in vivo live cell imaging platform...•ate imaging of fast cellular-level dynamics in live tissue ....

All-in-OneIntraVital Microscopy

IntraVital Microscopy (IVM)In Vivo Live Cell Imaging Platform

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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




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.




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


Pinhole

Lens


ExcitationLight

2D RasterScanning



Sample

ExcitationLight


Lens





Pinhole

Lens


ExcitationLight

2D RasterScanning



Sample

ExcitationLight


Lens




Fluorescence from focus

Fluorescence from out-of-focus


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




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




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




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




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




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




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


100 μm

MФ/NФ (LysM) Transfected cell


100 μm

MФ/NФ (LysM) Exosome




IVIM Technology’s Key Application 2 : Repetitive Intravital ImagingDrug Efficacy Monitoring (Anti-cancer Drug)


■ 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


- 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


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


■ 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




IVIM Technology’s Key Application 3 : Cellular-level imaging of various tissues (Brain) Optimized for intravital imaging with In Vivo / Ex Vivo


■ 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


■ 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


■ Imaging system

■ IVM-CM

■ Imaging tool


■ 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




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

in vivo live cell imaging platform...•ate imaging of fast cellular-level dynamics in live tissue ....

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