Confocal Laser Scanning Microscope

FV3000FLUOVIEW

Next Generation FLUOVIEW for the Next Revolutions in Science

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FLUOVIEW FV3000 Laser Scanning Microscope
The FLUOVIEW FV3000 series is designed to meet some of the most difficult challenges in modern science. With
the high sensitivity and speed required for live cell and tissue imaging and the ease of use and flexibility required for
microplate imaging and complex screening protocols, the FV3000 series supports complete workflows from live cell
2D–6D (x,y,λ,z,t,p) imaging to image processing, such as deconvolution, and analysis.
With Olympus’ renowned optics at the heart of the system, the FV3000 laser scanning microscope features TruSpectral
detection technology for multichannel spectral imaging with high-sensitivity detection in multiple dynamic ranges, soeven dim signals can be separated. The FV3000 Red system further expands the multichannel imaging capabilities into
the near-infrared (NIR) by combining NIR-specialized high sensitivity detectors and lasers for multiplex imaging fromviolet to NIR. The precise galvanometer scanner combined with a silver-coated resonant scanner in the FV3000RS
system enables users to combine precision and high-speed imaging in their experiments. The optical path enables
macro-to-micro imaging from 1.25X to 150X magnification, enabling users to easily observe fine detail within the overall
context of the tissue. Higher resolution imaging is possible using either TruSight deconvolution or Olympus Super
Resolution (OSR). Simplify complex experiments with robust, intuitive automation, such as one-click cellSens macro
analysis for cell counting and segmentation analysis. TruAI deep-learning technology provides efficient segmentation
and accurate results.
NoviSight 3D cell analysis software also includes advanced statistical tools to advance your discovery.
With user-savable and selectable software workflows, the FV3000 system adjusts to individual needs. The
microscope's software makes it easy for facility managers to track the system usage of every user, improving efficiency
in multiuser environments.

2

Meeting Your Application Challenges
Advanced Hardware

High-Sensitivity
Multiplex Imaging
from Violet to NIR

3Macro-to-Micro
Imaging andSuper Resolution

Accurate TimeLapse Imaging

Adapt the User Interface for Your Workflows

1. Layout

2. Acquisition Condition

3. Acquisition

4. Viewer

5. Analysis• Select the layout
appropriate foryour experiment• Auto-setting by dyes

• Acquire multidimensional images• Reload the settings fromprevious experiments• Acquire multi-area images• Review images during
or after acquisition
using multiple display
methods• Various image analysis
menus are available

Increase Productivity
with High-Speed
Imaging• Acquire images using a complex
protocolSuperior Objectives• X Line• A Line• One-click macro analysis

Advanced Solutions• TruAI Deep Learning• 3D Cell Analysis

4Application: High-Sensitivity Multiplex FluorescenceAcquiring multicolor fluorescent images is important for analyzing the fine internal structure of cells and tissues and confirming protein
expression. The FV3000 system's TruSpectral detectors enable users to select the detection wavelength on each channel to optimize
signal detection for each individual fluorophore. The variable barrier filter mode provides simultaneous four-channel image acquisition,and up to 16 channels sequentially. The system's online or offline lambda scanning mode facilitates the separation of complex
fluorescence signals by defining characteristic spectral emission profiles for each fluorophore, enabling accurate spectral unmixing ofcomplex overlapping fluorescent signals.

Mouse mPFC labeled with glial fibrillary acidic protein (GFAP; astrocyte marker; yellow), calmodulin-dependent protein kinase II (CaMKII; pyramidal neuron marker; red),amphoterin-induced protein 1 precursor (AMIGO-1; neuronal membrane marker; cyan), parvalbumin (PV; inhibitory neuron marker; purple), ankyrin-G (AnkG; axon initial
segment marker; green), and nuclear yellow (nuclei marker; blue).
Image data courtesy of Stephanie Shiers, Ph.D. Candidate, and Theodore J. Price, Ph.D., Price Lab, Eugene McDermott Professor, Director, Center for Advanced Pain
Studies, Department of Neurobiology, School of Behavioral and Brain Sciences, University of Texas at Dallas.

Emission spectra of the six fluorophores used to label mouse medial prefrontal cortex sections. Even though the spectrum of each fluorophore is overlapping, the cross talkcan be minimized by optimizing detection wavelength by TruSpectral detectors.

5Adjustable slit

VPH

High-Sensitivity Multiplex Imaging

VPH

Efficient TruSpectral Detection System

The FV3000 series employs Olympus’ TruSpectral detection
technology. Based on patented volume phase hologram (VPH)
transmission and an adjustable slit to emitted light, the spectral
detection is highly efficient, enabling users to select the detection
wavelength of each individual channel at the spectral resolutionof 1 nm.

All the detectors in the FV3000 series have spectral imaging
capability. TruSpectral detection delivers up to three-foldTransmittance
Ratio of FV3000
FV1200 in
Detection
improved
overall transmission
and tosensitivity
witha high Pathsignalto-noise ratio, resulting in excellent multicolor confocal imaging
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capabilities. FV3000

Tube lensAdjustable slit

Transmittance Ratio
Transmittance Ratio

A Fully Spectral System with Sensitivity and Accuracy

FV1200TransmittanceRatio
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Multichannel TruSpectral Detection with SixteenChannel Unmixing
www.olympus-lifescience.com/video/fv3000_truspectral

High-Sensitivity Spectral Detector (HSD) with Cooled
GaAsP Photomultiplier Tubes Enhance Quantum Efficiency
The GaAsP PMTs in the FV3000 system's high sensitivity
detector enable you to view samples whose emission is too
Transmittanceof FV3000
to FV1200
Detection
weak toRatio
view withconventional
detectioninmethods.
ThePathGaAsP
PMT unit incorporates two channels with a maximum quantum
efficiency
FV3000 of 45% and Peltier cooling that reduces backgroundnoise
by 20% for high S/N ratio images under very low excitation
FV1200light.

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Quantum Efficiency

The Standard Quantum Efficiencies of Detector Technologies
Quantum Efficiency

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TruSpectral technology's efficient design and software enable
spectral detectors to run in multichannel mode for both live
and post-processing spectral unmixing with a multichannel
lambda mode. The multichannel mode facilitates constant
spectral unmixing during live cell experiments, separatingcomplex fluorescence during acquisition. With up to four
dynamic ranges from the four detection channels, brightand dim spectral signals can be separated by independently
adjusting the sensitivity of each detector.

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Application: FV3000 Red System for NIR Solutions
Near-infrared (NIR) fluorescence expands the possibilities for
multiplexing, gentle live cell imaging, and deeper imaging, while atsame time reducing autofluorescence.
The FV3000 Red system's detection optical path greatly improves
near-infrared transmittance. Moreover, it is equipped with NIR lasers,
such as 730 nm or 785 nm wavelengths, and 1–2 channels of NIRdedicated GaAs detectors (~890 nm), which enable six-channel
multiplexed imaging from 400 nm to 890 nm.
Simultaneous imaging of up-conversion fluorescence of nanoparticies
and conventional fluorescence is now possible by introducing longer
wavelengths lasers such as 808 nm and 980 nm.

Rat brain slice labeled with Hoechst (blue), anti-IBA1 (Alexa Fluor 488; green), anti-MAP2 (Alexa Fluor 594, yellow), anti-FOX3/NeuN (Alexa Fluor 647; red), and anti-MBP(Alexa Fluor 750; cyan). Images were acquired using a UPLXAPO10X objective with 405 nm, 488 nm, 561 nm, and 730 nm laser lines on GaAsP and GaAs detectors.
Maximum intensity projection in Z with TruSight deconvolution processing. Sample courtesy of EnCor Biotechnology.

Cos-7 cells labeled with DAPI(blue), anti-Tubulin (Alexa Fluor 488; green), Concalavalin A (Alexa Fluor 594; yellow), SiR-Actin (magenta) and anti-TOMM20 (Alexa Fluor 750;
cyan). Images were acquired using a UPLAPO60XOHR objective and 405 nm, 488 nm, 561 nm, 640 nm, and 730 nm laser lines on GaAsP and GaAs detectors. Maximum
intensity projection in Z with TruSight deconvolution processing. Sample courtesy of Dr. Jana Döhner, Dr. Urs Ziegler, University of Zürich.

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FV3000 Red System for NIR Solutions
Cooled GaAs PMT Detector for NIR Imaging

NIR Laser Diodes%!!
100

Excitation
Efficiency (%)!"#$%&'"()*%"*+&%,(-./

Quantum Efficiency (%)
Quantum
Efficiency (%)

5050
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30

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00
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75$#

50#!

25"#

0!
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Wavelength
Wavelength(nm)(nm)

Multi-alkali
PMT PMTMulti-alkali

800
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&!!
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'!!400#!!
500(!!600

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700

)!!
800

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Wavelength (nm)
+,-.

GaAsP
GaAsP PMT

GaAs PMT
GaAs PMT

DAPI

/0-

GFP

10-

RFP

,23456027896('$,23456027896$#!

Alexa Fluor 647

Alexa Fluor 750

GaAs photomultiplier tube (PMT) has a higher quantum
efficiency at 700 nm–890 nm range compared with aconventional GaAsP PMT or mutli-alkali PMT.

NIR laser diodes help reduce crosstalk caused by dye selection.
These laser diodes (LD730/785) are designed to be driven incontinuous wave (CW) mode, offering stability, low maintenance,
and a longer lifetime.

High-Transmittance Optics for NIR Imaging

TruFocus Red Z-Drift Compensator

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Aluminum coating
Aluminumcoating

Silver coatingSilver coating

Efficient NIR imaging requires high-quality optics. All the
FV3000 system's optical elements have a high transmission
from 400 nm to 1300 nm, including the galvanometer andresonant scanner, which are coated in silver rather thanthe conventional aluminum coating.

The TruFocus Red uses a very low intensity 830 nm diode laser
to identify the coverslip surface and maintain the focus positionof the sample. The unit works well for both live cell and multiwellplate imaging and covers all wavelengths from visible to the NIR,
including fluorescent proteins like iRFP.

Multiplexing with X Line High-Performance Objectives

Deep-Tissue Observation with Silicone Oil Immersion
Objectives in the NIR
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UPLSAPO30XSIRUPLSAPO30XSUPLSAPO30XSIRUPLSAPO30XSAward-winning X Line objectives work well for NIR imaging
as they are corrected for chromatic aberrations between
400–1000 nm. They also have a higher numerical aperture,excellent flatness, and very high transmittance from UV
to NIR, increasing the multiplexing capabilities

One advantage of imaging in the NIR range is its ability topenetrate deeper into the sample. This is because longer
wavelengths of light are scattered less by biological samples.
Combined with silicone oil immersion objectives (ne~1.40),
spherical aberration is significantly minimized, enabling higher
S/N images, even deep within living tissues.

8Application: Macro-to-Micro Imaging and Super Resolution
Life science research applications require users to observe regions of interest within the context of the larger tissue structure. A large
field of view is critical to being able to see a cell’s components in relation to the entire tissue sample. The FV3000 microscope‘soptical light path facilitates macro-to-micro observation from 1.25X to 150X. Image stitching enhances these applications even moreby enabling users to quickly locate target cells on the macro image and follow up with higher resolution imaging of the cell’s finestructures. For even greater resolution, Olympus Super Resolution technology (FV-OSR) can be coupled with this approach to provide
optimal macro-to-micro imaging performance.
1.25X Objective Single Shot Acquisition with Blind Unmixing

1.25X Objective Single Shot Acquisition

Mouse brain hemisection embedded for expansion microscopy (pre-expansion), labeled with secondary antibodies against GFP (Alexa Fluor 488, green), SV2 (Alexa Fluor
565, red) Homer (Alexa Fluor 647, blue). Sample courtesy of Dr. Ed Boyden and Dr. Fei Chen, MIT.

Objectives for Macro Observation

Objectives for Micro Observation

NA

FOV

NA

Theoreitical Resolution (XY)

PLAPON1.25X

0.04

14.4 mmUPLXAPO40XO

1.4

169 nmPLAPON2X

0.08

9.0 mmUPLXAPO60XO

1.42

166 nmUPLXAPO4X

0.16

4.5 mmUPLXAPO100XO

1.45

163 nmUPLAPO60XOHR

1.5

157 nmUPLAPO100XOHR

1.5

157 nmAPON100XHOTIRF

1.7

139 nm*Theoretical resolution is caluculated at 488 nm exciation and confocal aperture
1 AU.

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Macro-to-Micro Imaging and Super Resolution
TruSight Deconvolution
TruSight deconvolution image processing improves the sharpness and clarity of images by reducing optical blur and noise. The
optimized image processing mode for optical characteristics enables the restoration of confocal images acquired using the FV3000
microscope. TruSight deconvolution can also reduce noise for Olympus Super Resolution (FV-OSR) acquired images. TruSight
deconvolution uses a GPU for high-speed image processing.

Without TruSight

With TruSight

cLSM image without TruSight

With TruSight

HeLa cell. Blue: nuclear (DAPI), green: microtubule (Alexa Fluor 488),
red: mitochondria (MitoTracker Red)

Image of GATTA-SIM nanoruler (SIM 120B, GATTA quant GmbH)acquired by using UPLAPO60XOHR objective (NA: 1.5) a 0.8 AU
pinhole size. The fluorescent markers at both ends of the nanorulers
can be resolved by TruSight deconvolution.

Olympus Super Resolution (FV-OSR) Technology with Up to Four Simultaneous Channels
Olympus’ widely applicable super resolution method requires no special fluorophores and works for a wide range of samples. Ideal
for colocalization analysis, the Olympus Super Resolution imaging module can acquire four fluorescent signals either sequentiallyor simultaneously with a resolution of approximately 120 nm*1, nearly doubling the resolution of typical confocal microscopy. The
imaging module is easy to use with minimal user training and can be added to any FV3000 system, making it a truly accessible
method for achieving super resolution.

Beyond Deconvolving Confocal: Comparison of Confocal and Deconvolved FV-OSR Images
Confocal Image

Enlargement

Olympus Super Resolution with TruSight

Secondary antibody labels against GFP (Alexa Fluor 488, neurons)
and SV2 (Alexa Fluor 565, red).
Sample courtesy of Dr. Ed Boyden and Dr. Fei Chen, MIT.

Example of Actual Measurement of FWHM Using Φ40 nm Fluorescent Beads*2
Confocal (1 AU)

TruSight

FV-OSR

Lateral FWHM [nm]

202

120

120

Axial FWHM [nm]

465

292

405

Lateral projection

Axial projection*1

Subject to objective magnification, numerical aperture, excitation and emission wavelength, and experiment conditions.

*2

Images are captured by UPLAPO60XOHR (NA1.5) / ex 488 nm / Φ40 nm beads

10Application: Time-Lapse Imaging
Time-lapse imaging experiments require focus to be maintained throughout with less phototoxicity to the sample. Olympus’ TruFocus Z-driftcompensator helps each position stay in focus during an experiment despite changes in temperature or the addition of reagents. Additionally,
the FV3000 system’s high-sensitivity detector requires reduced laser intensity, and the resonant scanner reduces the laser illumination time,
reducing phototoxicity for more physiologically accurate imaging data.

3D time-lapse imaging of a mouse embryonic fibroblast labeled with silicon rhodamine docetaxel (Tubulin), imaged with a 100X silicone oil objective and
30 fps resonant scanning followed by cellSens deconvolution. Image data courtesy of Dr. Markus Delling, Harvard University.

www.olympus-lifescience.
com/fv3000-cell-divisionresonant/fv3000-celldivision-resonant

www.olympus-lifescience.
com/fv3000-dudi-158vxyt-0001_00000
NK-cell mediated cell killing after therapeutic anitbody application (blue). GFP labeled NK-cells (green). DAPI uptake marking dead cells (Red).
Image data courtesy of Dr. Yuji Mishima, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research.

Application: High-Speed Imaging
High-speed imaging is required to observe fast dynamic phenomena, such as a beating heart, blood flow, or calcium ion dynamics inside
cells. The FV3000RS hybrid scan unit uses a galvanometer scanner for precision scanning as well as a resonant scanner that is ideal for highspeed imaging of live physiological events. The resonant scanner is capable of speeds starting from 30 fps at FN18, up to 438 fps using clip
scanning. Users can switch between the galvanometer scanner and resonance scanner with a single click and use the sequence manger torecord each action for complex experiments.

Intensity Modulated Display of the CFP/YFP ratio result during spontaneous contractions of an in vitro cardiomyocyte.
Image data courtesy of Yusuke Nino and Atsushi Miyawaki, Cell Function Dynamics, Brain Science Institute of RIKEN.

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Accurate Time-Lapse Imaging
Maintain Focus with TruFocus Z-Drift Compensation

Stage Control for Multi-Area Time-Lapse, Microplate,and Stitching

The TruFocus ZDC system uses minimally-phototoxic infrared
light (laser class 1) to identify the location of the sample
plane. One-shot autofocus (AF) mode enables several focus
positions to be set as desired for thicker samples, enabling
efficient Z-stack acquisitions in multiposition experiments.
The continuous AF mode keeps the desired plane ofobservation precisely in focus, avoiding focus drift caused bytemperature changes or the addition of reagents, making it
ideal for measurements that require more stringent focusing.
Furthermore, the increased optical offset enables continuous
AF with plastic vessels or with dry objectives. The TruFocus
ZDC system is also compatible with silicone oil objectives,
enabling accurate time-lapse imaging.

Multi-area time-lapse and stitching provide robust and accurate
time-lapse data and enable users to generate detailed overview
images to see their data in context. The well navigator function
provides sophisticated, intuitive controls for a wide range ofcommercially available microplates as well as custom designed
cell culture dishes.

TruFocus Optical Path Diagram
IR Laser
Laser Reflection
Focal Plane

Desired Focus
Vessel Bottom

Photodiode

Offset Lens
Position

Dichroic
MirrorIR Laser Diode

www.olympus-lifescience.com/video/ix3_zdc2

Reduce Complexity with the Sequence Manger
With the Sequence Manager software module, complex protocols are handled with ease and accurate timing. Multiday time-lapse
experiments are controlled with microsecond scan accuracy and millisecond sequence execution accuracy. Various protocols, such as
time-lapse with different time intervals, switching between high and low magnification, switching between galvo and resonant scanners,
and photo-stimulation between imaging by FRAP or FRET (acceptor photobleaching), can be performed.

A

512 x 512 pixels

Scanning
size
0min

B

10min

20min

512 x 32 pixels

30min

30m30sEvery 10 minutes

Interval
time
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512 x 512 pixels

59m30s60min

Every 30 seconds

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Every 5 minutes

60 min90 minThe Sequence Manager enables variable time-lapse experiments with microsecond scanning accuracy.

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Increase Productivity with High-Speed Imaging
Galvanometer and Hybrid Galvo/Resonant Scanners

Optimized for Live Cell Imaging

Users have their choice of two different types of scan units:

Resonant scanning greatly reduces photobleaching and phototoxicity

galvanometer only with the FV3000 system or hybrid galvanometer/

compared to standard galvanometer scans by preventing theresonant with the FV3000RS system. The hybrid scan unit has a

excitation of fluorophores into triplet states that create reactive

galvanometer scanner for high-precision scanning, as well as aoxygen species. These features make live cell experiments moreresonant scanner that is ideal for high-speed imaging. With therobust and reliable. The FV3000 series has complete laser intensity

galvanometer scanner and Olympus super resolution technology

control from low to high range, enabling the system to use the(FV-OSR), users can obtain resolutions down to 120 nm with a highminimum required amount of laser power on samples. The optionalsignal-to-noise ratio. The galvanometer scanner also features flexible

laser power monitor provides consistent laser power during long-scanning options, including precise tornado scanning as well as

term time-lapse imaging across multiple days.

multipoint stimulation with 100 ms switching time. The galvanometerscanner can image up to 16 frames per second. By switching to the

No Compromise between Speed and Field of View

resonant scanner, users can capture 30 frames per second with a

Many high-speed scanning methods restrict the field of view, limiting

full field of view at 512 × 512 pixels. At 512 × 32 pixels, the resonant

their usefulness for examining large areas with multiple cells. The

scanner can capture up to 438 frames per second to capture critical

FV3000 series’ resonant scanner maintains a full 1X field of view,

live physiological events such as calcium ion flux.

even at a video rate of 30 frames per second. By clipping the Y axis,
additional speeds up to 438 frames per second can be achieved.

Other
commonscanners
Platelets bound to a thrombosis in the blood vessel of a mouse. Images taken at 30
fps in full frame by resonant scanner with 2 CH GaAsP PMTs.
Image data courtesy of Dr. Takuya Hiratsuka, Dr. Michiyuki Matsuda, Graduate Schoolof Biostudies, Kyoto University.

FV3000 series
FN 18, 8 KHz

Most resonant scanners force a trade-off between speed and field of view. FLUOVIEW
systems are optimized to maintain the field of view with even signal intensity so dynamic
samples (e.g., calcium imaging) can be seen in the broad context of their cells andtissues.
The image above shows examples of the clipped fields of view required in other resonant
scanning systems.

A431 cells fixed with methanol labeled with Abcam Anti-ERK1 + ERK2 antibody (Alexa
Fluor 488) ab208564 and Anti-alpha Tubulin antibody (Alexa Fluor 594) ab195889 and
DAPI.
Sample courtesy of Abcam.

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Ratio Imaging and Intensity Modulated Display (IMD)

Rolling Average Processing

The FV3000 microscope's ratio imaging analysis function includes

High-speed scanning at low laser power to avoid phototoxicity

an intensity-modulated display (IMD) function in the software

often decreases the signal-to-noise ratio. With rolling average post-that displays quantitative fluorescence ratio changes during bothprocessing, users have the flexibility to adjust high-speed time-lapse

standard and high-speed acquisitions. This function is particularly

images while maintaining the time scale and keeping the original data.

useful for calcium and FRET imaging where a pure ratio display
provides poor contrast in background areas.
Ratio (ex405/ex488)

High
Raw 30 fps data acquired at low laser
power (0.05%, 488 nm).

Rolling average processing (10 frame) on
30 fps data acquired at low laser power.

Object Tracking

Low

In time-lapse imaging, moving objects can be automatically

10 μM CCCP treatment

detected, tracked, and analyzed. cellSens software's tracking
function provides a powerful and intuitive tool to quantify dynamic

Ratio (ex405/ex488)

processes such as cell movement and division.
Tracking object
1

tsGFP1-mito reveals heterogeneity in mitochondrial thermogenesis in HeLa cells.
Ratio images (ex 405 nm/ex 488 nm) in tsGFP1-mito-expressing cells shown before andafter CCCP treatment at 37 °C. Scale bars indicate 10 μm (whole image) and 3 μm (inset).
Image data courtesy of Shigeki Kiyonaka Ph.D., Yasuo Mori Ph.D., Molecular Biology Field,
Department of Synthetic Chemistry and Biological Chemistry, Kyoto University.

Time coded2

3

1
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Time-dependent change in the intensity of cells

Life Science Analysis
The cellSens Life Science Analysis module enables analysis of images from FRAP or FRET experiments. The FRAP module includes single aswell as double exponential fitting. This module helps to calculate τ/2 and mobile/immobile fractions.
The FRET module enables ratio imaging processing and measurement of FRET efficiency from acceptor photobleaching and sensitizes
emission. Several computational methods, including Gordon (1998), Xia (2011), and Elangovan (2003) are available.
Example of FRET analysis (acceptor photobleaching)

Acceptor ImageFRET Efficiency Image

Pre
BleachGray

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Donor Image

Example of FRAP analysis

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14

Application: Deep Tissue Observation with Silicone Oil Objectives
Olympus silicone oil immersion objectives deliver excellent performance for live cell imaging. The refractive index of silicone oil is close tothat of living tissue, enabling high-resolution observation deep inside living tissue with minimal spherical aberration. This refractive index
match delivers an ideal focal volume, resulting in perfect volume reconstruction and enabling high-resolution confocal imaging of large livingorganisms. The FV3000 system‘s 3D construction software enables simultaneous image acquisition and real-time 3D rendering to visualize
the sample in its entirety.

www.olympus-lifescience.com/fv3000-spheroid-animation

A spheroid image of an NMuMG cell line expressing Fucci2.
Image data courtesy of Atsushi Miyawaki, Cell Function Dynamics, Brain Science
Institute of RIKEN.

3D image of chick ciliary ganglion cleared by tissue clearing reagent.
Image data courtesy of Dr. Ryo Egawa. Tohoku University Graduate Schoolof Life Science.

3D observation of biliary tree structures in mouse liver.
Image data courtesy of Dr. Hajime Okada, Division of Mammalian Development,
Genetic Strains Research Center, National Institute of Genetics, and Dr. Tohru
Itoh, Laboratory of Stem Cell Therapy, Institute for Quantitative Biosciences, The
University of Tokyo.

NK cell line KHYG-1 (green) changing shape while attacking and killing HT-29
tumor cells labeled with cetuximab (blue). PI uptake (red) indicates cell death.
Image data courtesy of Dr. Yuji Mishima, The Cancer Chemotherapy Center of
JFCR.

https://www.olympus-lifescience.com/applications/cleared-mouse-liver/15

https://static3.olympus-lifescience.com/data/Video/Librar
y/3Dimageofchickciliaryganglionclearedbytissueclearingre
agent_480.mp4?rev=64AE

https://www.olympus-lifescience.com/applications/spheroid_3d_imaging/Superior Objectives
A Line: Silicone Oil Immersion Objectives for Live Cell Imaging Deliver High-Resolution Observation at Depth
Olympus offers four high NA silicone oil immersion objectives that deliver excellent performance for live cell imaging. The refractive index
of silicone oil (ne≈1.40) is close to that of living tissue (ne≈1.38), enabling high-resolution observations deep inside living tissue withminimal spherical aberration caused by refractive index mismatch. Silicone oil does not dry out or harden, so the need to refill the oil iseliminated, making it ideal for extended time-lapse observations.
Refractive Index Is Important with Deep Tissue Observation
Working Distance Numerical Aperture(WD) [mm](NA)

Objectives

UPLSAPO30XS

0.8

1.05

UPLSAPO40XS

0.3

1.25

UPLSAPO60XS2

0.3

1.3

UPLSAPO100XS

0.2

1.35

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

Samplene≈1.38

Immersionoilne≈1.52

Silicone oil
ne≈1.40

Cover glassne≈1.52

Oil immersion objective

Silicone oil immersion objective

When working with an oil immersion objective,the difference between the refractive index of
the samples and immersion oil results inspherical aberration in deep tissue, causing
resolution to deteriorate and fluorescence tobecome dim.

When working with a silicone oil immersion
objective, the difference between the refractive
index of the samples and silicone oil is minimal,
so it achieves brighter fluorescence images
with higher resolution for deep tissue
observation.

5μm

5μm

35 µm

35 µm

UPLSAPO60XS2（NA 1.3, W.D. 0.3mm, silicone oil ne = 1.4）

UPLSAPO60XO（NA 1.35, W.D. 0.15mm, immersion oil ne = 1.52）

Scal eA2-treated neocortex acquired with UPLSAPO60XS2 and UPLSAPO60XO objectives (predecessor of the UPLXAPO60XO).
Image data courtesy of Motokazu Uchigashima, M.D., Ph.D., Masahiko Watanabe, M.D., Ph.D., Department of Anatomy, Hokkaido University Graduate School of Medicine.

A Line PLAPON60XOSC2:
Enhance the Reliability of Colocalization Analysis with a Super-Corrected Apochromat Objective
This oil immersion objective minimizes chromatic aberration to
the utmost limit in the 405–650 nm spectrum. 0.1 µm or lessaxial chromatic aberration is guaranteed, and every objective
is delivered with its measured data sheet. Furthermore, various
aberrations are compensated even at 405 nm, and high-quality
homogeneous 405 nm images can be achieved. This objective
is used for rigorous colocalization analysis or super resolution.

Performance Comparison of the PLAPON60XOSC2 and UPLXAPO60XO
PLAPON
60XOSC2

On axis verticalchromatic aberration(Z direction)

Approx.
0 µm

UPLXAPO
60XO
Approx.
0.2 µmSuper-Corrected Apochromat 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.

Off axis lateralchromatic aberration(X-Y direction)
Approx. 0.05 µm

Approx. 0.15 µm

Comparison of chromatic aberration measured by the FLUOVIEW FV3000
microscope using TetraSpeck Microsphere.
Cyan: 405 nm excitation, Magenta: 640 nm excitation.

16

TruAI Deep-Learning Technology

AI

Experiments often require extracting and analyzing data from microscope images. For accurate image analysis, segmentation—especiallythresholds based on intensity values or color—is used to extract the analysis targets from the images. But this can be time-consuming andaffect the sample condition. TruAI technology's next-generation image analysis using deep learning helps address these challenges.

(a)

The result of detecting nuclei (right) with the conventional method, fromfluorescence images (left) with extremely poor SNR due to weak excitation light.
You can see that the detection accuracy is low.

(b)

The result of detecting nuclei (right) using TruAI from a fluorescence image (left)
with extremely poor SNR due to weak excitation light. You can see that theaccuracy is much higher accuracy than (a)Autophagosome of mouse fibroblast (left), and autophagosomes recognized by TruAI (right).

Prediction of glomeruli positions on a mouse kidney section using TruAI (blue). TruAI captures and detects the glomeruli features (right).

17

NoviSight 3D Cell Analysis Software
NoviSight 3D cell analysis software eases the analysis of 3D images of spheroids and organoids in microplates that are acquired using the
FV3000 laser scanning microscope. The software’s True 3D technology enables you to easily measure and count 3D objects in spheroids
and organoids. By improving your observation efficiency as well as the accuracy and speed of your 3D analysis, NoviSight software increases
the speed of discovery.

Fast, Precise Object Detection
With numerous detection algorithms designed for 3D samples, NoviSight software can quantify cell activity and interactions in three
dimensions. With precise detection, the software analyzes objects of interests to provide morphology and spatiotemporal parametersin 3D space.

Spheroids

Nuclei

Organelle (Autophagosome)

Intuitive User Interface Reduces Your Analysis TimeAll the data you need—recognition, analysis, and statistical results—are in one convenient location. The original images are paired with thequantitative data for easy validation and interpretation. The data is easily exported as a CSV or FCS file for further analysis.
Image

Graph

Image

Get 2D or 3D views of your samples. Locate
objects in 2D within an image plane or switch to 3D
to explore the entire spheroid.
Graph

The scatter plot makes it easier to classify objects.
You can click and select an individual point, which
will then bring up an image of that object.
Gallery

Observe the details in each region of an object at
a glance. Visualize how classification is working by
highlighting two areas in the scatter plot, such asmorphology differences in the nuclei, and display
the resulting galleries side by side.
Statistics

View quantitative results numerically or displayedon a heat map.
Gallery

Statistics

18

X Line High-Performance Objectives
X Line: Breaking Barriers to Improve Confocal Image Quality
Olympus X Line objectives provide improvements in three important areas: a higher numerical aperture (NA) to acquire brighter and higher
resolution images, expanded image flatness for smoother image stitching and efficient image analysis, and a wide chromatic aberration
correction range covering 400 nm–1000 nm wavelengths. X Line objectives can acquire multicolor images with high accuracy and precision.

Comparing the focus position in the 400-1000 nm wavelenghth range

Conventional objectives

X Line1X Zoom

6X Zoom

UPLXAP40X0 X Line objective (NA 1.40)

Selection Guide for X Line Objectives

19

Objectives

Working Distance (WD) [mm]Numerical Aperture (NA)UPLXAPO4X

13

0.16

UPLXAPO10X

3.1

0.4

UPLXAPO20X

0.6

0.8

UPLXAPO40X

0.18

0.95

UPLXAPO40XO

0.13

1.4

UPLXAPO60XO

0.15

1.42

UPLXAPO100XO

0.13

1.45

UPLXAPO60XOPH

0.15

1.42

UPLXAPO100XOPH

0.13

1.45

Choose the Frame That Suits Your Application
Inverted microscope• Used to observe cells cultured in a vessel.
•	TruFocus Z-drift compensation helps ensure the sample remains
in focus during time-lapse observations.
• Maintain the environmental conditions of cultured cells by adding
a stage-top or full enclosure incubator.

Upright microscope (configured for imaging)• Optimized for fixed tissue and glass slide specimens.
• Motorized nosepiece precisely maintains the focus position.
• Motorized seven-position nosepiece and condenser enable
automated transitions from low to high magnification.

Upright microscope (configured for electrophysiology)• Ample space around the objectives enables patch-clamp
devices to be installed.
• Add additional space by lowering the stage position forexperiments that require large sample handling.
• Swing and slider nosepieces are available so objectives can beeasily changed without interfering with the patch-clamp set-up.
• Trigger I/O is possible using the FV30-ANALOG to synchronize
with electrophysiology equipment.

20Modular Units Designed for Your Applications
Scanners

Laser Combiners

Other Equipment
Available upgrade options: field-upgradable
laser-based autofocus, fast and precise
motorized stage control, analog input/output
and TTL synchronization, and a convenientantivibration platform.

Hybrid Scan Unit (Resonant/Galvanometer)
The hybrid scanner combines the capabilities
of a galvanometer scanner with a resonant
scanner for high-speed imaging in the full fieldof view at 30 fps and up to 438 fps at 512 ×
32. The Sequence Manager makes it simple
to automatically switch between resonant andgalvanometer imaging in the same experiment.
Galvo Scan Unit
The galvanometer-only scanner provides
precision one-way scanning from 1 fps at 512
× 512, and up to 16 fps with bi-directionalscanning. High-speed multipoint stimulation
or detection experiments can travel between
multiple cells at over 100 Hz with data output
as high as 500 kHz.

Spectral Detectors

Main Laser Combiner
The main laser combiner is the heart of thelaser system. The combiner accommodates
four standard lasers with an option to add
a fifth laser or leave an open port to add
an additional three diode lasers via the sub
combiner.
Sub Laser Combiner
Add this optional combiner at any time withup to 3 diode lasers for a maximum of 7
laser lines in combination with the main laser
combiner.

TruFocus/IX3-ZDC2 Z-Drift Compensation
SystemThe TruFocus Z-drift compensator uses
minimally-phototoxic IR light to identify thelocation of the sample plane. The IX3-ZDC2 isalso compatible with silicone oil objectives andplastic-bottom vessels.

Illumination Units
The conventional illumination modules
are designed for long-duration time-lapse
experiments. Since light is introduced throughfiber delivery systems, no heat is transferred tothe microscope.
Ultrasonic Stage for the IX3/IX3-SSU
With low thermal drift for improved accuracy,
the ultrasonic stage can be controlled by both
software and the Touch Panel Control for fast,
reliable multi-area imaging.

High-Sensitivity Spectral Detector (GaAsP PMT)with TruSpectral Technology
The two-channel high-sensitivity spectral
detector (HSD) employs the same volume
phase holographic (VPH) technology as the
spectral detector (SD), with Peltier cooled
GaAsP PMTs and a high quantum efficiencyof 45% and detection up to 750 nm. This unit
can be combined with the two-channel SD
for a flexible dynamic range or a second twochannel HSD unit for powerful four-channel
sensitivity.
Spectral Detector (Multialkali PMT) with
TruSpectral Technology
The two-channel SD employs efficient VPH
transmission and an adjustable slit with 1–100
nm bandwidth from 400–800 nm detection.
The multialkali PMTs provide a broad dynamic
range for detection up to 800 nm.

21Light Source/U-HGLGPS
The pre-centered fluorescence illumination
source requires no adjustment and has anaverage lifespan of 2,000 hours.

Umbra unit/FV31-SPCOVThe umbra unit is designed for fluorescenceobservation under bright room conditions. It
efficiently blocks out room light and enables
clear fluorescence observation without theneed for a dark room.

Transmitted Detector
This unit combines an external transmitted
light photomultiplier detector and LED
conventional illumination for both laser
scanning and conventional transmitted
light Nomarski DIC observation. Users canundertake simultaneous multichannel confocal
fluorescence imaging and transmitted DIC
acquisition.

Remote correction collar unit/IX3-RCC
The remote correction collar unit enables
the user to easily adjust the correction collar
manually to improve image quality.

Specifications
FLUOVIEW FV3000 Laser Confocal Microscope Specifications
FV3000FV3000RS

Laser Light

Visible Light Laser

405 nm: 50 mW, 488 nm: 20 mW, 561 nm: 20 mW, 640 nm: 40 mW
One optional laser port for sub laser combiner or optional laser unit

Optional Laser

Sub Laser Combiner

Laser as follows (max. 3 laser units )445 nm: 75 mW, 514 nm: 40 mW, 594 nm: 20 mW, connected to main laser combiner

Single Laser Unit

445 nm: 75 mW, 514 nm: 40 mW, or 594 nm: 20 mW, directly connected to main laser combiner

Single NIR Laser Unit

730 nm: 30 mW, 785 nm: 100 mW, connected to scanner via optional port

Scanning Method

2 silver-coated galvanometer scanningmirrors

Galvanometer Scanner(Normal Imaging)

Scanning Resolution: 64 × 64 to 4096 × 4096 pixels
Scanning Speed (One Way): 512 × 512 with 1.1 s–264 s. pixel time : 2 µs–1000 µs
Scanning Speed (Round Trip): 512 × 512 with 63 ms–250 ms / 256 × 256 with 16 ms–125 ms
Optical Zoom: 1X–50X in 0.01X increments
Scan Rotation: Free rotation (360 degrees) in steps of 0.1 degree
Scanning Mode: PT, XT, XZ, XY, XZT, XYT, XYZ, XYλ, XYZT, XYλT, XYλZ, XYλZT
ROI scanning, rectangle clip, ellipse, polygon, free area, line, free line, and point; tornado mode only for stimulation

Scanner

Resonant Scanner(High-Speed Imaging)SpectralDetector
NIR Detector–

2 silver-coated galvanometer scanning mirrors
1 silver-coated resonant and 1 silver-coated galvanometer scanning mirrors

Scanning Resolution: 512 × 32 to 512 × 512 pixels
Scanning Speed: 30 fps at 512 x 512, 438 fps at 512 × 32
Optical Zoom: 1X–8X in 0.01X increments
Scanning Mode: XT, XZ, XY, XZT, XYT, XYZ, XYλ, XYZT, XYλT, XYZ, XYλZTROI Scanning, Rectangle Clip, Line

PinholeSingle motorized pinhole, pinhole diameter ø50 – 800 µm (1 µm steps)

Field Number (FN)

18

Dichromatic Mirror Turret

8 positions (high-performance DMs and 10/90 mirror)

Optional Unit for Scanner

Laser power monitor, optional laser port

Detector Module

Cooled GaAsP photomultiplier (high-sensitivity type) or Multi-Alkali photomultiplier, 2 channels

Spectral Method

Motorized Volume Phase Holographic transmission diffraction grating, motorized adjustable slit,
selectable wavelength bandwidth: 1–100 nm, wavelength resolution: 2 nm

Dichromatic Mirror Turret

8 positions (high-performance DMs and mirror)

Detector Module

GaAs photomultiplier tube, 1 channel or 2 channels with filter cube

Fluorescence Illumination Unit

External fluorescence light source, fiber adaptor to optical port of scan unit, motorized switching between LSM light pathand fluorescence illumination

Transmitted Light Detector Unit

Module with integrated external transmitted light photomultiplier detector and LED lamp, motorized switching

Microscope
Inverted frame

Upright frame (for imaging)Microscope Frame

Motorized inverted microscope
IX83P2ZF

Motorized fixed stage upright microscope
BX63L

Upright frame (for electrophysiology)

Revolving Nosepiece

Motorized sextuple revolving nosepiece Motorized septuple revolving nosepiece Coded swing nosepiece
Coded slider nosepiece

Condenser

Motorized long working distance condenser Motorized universal condenser

Focus Stroke

Built-in motorized nosepiece focus
Stroke: minimum increment: 0.01 μm

Manual long working distance condenser

Software
Basic Features

GUI designed for darkroom environment. User-arrangeable layout.
Acquisition parameter reload features. Hard disk recording capability, adjust laser power and HV with Z-stack acquisition.
Z-stack with alpha blending, maximum-intensity projection, iso-surface rendering.

2D Image Display

Each image display: single-channel side-by-side, merge, cropping, live tiling, series (Z/T/λ), LUT: individual color setting,pseudo-color, comment: graphic and text input

3D Visualization and Observation

Interactive volume rendering: volume rendering display, projection display, animation display.
3D animation (maximum intensity projection method, α blending) 3D and 2D sequential operation function

Image Format

OIR image format
8/16-bit gray scale/index color, 24/ 32/ 48-bit color, JPEG/ BMP/ TIFF image functions, Olympus multi-tif format

Spectral Unmixing

Fluorescence spectral unmixing modes (up to 16 channels)

Image Analysis

Region and line measurements, Intensity plot over time/Z, Colocalization analysis

Statistical Processing

2D data histogram display

Optional Software

Motorized-stage control / Mapping and multipoint stimulation / Sequence manager / Virtual channel acquisition /Microplate navigation / Remote development kit / Super resolution imaging (FV-OSR) / Digital camera control function /TruSight Deconvolution / FRET&FRAP analysis / Automatic object measurement and classification / Object tracking / TruAI
deep-learning technology / NoviSight 3D cell analysis

22

Image data are courtesy of the following institutions:Cos-7 cells labeled with DAPI (blue), anti-Tubulin (Alexa Fluor 488; green),
Concalavalin A (Alexa Fluor 594; yellow), SiR-Actin (magenta) and anti-TOMM20(Alexa Fluor 750; cyan). Images were acquired using a UPLXAPO60XO objective
and 405 nm, 488 nm, 561 nm, 640 nm, and 730 nm laser lines on GaAsP and
GaAs detectors. Maximum intensity projection in Z with TruSight deconvolution
processing. (cover)Sample courtesy of Dr. Jana Döhner, Dr. Urs Ziegler, University of Zürich.
Immunocytochemistry (peripherin; green; neuronal cell bodies and axons) in
combination with in situ hybridization (CALCA mRNA; red and P2RX3 mRNA;blue) of cultured mouse dorsal root ganglion neurons. (top left, page 1)
Image data courtesy of Stephanie Shiers, Price Lab, University of Texas at Dallas.
Live culture of Drosophila (fruit fly) brains. Microtubule marker which marks
neurons in the brain, and histone marker which marks nuclei. (top right, page 1)
Image data courtesy of Martin Hailstone, Oxford University.
Mouse brain tissue section was labelled with trans-synaptic viral tracer toinvestigate neural pathways that link the visual systems in the mouse. The varying
colours reflect the changing depths of neuronal processes. (center, page 1)
Image data courtesy of Arthur Chien, Macquarie University.
Fruit fly 3rd instar larvae brain lobe. Neuroblasts and ganglion mother cells are red
and pink, respectively. Actin is green. DNA is blue. Wolbachia bacteria are green
dots within cells. (bottom left, page 1)
Image data courtesy of Anton Strunov, Medical University of Vienna.
5 μm sagittal cryosection of E14.5 mouse embryo, stained with TSA reagents.
(page 2)
Image data courtesy of Dr. Guan Yang and Prof. Xiao Yang,Genetic Laboratoryof Development and Diseases, Beijing Institute of Biotechnology, AMMS.

•
is ISO14001 certified.
•is ISO9001 certified.
• Illumination devices for microscope have suggested lifetimes.
Periodic inspections are required. Please visit our website for details.

www.EvidentScientific.com• This product is designed for use in industrial environments for the EMC performance. Using it in a residential
environment may affect other equipment in the environment.
• All company and product names are registered trademarks and/or trademarks of their respective owners.
• Images on the PC monitors are simulated.
• Specifications and appearances are subject to change without any notice or obligation on the part of the manufacturer.

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