using only one probe
3 Spectral data provided by probe maker
Integrator (1)
Integrator (2)
Pixel time
1
4
7
10
13
16
19
(Channel)
22
25
28
31
Integration
Hold
Reset
Two integrators work in parallel when the optical signal is read to ensure there are no discrepancies.
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Unmixing of multiple fluorescence
Confirmation of GFP expression
Because wavelength resolution and range are freely selectable, scanning of a fluorescence
protein with a wide wavelength range from blue to red such as CFP/GFP/YFP/Ds Red is
possible at one time. Reference data allows unmixing and display of each color.
In conventional confocal observation, fluorescence is visualized as fluorescence intensity
in a certain wavelength range. The spectral detector allows the confirmation of detailed
wavelength characteristics of the fluorescence. The C1si’s spectral detector enables the
slight color differences to be confirmed as wavelengths through sensitivity correction.
Fluorescence
unmixing
Specimen: HeLa cell in which nucleus is labeled with CFP, actin-related protein (Fascin) labeled with GFP, Golgi body labeled
with YFP, and mitochondria labeled with DsRed. Spectral image captured with 408nm and 488nm laser exposure (left). The
fluorescence spectra of the captured image are unmixed using reference spectra (right).
Specimen courtesy of Dr. Kaoru Kato and Dr. Masamitsu Kanada, Neuroscience Research Institute, The National Institute of
Advanced Industrial Science and Technology (AIST)
Fluorescence
unmixing
The correspondence of the spectral curve (blue) of ROI2 in the
image and the reference curve (green) of eGFP proves that
GFP is expressed in ROI2
Unmixing red fluorochromes
Red fluorochromes, which had previously posed a challenge, are now simple to unmix.
Frame lambda
Fluorescence
unmixing
Spectra for ROI 1 and 2 corresponding to the image on the right
Rhodamine’s fluorescence spectral peak is at approximately 579nm,
while that for RFP is approximately 600nm. RFP’s fluorescence is
weaker than Rhodamine’s, but their spectra are cleanly unmixed.
Specimen: Arabidopsis proteoglycan and fused protein of GFP. Spectral image captured with 488nm laser exposure (left). Once
the image is unmixed using reference spectra for autofluorescence (ROI1) and GFP, GFP is indicated in green and
autofluorescence is indicated in red (right).
Specimen courtesy of Assistant Prof. Toshihisa Kotake, Laboratory of Developmental Biology, Department of Life Science,
Graduate School of Science and Engineering, Saitama University
Specimen: actin of HeLa cell that has RFP expressed in the nucleus is stained with Rhodamine. Spectral image in the 550630nm wavelength range captured at 2.5nm wavelength resolution with 543nm laser exposure (left). RFP indicated in red and
Rhodamine indicated in green (right) in the image after fluorescence unmixing.
Specimen courtesy of Dr. Yoshihiro Yoneda and Dr. Takuya Saiwaki, Faculty of Medicine, Osaka University
Using this function allows the user to acquire either wide continuous or bands of spectra by
sequentially acquiring individual narrower spectra regions. The laser and the spectral range
acquired can be individually set for each sequence in the series, the final single spectra is a
result of combining all spectra in the sequence.
Frame lambda setting panel
Image and spectral curve obtained with frame lambda function. 410-740nm spectral range captured with
408nm, 488nm and 561nm lasers is used.
Segmentation
Unmixing autofluorescence of multi-stained samples
Fluorescence unmixing makes it possible not only to separate closely overlapping
fluorescence spectra such as CFP and YFP but also to eliminate autofluorescence of cells,
which until now was difficult.
By spectral imaging with C1si, wavelength information of the entire range can be
obtained in a single scan. Therefore, it is no longer necessary to acquire only the
limited wavelength range or reshoot other ranges during the imaging session.
Consequently, there is minimized photo-toxicity to the specimen.
After spectral imaging, images that are filtered (segmented) with any desired
wavelength range easily displayed.
Segmented image of the spectral image obtained with the frame lambda function
Fluorescence
unmixing
Reprint from Cell Imaging Press Vol. 3
12
Specimen: Zebrafish egg stained with cadherin-GFP and DAPI. Spectral image captured with 408nm and 488nm laser exposure
(left). After unmixing using reference spectra for autofluorescence (ROI1), GFP and DAPI, the autofluorescence in the image is
eliminated (right).
Specimen courtesy of Dr. Tohru Murakami, Neuromuscular and Developmental Anatomy, Gunma University Graduate School of
Medicine
Segmentation setting panel
The spectral image of stained actin (Alexa 488), mitochondria (Mito Tracker Organe) and nucleus (DAPI) of
HeLa cell, captured with the frame lambda function, is post-acquired in the three wavelength ranges of 420480nm, 500-530nm, and 570nm and longer using the segmentation function.
Specimen courtesy of Dr. Yoshihiro Yoneda and Dr. Takuya Saiwaki, Faculty of Medicine, Osaka University
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The system expandability of the C1 series meets the needs
of today’s research
Time-lapse acquisition of spectral images
To meet the requirements of constantly evolving live cell imaging, Nikon provides a wide array
of microscopes, accessories and software. Outstanding expandability of the C1 series delivers
optimal solutions for live cell observation.
True spectral FRET analysis
FRET (Fluorescence Resonance Energy Transfer) analysis using true spectral
imaging allows three dimensional analysis with high S/N ratio and high
spatial resolution as well as easy determination of FRET by real-time
detection of spectral changes derived by FRET.
Also, even when spectra of donor and acceptor are overlapped like CFP and
YFP, unmixing using reference data enables detection of detailed intensity
changes and ratio analysis of fluorescence signals (YFP/CFP) without bleed
through.
Acquisition of spectral image (XYT λ)
Fluorescence unmixing
Spectral image in the 460-620nm range captured at 5nm wavelength
resolution using a spectral detector enables observation of fluorescence
wavelength changes.
Spectral FRET analysis is possible by unmixing using reference data of CFP and YFP. Twodimensional change (FRET) of intracellular Ca2+ concentration is easily determined from
spectral data without acceptor bleaching.
Before ATP stimulation
True color image
CLEM (Controlled Light Exposure Microscopy) system
What is CLEM?
The CLEM system senses the existence of fluorescence in a specimen and
exposes the laser in areas with fluorescence by controlling the laser pixel by
pixel. Because the laser is not used where excitation light is not needed,
laser exposure is minimized and live cell phototoxicity is drastically reduced.
Features
8 sec after ATP stimulation
Laser on
• Because the laser is switched off in areas that do not emit fluorescence,
exposure light is reduced and the fading speed of fluorescence labels is
decreased twofold to fourfold. Phototoxicity of cells is also reduced. (See
diagram below )
Scan
Laser off
Laser off
Fluorescent sample
• CLEM switches off the laser in areas with sufficient fluorescence and
calculates pixel brightness. This extends the apparent dynamic range of
fluorescence intensity, allowing an image with both extremely weak and
bright fluorescence to be displayed without saturation.
Extending dynamic range
CLEM off
CLEM on
Reducing phototoxicity
CLEM off
Fluorescence intensity
Spectral analysis
100%
FRET image after spectral unmixing. CFP is indicated in blue and YPF indicated in green.
CLEM on
CLEM off
50%
Five-dimensional analysis (XYZT λ)
CLEM on
Time-lapse changes (T) and spectra (λ) in three-dimensional space (XYZ) can
be analyzed.
Before ATP stimulation
8 sec after ATP stimulation
yz
0%
0
3000
720
Time (sec)
yz
HeLa cell
xy
xy
Specimen: HeLa cells loaded with Rhodamine 123 are observed with CLEM on and off. The graph shows the
fluorescence intensity change and scan time, and the reduction of fading by CLEM.
Images courtesy of Dr. Takashi Sakurai, Photon Medical Research Center, Hamamatsu University School of
Medicine
xz
xz
Reference
R.A. Hoebe, C.H. Van Oven, T.W.J. Gadella Jr, P.B. Dhonukshe1, C.J.F. Van Noorden & E.M.M. Manders,
“Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging.” Nature Biotechnology P1-5 (2007)
True color image and spectral analysis of CFP and YFP. Spectral curve in ROI. Left
peak indicates CFP and right peak indicates YFP respectively. After ATP stimulation,
peak of CFP drops and peak of YFP rises due to FRET.
Reprint from Cell Imaging Press Vol. 1
True spectral Kaede analysis
Multi-point observation system
Changes of Kaede fluorescence protein over time can be recorded as spectral
changes. Not only color change from green to red but also slight spectral
changes can be captured.
Before 405nm optical stimulation
Specimen: three-dimensional reconstruction image of juxtaglomerular cell of mouse olfactory bulb
shows diversity of three-dimensional structure captured with confocal laser microscope. 50mm-thick
coronal section is multiply immunostained with anti-calbindin antibody (mouse monoclonal antibody,
FITC label, green) and anti-tyrosine hydroxylase antibody (mouse monoclonal antibody, Cy3 label, red).
The image captured with CLEM on allows volume rendering without saturation even if the image has
fluorescence intensity difference. In the image captured with CLEM off, saturation occurs in the volume
rendering process.
Specimen courtesy of Assistant Prof. Kazunori Toida, Department of Anatomy and Cell Biology, Institute
of Health Biosciences, the University of Tokushima Graduate School
Use of optional motorized stage enables easy multi-point observation including multi-point time-lapse, multi-point XYZ and multi-point four-dimensional (XYZT)
observations.
Basic concept of multi-point XYZ imaging
After 405nm optical stimulation
Specimen: fluorescent protein vector “CoralHue® Kaede” of Amalgaam Co., Ltd. expressed in HeLa cell. Fluorescence in the 500-660nm range is
obtained at 5nm wavelength resolution using the real-time spectral method. During this process, ROI1 is optically stimulated by 405nm laser. Spectral
graphs show photoconversion of fluorescence from 520nm before optical stimulation to 580nm after stimulation.
Specimen courtesy of Amalgaam Co., Ltd.
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Multi-point setting panel
15
Confocal microscope with Perfect Focus System
Multi-mode imaging system—TIRF-C1
• Motorized inverted microscopes Ti-E and TE2000-E feature real-time focus
maintaining system—Perfect Focus System (PFS)—continuously corrects
focus drift during lengthy time-lapse observation and when reagents are
being added.
• The spectra of a broad 320nm range can be obtained at one time with a
spectral detector allowing stable time-lapse observation of the spectra.
• By using an optional stage incubator, lengthy time-lapse observations are
possible.
Focus drift during long-term time-lapse imaging
Focus drift resulting from temperature change during longterm imaging is eliminated.
Confocal image
The laser TIRF system and the confocal microscope system can be mounted
simultaneously on the inverted microscope Ti-E or TE2000-E. The laser TIRF
system incorporates an epi-fluorescence module. Switching between the two
light sources and adjustment of alignment are easy.
This shows the basal portion of the cell. A
clear band of substantial F-actin (red) is
shown at the leading edge of the cell, which
is migrating toward the right side. Paxillin
molecules are green. Stress fibers are facing
the rear of the cell.
Focus drift when adding reagents
Focus drift resulting from sudden temperature change when
adding reagents is eliminated, therefore improving the
reliability of fluorescence intensity change measurement data.
Focus drift during multi-point time-lapse
observation (using motorized XY stage)
TIRF image
Strong and clear fluorescence derived from
paxillin is observed in the evanescent field.
The focal adhesions existing at the portion of
cells in contact with the coverglass were
clearly confirmed.
Focus drift caused by stage movement is eliminated.
Focus drift when capturing three-dimensional
images
Focus position is maintained in real time, eliminating the need
to take extra images in anticipation of focus drift. This reduces
photobleaching and damage to live cells.
Concept of the Perfect Focus System
Specimen
Digital camera is an option.
Offset plane
Medium
Interface
Confocal patch-clamp imaging system FN1-C1
Stage
Stage
Oil, water
Coverslip
Objective
Perfect Focus System
LED lightsource and
detector
Weak near-IR light
Perfect Focus System with motorized sextuple DIC nosepiece (Ti-E)
Monitor light
XZT recording of mitochondria by confocal microscopy
PFS off
Specimen: mouse bone marrow stroma cell (ST2 cell). After fixing in 4% formaldehyde, cells were
treated with 0.25% Triton X-100 before double staining with paxillin antibodies and TRITC-phalloidin.
Images courtesy of Assistant Prof. Shuichi Obata, Kitasato University
Optical offset
Confocal observation with FN1 (fixed stage microscope) is highly beneficial
in vivo imaging. The FN1 allows high-resolution imaging of deeper areas of
cells with the excellent patch-clamp operability of
FN1 and the performance of Plan 100x (NA 1.1)
objectives. Also, elimination of autofluorescence in
vivo, which until now has been difficult, can be
easily achieved.
Observed light
Camera
PFS on
Sequential XZ sectional images taken by changing the Z-axis
position. With PFS off, strong photobleaching occurs due to
frequent (65) scanning in anticipation of focus drift. With PFS on,
scanning is less frequent because of the elimination of focus drift,
thereby reducing photobleaching
Fluorescence image
DIC image
Specimen: GABA ERGIC neuron (green: GFP) and potassium channel protein (red: YFP) in a cerebellar slice
Images courtesy of Dr. Thomas Knöpfel, Team Leader, Laboratory for Neuronal Circuit Dynamics, Brain
Science Institute, RIKEN
Reprint of Cell Imaging Press Vol. 2
Specimen: HeLa cells stained with Rhodamine 123
Objective: CFI Plan Apo VC 60x water dipping, NA 1.20
Stage incubation system INU series
Optional imaging software NIS-Elements series
Nikon’s original NIS-Elements imaging software enables advanced analysis
using images captured by the C1 series confocal microscope. It allows object
counting, measurement of area and brightness, and creation of histogram.
Number
Temperature of the stage, water bath, cover, and objective lens is controlled,
allowing living cells to be maintained for a long period. A transparent glass
heater prevents condensation, and loss of focus due to heat expansion on the
stage surface is prevented, making this system ideal for lengthy time-lapse
imaging applications.
Manufactured by Tokai Hit Co., Ltd.
FN1-C1
Intensity distribution measurement
Operation panel
16
Nuclei of HeLa cells are stained with DAPI. The graph shows their intensity distribution and number.
17
Confocal image gallery
Sliced hippocampus of a transgenic rat (image of a nerve in the spine)
Image courtesy of Dr. Hu Qian, Chinese Academy of Science
GFP expressed in the whole tail of drosophila sperm. Anterior pole of the
egg is indicated in red (pseudo color) and GFP indicated in green after
unmixing the autofluorescence spectrum of the egg and spectrum of sperm
(GFP).
Images courtesy of Director and Professor Masatoshi Yamamoto, Drosophila
Genetic Resource Center, Kyoto Institute of Technology
Argulus acetabulum
Specimen courtesy of School of Environmental Sciences and Development,
North-West University, South Africa
Autofluorescence of thrips, 408nm/488nm/543nm excitation
Image courtesy of Dr. Steve Cody, Ludwig Institute for Cancer Research
Three-dimensional reconstruction image of mouse hippocampus (GFP:
inhibitory neurons, green, YFP: excitatory neurons, magenta) through
volume rendering after spectra unmixing
Image courtesy of Dr. Masayuki Sekiguchi, Department of
Degenerative Neurological Diseases, National Institute of
Neuroscience, National Center of Neurology and Psychiatry, Japan
64-celled (32 cells) embryo of Branchiostoma belcheri stained with
anti α tubulin antibody and DAPI
Image courtesy of Prof. Kinya Yasui, Assistant Prof. Kunifumi Tagawa,
Marine Biological Laboratory, Hiroshima University Graduate School
of Science
Three-dimensional reconstruction image of nerve through volume
rendering after unmixing spectra of nerve (GFP) and peripheral tissue
(autofluorescence) of the 188µm-thick sliced hippocampus image
(spectral imaging with 5nm wavelength resolution, 500-660nm)
Specimen courtesy of Professor Shigeo Okabe and Tatsuya Umeda,
Department of Cell Biology, School of Medicine, Tokyo Medical and
Dental University
Living mouse egg, stained with Hoechst3342 (nucleus) and
MitoTrakerOrange (mitochondria) and overlaid with DIC image
Image courtesy of Dr. Atsuo Ogura and Dr. Hiromi Miki, RIKEN Tsukuba
Institute, RIKEN BioResource Center, BioResource Engineering Division
Fungus spore
Specimen courtesy of Prof. Rudi Verhoeven, Department of Plant
Sciences, University of the Free State Bloemfontein, South Africa
Comparison before (left) and after (right) exposure of anticancer agent to T47D breast cancer cell. Blue: nucleus, green:
actin, red: mitochondria
Specimen courtesy of Dr. Mitsuhiro Kudo, Department of Pathology, Integrative Oncological Pathology,
Nippon Medical School
DAPI, Alexa 488, Alexa 546
Specimen courtesy of Dr. Ulf Ahlgren, Umea University,
Sweden
18
Kaede expression localized to the mitochondria within Arabidopsis leaf
Top: true color image before (left) and after (right) optical stimulation
Bottom: Kaede-green and Kaede-red after unmixing the above image using reference data
of autofluorescence, Kaede-green and Kaede-red. Image before (left) and after (right)
optical stimulation
Specimen courtesy of Assistant Professor Shinichi Arimura, Graduate School of Agricultural
and Life Sciences, The University of Tokyo
Cells of an onion root, Hoechst33258, OregonGreen488
Image courtesy of Dr. Yoshinobu Mineyuki, Department of Life
Science, Graduate School of Life Science, University of Hyogo
Actin of HeLa cell that has YFP expressed in nucleus is
stained with Alexa 488. Alexa 488 is indicated in green
and YFP is indicated in red after unmixing using
fluorescence reference data.
Specimen courtesy of Dr. Yoshihiro Yoneda and Dr.
Takuya Saiwaki, Faculty of Medicine, Osaka University
19
System diagram
Scanning Head
(C1plus/C1si-Ready/C1si)
Laser Fiber
Optical Fiber
Optical Fiber
1st Dichroic Mirror
(C1si only)
Fluorescence Filter Block
Ring Adapter L/S
Spectral Detector
C1 Adapter
Inverted Microscope Ti-E/U
Standard Epi-fl Detector
(2-PMT or 3-PMT)
L4
3-laser Unit
C1 Adapter
90i Microscope with
Digital Imaging Head
80i Microscope with
Laser-safe Trinocular Tube
L3
L2
L1
4-laser Unit
(Equipped with AOTF)
Fixed Stage Microscope FN1
with Laser-safe Trinocular Tube
AOM Controller
CLEM Control Unit
Software
PC
Monitor
Motorized
PFS nosepiece
Z-focus Module (for Ti-U)
Z-focus Module
Z-focus Module
Diascopic Detector (Manual/Motorized)
Controller
20
21
Specifications
Recommended layout
C1 plus
Combination with the Inverted Microscope Ti-E/U with 4-laser Unit
Laser light source
Laser wavelength
380
700
650
(785)
(2400 or 2900)
L4
200
L3
L2
L1
852
510
Confocal pinhole
Standard fluorescence
detector
W
Maximum loading number
Laser control
Laser shutter
Variable
Number of channels
W=1500mm (two 19-inch monitors)
W=1000mm (24-inch monitor)
レーザユニット
Scanning specifications
for a standard fluorescence
detector
Standard
Epi-fl
Detector
4-laser Unit
Scanning
Head
Display mode
Scanning speed
Scanning mode
(1420)
L4 L3 L2 L1 POWER
700
Controller
430
Spectral Detector
Spectral detector
Note 1) Computer table size is for reference only.
Note 2) Spectral detector is unnecessary for C1plus and C1si-Ready.
Scanning specifications
for a spectral detector
Combination with the Upright Microscope ECLIPSE 80i/90i with 3-laser Unit
380
700
650
200
510
852
W
W=1500mm (two 19-inch monitors)
W=1000mm (24-inch monitor)
Scanning Head
Standard
Epi-fl Detector
(1490)
3-laser Unit
700
Controller
430
Spectral detector
AOM Controller
22
Note 1) Computer table size is for reference only.
Note 2) Spectral detector is unnecessary for C1plus and C1si-Ready.
Data acquisition for applications
C1si with 4-laser unit
3
AOM
4
AOTF
3 channels
457(440), 408/514, 405/488, 457(440)/514, 488/543,
488/594, 408/488/543, 405/488/561, 488/543/633
457(440), 488, 405/488, 408/514, 457(440)/514, 488/543,
488/594, 408/488/543, 408/488/561, 488/543/633
457(440), 488, 405/488, 408/514, 457(440)/514, 488/543,
488/594, 408/488/543, 408/488/561, 488/543/633
3 channels
457(440), 405/488, 408/514, 457(440)/514, 488/543,
408/488/543, 405/488/561, 405/488/543/640,
405/488/561/640, BS20/80
457(440), 488, 405/488, 408/514, 457(440)/514, 488/543,
405/488/543, 405/488/561, 488/543/640, 488/561/640,
405/488/543/640, 405/488/561/640
160x16 to 2048x2048 pixels
Standard: 1fps (512x512 pixels)
Bi-directional scanning: 1.4fps (512x512 pixels)
2D: X-Y, X-T
3D: X-Y-Z, X-Y-T
4D: X-Y-Z-T
ROI scan (AOM necessary)
Multi-point time-lapse within single screen (X-Y-Z-T-Point)
Multi-point time-lapse (X-Y-Z-T-Point, motorized YX stage necessary)
Point scan
Scan rotation (-90 to 90°, 1° step)
FRET, FLAP, FLIP
Compatible (when laser is controlled by optional AOM)
—
—
—
—
—
—
—
—
(820)
(2400 or 2900)
Data acquisition for applications
CLEM compatibility
Number of channels
1st dichroic mirror
Corresponding wavelength
Wavelength resolution
Minimum wavelength step
Display mode
Scanning speed
Scanning mode
C1si with 3-laser unit
BD laser (405nm, 36mW, variable)
Laser diode (440nm, 20mW, variable)
Ar laser (488nm, 10mW)
Ar laser (488nm, 25mW)
Ar laser (488nm/514nm, 40mW)
Solid-state laser (488nm, 20mW)
G-HeNe laser (543nm, 2mW random polarization)
Solid-state laser (561nm, 10mW)
Y-HeNe laser (594nm, 2mW)
R-HeNe laser (633nm, 5mW)
4
AOM/AOTF/Halving laser controller
Motorized mechanical shutter (each laser)
Motorized switching
2 channels/3 channels
457(440), 408/514, 405/488, 457(440)/514, 488/543,
488/594, 408/488/543, 405/488/561, 488/543/633
—
1 channel (motorized or manual)
1x-1000x (continuous variable)
Square inscribed in a ∅ 18mm circle
12 bits
Upright type
ECLIPSE 80i/90i
Inverted type
ECLIPSE Ti-E/U, ECLIPSE TE2000-E/U/PFS
Fixed stage type
ECLIPSE FN1
Z-axis control
Built-in microscope motor
ECLIPSE 90i, ECLIPSE Ti-E, ECLIPSE TE2000-E/PFS
External motor
Stepping motor, 50nm step
Control computer
OS
Windows ® XP Professional
Interface
Ethernet
Analysis functions
2D, 3D, 4D, time-lapse, etc.
Power
BD laser (408nm, 38mW/440nm, 15mW)
15W (single phase AC 100V, 0.15A, with earth)
Ar laser
1500W (single phase AC 100V, 15A, with earth)
Solid-state laser (488nm, 20mW)
140W (single phase AC 100V, 1.4A, with earth)
G-HeNe laser (543nm, 1/2mW)
40W (single phase AC 100V, 0.4A, with earth)
Y-HeNe laser (594nm, 2mW)
40W (single phase AC 100V, 0.4A, with earth)
Solid-state laser (561nm, 10mW)
40W (single phase AC 100V, 0.4A, with earth)
R-HeNe laser (633nm, 10mW)
40W (single phase AC 100V, 0.4A, with earth)
LD laser (640nm, 10mW)
15W (single phase AC 100V, 0.15A, with earth)
Confocal system
830W (single phase AC 100V, 8.3A, with earth)
(PC, monitor, C1 controller, AOM controller)
Fluorescence microscope
630W (Ti-E)
Installation condition
Temperature 5-35°C, humidity 65% (RH) or less (non-condensing)
Please ask Nikon or your local distributor about combining laser types.
BD laser (405nm, 36mW, variable)
Laser diode (440nm, 20mW, variable)
Ar laser (488nm, 10mW)
Ar laser (488nm, 25mW)
Ar laser (457nm/477nm/488nm/514nm, 40mW)
Solid-state laser (488nm, 20mW)
G-HeNe laser (543nm, 1mW)
Solid-state laser (561nm, 10mW)
Semiconductor laser (640nm, 10mW)
Compatible
Not compatible
32 channels
20/80 Beam Splitter
400 -750nm
2.5/5/10nm (switchable)
0.2nm
160x160 to 1024x1024 pixels
Standard: 0.5fps (512x512 pixels, 32-channel simultaneous recording)
3D: X-Y-λ
4D: X-Y-Z-λ, X-Y-t-λ
5D: X-Y-Z-t-λ
Multi-point time-lapse within single screen (X-Y-Z-T-λ-Point)
Multi-point time-lapse scan (X-Y-Z-T-λ-Point, motorized YX stage necessary)
ROI scan (AOTF or AOM necessary)
Point scan
Scan rotation (-90 to 90°, 1° step)
FRET, FLAP, FLIP, etc.
Diascopic detector
Optical zoom
FOV
Image bit depth
Compatible microscopes
Spectra, Fluorescence unmixing, 2D, 3D, 4D, time-lapse, etc.
910W (single phase AC 100V, 9.1A, with earth)
(PC, monitor, C1 controller, 4-laser unit )
23
Basic Principle of Confocal Microscopy
•Extremely high resolving power in the Z-axis direction (depth) makes confocal observation ideal
for observing thick specimens such as embryos and eggs.
•Fluorescent-dyed specimens can be rendered in 3D.
•Extremely high S/N ratio images are obtainable.
Detector
Pinhole
Dichroic Mirror
Laser
X
Y
Scanning Mirror
Non-confocal microscope image
Objective
Confocal microscope image
With confocal pinhole observation, high S/N ratio images can be captured that have superior Zaxis resolution compared to that of ordinary fluorescence (non-confocal) observation. Minute
structures deep within a thick specimen are clearly visible.
Specimen
Focal Plane
Images and specimens courtesy of:
Cover image (top): Mouse’s whole brain stained with mCB (mouse monoclonal anti-calbindin: green), rPV (rabbit polyclonal
anti-parvalbumin: red) and nucleus (Hoechst: blue)—Assistant Prof. Kazunori Toida, Department of Anatomy and Cell Biology,
Institute of Health Biosciences, the University of Tokushima Graduate School
Cover image (middle): Three-dimensional reconstruction image of mouse hippocampus (GFP: inhibitory neurons, green,
YFP: excitatory neurons, magenta) through volume rendering after spectra unmixing—Dr. Masayuki Sekiguchi, Department of
Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Japan
Cover image (bottom): HepG2 cells infected with dengue virus. Fluorescence reagent: Dengue virus (FITC), Nuclei (DAPI),
Clathrin (Texas Red)—Ang Firzan and Dr. Justin Chu, Department of Microbiology, National University of Singapore.
The AOTF incorporated into the 4-laser unit and the
AOM optionally incorporated into the 3-laser unit are
classified as controlled products (including provisions
applicable to controlled technology) under foreign
exchange and trade control laws. You must obtain
government permission and complete all required
procedures before exporting this system.
Specifications and equipment are subject to change without any notice or obligation
on the part of the manufacturer. June 2008 ©2008 NIKON CORPORATION
WARNING
TO ENSURE CORRECT USAGE, READ THE CORRESPONDING
MANUALS CAREFULLY BEFORE USING YOUR EQUIPMENT.
* Monitor images are simulated.
Company names and product names appearing in this brochure are their registered trademarks or trademarks.
NIKON CORPORATION
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phone: +81-3-3773-8973 fax: +81-3-3773-8986
http://www.nikon-instruments.jp/eng/
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phone: +1-631-547-8500; +1-800-52-NIKON (within the U.S.A.only)
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