Biological confocal laser scanning microscope

Images are courtesy of the following institutions:“Brainbow” mouse brain stem
Courtesy of the laboratories of Jeff W. Lichtman and Joshua R., Sanes Harvard University MCB Department and the Center for Brain Science (cover page)

FV1200Glandular and non-glandular leaf hairs (trichomes) of Pelargonium
Courtesy of Dr Ferhan Ayaydin, Cellular Imaging Laboratory, Biological Research Center, Szeged, Hungary (1, on page 1)
Pilidium larva of Micrura alaskensis
Courtesy of Dr Svetlana Maslakova of the University of Washington and Dr. Mikhail V Matz of the Whitney Laboratory for Marine Bioscience, University of Florida (2, on the page 1)

FLUOVIEW

CFP and YFP labelling of glycerol-cleared fruit fly brain taken with 30x silicone objective
Courtesy of Dr.Hidehiko Inagaki, Anderson lab, California Institute of Technology (3, on page 1)
Cultured nerve cells derived from the mouse hippocampus
Courtesy of Dr Koji Ikegami, Dr. Mitsutoshi Setou, Molecular Geriatric Medicine, Mitsubishi Kagaku Institute of Life Sciences (5, on page 1, bottom of page 2)
Drosophila, Stage 14
Courtesy of Dr Tetsuya Kojima, Laboratory of Innovational Biology, Department of Integrated Biosciences Graduate School of Frontier Sciences, University of Tokyo (top of page 2)

Art. code: E0438442 · Printed in Germany 10/2012

High-performance laser scanning microscope forlive cell imaging, combining accuracy, sensitivity and laser stimulation

The manufacturer reserves the right to make technical changes without prior notice.

Postbox 10 49 08, 20034 Hamburg, Germany
Wendenstrasse 14–18, 20097 Hamburg, Germany
Phone: +49 (0)40 237 730, Fax: +49 (0)40 230 761
Email: microscopy@olympus-europa.com

Reliability and flexibility

FV1200 (IX83 configuration)

1 2

THE FLUOVIEW FV1200: HIGH-QUALITY LIVE CELL IMAGING
WITH HIGH-LEVEL RELIABILITY
The FLUOVIEW FV1200 biological laser scanning microscope builds on renowned Olympus optics, enhancing sensitivity througha new galvanometer coating and GaAsP detector technology. With the new IX83 microscope, the FV1200 has been optimised forsome of the most challenging live cell imaging experiments, implementing real-time Z-drift compensation and touch panel control.
From the high-resolution, confocal observation of fixed samples, with up to 5 simultaneous fluorescent detection channels, throughto high-speed fluorescent measurements and the simultaneous stimulation of living cells, the FV1200 offers advances in confocal
system performance while providing the speed and sensitivity required for live cell imaging, with minimal risk of damage to living
specimens.
What’s more, the FLUOVIEW FV1200 supports an array of optional functions – such as the ability to measure cellular molecular
diffusion coefficients – extending the exceptional performance from visualisation and stimulation through to precision measurement.
3
4 5

FV1200 (BX61WI configuration)

1

2

System overview

EXCELLENT PRECISION, SENSITIVITY AND STABILITYFLUOVIEW FV1200 ENABLES PRECISE, BRIGHT IMAGING WITHMINIMUM PHOTOTOXICITY

Main scanner

GaAsP detector

SIM scanner
BarrierfilterMicroscope

PMT
Grating

Laser combiner

PMT
Grating

LD635

PMT

LD559/HeNe(G)543
LD473/multi Ar

Broadband fibre

Confocal pinhole

Specimen
Galvanometerscanning mirrors

AOTF

UIS2 objectives

LD440
LD405

AOTF
Broadband fibre

Galvanometerscanning mirrors

Laser combiner/fibre

Scanners and detection system

Features of the NEW IX83

Diode laser
Greater stability, longer service life and lower operating costs areachieved using diode lasers.

Choice of main scanner
Select the scanner to match the purpose at hand by choosing either
the spectral scan unit that achieves 2 nm resolution for high-precision
spectroscopy or the filter scan unit featuring high-quality filters.

Discover improved expandability and rigidity with the IX83
The Z-drive guide with high thermal rigidity is installed near the
revolving nosepiece to further augment the stability of the IX83 in
the face of heat and vibration, and improve the results of time-lapse
imaging. Furthermore, when combined with the IX3-ZDC Z-driftcompensator and the motorised stage, high-precision multipoint
time-lapse imaging is made possible without the risk of focus drift or
misalignment.

Laser compatibility
Diode laser: 405 nm, 440 nm, 473 nm, 559 nm, 635 nm
Gas laser; Multi Ar laser (458 nm, 488 nm, 515 nm)
HeNe (G) laser (543 nm)
Broadband fibre
Broadband fibre connection for 405–635 nm lasers, to achieve an
ideal point light source with minimal colour shift and position shift
between images.
Laser combiner – two versions available•Dual fibre-type combiner for observation and simultaneous
photostimulation•Single fibre-type combiner for observation and sequential
photostimulation

High-performance detection system
High performance and high S/N ratio optical performance are achieved
through the smooth integration of a pupil projection lens, a high-performance
photomultiplier tube, silver-coated galvanometer scanning mirrors withhigh reflectance across a broad range of wavelengths, and an analogue
processing circuit that reduces noise 10090
to an absolute minimum. Furthermore, 80
7060
because the system enables image50
acquisition of this quality with minimal 40
30
Silver coating
20
Aluminum coating
laser power, phototoxicity is also
10
0
400 500 600 700 800 900 100 1100
significantly reduced.
Reflectance [%]Laser feedback control
Scanner unit is equipped with laser power monitor for feedback
control, ensuring stable laser output.

Wavelength [nm]Comparison of galvano mirrors, silver vs. aluminium
High-sensitivity detector* Reflectance of two galvanometer scanning mirrors
A high-sensitivity detector employing
gallium phosphide (GaAsP) is also available as an option.

Optical system
UIS2 objectives
Olympus UIS2 objectives offer world-leading, infinity-corrected optics
that deliver unsurpassed optical performance over a wide range of
wavelengths.

3

Pupil projection
lensHigh S/N ratio objectives with suppressed autofluorescence
Olympus offers a range of high numerical aperture objectives withimproved fluorescence S/N ratio, including objectives with silicone
immersion, exceptional correction for chromatic aberration, totalinternal reflection fluorescence (TIRF), and oil and water immersion
objectives.

Cantilevered
drive guide
The cantilevered structurewith a Z-drive guide locatednear the nosepiece offers highresistance to thermal drift.

Switch observation methods with a tap of the touch panel
A single tap is all it takes
to manage changes inmagnification, switch between
optical elements, and makeadjustments to illumination. Not only
does the controller make it a cinch
to carry out complex microscope
operations, it can also save settings
for observation modes.

The U-MCZ controller executes procedures from a preferred position
The controller allows monitor
observation to be executed inyour preferred position and mode,
while simple key arrangement
allows confident control – evenunder darkroom conditions.

The U-HGLGPS fluorescence illumination source minimises theimpact of lamp heat to both microscope and specimen
Featuring a high-pressure mercury lamp with an average life of 2,000
hours, this user-friendly fluorescence illumination source incorporates
a low chromatic aberration adapter that cleverly compensates whenswitching between excitation wavelengths.

4High sensitivity detection

A STEP UP IN SENSITIVITY
THE FV1200 CAPTURES SUBTLE CHANGES IN LIVE CELLS, WITH HIGHLYSENSITIVE DETECTION IMMEDIATELY FOLLOWING PHOTO STIMULATION

The high-sensitivity GaAsP detector module

High performance across a wide range of wavelengths
Galvanometer scanning mirrors on the main scanner feature an anti-oxidative silver coating that increases reflection efficiency for excitationand emission filters from 5% to 15% in the visible spectrum, and by a maximum of 22% in the near-infrared spectrum. The standard,
onboard multi-alkali photomultiplier tubes with a high dynamic range can also be combined with the optional, ultra high-sensitivity GaAsP
photomultiplier tubes to further increase the freedom for experimental set-ups across a broad range of wavelengths.

Cooled GaAsP Photodetector
Ultra-high sensitivity detector with GaAsP photomultipliertubes further enhances quantum efficiency
The ultra high-sensitivity detector makes it possible to view samples
that were simply too dim to view with conventional equipment. The
GaAsP PMT incorporates 2 channels and combines the images
with a further 3 built-in channels, as well as the channel transmitted
from the detector. Maximum quantum efficiency is 45%, Peltiercooling holds noise down by 20%, and high S/N ratio images can beobtained under exceptionally low excitation light.

Two versions of light detection system that set new quality standards
Spectral-based detection
High performance
Spectral detection using gratings for 2 nm wavelength
resolution and image acquisition matched to fluorescencewavelength peaks. User-adjustable bandwidth of emission
spectrum for acquiring bright images with minimal crosstalk.

Standard quantum efficiencies of detector technologies

Precise spectral imaging
The spectral detection unit uses a grating method that offers
linear dispersion compared with prism non-linear dispersion.
The unit provides a uniform 2 nm wavelength resolution
across the entire detection spectrum and high-performance
photomultiplier tube detectors. Fluorescence separation
can be achieved through unmixing, even when crosstalk is
generated by multiple fluorescent dyes with similar peaks.
A standard third filter channel is provided without a grating,allowing researchers greater flexibility and sensitivity.

40

GaAsP PMT

QE [%]

30
20
10
0
400

Multi-Alkali PMT
500

600
700
Wavelength [nm]

800

900

EGFP–EYFP fluorescence separation
2,600
2,400

EGFP (dendrite)—EYFP (synapse) XYλ
Wavelength detection range: 495 nm–561 nm in 2 nm steps
Excitation wavelength: 488 nm

SIM scanner allows simultaneous photostimulation during time-lapse imaging

2,200
2,000

EYFP

1,800
Intensity

Courtesy of Dr Shigeo Okabe
Department of Anatomy and Cell Biology,
Tokyo Medical and Dental University

SIM scanner unit

1,600
1,400

EGFP

1,200
1,000
800
600
400
496

500

504

508

512

516

520

524

528

Wavelength

532

536

540

544

548

552

Dedicated scanner for photostimulation
The combination of the main and photostimulation scanner provides
essential flexibility for tracking the diffusion or the transport offluorescence-labelled molecules or for marking specific live cells.The
dual-fibre laser combiner makes it possible to use imaging lasers forphotostimulation.

Simultaneous photostimulation and imaging
Performs simultaneous photostimulation and imaging to acquire
images of immediate cell responses to stimulation in photobleachingexperiments.

Filter-based detection
Enhanced sensitivity
Three-channel scan unit with detection system featuring hard coated filter base. Hightransmittance and high S/N ratio optical performance is achieved through the integration of apupil projection lens within the optics, and the use of a high-performance photomultiplier andan analogue processing circuit with minimal noise.

80

AOTF

LD635

60

LD559/HeNe(G)543

40

AOTF

LD473/Multi Ar

20

LD440

0
480 500

520

540

560 580 600 620
Wavelength (nm)Conventional mirror unit

5

Branching of laser in laser combiner.

DM488/543/633 comparison
100
Transmittance (%)

High-performance filters deliver
outstanding separation
Special coatings deliver exceptionally sharptransitions to a degree never achieved before,
for acquisition of brighter fluorescence images.

Lasers are used for both imaging and photostimulation.

640 660 680

700

LD405

High-performance mirror unit

6

Accuracy

ENHANCED RELIABILITY FOR LIVE CELL IMAGING MEETS DEMANDS FORDEEPER 3D STRUCTURING, TIME-LAPSE IMAGING AND
PRECISION MEASUREMENT

Silicone immersion objectives for live cell imaging deliver high-resolution observation at depth

Maintain high-precision focus through extended time-lapse imaging

Silicone immersion objective

Z-drift compensation system

High-resolution silicone immersion objective
Silicone immersion objectives can be designed with a larger
numerical aperture (NA) than water immersion objectives, increasing
image resolution and brightness.
Complete the range with the UPLSAPO40XSThis new objective with intermediate magnificationand high NA performance supports continuous
focus with the IX3-ZDC. Continuous high-resolution
observation during extended time-lapse imaging.
Magnification: 40x
NA: 1.25 (silicone oil immersion)
W.D.: 0.3 mm
Cover glass thickness: 0.15–0.19 mm
Operation temperature: 23 ºC–37 ºC

UPLSAPO30XS: for a broader view and greater depth

The IX3-ZDC Z-drift compensator offers a range of functions
for autofocusing
The IX3-ZDC uses low phototoxicity IR light to detect the correct focus
position, as set by the user. One-shot AF mode allows several focus
positions to be set as desired for deeper samples, enabling efficientZ-stack acquisition in multi-position experiments. Continuous AF mode
keeps the desired plane of observation precisely in focus, avoiding
focus drift caused by temperature changes due to perfusion or reagent
addition, and making it ideal for measurements such as TIRF that
requires more stringent focusing.

Magnification: 30x
NA: 1.05 (silicone oil immersion)
W.D.: 0.8 mm
Cover glass thickness: 0.13–0.19 mm
Operation temperature: 23 ºC–37 ºC

UPLSAPO60XS: for 3D with superior resolution
Magnification: 60x
NA: 1.30 (silicone oil immersion)
W.D.: 0.3 mm
Cover glass thickness: 0.15–0.19 mm
Operation temperature: 23 ºC–37 ºC

IX3-ZDC optical path diagram

OffsetOffset lens
Cover glass
Objective

ZDC one-shot function detects focus fast, even in highmagnification observation
IX3-ZDC focus detection and tracking can be performed via theinnovative touch panel independent of software. There’s also a focus
search function supported by a cell-safe, near-infrared laser enabling
instant focusing on samples.

SIL300CS-30CC: for extended time-lapse imaging
Refractive index: ne=1.406, 23 ºC
Net 30 ml
Low autofluorescence

Refractive index is important with deep tissue observation

Cell

AF sensor

IX3-ZDC optics

In deep tissue observation, image quality depends on keeping the refractive index of
the sample and the immersion medium as close to each other as possible.

Samplene≈1.38
Water
ne≈1.33

Scanning unit

Cover glassne≈1.52

Silicone oil
ne≈1.40
XY: 466 µm x 224 µm (777 x 374 pixels)

Water immersion objective
When working with a water
immersion objective, the difference
between the refractive index of thesamples and the water results inspherical aberration in deep tissue,
causing resolution to deteriorate andfluorescence to become dim.

Silicone immersion objective
When working with a silicone
immersion objective, the difference
between the refractive index of thesamples and silicone oil is minimal.
So it achieves brighter fluorescence
images with higher resolution fordeep tissue.

Confocal image of a Drosophila embryo at stage 11
expressing the tracheal marker trh-LacZ (Cy3, red)and the cell membrane marker DIg (Alexa488,
green). Enlarged view shows invaginating tracheal
placode.
Courtesy of Dr Takefumi Kondo, Dr Shigeo Hayashi,Laboratory for Morphogenetic Signaling,
RIKEN Center for Developmental Biology

Rigidity
XY: 120 µm x 90 µm (800 x 600 pixels)
Z: 21 µm (42 slices)Enhance the reliability of colocalisation analysis with the low chromatic aberration objective
Low chromatic aberration objective
Acquire and analyse colocalisation imaging with the
PLAPON60XOSC
This oil immersion objective minimises lateral and axial chromaticaberration in the 405–650 nm spectrum, while supporting thereliable acquisition and measurement of colocalisation images withsuperior positional accuracy. The objective
also compensates for chromatic aberration
through near infrared of up to 850 nm,
making it an optimal choice for near infrared
fluorescence observation.
Low chromatic aberration objective
Magnification: 60x
NA: 1.4 (oil immersion)
W.D.: 0.12 mm
Chromatic aberration compensation range:
405–650 nm
Optical data provided for each objective.

7

Performance comparison of PLAPON 60×OSC and UPLSAPO 60×O

PLAPON
60×OSC
Axial chromatic aberration (Z-direction)
Compared for PSF fluorescent beads(405 nm, 633 nm)

Approx.
0 µm

UPLSAPO
60×O
Approx.
0.5 µmTackle the conflicting requirements of expandability andrigidity with the IX3
A Z-drive guide installed near the revolving nosepiece combineshigh thermal rigidity with the stability of a wrap-around structure to
significantly reduce the impact of heat and vibration and improve thequality of time-lapse imaging. Integration with the IX3-ZDC
Z-drift compensator permits
imaging without focus drift or
misalignment, even throughtemperature changes due tothe addition of reagents or aperfusion device. Furthermore,combination with a motorised
stage that enables multipoint
registration makes high-precisionmultipoint time-lapse imaging
possible.

Periodic damping

Thermal drift displacement

IX81
IX81

IX83
IX83
0

50

100

150

200 (min)

0.0

0.5

1.0

1.5

2.0 (s)Lateral chromatic aberration (X-Y direction)
Compared for PSF fluorescent beads(405 nm, 488 nm, 633 nm)

Approx. 0.1 µm

Square frame for increased
rigidity

Approx. 0.2 µm

3D image

IX83 : two-deck system + IX3-ZDC

Tubulin in Ptk2 cells labelled with two colours(405 nm, 635 nm) and compared* UPLSAPO40XS: scheduled to be available 2013.

8

Applications

USER-FRIENDLY SOFTWARE TO SUPPORTYOUR RESEARCH

MULTI-DIMENSIONAL TIME LAPSE

Image acquisition by application
User-friendly icons offer quick access tofunctions, for image acquisition according
to the application (XYZ, XYT, XYZT, XYλ,XYλT).

Multi-dimensional time lapse imaging with outstanding positional accuracy
The FLUOVIEW FV1200 can be used for ideal multi-dimensional time-lapse imaging during confocal observation, using multi-area time-lapse
software to control the Motorised XY stage and IX3-ZDC Z-drift compensator.
Significantly improved multi-point time-lapse throughput
Equipped with motorised XY stage for repeated image acquisition from multiple points scattered across a wide area. The system efficiently
analyses changes over time of cells in several different areas, capturing large amounts of data during a single experiment for increased
efficiency. Microplates can be used to run parallel experiments, which significantly improves throughput for experiments that require long-term
observation.
Point 2
Point 3Time controller
Precisely synchronises different experimental
protocols including FRAP, FLIP and FRET
through acceptor photobleaching and time
lapse. Save and reopen settings for later use.

Point 1
Focal plane 4
Focal plane 3
Focal plane 2
Focal plane 1
Point 5

Point 4

Point 6

Configurable emission wavelength
Select the dye name to set the optimalfilters and laser lines.

Wide choice of scanning modes
Several scanning modes available,
including ROI, point and high-speed
bi-directional scanning.

Reuse function
Open previously configured scanning
conditions and apply them to new orsubsequent experiments.
Supports repeated image
acquisition from multiple areas
in a single microplate well.
Multi-point time-lapse software

Dark application skin
The use of the dark application skin mode
minimises the influence of the screen
brightness for the imaging process.

Configurable excitation laser power
Easily adjust the optimum laser power foreach specimen (live cells and fixed
specimens).

Maintain cell activity over a long period
CO2 incubator control keeps the environment inside the tissue culture dish completely stable. The environment is maintained precisely at
37 °C,with 90% humidity and 5% CO2 concentration.

0s1000 s2000 s3000 s4000 s5000 s6000 s7000 sHuman lymphoblast cells TK6
Courtesy of Masamitsu Honma, Dir.
Biological Safety Research Center Div. of Genetics and Mutagenesis I, National Institute of Health Sciences

9

10

Inte

1,400
1,200
1,000
800
600

Applications

400
200

SIMULTANEOUS PHOTOSTIMULATION0010,000 20,000 30,000 40,000 50,000 60,000 70,000 80,000 90,000 100,000
Time (ms)

750
700
650

Intensity

600
550
500
450
400
350
300

Uncaging
250
0
10,00020,00030,00040,00050,00060,00070,00080,000A 405 nm laser is optional for uncaging with the SIM scanner system. Caged compounds can be uncaged point by point,Timeor(ms)within a region ofinterest, while the main scanner of the FV1200 captures images of the response with no time delay.

Combined photostimulation and imaging with microsecond precision control
The SIM scanner system combines the main scanner with a photostimulation scanner. Control of the two independent beams enables
simultaneous stimulation and imaging in order to capture reactions during stimulation. Multi-stimulation software is used to continuously
stimulate multiple points with laser light for the simultaneous imaging of the effects of stimulation on the cell.

2,000
1,900
1,800
1,700

FLIP – fluorescence loss in photobleaching
Fluorescence loss in photobleaching (FLIP) combines imaging with the continuous bleaching of a specific region to observe the diffusion ofa target protein within a cell. The changes in the image over time make it possible to observe the location of structural bodies that inhibit the
diffusion of the molecule.

1,600
1,500
Intensity

1,400
1,300
1,200
1,100
1,000
900
800
700
600
500

Caged glutamate
Fluorescent calcium indicator Fluo-3 in HeLa cells. Image acquisition at 1-second intervals.
Using the caged compound Bhcmoc-Glutamate, an increase in the calcium ion concentration inside the cell can beobserved in response to glutamate stimulation, released via 405 nm laser illumination.
Data courtesy of Dr Hiroshi Hama, Dr Atsushi Miyawaki
RIKEN Brain Science Institute Laboratory for Cell Function Dynamics
Caged compound Bhcmoc-Glutamate presented by Dr Toshiaki Furuta,
Department of Science, Toho University

3,000
2,800

0

5,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000 45,000 50,000 55,000Time (ms)

2,600
2,400
2,200

Multi-stimulation software

Intensity

2,000
1,800
1,600
1,400
1,200
1,000
800
600
400

Specimen: HeLa cell, GFP (free), 488 nm excitation (multi-argon laser)
Image acquisition time: 100 ms bleach time: 100 s continuously, 405 nm bleaching

200
0

0

10,000 20,000 30,000 40,000 50,000 60,000 70,000 80,000 90,000 100,000
Time (ms)Intensity

FRAP—Fluorescence Recovery after Photobleaching
Exposure of fluorescently labelled target proteins to strong laser light causes their fluorescence to fade750locally. Fluorescence recovery after
700
photobleaching (FRAP) is used to observe the gradual recovery of fluorescence intensity caused by protein
diffusion from the area
650
surrounding the bleached region. By examining the resulting images, it is possible to characterise the diffusion
speed of the molecule, and the600
speed of binding and release between the molecule and cell structures.
550

High-speed multipoint scans
Users can designate the number ofpoints on an image for light stimulation.
Stimulation timing, duration and intervals
can be defined in the magnitude of µs andthe user can programme the experimentwith continuous or pulse stimulation. The
same software also provides features thatallows extended multiple points surrounding
one single point to cover a small area.

500

Fluorescent intensity

3,000
450
2,800
400
2,600

Time

2,200
300
2,000
250
1,800
1,600

0

10,00020,00030,0001,400

40,000Time (ms)50,00060,00070,00080,000

1,200
1,000

Time

Time

Intensity

Example: fluorescencerecovery with interactions
1,800
200
1,700
If the protein
is0 strongly bound to a structure, or forms part
1,600 010,000 20,000 30,000 40,000 50,000 60,000 70,000 80,000 90,000 100,000
of a large protein
complex, the bleached
Time (ms) region recovers its
1,500
1,400
fluorescenceat a slower rate relative to the unbound state.

Fluorescent intensity

Fluorescent intensity

Example: fluorescence recovery without interactions
If the protein can freely diffuse, the bleached region recovers
its fluorescence at a high speed due to Brownian motion.

800
2,000
600
1,900
400

1,300
1,200
1,100
750
1,000
900
700
800

Intensity

Fluorescent intensity

700
650
600
600
500

Specimen: Hippocampal neurons, Shank-GFP stain, 488 nm excitation (multi-argon laser)
Image acquisition time: 100 ms Bleach time: 80 ms, 488 nm excitation (Sapphire 488 laser)
Data courtesy of Dr Shigeo Okabe,
Department of Anatomy and Cell Biology, Tokyo Medical and Dental University

1050.01000.0
950.0
900.0
850.0
800.0
750.0
700.0
650.0
600.0
550.0
500.0
450.0
400.0
350.0

550 0
500

300.0

5,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000 45,000 50,000 55,000Time (ms)

250.0
200.0
150.0
100.0
50.0

450

Time

Mapping scans
Light stimulation can be applied to arectangular region of interest. Software
control of the stimulation of each point
assures that neighbouring points will not
be excited. This allows the user to observe
the reaction of a sample more accurately.
Changes in intensity from those points canbe processed as a mapped image or graph.

Intensity

Intensity

2,400
350

0

1000 2000 3000 4000 5000 6000 7000 8000 9000 100001100012000
Time(µs)

400
350
300
250
0

10,00020,00030,00040,000Time (ms)50,00060,00070,00080,000

2,000

11

1,900
1,800
1,700
1,600
1,500

12

Applications

DIFFUSION MEASUREMENT PACKAGE

3D MOSAIC IMAGING

Diffusion measurement package extends analytical capabilities
This optional software module enables data acquisition and analysis to investigate molecular interactions and concentrations by calculating the
diffusion coefficients of molecules within the cell. Diverse analytical methods (RICS/ccRICS, point FCS/point FCCS and FRAP) cover a wide range ofmolecular sizes and speeds.

High-level magnification with high resolution for the broad-scope imaging of large-scale specimens
Mosaic imaging is performed using a high-magnification objective to acquire continuous 3D (XYZ) images of adjacent fields of view using the
motorised stage and utilising proprietary software to assemble the images. The entire process, from image acquisition to tiling, can be fully
automated.

RICS – raster image correlation spectroscopy
Raster image correlation spectroscopy (RICS)
is a new method for analysing the diffusion andbinding dynamics of molecules in one complete
image. RICS uses a spatial correlation algorithm
to calculate diffusion coefficients and thenumber of molecules in specified regions.
Cross correlation RICS (ccRICS) characterises
molecular interactions using fluorescenselabelled molecules in two colours.

RICS analytical method

Results of analysis(diffusion coefficient andmolecule count)

LSM image

Spatial correlation

Mosaic imaging for 3D XYZ construction
Composite images are quickly and easily prepared using the stitching function, to form an image over a wide area. 3D construction can also
be performed by acquiring images in the X, Y and Z directions. Tiled images can be enlarged in sections without losing resolution. Particularly
useful for “connectome” or “brain mapping” or similar projects requiring large-area scanning at high resolution. Tiling functions include true
stitching and smoothing options for improved seamless images.
CNS markers in normal mice
Objective : PLAPON60X
Zoom
: 2x
Image acquisition numbers (XY): 32 x 38, 48 slices for each image

Theoretical formula used
for fitting calculation

Courtesy of Dr Mark Ellisman PhD, Hiroyuki Hakozaki,
MS Mark Ellisman
National Center for Microscopy and Imaging Research (NCMIR),
University of California, San Diego

Comparison of diffusion coefficients for EGFP fusion proteins near to cell membranes and in cytoplasm
At cytoplasmic membrane
In cytoplasm
RICS can be used to designate and analyze regions of interest based
Diffusion coefficient: D=0.98 μm2/s
Diffusion coefficient: D=3.37 μm2/s
on acquired images.
EGFP is fused with protein kinase C (PKC) for visualisation, using live
cells to analyse the translocation with RICS. The diffusion coefficient
close to cell membranes was confirmed to be lower than in cytoplasm,
after stimulation with phorbol myristate acetate (PMA). This is thought
to arise from the mutual interaction between PKC and cell membrane
molecules in cell membranes.
In addition to the localisation of molecules, RICS analysis cansimultaneously determine changes in the diffusion coefficient, for a
detailed analysis of various intracellular signalling proteins.

Sample image:
HeLa cells expressing EGFP fusion PKC (after PMA stimulation)

FRAP analysis
The Axelrod analytical algorithm is used as a FRAP analysis method. The algorithm is used to calculate diffusion coefficients and the
proportions of diffusing molecules.
Analytical methods
according to molecule
diffusion speeds

Small molecules
in solution

Proteins
in solution

Diffusion ofproteins
in solution

Lateral diffusionin cell membrane

Proteintrafficking

Oligomers,
aggregation

Diffusion constant (m2/s)

> 100

~ 100

1 ~ 100< 0.1< 0.01<< 0.001(membrane trafficking)(endosytosis)

Automated from 3D image acquisition to mosaic imaging
Multi-area time-lapse software automates the process from 3D
image acquisition (using the Motorised XY stage) to stitching.
The software can be used to easily register wide areas, and thethumbnail display provides a view of the entire image acquired
during the mosaic imaging process.

point FCS

capable rangeof measurement RICS

FRAP

13

Coordinate information

Thumbnail14

Expandability

ACCESSORY UNITS THAT SUPPORTAN ARRAY OF APPLICATIONS

5
40
300

350

The multi-combiner enables combinations withall of the following diode lasers: 405 nm,
440 nm, 473 nm, 559 nm and 635 nm.
The system can also be equipped with theconventional multiline Ar laser and HeNe(G)laser.
Dual typeThe multi-combiner outputs laser light with two
fibres. Light can be used for both observationand photostimulation.

Single type
Single-channel laser output. AOTF is standard
equipment.

Fluorescence illumination source/U-HGLGPS
The pre centred fluorescence illumination source
requires no adjustment and has an average
lifespan of 2,000 hours.

Transmitted light detection unit
External transmitted light photomultiplier detector and
100 W halogen conventional illumination, integrated forboth laser scanning and conventional transmitted light
Nomarski DIC observation. Motorised exchange between
transmitted light illumination and laser detection.
Simultaneous multichannel confocal fluorescence image
and transmitted DIC acquisition enabled.

Conventional illumination modules aredesigned for long-duration time-lapse
experiments. Since light is introduced throughfibre delivery systems, no heat is transferred tothe microscope.

Optional upgrade equipment for FV1200

TIRFM unit
Enables control of the necessary
volume of excitation light using FV1200
software. This unit enables TIRF imaging
using the laser light source used withthe confocal system.

15

500

550

LD

600

650

ECFP ●
furaRed ●EYFP ●
EGFP ●Cy2●
DiO ●YOYO-1 ●AzamiGreen ●fluo-4●
FITC●
RhodamineGreen ●OregonGreen488 ●Alexa Fluor 488●
AcridineOrange●
CalciumGreen-1 ●fluo-3●MagnesiumGreen ●Kaede●
SNARF-1●
PI ●CalciumOrange ●Cy3 ●MagnesiumOrange ●
Dil
●
TRITC
●
rhod-2 ●Alexa Fluor 546●
RhodaminePhalloidin●
DsRed2 ●Rhodamine Red-X●
Alexa Fluor 568
●
Cy3.5
●
MitoTracker●
X-rhod-1●
mCherry●
HcRed1●
Alexa Fluor 594 ●TexasRed●
Alexa Fluor 633●
TOTO-3●Cy5●Alexa Fluor 647●
Cy5.5

Illumination units

Achieve ultra-high sensitivity with low noise
thanks to the gallium arsenide phoshide(GaAsP) detector and the on-board Peltiercooling system.

450

5

63

700

750

800

700

750

800

Hoechst33258 ●DAPI ●
GFP-uv ●Laser systems

Ultra high-sensitivity detector/GaAsP
photomultiplier tubes

400

LD

e
n
on
on
eN
rg
rg
go
H
r
A
A
A
ti
ti
en
lti
ul
ul
re
LD M LD Mu
G LD
M
0
8 3
3 9
5
8
44 45 47 48
54 55
51

4th channel detector unit
Attaches to the optional port of either thefilter or spectral-type scanning unit andis used as a 4th confocal fluorescence
detection channel. This is a filter-based
fluorescence detection unit.

Fibre port for fluorescence output
Confocal fluorescence emission canbe introduced into an external device
via a fibre delivery system. Fibre port
equipped with FC connector (fibredelivery system not included).

SIM Scanner
Second dedicated scanner for photostimulation,synchronised to the FV1200 main scanner for
simultaneous photostimulation and confocal
image acquisition. Independent fibre-optic laser
introduction port. Dichromatic mirror within
motorised optical port of the scan unit requiredfor introduction of laser into main scanner.

IX3-ZDC/Z-drift compensator
Focal drift compensation for long timelapse imaging.

CO2 stage top incubator*1
Precise controls maintain a constant environment within the dish orwell plate, controlling temperature,humidity and CO2 concentration.

Motorised XY stage*1
This motorised stage supports wellplates, 35 mm diameter dishes, andslide chambers, and also comescomplete with a universal sample holder.

* Requires IX83 microscope. For information
about ZDC-compatible objectives, contact
your Olympus dealer.

(Manufactured by Tokai Hit CO., Ltd).

300*1 Available in the near future.

350

400

450

500

550

* Selected fluorescence dyes; white dot shows the absorption maximum, graphs show the dye emission spectra.

600

650

●

16

FLUOVIEW FV1200 major specifications

FLUOVIEW FV1200 system diagram

Violet/visible light laser

E
U-HGLGPS
130 W mercury lamp
housing with fiber

Scanning unit for IX83
(Spectral or filter-type
detector system)

A

CO2 incubator **Laser light

Fibre

Motorised XY stage **G

AOTF laser combiner

Scanner module

F
Detector module

LD635 laser
LD559 laser

AOTF laser combiner(Single-fibre type)

D

B

A

Scanning anddetection

B

559 nmHeNeG laser

Multi Ar laser
458, 488, 515 nm

Cover*

IX83
Research inverted microscope
IX3-ZDC
Z-drift compensator

543 nmSelect either laser

AOTF laser combiner(Dual-fibre type)LD473 laser

Scanning modes

PinholeB

473 nmSelect either laser

Field number (NA)
Optical zoom
Z-drive
Transmitted light detector unit
Motorised microscope

TIRFM unit*

C

Scanning unit for BX61WI, BX61
(Spectral or filter-type detector system)

LD440 laser*

G

Microscope

440 nm

LD405 laser*

SIM scanner*

GF

405 nmPhoto detection method
Scanning method

Fluorescence illumination unit
Control unit

System control

Power supply unit
Display
Sim scannerD

IR laser*

D

B

A

Optional unitC

Ultra high-sensitivity detector
Fourth confocal detector
Fibre port for fluorescence

Transmitted light detection unit

E

E

Fibre port for
fluorescence
output*Software

BX61WI
BX61
Upright motorised microscope

F

Image acquisition

FV power supply*

Microscope
control unit

4th channel
detector unit*

TIRFM unit

Programmable scan controller
2D image display

F

Software3D visualization and observation

Basic software
Gallium arsenide
phosphide (GaAsP)
detector unit*F
FV power
supply unit

FV control
unit

Review station software*

Image format

Diffusion measurement package*

Spectral unmixing

Multi-stimulation software*

Image processing

MonitorMulti-area time-lapse software*

Image analysis
Statistical processing* Optional unit
* Available in the near future

Optional software

Objectives for BX2 and IX3Objectives for fixed stage upright microscope(using U-UCD8A-2, IX3-LWUCDA and U-DICTS)
Model

NA

W.D.
(mm)

Cover glass
thickness

UPLSAPO4X
UPLSAPO10X2UPLSAPO20X

0.16
0.40
0.75

13
3.1
0.6

—
0.17
0.17

UPLSAPO20XOUPLSAPO30XSUPLSAPO40X2UPLSAPO40XS*1UPLSAPO60XOUPLSAPO60XWUPLSAPO60XSUPLSAPO100XO
PLAPON60XO
PLAPON60XOSCUPLFLN40XO
APON60XOTIRFAPON100XHOTIRFUAPON100XOTIRFUAPON150XOTIRF

0.85
1.05
0.95
1.25
1.35
1.20
1.30
1.40
1.42
1.40
1.30
1.49
1.65
1.49
1.45

0.17
0.8
0.18
0.3
0.15
0.28
0.3
0.12
0.15
0.12
0.2
0.1
0.1
0.1
0.08

—
0.13–0.19
0.11–0.23
0.15–0.19
0.17
0.13–0.21
0.15–0.19
0.17
0.17
0.17
0.17
0.13–0.19
0.15
0.13–0.19
0.13–0.19

17

Normal scan: 64 x 64, 128 x 128, 256 x 256, 320 x 320, 512 x 512, 640 x 640, 800 x 800, 1024 x 1024, 1600 x 1600, 2048 x 2048, 4096 x 4096
Clip rectangle scan, clip ellipse scan, polygon clip scan, line scan, free-line scan, point scan, real-time image
2-dimension: XY, XZ, XT and X λ3-dimension: XYZ, XYT, XY λ, XZT, XT λ and XZ λ4-dimension: XYZT, XZT λ and XYT λ
5-dimension: XYZT λTime controller function
Each image display: single-channel side-by-side, merge, cropping, live tiling, live tile, series (Z/T/λ), LUT: individual colour setting, pseudo-colour, comment: graphic and text inputInteractive volume rendering: volume rendering display, projection display, animation displayed (save as OIF, AVI or MOV format)
Free orientation of cross-section display
3D animation (maximum intensity projection method, SUM method)
3D and 2D sequential operation function
OIB/ OIF image format
8/16-bit gray scale/index color, 24/ 32/ 4-bit colour, JPEG/ BMP/ TIFF/ AVI/ MOV image functions
Olympus multi-tif format
2 fluorescence spectral unmixing modes (normal and blind mode)
Filter type: sharpen, average, DIC sobel, median, shading, laplacianCalculations: inter-image, mathematical and logical, DIC background leveling
Fluorescence intensity, area and perimeter measurement, time-lapse measurement
2D data histogram display, colocalisation
Review-station software, offline FLUOVIEW software for data analysis.
Motorised stage control software, diffusion measurement package, multi-stimulation software, multi-area time-lapse software(using WI-UCD, WI-DICTHRA2)
ImmersionliquidOil
Silicone
Silicone
Oil
Water
Silicone
Oil
Oil
Oil
Oil
Oil
Oil
Oil
Oil

Correction
ring

3
3
3
3
3

3
3
3

Condenser for Condenser for
IX3
BX2
IX3-LWUCDA
U-UCD8A-2
optical element optical element

U-DICTSposition

U-DIC10
U-DIC20

IX2-DIC10IX2-DIC20

normalnormalU-DIC20
U-DIC60HC
U-DIC40
U-DIC40
U-DIC60
U-DIC60
U-DIC60
U-DIC100
U-DIC60
U-DIC60
U-DIC40
U-DIC60
U-DIC100
U-DIC100
U-DIC100

IX2-DIC20
IX2-DIC30
IX2-DIC40
IX2-DIC40
IX2-DIC60
IX2-DIC60
IX2-DIC60
IX2-DIC100
IX2-DIC60
IX2-DIC60
IX2-DIC40
IX2-DIC60
IX2-DIC100
IX2-DIC100
IX2-DIC100

normalnormalnormalBFP1
BFP1
normalnormalnormal
BFP1
BFP1
BFP1
BFP1
normalnormalnormal

*1 Scheduled to be available in 2013

Model

NA

W.D. (mm)DIC prism

MPLN5X

0.10

20.00

—

UMPLFLN10XW

0.30

3.50

WI-DIC10HR

UMPLFLN20XW

0.50

3.50

WI-DIC20HRLUMPLFLN40XW

0.80

3.30

WI-DIC40HRLUMPLFLN60XW

1.00

2.00

WI-DIC60HR

LUMFLN60XW

1.10

1.5

WI-DIC60HRXLUMPLFLN20XW

1.00*2

2.0

WI-DICXLU20HR*

Revolving
nosepiece
WI-SSNP,
WI-SRE3
WI-SSNP,
WI-SRE3
WI-SSNP,
WI-SRE3
WI-SSNP,
WI-SRE3
WI-SSNP,
WI-SRE3
WI-SSNP,
WI-SRE3
WI-SNPXLU22 Note: these conditions are not met in confocal microscopy

Dimensions, weight and power consumption
Microscope with BX61/BX61WI
scan unit
IX83
FluorescenceLamp
illumination unit power supply
Transmitted light detection unit
Microscope control unit
FV power supply unit
FV control unit
19 inches, dual(value per monitor)
Display
29.7 inches
Power supply unit for laser combiner
Laser combiner (with ar laser heads)
Laser combiner (without ar laser heads)
LD559 laser power supply
Multi ar laser power supply
Hene(g) laser power supply

Dimensions (mm)320 (W) x 580 (D) x 565 (H)
385 (W) x 835 (D) x 755 (H)
180 (W) x 320 (D) x 235 (H)
90 (W) x 270 (D) x 180 (H)
170 (W) x 330 (D) x 130 (H)
125 (W) x 332 (D) x 216 (H)180 (W) x 328 (D) x 424 (H)136 (W) x 380 (D) x 329 (H)Recommended FV1200 system set-up (IX83, BX61, BX61WI)
Weight (kg)
41
59
6.7
3.0
5.9
5.2
7.5
8.5

Power consumption

AC 100-240 V 50/60 Hz 1.6 A—
AC 100-120/220-240 V 50/60 Hz 3.5 A/1.5 A
AC 100-120/220-240V 50/60 Hz 4.0 A/2.0 A
AC 100/240 V 50/60 Hz 600 W

363 (W) x 216 (D) x 389.5–489.5 (H)5.9

AC 100-120/200-240 V 50/60 Hz 0.65 A/0.4 A

694 (W) x 276 (D) x 489–589 (H)210 (W) x 300(D) x 100 (H)
514 (W) x 504 (D) x 236 (H)
514 (W) x 364 (D) x 236 (H)
200 (W) x 330 (D) x 52 (H)162 (W) x 287 (D) x 91 (H)
130 (W) x 224 (D) x 62 (H)

13.0
4.0
45
40
1.2
4.4
1.8

AC 100-240 V 50/60Hz 2.0 A
AC 100-120/200-240 V 50/60 Hz 2.0 A/1.0 A——
AC 100-240 V 50/60 Hz 30 W
AC 100-240 V 50/60 Hz 20 A
AC 100-120 V 50/60 Hz 0.45 A

—

1400

635 nmSpectral version
Filter version
LD lasers: 405 nm: 50 mW, 440 nm: 25 mW, 473 nm: 15 mW, 559 nm: 15 mW, 635 nm, 20 mW
Multiline Ar laser (458 nm, 488 nm, 515 nm, Total 30 mW), HeNe(G) laser (543 nm, 1 mW)
Visible light laser platform with implemented AOTF system, ultra fast-intensity modulation with individual laser lines, additional shutter control
Continuously variable (0.1%–100%, 0.1% increment), REX: Capable of laser intensity adjustment and laser wavelength selection for each region
Broadband type (400 nm–650 nm)
Standard: 3 laser ports, violet to IR
Excitation dichromatic mirror turret, 6 position (High performance DMs and 20/80 half mirror), Dual galvanometer mirror scanner (X, Y)
Motorised optical port for fluorescence illumination and optional module adaptation, Adaptation to microscope fluorescence condenser
Standard: 3 confocal channels (3 photomultiplier detectors)
Additional optional output port light path available for optional units
Standard: 3 confocal channels (3 photomultiplier detectors)
6 position beamsplitter turrets with CH1 and CH2
Additional optional output port light path available for optional units
CH1 and CH2 equipped with independent grating and slit for fast and flexible spectral detection
6-position beamsplitter turrets with CH1 and CH2
Selectable wavelength bandwidth: 1–100 nm
CH1 to CH3 each with 6-position barrier filter turret (high-performance filters)
Wavelength resolution: 2 nm
Wavelength switching speed: 100 nm/ms
CH3 with 6 position barrier filter turret
2 detection modes: analogue integration and hybrid photon counting
2 silver-coated galvanometer scanning mirrors
Scanning speed: 5 12 x 512 (1.1 s, 1.6 s, 2.7 s, 3.3 s, 3.9 s, 5.9 s, 11.3 s, 27.4 s, 54.0 s)
bi-directional scanning: 256 x 256 (0.064 s, 0.129 s), 512 x 512 (0.254 s)
X,Y,T,Z
X,Y,T,Z,Line scanning: straight line with free orientation, free line, Point scanningLine scanning: straight line with free orientation, free line, Point scanning
Single Motorised pinhole
Single motorised pinhole
Pinhole diameter ø 50–300 µm (1 µm step)
Pinhole diameter ø 50–800 µm (1 µm step)18
1x–50x in 0.1x increment
Integrated motorised focus module of the microscope, minimum increment of 0.01 µm or 10 nm
Module with integrated external transmitted light photomultiplier detector and 100 W halogen lamp, motorised switching, fibre adaptation to microscope frame
Inverted IX83 (IX83P2ZF), Upright BX61, Upright focusing nosepiece and fixed-stage BX61WIExternal fluorescence light source with motorised shutter, fibre adaptation to optical port of scan unit
Motorised switching between LSM light path and fluorescence illumination
OS: Windows 7 Professional (English version), CPU: Intel Xeon E5-1620 (3.60 GHz) or higher, Memory: 8 GB (2 GB x 4), Hard disk: 1 TB or more for data storage,
Dedicated I/F board: built-in control unit, Graphics board: NVIDIA Quadro 600, Optical drive: DVD ± R/RW Super-Multi
Galvo control boards, scanning mirrors and gratings, real-time controller
Galvo control boards, scanning mirrors
SXGA 1280 x 1024, dual 19-inch (or larger) monitors or WQUXGA 2560 x 1600, 29.7-inch monitor
2 galvanometer scanning mirrors, pupil projection lens, built-in laser shutter, 1 laser port, fibre introduction of near-UV diode laser or visible light laser
Optional: 2nd AOTF laser combiner
Available laser: 405–635 nm.
Motorised penetration ratio adjustment.
Automatic optical setting for TIRFM objectives
Cooled GaAsP-PMT 2 channels
Module with photomultiplier detector, barrier filter turret, beamsplitter turret mounted with 3rd CH light path
Output port equipped with FC fibre connector (compatible fibre core 100–125 µm)

680

1200
1880
Depth: 990(Unit: mm)

18