IntraVital (IVM) In Vivo Live Imaging Platform

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All-in-One IntraVital Microscopy Contents

Introduce··························································································································································· 4p

Technology :

IntraVital Microscopy ················································································· 5p

Key Advantages ··········································································································· 10p

Product Lines ························································································································· 14p

Key Applications ········································································································ 20p

Specifications ························································································································ 26p IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

IVIM Technology : Why IntraVital Microscopy (IVM) ? Global No. 1 Solution Provider of IntraVital Microscopy IntraVital Microscopy (IVM) is In Vivo Cellular-level 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 Macroscopic Imaging & Analysis at the Organ / Organism level observations in natural physiological in vivo microenvironments. Using intravital microscopy, in vivo visualizations of gene expression, protein activity, cell trafficking, IntraVital Microscopy 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. Microscopic Imaging & Anlaysis at Cellular / Molecular Level However, up to now, individual users have had to improvise the required functions for each of the - 3D cellular & molecular imaging -Monitoring cellular dynamics intravital imaging applications for the various organs with conventional standalone microscope, which 30 min 180 min 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. / Vessel Vessel / RBC / NФ B cell / T cell / Vessel MФ & NФ / Inflammatory protein • 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 - In vivo delivery monitoring imaging applications. Liver Retina Day1 Day6 Day13

• 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 - apparatus optimized for integration into animal stage NP Vessel RBC NP Gene - Animal motion stabilization apparatus customized for various organs Liver 00:00 00:33 04:54 Liver - 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 ICG ICG - High-precision Animal motion compensation enabled by optimized registration algorithm and ICG ICGKupffer cell & DC ICGMΦ & myeloid DC accelerated by GPU-assisted parallel computing - Image quality optimization by a comprehensive processing algorithm • IntraVital Microscopy (IVM) enables dynamic real-time imaging of various cellular-level dynamics such as The world's first All-in-One intravital microscopy platform from IVIM Technology is a key solution cell trafficking, cell-cell / cell-microenvironment interaction and various physiological responses to stimuli that can explore complex dynamic behaviors of numerous cells inside a living body and serve as next inside the living body in vivo, providing novel insights in the processes of human disease development. generation core technology to elucidate unknown pathophysiology of various human diseases and • For drug development, IVM enables a direct analysis of drug delivery to target tissues and cells and the discover new cures for them. efficacy of new therapeutic candidates by the intended MOA (Mode of Action) at a microscopic cellular- level in various preclinical models of human diseases.

04 IVIM Technology IntraVital Microscopy 05 IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

Why IntraVital Microscopy (IVM) ? Why IntraVital Microscopy (IVM) ? IntraVital Microscopy (IVM) is a Unique Solution for In Vivo Live Cell Imaging ! All-in-One IVM Platform

Bone Marrow Brain Skin Lung Tumor Pancreas Spleen Conventional Approach

Image processing Animal manipulation Individual user have had to improvise the required functions for each of the intravital Retina Small Intestine imaging applications with conventional standalone microscope. •High degree of technical difficulty for non-expert individual user Trachea Colon •Non-optimized intravital imaging performance •Limited applicability to various organs •Difficult to standardize intravital imaging In vivo fluorescence labeling Imaging chamber procedure

Esophagus Heart Liver Kidney Mammary Tissue Lymph Node Prostate •Difficult to establish universal protocol for experiment •Limited reproducibility and productivity

+ Thymus, Thyroid gland, Adipose Tissue, Lymphatics, Microcirculation … etc. 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, IVIM’s All-in-One Packaged Platform gastrointestinal tracts, retina, , , peripheral lymph node, prostate, thymus, All-in-One IVM Platform thyroid gland, adipose tissue, blood and lymphatic vessels, etc. New! All-in-One •All-in-One single-box packaged IntraVital Microscopy (IVM) IntraVital Microscopy In addition, to handle various imaging needs raised by researchers for a wide range of biomedical system studies, detailed experimental protocols have been established for high-quality intravital image •Fully integrated with key functionalities for imaging of live animal model acquisition. •Unified animal stage to accommodate various accessories for Subsequent quantitative analysis of various cellular-level dynamics and physiological alterations have wide-range of intravital imaging applications been established. •User-friendly design for easy and smooth operation Optimized In Vivo System For drug discovery and development applications, IVIM Technology’s All-in-One intravital microscopy •Sub-µm in vivo imaging resolution system can serve as a highly valuable and versatile tool. IVIM Technology’s All-in-One intravital confocal / •Ultra-high speed in vivo imaging (Max. 100 fps @ 512x512 • Target identification and validation in natural in vivo microenvironments two-photon microscopy system (IVM-C/M/CM/MS) pixels) is extensively optimized and carefully engineered to •4-color simultaneous confocal / two-photon microscopic • Confirmation of MoA (Mode of Action) and PoC (Proof of Concept) of new therapeutics by direct provide superb performance in the intravital imaging in vivo imaging in vivo cellular observation •Integrated animal motion compensation • Monitoring of in vivo drug delivery, distribution, retention and in situ efficacy at target tissues and of live animal models, in vivo. organs

06 IVIM Technology IntraVital Microscopy 07 IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

Two-Photon Microscopy (IVIM Technology : IVM-M, CM, MS) ■ High-resolution optical sectioning imaging of in vivo tissue IVIM Technology’s Confocal / Two-Photon Technologies • Fluorescence signal is intrinsically generated only at the focus Enable High-resolution In Vivo Imaging ■ Deeper tissue imaging with longer-wavelength near-infrared (NIR) fs-pulse laser for two-photon / multi- photon excitation (IVIM Technology : IVM-C, CM) ■ Capable of label-free, non-linear multi-harmonic generation imaging (SHG, THG) ■ No photo-bleaching at non-focal plane : reduced phototoxicity ■ High-resolution optical sectioning imaging of in vivo tissue ■ Ideal for long-term 3D intravital 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 Single-Photon Two-Photon ■ Ultrafast precise Raster scanning of multi-color excitation laser-beam focus Excitation Excitation Second Harmonic Generation (SHG) Excited Imaging of in vivo tissue • Video-rate imaging of fast cellular-level dynamics in live tissue in situ state Fibrosis ■ Ideal for high-sensitivity multi-color intravital imaging of in vivo tissue

Widefield Image Optical Sectioning Image Ground state Fluorescence signal

Photobleaching Patterns Confocal Microscopy Two-Photon Microscopy Coverslip Photobleached Fluorophore Light Path

Excitation Laser Fluorescence Fluorescence ExcitationBeamExcitation Scanning LaserExcitation Laser Laser FluorescenceFluorescencefromFluorescence focus FluorescenceFluorescencefromFluorescence out-of-focus Beam ScanningBeamBeam Scanning Scanning from focusfromfrom focus focus from out-of-focusfromfrom out-of-focus out-of-focus

DetectorDetectorDetector DetectorDetectorDetector DetectorDetectorDetector Confocal Microscopy Two-Photon Microscopy Pinhole PinholePinhole ■ Single-photon excitation ■ Two-photon excitation

Lens Lens Lens ■ Point scanning + Pinhole ■ Point Scanning + No Pinhole ■ Optical sectioning : ■ Optical sectioning : Fluorescence signal from out-of-focus is blocked Fluorescence signal is intrinsically generated only Dichroic BeamDichroicDichroic Beam Beam Splitter SplitterSplitter at the focus ■ Imaging Depth : ■ Imaging Depth : 100-200 µm 250-1000 µm ExcitationExcitationExcitation ExcitationExcitationExcitation ExcitationExcitationExcitation Light Light LightObjectiveObjectiveObjective Light Light Light Light Light Light ■ Continuous-wave solid-state laser with flexible ■ Femto-second pulsed laser tunable at near- Lens Lens Lens choice of wavelength at the range from ultraviolet infrared (NIR) wavelength range 2D Raster2D Raster2D Raster FluorescenceFluorescenceFluorescence FluorescenceFluorescenceFluorescence Sample SampleSample (from out-of(from out-of(from out-of (UV) and visible (VIS) to near-infrared (NIR) ScanningScanningScanning (from focal(from plane) focal(from plane) focal plane) -focal plane) -focal plane) -focal plane) ■ Descanned confocal detector ■ Non-descanned detector (NDD) ■ Easy & efficient, multi-color 3D intravital imaging ■ Deeper-tissue 3D intravital Imaging

08 IVIM Technology IntraVital Microscopy 09 IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

IVIM Technology’s Key Advantage 1 : IVIM Technology’s Key Advantage 2 : Ultrafast Scanning with Uniform Illumination Animal Motion Compensation

■ IVIM Technology - Integrated Motion Artifact Compensation Ultrafast Uniform Laser-beam Scanning - Automatic hassle-free high-precision motion compensation

IVIM Technology's Ultrafast Conventional Resonant - Immediate acquisition of motion-compensated imaging results by GPU-assisted Polygon Scanning Galvo Scanning 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)

In Vivo Imaging IVIM Technology’s Advanced of Moving Live Tissue Motion Compensation Frame #2 Corrected Frame #3 Corrected Corrected Frame #1 ■ IVIM Technology’s All-in-One intravital microscopy system is equipped with ultrafast Frame #4 Frame #4 Corrected rotating polygonal mirror scanner A Frame #2 •Enables ultra-high speed in vivo imaging (max. 100 fps @ 512x512 pixels) Frame #1 A •Achieves uniform excitation illumination over entire imaging field of view (FOV) A A - No reduced fluorescence signal and signal-to-noise ratio (SNR) at center area of FOV AFrame #3 - No excessive photobleaching at edge area of FOV Cellular object ‘A’ appeared as - Uniform high signal-to-noise ratio over entire FOV moving object → Motion artifact - Improved image quality without wasting excessive photons Blurred In Vivo Image Sharpened In Vivo Image Motion Artifact Motion Compensation

10 IVIM Technology IntraVital Microscopy 11 IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

IVIM Technology’s Key Advantage 3 : All-in-One Packaged Platform Software IVM Engine

IntraVital Microscopy IVM-C / M / CM / MS · Inhalation anesthesia Superb · Imaging chamber & holder Hardware · In vivo animal stage

Optimized Software IVM Engine & Viewer Expert Solution for Intravital Imaging · Animal Motion compensation Expert · Consulting for intravital imaging · Z-stack / Mosaic / Multi-position · Optimization of intravital imaging Research · Modeling & Intravital imaging Service IVM Viewer · Data processing

Hardware

All-in-One IntraVital Microscopy Accessories for Intravital Imaging

Body temp. control system Inhalation anesthesia

Research Service

Stereotactic mount Imaging chamber & holder Step Consulting for Sample Demonstration Optimization of 1 In Vivo Live Cell Imaging & Test Imaging Experimental Plan

Step Creating Animal Models IVM Imaging Image Processing of of Various Organs & Analysis 2 Various Human Diseases in Mouse Model in Cellular Level

12 IVIM Technology IntraVital Microscopy 13 IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

IVIM Technology’s Product Lines :

■ IVM-C (Confocal) Key features of IntraVital Microscopy (IVM) ■ IVM-M (Two-Photon) ■ Superb performance and unique functionality for in vivo imaging of ■ IVM-CM (Confocal & Two-Photon) living animal ■ IVM-MS (Two-Photon Smart Ver.) •Ultra High-speed Imaging (max. 100 fps - 512x512 pixels) •4D Animal Motion Compensation (X,Y,Z & Time) ■ Research Service - Intravital Imaging •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

14 IVIM Technology IntraVital Microscopy 15 IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

IVIM Technology’s Product Lines :

IVM-C (Confocal) IVM-CM (Confocal & Two-Photon)

IVM-C is the All-in-One IntraVital Confocal Microscopy System, optimized for in vivo imaging IVM-CM is the All-in-One IntraVital Confocal / Two-Photon Microscopy System, optimized for in vivo experiments. Especially, because it is equipped with 4 different wavelength lasers and 4 high-sensitivity imaging experiments. Especially, because both of the confocal and two-photon microscopes are confocal detectors, IVM-C is the optimal system to observe highly diverse, dynamic multi-cellular integrated into a single-box packaged system, IVM-CM provides the ultimate versatile functionality of behaviors in live animals simultaneously with 4 different fluorescence colors. the IVM-C and IVM-M in one system.

•Easy & highly efficient multi-color simultaneous imaging •Dual-mode IVM-C (Confocal) / IVM-M (Two-Photon) imaging capability •High flexibility in selection of laser / detector wavelength •One-click automated transition between confocal and two-photon imaging mode •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-M (Two-Photon) IVM-MS (Two-Photon Smart Ver.)

IVM-M is the All-in-One IntraVital Two-Photon Microscopy System, optimized for in vivo imaging IVM-MS 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, experiments and equipped with a new compact high-efficiency fs-pulse laser module. Especially, IVM-M is the optimal system for users who need to conduct deeper-tissue imaging using less-scattering because it integrates a compact high-stability maintenance-free fs-pulse laser into a single box, the NIR wavelength. Full control functionality of the fs-laser system is integrated in the two-photon imaging IVM-MS is the ideal solution for customers in need of a two-photon microscope with limited resources software for user convenience with various automation algorithms. of space and budget.

•Laser wavelength tuning for optimal two-photon excitation of a wide-range of fluorescence agents •Less expensive, fully-automated fs-laser system •Deeper tissue imaging with a longer-wavelength NIR fs-laser system •Space saving with no additional fs-laser box configuration • Capable of label-free, non-linear multi-harmonic generation imaging •Simple hand-free turn-key operation of the fs-laser for two-photon excitation •Motion compensation and ROI tracking for long-term in vivo imaging •Easy maintenance and management without liquid cooling system requirements

16 IVIM Technology IntraVital Microscopy 17 IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

IVIM Technology’s Product Lines : ■ Key Research Service Research Service – Intravital Imaging - In vivo 4D cell imaging, tracking & interaction monitoring - In vivo visualization of dynamic molecular & cellular mechanisms Step1 : Consulting, Testing & Planning - In vivo imaging analysis of novel drug compound efficacy & action Consulting for Sample Demonstration Optimization of - In vivo imaging of drug delivery to target tissues & cells In Vivo Live Cell Imaging & Test Imaging Experimental Plan - 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, •Fluorescence protein (FP) reporter mouse esophagus, bone marrow, thymus, etc.) In Vivo •Injection of antibody-fluorophore conjugate Fluorescence (vasculature, lymphatics, various immune cells, stromal cells) Labeling •Chimeric mouse generation by bone marrow transplantation Live Cell Intravital Imaging •Adoptive fluorescent cell transfer ■ In vivo cell dynamics of migration, interaction, activation, etc. ■ In vivo time-lapse, multi-position, mosaic, z-stack imaging Tissue •In vivo / in vitro / ex vivo tissue sampling Preparation •Choice of optimal mouse model Lymph Node Bone Marrow Skin for Imaging •Optimization of surgical procedures for tissue preparation

• Imaging Imaging time-point & duration Parameter •Required numbers of mouse models Establishment •Imaging method (3D, large-area mosaic, time-lapse imaging) Endothelial cell T cell BM cell Nuclei Inflammatory protein FRC Sinus Neutrophil / Macrophage

Step 2 : Intravital Imaging & Analysis In Vivo Drug Efficacy Monitoring Creating Animal Models of IVM Imaging of Various Organs Image Processing & Analysis ■ Immune cell recruitment ■ Proliferation & migration Various Human Diseases in Mouse Model in Cellular Level Skin 0hr 3hr 6hr Bone marrow 0 min 6 min

••Xenograft & syngeneic cancer model using fluorescent cancer cell lines Various Mouse ••Acute & chronic inflammation (systemic injection, organ / tissue injury, Models ischemia-reperfusion injury) 18 min 24 min

••Chimera model (stem cell transplantation, adoptive cell transfer) Inflammatory Stem cell protein Nuclei Bone marrow ••Compensation of motion artifacts ••Maintenance of tissue homeostasis Lymph node T cell EC ••Usage of appropriate anesthesia ••Blood circulation maintenance Maintenanceduring ■ Angiogenesis Imaging method ••Minimizing photobleaching ••Body temperature maintenance, 37°C Skin Kidney 0 sec 48 sec 222 sec

••Cell dynamics (cell movement, motility, cell trafficking, homing) ■ Tumor metastasis Image-based ••Cell distribution, cell death / survival Mammary fat pad Cellular-level Analysis ••Cell-microenvironment, cell-cell interaction ••Delivery & accumulation of drug into target organs Vessel Cancer Vessel Pancreatic cell Vessel Cancer

18 IVIM Technology IntraVital Microscopy 19 IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

IVIM Technology’s Key Application 1 : -16 -1 0 +2 Time (hr) -16 0 +1 +3 Time (hr)

In Vivo 4D Dynamic Live Cell Imaging Target Shaving Target Cell Time-lapse Target Shaving Exosome Time-lapse Transfection cell inj. imaging exosome injection imaging labeling (Cell recruitment labeling (Cell recruitment Drug Target Identification & Validation (Anti-Inflammation) at each time-point) at each time-point)

30 min 60 min 90 min 0 min 90 min 120 min ■ Imaging Target & Imaging Method

■ Objective : Identification & validation of anti-inflammatory drug target Empty Vector Empty Vector 100 μm 100 μm - Monitoring of pro-inflammatory effect of target protein - Monitoring of pro-/anti-inflammatory effect of target overexpression & inhibition

■ Quantification : Changes in immune cellular dynamics

Target transfected Target MФ/NФ (LysM) transfected Target MФ/NФ (LysM) Transfected cell Exosome - Immune cell recruitment & motility : Immune cell number & movement Journal of Cell Biology, 216(7):2201-2216 (2017) Journal of Cell Biology, 216(7):2201-2216 (2017) - Phagocytic efficacy : Colocalization of fluorescent signal of immune cell & pathogen

■ Imaging method : Intravital time-lapse, z-stack imaging of animal skin Recommended Solutions

■ Imaging system ■ Fluorescent Labeling Objective lens ■ IVM-C ■ Transgenic mice : Intrinsic fluorescent protein (GFP) in immune cells

-16 0 +1 +6 Time (hr) -16 0 +1 +3 Time (hr)

Glass Target Shaving Target Pathogen Shaving Target Time-lapse imaging Time-lapse z-stack heater protein protein (Cell recruitment & cell labeling protein & imaging labeling injection motility at each time-point) pathogen (Phagocytosis) injection Plate heater Rectal probe

30 min 180 min 360 min Motorized XYZ stage Saline Target protein

BSA MФ/NФ (LysM) ■ Imaging tool Pathogen BSA ■ In vivo animal stage 100 μm 100 μm 100 μm ■ 4‌ Channel body and tissue temperature control system

100 μm - User-friendly S/W

Target protein Target - Temperature control with feedback MФ/NФ (LysM) from rectal probe Target protein 100 μm 100 μm 100 μm Journal of Cell Biology, 216(7):2201-2216 (2017) Nature Microbiology, 2:16191 (2016) - Glass heater & plate heater - 2 temperature indicators for monitoring tissue temperature

20 IVIM Technology IntraVital Microscopy 21 IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

IVIM Technology’s Key Application 2 : 0 +4 +5 +6 Time (day) 0 +7 +10 +13 Time (day)

Repetitive Intravital Imaging Z-stack mosaic Z-stack mosaic st nd rd st nd rd 1 2 3 imaging 1 2 3 imaging (Vasculature) (Vasculature)

Drug Efficacy Monitoring (Anti-cancer Drug) Tumor cell Lead compound Tumor cell Lead compound inoculation & 1st 2nd or vehicle treatment inoculation & 1st 2nd 3rd 4th 5th 6th 7th or vehicle dorsal skinfold (Daily, IV inj.) dorsal skinfold treatment ■ Imaging Target & Imaging Method chamber implantation chamber implantation (Daily, IV inj.)

■ Objective : Validation of anti-cancer lead compounds Vascular density Day7 Day13 ■ Quantification 1 : Changes in tumor cell apoptosis Vessel density - Apoptotic cell numbers : The number of Annexin-V+ cells / Total Vehicle Before After treatment

■ Quantification 2 : Changes in tumor vessel characteristics, vessel normalization Apoptotic cells (Annexin-V) 100 μm - Vessel dilation : Average vessel diameter

- Vessel density : Area ratio, CD31+ vessel area / Total area Vehicle - Vessel permeability : Area ratio, Dye leak area / Tissue area 50 μm Lead compound Vessel (CD31) Normal Morphology Permeability Annexin-V Vessel dilation

Ineffective Day7 Day13 Vessel PS dilation Lung carcinoma (GFP) Early-stage Apoptosis Drug candidate

CD31 Vehicle Carcinoma CD31 Carcinoma (GFP) Dextran Effective 50 μm

Characteristic of apoptotic cells Characteristic of tumor vessels · Cell shrinking, chromatin condensation, nuclear fragmentation · Abnormal vascular morphology · Loss of membrane integrity (exposure of phosphatidylserine) · Increase in vessel diameter (vessel dilation) Vessel (CD31) · Membrane blebbing, shedding of apoptotic bodies · Increased vascular permeability Lead compound LLC (GFP) - Imaging method :

■ Repetitive intravital imaging of identical site Recommended Solutions ■ Z-stack, mosaic imaging of tumor cells or vasculatures Rectal ■ Imaging system probe Objective ■ Fluorescent Labeling lens ■ IVM-C - Cancer cell labeling ■ Imaging tool ■ Intrinsic fluorescent protein (GFP/RFP) expressing stable cancer cell line ■ FP expression in cancer cells : Nuclei (H2B) only, or cytosol & nuclei of cells ■ In vivo animal stage Imaging ■ 4‌ Channel body and tissue - Vessel (vascular endothelial cell) labeling : chamber temperature control system ■ Intravenous injection of anti-CD31 antibody conjugated with fluorescent dye Chamber holder ■ Imaging chamber & holder - Apoptotic cell labeling : Plate heater - Dorsal‌ skinfold chambers for each imaging ■ Intravenous injection of Annexin-V conjugated with fluorescent dye organ (tumor, surrounding microenvironment)

22 IVIM Technology IntraVital Microscopy 23 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 Neuron (Thy1-GCaMP3) (CD11c) Vessel (CD31) Vessel (CD31) ■ Imaging Target & Imaging Method

■ Objective : Identification & validation of drug targets Antibody -12 Neuron labeling (Thy1-GCaMP3) ■ Quantification : Immune cell distribution & target uptake in brain tissue - Target cell numbers in parenchyma, vessel Sampling - Target cell motility changes before and after treatment 0 - Colocalization of immune cell & target material Optical clearing ■ Fluorescent Labeling +6-24 ■ Mosaic Z-stack Transgenic mice : Intrinsic fluorescent protein (GFP) in immune cells imaging (Cell distribution & ■ Vessel (vascular endothelial cell) labeling : material uptake) +9-27 - Intravenous injection of anti-CD31 antibody conjugated with fluorescent dye Microglia (CD11c) - Dextran conjugated fluorescent dye Time (hr) Vessel (CD31) ■ Intravital Brain Imaging

■ Imaging method : - Repetitive intravital imaging of identical site Recommended Solutions - Z-stack, mosaic imaging of immune cells, vessel ■ Imaging system

-21 days 0 hr 6 hr Time Objective lens ■ IVM-CM

■ Imaging tool Cranial window Material treatment Vessel Time-lapse, z-stack, mosaic implantation labeling imaging ■ In vivo animal stage (Cell distribution & Cranial movement) Window ■ 4‌ Channel body and tissue temperature control system

■ Imaging chamber & holder Stereotactic Plate heater Mount - Cranial window for brain imaging - Stereotactic mount LysM LysM (T1/T2) CD31 LysM (T1/T2) CX3CR1 (T1/T2) CD31 Dextran 50 μm CD31 CD31 50 μm

24 IVIM Technology IntraVital Microscopy 25 IntraVital Microscopy (IVM) In Vivo Live Cell Imaging Platform

Specifications IVM Series (IVM-) C M CM MS · Max. 4 laser unit (405, 420, 445, 473, 488, 505, 514, 532, 561, 633, 642, 660, 685, 705 Confocal Laser Unit 730, 785 nm)

· Ti:Sapphire laser Tunable Two-Photon · Wavelength : 690-1050 nm, Pulse width < 75 fs, Rep. rate: 80 MHz Laser Laser Unit · Avg. power > 2.5 W, Dispersion compensation: 0 to -49,000 fs2 · Air cooled fs-fiber laser system Compact Two-Photon · Wavelength : 920 nm, Pulse width < 100 fs, Rep. rate: 80 MHz Laser Unit Work table Anti-vibration table · Avg. power > 1 W, Dispersion compensation: 0 to -30,000 fs2 · Wavelength: 185 - 900 nm (DAPI, CFP, GFP, YFP, RFP, Cy5, Cy5.5, etc.) Confocal Detector · 4 Ultra-broadband high SNR PMTs (UV to Near IR, Ultra High Sensitivity, Low Dark Current) · 25-2000 μm variable pinhole (16 steps) Fluorescence · Wavelength: 185 - 760 nm (DAPI, CFP, GFP, YFP, RFP, Cy5, Cy5.5, etc.) Two-Photon Detector IVIM Technology Detector · 4 High quantum efficiency PMTs (UV to Near IR, Ultra High Sensitivity, Low Dark Current)

Variable Emission · 6 or 2 emission filters can be mounted on each of four detectors 750 mm IVM-C / MS Filter (optional)

· Polygonal mirror (Fast axis scanning, Max. 66 kHz) Scanner

1,000 mm ScanHead 650 x 910 x 550 · Galvano scanner (Slow axis scanning, Max. 200 µs/step) (W x D x H) · Max. 6 objectives are mountable on motorized turret (1X – 100X) Objectives Imaging Head · Compatible for commercial objectives (RMS or M25) 1,000 mm FOV · 100 x 100 µm2 - 10 x 10 mm2 Image 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) 750 mm · Travel Range: 50,000 x 50,000 x 75,000 µm (XYZ) 3D Stage · Micromanipulation (Max. 0.2 µm resolution)

· Flexible-design universal specimen holder can be mounted

· U-shape window bracket for imaging inner organs · (optional) Homeothermic warming system with plate heater and body temperature probe Sample Stage · (optional) Small animal inhalation anesthesia system In Vivo Specimen Holder (optional) Long term imaging holders for transplanted window chamber - Stereotactic mount for cranial window Work table Anti-vibration table Laser control units - Ring-type window holder for abdominal imaging window

Ex Vivo · A single glass slide or culture dishes In Vitro

· 3-axis independent control of stage position & translation speed IVIM Technology fs-pulsed Laser Control Unit Jog Dial fs-pulsed (Cooler) · Motorized turret control for objective lens change Laser (Head) IVM-M / CM · XY motion compensation : Averaged image acquisition with motion artifact compensation 750 mm 369 x 780 x 189 · Z motion compensation : Image-based sample Z position adjustment for long-term intravital 4-D In Vivo Imaging (W X D X H) Motion microscopic imaging & sample tracking (Feedback-loop automatic stage control) 650 x 910 x 550 1,000 mm Motion Compensation Correction · T motion compensation : Image-based image XY position adjustment for long-term intravital (W x D x H) & Tracking fs-pulsed Laser microscopic imaging & sample tracking (Feedback-loop automatic stage control) (Controller) · Combination of above three compensation for 4D in vivo motion compensation 1,000 mm · Independent 4 single channel display (RGBA channel) Image Display · Overlay channel display (Selection among RGBA channel)

1,200 mm 500 mm In Vivo · Averaged image with motion artifact compensation Image Acquisition · Continuous real-time video recording and display

· Mosaic imaging (XY), Z-stack imaging (Z), Time-lapse imaging (T) Software In Vivo · Time-lapse imaging at Multi-position (T-M) Imaging Mode · Time-lapse & Z-stack imaging (TZ) · Time-lapse & Z-stack imaging at Multi-position (TZ- M)

· Contrast / Brightness control, Histogram / Level adjustment Image Adjustment · Pseudo color setting, Channel splitting, Color mapping · Zoom, Cut, Crop, Rotation, Invert, Annotation

26 IVIM Technology IntraVital Microscopy 27 [email protected] WWW.SCINTICA.COM