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All-in-OneIntraVital Microscopy
IntraVital Microscopy (IVM)In Vivo Live Cell Imaging Platform
BY
Contents
Introduce ···· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 4p
Technology : IntraVital Microscopy ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 5p
Key Advantages ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 10p
Product Lines ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 14p
Key Applications ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 20p
Specifications ···· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 26p
04 05IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
Intravital microscopy enables dynamic 3D cellular-level imaging of various biological processes in
living animals in vivo. It enables scientists to directly verify hypotheses derived from ex vivo or in vitro
observations in natural physiological in vivo microenvironments.
Using intravital microscopy, in vivo visualizations of gene expression, protein activity, cell trafficking,
cell-cell / cell-microenvironment interaction and various physiological responses to stimuli have been
accomplished providing novel insights, which have been impossible to obtain with conventional static
2D observation of ex vivo or in vitro samples.
However, up to now, individual users have had to improvise the required functions for each of the
intravital imaging applications for the various organs with conventional standalone microscope, which
resulted in non-optimal imaging performance and limited applicability.
IVIM Technology’s All-in-One intravital confocal / two-photon microscopy system (IVM-C/M/CM/MS) is
extensively optimized and carefully engineered to provide superb performance in the intravital imaging
of live animal models in vivo.
• IVIM Technology’s IVM series integrates expertly-selected best-suited optical, opto-mechanical and
opto-electrical components, ensuring optimal imaging performance for a wide range of intravital
imaging applications.
• Key indispensable functionalities for intravital imaging are fully integrated into the All-in-One system
with attentive design for smooth and easy operation.
- Body temperature monitoring and automated regulation system
- Anesthesia apparatus optimized for integration into animal stage
- Animal motion stabilization apparatus customized for various organs
- Implantable imaging window apparatus customized for longitudinal observation of various organs
• Versatile intravital imaging software with unique features
- Ultrafast image acquisition to capture fast real-time dynamics
- High-precision Animal motion compensation enabled by optimized registration algorithm and
accelerated by GPU-assisted parallel computing
- Image quality optimization by a comprehensive processing algorithm
The world's first All-in-One intravital microscopy platform from IVIM Technology is a key solution
that can explore complex dynamic behaviors of numerous cells inside a living body and serve as next
generation core technology to elucidate unknown pathophysiology of various human diseases and
discover new cures for them.
• IntraVital Microscopy (IVM) enables dynamic real-time imaging of various cellular-level dynamics such as
cell trafficking, cell-cell / cell-microenvironment interaction and various physiological responses to stimuli
inside the living body in vivo, providing novel insights in the processes of human disease development.
• For drug development, IVM enables a direct analysis of drug delivery to target tissues and cells and the
efficacy of new therapeutic candidates by the intended MOA (Mode of Action) at a microscopic cellular-
level in various preclinical models of human diseases.
IVIM Technology :Global No. 1 Solution Provider of IntraVital Microscopy
Why IntraVital Microscopy (IVM) ?IntraVital Microscopy (IVM) is In Vivo Cellular-level Imaging Platform.
Macroscopic Imaging & Analysis at the Organ / Organism level
Microscopic Imaging & Anlaysis at Cellular / Molecular Level
- 3D cellular & molecular imaging
- In vivo delivery monitoring
- Monitoring cellular dynamics
IntraVital Microscopy
Stem cell / Vessel Vessel / RBC / NФ B cell / T cell / Vessel MФ & NФ / Inflammatory protein
30 min 180 min
ICG ICG ICG
00:00Liver
Liver Retina
00:33 04:54
NP Vessel RBC GeneNP
Day1 Day6 Day13
Liver
ICG Kupffer cell & DC
ICG MΦ & myeloid DC
06 07IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
IVIM’s All-in-One Packaged Platform
Why IntraVital Microscopy (IVM) ?IntraVital Microscopy (IVM) is a Unique Solution for In Vivo Live Cell Imaging !
Why IntraVital Microscopy (IVM) ?All-in-One IVM Platform
+ Thymus, Thyroid gland, Adipose Tissue, Lymphatics, Microcirculation … etc.
Bone Marrow
Liver
Retina
Trachea
Esophagus Heart
Brain
Kidney
Skin
Mammary Tissue
Lung
Lymph Node
Tumor Pancreas Spleen
Prostate
Small Intestine
Colon
IVIM Technology’s All-in-One intravital confocal / two-photon microscopy system (IVM-C/M/CM/
MS) has been designed to provide expandability and flexibility for highly diverse intravital imaging
application of various tissues and organs including the brain, liver, spleen, kidney, pancreas, lung, heart,
gastrointestinal tracts, retina, skeletal muscle, bone marrow, peripheral lymph node, prostate, thymus,
thyroid gland, adipose tissue, blood and lymphatic vessels, etc.
In addition, to handle various imaging needs raised by researchers for a wide range of biomedical
studies, detailed experimental protocols have been established for high-quality intravital image
acquisition.
Subsequent quantitative analysis of various cellular-level dynamics and physiological alterations have
been established.
For drug discovery and development applications, IVIM Technology’s All-in-One intravital microscopy
system can serve as a highly valuable and versatile tool.
• Target identification and validation in natural in vivo microenvironments
• Confirmation of MoA (Mode of Action) and PoC (Proof of Concept) of new therapeutics by direct in vivo cellular observation
• Monitoring of in vivo drug delivery, distribution, retention and in situ efficacy at target tissues and organs
All-in-One IVM Platform
•All-in-One single-box packaged IntraVital Microscopy (IVM) system
•Fully integrated with key functionalities for imaging of live animal model
•Unified animal stage to accommodate various accessories for wide-range of intravital imaging applications
•User-friendly design for easy and smooth operation
Optimized In Vivo System
•Sub-µm in vivo imaging resolution
•Ultra-high speed in vivo imaging (Max. 100 fps @ 512x512 pixels)
•4-color simultaneous confocal / two-photon microscopic in vivo imaging
•Integrated animal motion compensation
IVIM Technology’s All-in-One intravital confocal /
two-photon microscopy system (IVM-C/M/CM/MS)
is extensively optimized and carefully engineered to
provide superb performance in the intravital imaging
of live animal models, in vivo.
All-in-OneIntraVital Microscopy
New!
Conventional Approach
Imaging chamberIn vivo fluorescence labeling
Image processing Animal manipulation
•High degree of technical difficulty for
non-expert individual user
•Non-optimized intravital imaging performance
•Limited applicability to various organs
•Difficult to standardize intravital imaging
procedure
•Difficult to establish universal protocol for
experiment
•Limited reproducibility and productivity
Individual user have had to improvise the
required functions for each of the intravital
imaging applications with conventional
standalone microscope.
08 09IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
IVIM Technology’s Confocal / Two-Photon Technologies Enable High-resolution In Vivo Imaging
Confocal Microscopy (IVIM Technology : IVM-C, CM)■ High-resolution optical sectioning imaging of in vivo tissue
• Rejection of fluorescence signal from out-of-focal volume by confocal pinhole
• High contrast, low background, high quality in vivo imaging
■ Ultrafast precise Raster scanning of multi-color excitation laser-beam focus
• Video-rate imaging of fast cellular-level dynamics in live tissue in situ
■ Ideal for high-sensitivity multi-color intravital imaging of in vivo tissue
Two-Photon Microscopy (IVIM Technology : IVM-M, CM, MS)■ High-resolution optical sectioning imaging of in vivo tissue
• Fluorescence signal is intrinsically generated only at the focus
■ Deeper tissue imaging with longer-wavelength near-infrared (NIR) fs-pulse laser for two-photon / multi-photon excitation
■ Capable of label-free, non-linear multi-harmonic generation imaging (SHG, THG)
■ No photo-bleaching at non-focal plane : reduced phototoxicity
■ Ideal for long-term 3D intravital imaging of in vivo tissue
Widefield Image Optical Sectioning Image
Confocal Microscopy■ Single-photon excitation
■ Point scanning + Pinhole
■ Optical sectioning :
Fluorescence signal from out-of-focus is blocked
■ Imaging Depth :
100-200 µm
■ Continuous-wave solid-state laser with flexiblechoice of wavelength at the range from ultraviolet (UV) and visible (VIS) to near-infrared (NIR)
■ Descanned confocal detector
■ Easy & efficient, multi-color 3D intravital imaging
Two-Photon Microscopy ■ Two-photon excitation
■ Point Scanning + No Pinhole
■ Optical sectioning : Fluorescence signal is intrinsically generated onlyat the focus
■ Imaging Depth :
250-1000 µm
■ Femto-second pulsed laser tunable at near-infrared (NIR) wavelength range
■ Non-descanned detector (NDD)
■ Deeper-tissue 3D intravital Imaging
Excitation LaserBeam Scanning
Pinhole
Lens
Dichroic BeamSplitter
ExcitationLight
2D RasterScanning
Fluorescence(from focal plane)
Fluorescence(from out-of -focal plane)
Sample
ExcitationLight
ExcitationLightObjective
Lens
Detector Detector Detector
Fluorescencefrom focus
Fluorescencefrom out-of-focus
Excitation LaserBeam Scanning
Pinhole
Lens
Dichroic BeamSplitter
ExcitationLight
2D RasterScanning
Fluorescence(from focal plane)
Fluorescence(from out-of -focal plane)
Sample
ExcitationLight
ExcitationLightObjective
Lens
Detector Detector Detector
Fluorescencefrom focus
Fluorescencefrom out-of-focus
Excitation LaserBeam Scanning
Pinhole
Lens
Dichroic BeamSplitter
ExcitationLight
2D RasterScanning
Fluorescence(from focal plane)
Fluorescence(from out-of -focal plane)
Sample
ExcitationLight
ExcitationLightObjective
Lens
Detector Detector Detector
Fluorescencefrom focus
Fluorescencefrom out-of-focus
Fluorescence from focus
Fluorescence from out-of-focus
Excitation LaserBeam Scanning
Single-PhotonExcitation
Excitedstate
Groundstate
Fluorescence signal
Two-PhotonExcitation
Photobleaching Patterns
Coverslip Light Path
Two-Photon MicroscopyConfocal Microscopy
Photobleached Fluorophore
Second Harmonic Generation (SHG) Imaging of in vivo tissue
Fibrosis
10 11IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
IVIM Technology’s Key Advantage 1 : Ultrafast Scanning with Uniform Illumination
IVIM Technology’s Key Advantage 2 : Animal Motion Compensation
■ IVIM Technology - Integrated Motion Artifact Compensation
- Automatic hassle-free high-precision motion compensation
- Immediate acquisition of motion-compensated imaging results by GPU-assisted parallel computing for acceleration of algorithm processing
- Synergistic effect with ultrafast intravital imaging
- Guarantee optimal resuls in wide-range of spatiotemporal tissue movement from slowly moving tissues (e.g. abdominal organs such as liver, kidney, spleen) to rapidly moving tissues (e.g. thoracic organs such as heat, lung)
■ IVIM Technology’s All-in-One intravital microscopy system is equipped with ultrafast rotating polygonal mirror scanner
•Enables ultra-high speed in vivo imaging (max. 100 fps @ 512x512 pixels)
•Achieves uniform excitation illumination over entire imaging field of view (FOV)
- No reduced fluorescence signal and signal-to-noise ratio (SNR) at center area of FOV
- No excessive photobleaching at edge area of FOV
- Uniform high signal-to-noise ratio over entire FOV
- Improved image quality without wasting excessive photons
10 IVIM Technology
Ultrafast Uniform Laser-beam Scanning
IVIM Technology's Ultrafast Polygon Scanning
Conventional Resonant Galvo Scanning
AFrame #1
Frame #2
Frame #4
Frame #3
AAA
Corrected Frame #3
Corrected Frame #4 Corrected
Frame #2
Corrected Frame #1
A
IVIM Technology’s Advanced Motion Compensation
Sharpened In Vivo Image Motion Compensation
In Vivo Imaging of Moving Live Tissue
Blurred In Vivo Image Motion Artifact
Cellular object ‘A’ appeared as moving object → Motion artifact
12 13IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
IVIM Technology’s Key Advantage 3 : All-in-One Packaged Platform
Hardware
All-in-One IntraVital Microscopy Accessories for Intravital Imaging
Research Service
Consulting for In Vivo Live Cell Imaging
Step
1
Step
2
Creating Animal Models of
Various Human Diseases
IVM Imaging of Various Organs in Mouse Model
Image Processing& Analysis
in Cellular Level
Sample Demonstration & Test Imaging
Optimization ofExperimental Plan
Body temp. control system Inhalation anesthesia
Imaging chamber & holder Stereotactic mount
SuperbHardware
Optimized Software
ExpertResearchService
IVM Engine & Viewer
· Animal Motion compensation
· Z-stack / Mosaic / Multi-position
IntraVital MicroscopyIVM-C / M / CM / MS
· Inhalation anesthesia· Imaging chamber & holder· In vivo animal stage
Expert Solution for Intravital Imaging
· Consulting for intravital imaging· Optimization of intravital imaging· Modeling & Intravital imaging· Data processing
IVM Viewer
SoftwareIVM Engine
14 15IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
IVIM Technology’s Product Lines :
■ IVM-C (Confocal)
■ IVM-M (Two-Photon)
■ IVM-CM (Confocal & Two-Photon)
■ IVM-MS (Two-Photon Smart Ver.)
■ Research Service - Intravital Imaging
Key features of IntraVital Microscopy (IVM)
■ Superb performance and unique functionality for in vivo imaging of
living animal
•Ultra High-speed Imaging (max. 100 fps - 512x512 pixels)
•4D Animal Motion Compensation (X,Y,Z & Time)
•Automatic, Hassle-free, GPU-accelerated processing
■ World’s 1st All-in-One Packaged IntraVital Microscopy for live animal
model
•Single box-type IVM System
•Fully Integrated for in vivo imaging
•In vivo maintenance unit / in vivo animal stage
- Monitoring & homeostatic regulation of animal vitality
•4-color simultaneous imaging (Confocal / Two-Photon modes)
■ IVIM Technology’s All-in-One IntraVital Microscopy system has been
verified to be used without any restriction on various organs
16 17IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
IVM-C (Confocal)
IVM-C is the All-in-One IntraVital Confocal Microscopy System, optimized for in vivo imaging
experiments. Especially, because it is equipped with 4 different wavelength lasers and 4 high-sensitivity
confocal detectors, IVM-C is the optimal system to observe highly diverse, dynamic multi-cellular
behaviors in live animals simultaneously with 4 different fluorescence colors.
•Easy & highly efficient multi-color simultaneous imaging
•High flexibility in selection of laser / detector wavelength
•Customizable design for modifications according to the customer’s requests and accommodations for
future updates
•Applicability for conventional ex vivo, in vitro and in vivo imaging
IVM-CM (Confocal & Two-Photon)
IVM-CM is the All-in-One IntraVital Confocal / Two-Photon Microscopy System, optimized for in vivo
imaging experiments. Especially, because both of the confocal and two-photon microscopes are
integrated into a single-box packaged system, IVM-CM provides the ultimate versatile functionality of
the IVM-C and IVM-M in one system.
•Dual-mode IVM-C (Confocal) / IVM-M (Two-Photon) imaging capability
•One-click automated transition between confocal and two-photon imaging mode
IVM-M (Two-Photon)
IVM-M is the All-in-One IntraVital Two-Photon Microscopy System, optimized for in vivo imaging
experiments. Especially, because it is equipped with a fully-automated tunable fs-pulse NIR laser system,
IVM-M is the optimal system for users who need to conduct deeper-tissue imaging using less-scattering
NIR wavelength. Full control functionality of the fs-laser system is integrated in the two-photon imaging
software for user convenience with various automation algorithms.
•Laser wavelength tuning for optimal two-photon excitation of a wide-range of fluorescence agents
•Deeper tissue imaging with a longer-wavelength NIR fs-laser system
•Capable of label-free, non-linear multi-harmonic generation imaging
•Motion compensation and ROI tracking for long-term in vivo imaging
IVM-MS (Two-Photon Smart Ver.)
IVM-MS is the All-in-One IntraVital Two-Photon Microscopy System, optimized for in vivo imaging
experiments and equipped with a new compact high-efficiency fs-pulse laser module. Especially,
because it integrates a compact high-stability maintenance-free fs-pulse laser into a single box, the
IVM-MS is the ideal solution for customers in need of a two-photon microscope with limited resources
of space and budget.
•Less expensive, fully-automated fs-laser system
•Space saving with no additional fs-laser box configuration
•Simple hand-free turn-key operation of the fs-laser for two-photon excitation
•Easy maintenance and management without liquid cooling system requirements
IVIM Technology’s Product Lines :
16 17 IVIM Technology IntraVital Microscopy
18 19IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
IVIM Technology’s Product Lines : Research Service – Intravital Imaging
In Vivo Drug Efficacy Monitoring
Step1 : Consulting, Testing & Planning
Step 2 : Intravital Imaging & Analysis
Consulting for In Vivo Live Cell Imaging
Sample Demonstration & Test Imaging
Optimization ofExperimental Plan
In Vivo Fluorescence
Labeling
•Fluorescence protein (FP) reporter mouse
•Injection of antibody-fluorophore conjugate (vasculature, lymphatics, various immune cells, stromal cells)
•Chimeric mouse generation by bone marrow transplantation
•Adoptive fluorescent cell transfer
■ Key Research Service- In vivo 4D cell imaging, tracking & interaction monitoring
- In vivo visualization of dynamic molecular & cellular mechanisms
- In vivo imaging analysis of novel drug compound efficacy & action
- In vivo imaging of drug delivery to target tissues & cells
- In vivo imaging of various organs in mouse model (liver, lymph node, spleen, skin, retina, lung, brain, colon, pancreas, small intestine, prostate, kidney, heart, trachea, esophagus, bone marrow, thymus, etc.)
Tissue Preparationfor Imaging
•In vivo / in vitro / ex vivo tissue sampling
•Choice of optimal mouse model
•Optimization of surgical procedures for tissue preparation
Imaging Parameter
Establishment
•Imaging time-point & duration
•Required numbers of mouse models
•Imaging method (3D, large-area mosaic, time-lapse imaging)
Creating Animal Models of Various Human Diseases
IVM Imaging of Various Organs in Mouse Model
Image Processing & Analysis in Cellular Level
Various Mouse Models
•Xenograft & syngeneic cancer model using fluorescent cancer cell lines
•Acute & chronic inflammation (systemic injection, organ / tissue injury, ischemia-reperfusion injury)
•Chimera model (stem cell transplantation, adoptive cell transfer)
Image-based Cellular-level
Analysis
•Cell dynamics (cell movement, motility, cell trafficking, homing)
•Cell distribution, cell death / survival
•Cell-microenvironment, cell-cell interaction
• Delivery & accumulation of drug into target organs
Maintenanceduring Imaging
•Compensation of motion artifacts
•Usage of appropriate anesthesia method
•Body temperature maintenance, 37°C
•Maintenance of tissue homeostasis
•Blood circulation maintenance
•Minimizing photobleaching
Lymph Node Bone Marrow Skin
Endothelial cell T cell FRC
BM cell Nuclei Sinus
Inflammatory proteinNeutrophil / Macrophage
Vessel Cancer Vessel Pancreatic cell
KidneySkin
Skin
Inflammatoryprotein
0hr 3hr 6hr 0 min
18 min
6 min
24 min
0 sec 48 sec 222 sec
Bone marrow
Stem cellNucleiBone marrow
Lymph node T cell EC
Mammary fat pad
Vessel Cancer
■ Immune cell recruitment ■ Proliferation & migration
■ Tumor metastasis
■ Angiogenesis
■ In vivo cell dynamics of migration, interaction, activation, etc.
■ In vivo time-lapse, multi-position, mosaic, z-stack imaging
Live Cell Intravital Imaging
20 21IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
IVIM Technology’s Key Application 1 : In Vivo 4D Dynamic Live Cell Imaging Drug Target Identification & Validation (Anti-Inflammation)
Recommended Solutions
■ Imaging system
■ IVM-C
■ Imaging tool
■ In vivo animal stage
■ 4 Channel body and tissue temperature control system
- User-friendly S/W
- Temperature control with feedback from rectal probe
- Glass heater & plate heater
- 2 temperature indicators for monitoring tissue temperature
Glass heater
Rectal probe
Objective lens
Plate heater
Motorized XYZ stage
■ Imaging Target & Imaging Method
■ Objective : Identification & validation of anti-inflammatory drug target
- Monitoring of pro-inflammatory effect of target protein
- Monitoring of pro-/anti-inflammatory effect of target overexpression & inhibition
■ Quantification : Changes in immune cellular dynamics
- Immune cell recruitment & motility : Immune cell number & movement
- Phagocytic efficacy : Colocalization of fluorescent signal of immune cell & pathogen
■ Imaging method : Intravital time-lapse, z-stack imaging of animal skin
■ Fluorescent Labeling
■ Transgenic mice : Intrinsic fluorescent protein (GFP) in immune cells
Targ
et p
rote
inBS
A
Journal of Cell Biology, 216(7):2201-2216 (2017)
BSA
30 min 180 min 360 min
100 μm 100 μm 100 μm
100 μm100 μm100 μm
MФ/NФ (LysM) Target protein
Nature Microbiology, 2:16191 (2016)
Saline Target protein
MФ/NФ (LysM) Pathogen
100 μm
-16 0 +1 +6 Time (hr)
Targetproteinlabeling
Targetprotein
injection
Time-lapse imaging(Cell recruitment & cell
motility at each time-point)
Shaving
-16 0 +1 +3 Time (hr)
Pathogenlabeling
Targetprotein & pathogeninjection
Shaving Time-lapse z-stack imaging
(Phagocytosis)
Journal of Cell Biology, 216(7):2201-2216 (2017) Journal of Cell Biology, 216(7):2201-2216 (2017)
-16 0 +1 +3 Time (hr)
Targetexosomelabeling
Exosomeinjection
Shaving Time-lapseimaging
(Cell recruitmentat each time-point)
-16 -1 0 +2 Time (hr)
TargetTransfection
Targetcell
labeling
Shaving Cellinj.
Time-lapseimaging
(Cell recruitmentat each time-point)
Targ
et tr
ansf
ecte
d
Targ
et tr
ansf
ecte
d
Empt
y Ve
ctor
Empt
y Ve
ctor
30 min 60 min 90 min
100 μm
MФ/NФ (LysM) Transfected cell
0 min 90 min 120 min
100 μm
MФ/NФ (LysM) Exosome
22 23IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
IVIM Technology’s Key Application 2 : Repetitive Intravital ImagingDrug Efficacy Monitoring (Anti-cancer Drug)
■ Imaging Target & Imaging Method
■ Objective : Validation of anti-cancer lead compounds
■ Quantification 1 : Changes in tumor cell apoptosis
- Apoptotic cell numbers : The number of Annexin-V+ cells / Total
■ Quantification 2 : Changes in tumor vessel characteristics, vessel normalization
- Vessel dilation : Average vessel diameter
- Vessel density : Area ratio, CD31+ vessel area / Total area
- Vessel permeability : Area ratio, Dye leak area / Tissue area
- Imaging method :
■ Repetitive intravital imaging of identical site
■ Z-stack, mosaic imaging of tumor cells or vasculatures
■ Fluorescent Labeling
- Cancer cell labeling
■ Intrinsic fluorescent protein (GFP/RFP) expressing stable cancer cell line
■ FP expression in cancer cells : Nuclei (H2B) only, or cytosol & nuclei of cells
- Vessel (vascular endothelial cell) labeling :
■ Intravenous injection of anti-CD31 antibody conjugated with fluorescent dye
- Apoptotic cell labeling :■ Intravenous injection of Annexin-V conjugated with fluorescent dye
Characteristic of apoptotic cells
· Cell shrinking, chromatin condensation, nuclear fragmentation
· Loss of membrane integrity (exposure of phosphatidylserine)
· Membrane blebbing, shedding of apoptotic bodies
Characteristic of tumor vessels
· Abnormal vascular morphology
· Increase in vessel diameter (vessel dilation)
· Increased vascular permeability
Recommended Solutions
Imaging chamber
Objective lens
Rectal probe
Chamber holder
Plate heater
0 +4
1st
1st
2nd
2nd
3rd
+5 +6 Time (day)
Tumor cellinoculation &
dorsal skinfoldchamber implantation
Z-stack mosaicimaging
(Vasculature)
Lead compoundor vehicle treatment
(Daily, IV inj.)
0
1st
1st
2nd
2nd 3rd 4th 5th 6th 7th
3rd
+7 +10 +13 Time (day)
Tumor cellinoculation &
dorsal skinfoldchamber implantation
Lead compoundor vehicle treatment
(Daily, IV inj.)
Z-stack mosaicimaging
(Vasculature)
Inef
fect
ive
Drug
can
dida
teEf
fect
ive
Vehi
cle
After treatmentBeforeApoptotic cells (Annexin-V
)Lung carcinom
a (GFP)
50 μm
Lead
com
poun
d
Vascular densityDay7 Day13
Vehi
cle
Vesseldensity
Vessel (CD31)
100 μm
Lead
com
poun
d
Vessel dilationDay7 Day13
Vehi
cle
Vessel dilation
50 μm
Vessel (CD31) LLC (GFP)
■ Imaging system
■ IVM-C
■ Imaging tool
■ In vivo animal stage
■ 4 Channel body and tissuetemperature control system
■ Imaging chamber & holder
- Dorsal skinfold chambers for each imaging organ (tumor, surrounding microenvironment)
Normal
PS
Early-stage Apoptosis
Annexin-VMorphology
CD31 Carcinoma (GFP)
CD31 CarcinomaDextran
Permeability
24 25IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
IVIM Technology’s Key Application 3 : Cellular-level imaging of various tissues (Brain) Optimized for intravital imaging with In Vivo / Ex Vivo
■ Imaging Target & Imaging Method
■ Objective : Identification & validation of drug targets
■ Quantification : Immune cell distribution & target uptake in brain tissue
- Target cell numbers in parenchyma, vessel
- Target cell motility changes before and after treatment
- Colocalization of immune cell & target material
■ Fluorescent Labeling
■ Transgenic mice : Intrinsic fluorescent protein (GFP) in immune cells
■ Vessel (vascular endothelial cell) labeling :
- Intravenous injection of anti-CD31 antibody conjugated with fluorescent dye
- Dextran conjugated fluorescent dye
■ Intravital Brain Imaging
■ Imaging method :
- Repetitive intravital imaging of identical site
- Z-stack, mosaic imaging of immune cells, vessel
Recommended Solutions
■ Imaging system
■ IVM-CM
■ Imaging tool
■ In vivo animal stage
■ 4 Channel body and tissue
temperature control system
■ Imaging chamber & holder
- Cranial window for brain imaging
- Stereotactic mount
-21 days 0 hr 6 hr Time
Cranial windowimplantation
Material treatment Vessellabeling
Time-lapse, z-stack, mosaic imaging
(Cell distribution & movement)
LysMCD31Dextran
LysM (T1/T2)CD31
CX3CR1 (T1/T2)CD3150 μm 50 μm
LysM (T1/T2)CD31
-12Antibody labeling
Sampling
Optical clearing
Mosaic Z-stackimaging
(Cell distribution & material uptake)
0
+6-24
+9-27
Time (hr)
Neuron (Thy1-GCaMP3)Vessel (CD31)
Microglia (CD11c) Vessel (CD31)
Neuron(Thy1-GCaMP3)
Microglia (CD11c) Vessel (CD31)
Objective lens
CranialWindow
StereotacticMount Plate heater
26 27IVIM Technology IntraVital Microscopy
IntraVital Microscopy (IVM)
In Vivo Live Cell Imaging Platform
Specifications IVM Series (IVM-) C M CM MS
Laser
Confocal Laser Unit· Max. 4 laser unit (405, 420, 445, 473, 488, 505, 514, 532, 561, 633, 642, 660, 685, 705
730, 785 nm)
Tunable Two-PhotonLaser Unit
· Ti:Sapphire laser
· Wavelength : 690-1050 nm, Pulse width < 75 fs, Rep. rate: 80 MHz
· Avg. power > 2.5 W, Dispersion compensation: 0 to -49,000 fs2
Compact Two-PhotonLaser Unit
· Air cooled fs-fiber laser system
· Wavelength : 920 nm, Pulse width < 100 fs, Rep. rate: 80 MHz
· Avg. power > 1 W, Dispersion compensation: 0 to -30,000 fs2
Fluorescence Detector
Confocal Detector· Wavelength: 185 - 900 nm (DAPI, CFP, GFP, YFP, RFP, Cy5, Cy5.5, etc.)
· 4 Ultra-broadband high SNR PMTs (UV to Near IR, Ultra High Sensitivity, Low Dark Current)
· 25-2000 µm variable pinhole (16 steps)
Two-Photon Detector· Wavelength: 185 - 760 nm (DAPI, CFP, GFP, YFP, RFP, Cy5, Cy5.5, etc.)
· 4 High quantum efficiency PMTs (UV to Near IR, Ultra High Sensitivity, Low Dark Current)
Variable EmissionFilter (optional)
· 6 or 2 emission filters can be mounted on each of four detectors
ScanHead Scanner· Polygonal mirror (Fast axis scanning, Max. 66 kHz)
· Galvano scanner (Slow axis scanning, Max. 200 µs/step)
Imaging Head Objectives· Max. 6 objectives are mountable on motorized turret (1X – 100X)
· Compatible for commercial objectives (RMS or M25)
Image
FOV · 100 x 100 µm2 - 10 x 10 mm2
Pixel Resolution · Max. 2,048 x 2,048 pixels
Imaging Speed · 30 fps @ 512 x 512 pixels (Max. 100 fps), 15 fps @ 1,024 x 1,024 pixels (Max. 50 fps)
Sample Stage
3D Stage· Travel Range: 50,000 x 50,000 x 75,000 µm (XYZ)
· Micromanipulation (Max. 0.2 µm resolution)
Specimen Holder
· Flexible-design universal specimen holder can be mounted
In Vivo
· U-shape window bracket for imaging inner organs
· (optional) Homeothermic warming system with plate heater and body temperature probe
· (optional) Small animal inhalation anesthesia system
(optional) Long term imaging holders for transplanted window chamber
- Stereotactic mount for cranial window
- Ring-type window holder for abdominal imaging window
Ex Vivo
In Vitro· A single glass slide or culture dishes
Control Unit Jog Dial· 3-axis independent control of stage position & translation speed
· Motorized turret control for objective lens change
Motion Correction
4-D In Vivo Imaging
Motion Compensation
& Tracking
· XY motion compensation : Averaged image acquisition with motion artifact compensation
· Z motion compensation : Image-based sample Z position adjustment for long-term intravital
microscopic imaging & sample tracking (Feedback-loop automatic stage control)
· T motion compensation : Image-based image XY position adjustment for long-term intravital
microscopic imaging & sample tracking (Feedback-loop automatic stage control)
· Combination of above three compensation for 4D in vivo motion compensation
Software
Image Display· Independent 4 single channel display (RGBA channel)
· Overlay channel display (Selection among RGBA channel)
In Vivo
Image Acquisition
· Averaged image with motion artifact compensation
· Continuous real-time video recording and display
In Vivo
Imaging Mode
· Mosaic imaging (XY), Z-stack imaging (Z), Time-lapse imaging (T)
· Time-lapse imaging at Multi-position (T-M)
· Time-lapse & Z-stack imaging (TZ)
· Time-lapse & Z-stack imaging at Multi-position (TZ- M)
Image Adjustment· Contrast / Brightness control, Histogram / Level adjustment
· Pseudo color setting, Channel splitting, Color mapping
· Zoom, Cut, Crop, Rotation, Invert, Annotation
Work table
1,000 mm
1,200 mm 500 mm
1,00
0 m
m
750
mm
Anti-vibration table Laser control units
IVIM Technology
IVM-M / CM
650 x 910 x 550(W x D x H)
fs-pulsed Laser (Head)
369 x 780 x 189(W X D X H)
fs-pulsed Laser(Cooler)
fs-pulsed Laser(Controller)
Work table
750
mm
750 mm
1,000 mm
1,00
0 m
m
Anti-vibration table
IVIM Technology
IVM-C / MS
650 x 910 x 550(W x D x H)
[email protected] WWW.SCINTICA.COM