SI8000 Live Cell Imaging System

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1 SI8000 Live Cell Imaging System Sony Biotechnology Inc.

2 SI8000 Cell Motion Imaging System The Sony SI8000 Live Cell Imaging System detects and quantifies cellular motion using proprietary video processing technology. Data is captured with a high-performance video camera and evaluated in a stain free, noninvasive label-free environment. Observation is possible on standard culture plates used in the lab to maintain the physiological environment of cell cultures. Cell behavior and evaluation can be realized without staining. The system s core technology uses high speed video imaging with a unique motion vector software to capture the motion of cells with high temporal and spatial fidelity. Motion detection points can be set by the user to capture the characteristics of moving images. Once captured, the software analyzes how each detection point moves in the movie and displays it as a motion vector. Unlike other systems where motion is inferred from impedance or electrophysiological measures, the SI8000 records actual cellular motion, delivering more precise information that is less susceptible to false negatives and false positives. The system is available in Cardio and Research models. The Cardio model enables quantification of cardiomyocytes using contraction and relaxation speed, average deformation distance, as well as contraction and relaxation duration. The Research model enables observation of live cell behaviors for a broad spectrum of applications such as viability and migration assays. Each model is available in basic or full configurations. High speed video recording that provides a deeper depth of data Noninvasive label free motion analysis that provides data precision User-friendly software that provides high temporal and spatial resolution for greater depth and precision of data Measures the actual movement of cells instead of inferring motion from electrophysiology, impedance, or invasive dyes

3 Common Software Functions Cardio and Research software include functions that let users detect cells motion, analyze a region of interest and visualize data via map, plot or graph. Visualization The sophisticated Motion Vector algorithm yields precise visualization of motion parameters. Power Spectral Density (PSD) analysis provides visualization of the speed of motion in single cells or across cell cultures. PSD is displayed in Cardiomyocytes (left) and neuron culture (right.) H Speed L H PDS LOW L 3

4 Visualize Cell Motion Motion detection points capture the characteristics of moving images in a movie and displays the characteristics as a motion vector. Motion Vector Analysis High spatial and temporal resolution enables high fidelity detection of the motion of cells (red arrow). The analysis software searches for luminance similarity using the block matching method from one frame to the next to accurately calculate displacement vectors, quantifying the distance the cells moved for richer and more precise data. Current frame Previous frame Search area 4

5 Cardio Model Software Functions The Cardio Model has a number of specialized capabilities for analyzing the beating motion of cardiomyocytes. Graphic outputs include heat maps of contraction, relaxation, propagation, and isochrone maps that detect abnormalities in cardiac cells as well as cellular and sub-cellular motion visualization. Quantification In Cardiomyocytes, a beat profile is obtained from Regions of Interest (ROIs). ROIs can be positioned over single cells or clusters of cells. From the beat profile, contraction and relaxation parameters can be calculated. A sophisticated Motion Vector algorithm allows for highly accurate propagation analysis and quantification for a richer and deeper understanding of results. Overlay of the beat profiles showing contraction (first peak) and relaxation (second peak). The high speed camera enables precise detection of both contraction and relaxation of a beating cardiomyocyte for more precise indication of beat parameters. A series of beat profiles showing contraction (first peak) and relaxation (second peak). By measuring both contraction and relaxation, the SI8000 provides a complete profile of the actual contraction unlike other systems that infer contraction parameters from other measures. 5

6 Contractile motion of single cardiomyocyte derived from hips cell (hips-cm) This data shows single cell beat profiles from single cardiomyocytes. A beat profile consists of the contract and relaxation of a beating cardiomyocyte. The SI8000 lets researchers visualize the spread of contraction throughout the cardiomyocyte and obtain valuable data regarding the mechanisms of contraction. Researchers can also visualize the spread of contraction across single cells for a deeper understanding of the mechanisms of cardiomyocyte contractility. Phase contrast Vector Motion speed 30 1 [µm/s] A single cardiomyocyte is isolated (left) and its beat profile is obtained. The images in the middle and right show motion vectors and propagation of contraction respectively. This analysis demonstrates the high fidelity of motion analysis at the single cell level Average speed [μm/s] Average speed [μm/s] Time[s] Time[ms] The beat profile is obtained (left) as the cardiomyocytes contract and relax (bottom). The phases of the beat profile (top right) correspond to the contraction and relaxation of the cardiomyocyte (bottom right) Contraction Relaxation 6

7 Cardio Model Software Functions Quantification of cardiomyocytes contractility for Safety Pharmacology assays High speed video imaging analyzes cardiomyocyte contraction to detect subtle changes that can occur in response to compounds. The system delivers excellent insights for safety pharmacology assays. The phase contrast microscope and high speed video imaging improves spatial resolution of cardiomyocytes over systems that use fluorescent or voltage sensitive dyes, while providing precise temporal resolution similar to electrophysiological assays. High spatial and temporal resolution offers greater depth of data and more precision for safety pharmacology assays. By analyzing the actual contraction of cardiomyocytes, the SI8000 has the ability to detect subtle changes that may occur in response to a compound or drug. Single beat profile 12 contraction 8 4 relaxation Time [ms] The SI8000 supports a variety of beat parameters including beat rate and interval, contraction and relaxation velocity, shortening length (displacement), contraction-relaxation duration, synchronicity of contraction, propagation of contraction, and orientation of contraction direction. 7

8 Easily detect markers of arrhythmia The SI8000 detects both contraction and relaxation to detect subtle differences in the beat profile that may be missed by other systems. For example, if field potential duration is modified by a drug or compound, the system determines the precise effect a drug or compound has on contraction, relaxation, or both contraction and relaxation, to deliver more precise data for analysis. 20 E-4031 Average velocity 0 nm 50 nm Average velocity [μm/s] ms The data show the beat profile of stem cell derived cardio-myocytes in response to E-4031 reference compounds known to have an influence on field potential duration. By analyzing both contraction and relaxation, the SI8000 can identify changes in the beat profile even in the absence of changes in electrophysiology or changes in the field potential waveforms. 100 nm Time Time[s] Enlargement Field potential [mv] Time[s] Average velocity [μm/s] Field potential [mv] 500 ms This data shows simultaneous acquisition of multi-electrode array recordings (blue trace) and motion imaging (red trace). The red trace shown an Early After Depolarization (EAD) phenomenon in response to 100 nm E The SI8000 can reliably and accurately detect arrhythmic events such as EADs offering a true representation of the data. 8

9 Research Model Software Functions Research software detects and analyzes cellular behavior from intra-cellular level to tissue-level. It can detect and quantify cell motion at the submicron level allowing researchers to visualize and analyze the fine movement of target cells, at target size, and time intervals. The Sony A super resolution engine powers cell imaging and High Quality (HQ) mode delivers clear moving images. Easily Quantify Cell Movement The software enables a variety and wide range of motion to be evaluated from the cellular micro-level to the tissue macro-level. Macro and micro motion analysis allows users to select and set a tracking are for analysis. Analysis can be performed on a cell area. The software calculates the cell area parameters selected by the user. By selecting the tracking areas and direction of motion with trajectory analysis. The software quantifies the speed or distance of migrating cells. Frequency analysis in the software analyzes frequency of the cellular movement. Automatic Cell Recognition and Region of Interest (ROI) Setting 2D Mapping Using machine learning, the software can automatically recognize cells. This is accomplished by identifying target cells with the software and then registering them with the software. Once this is set up the software can also be set to look for target cells in a ROI of up to 6000 objects in the image area automatically. The system can search for multiple cells simultaneously to speed automatic identification. The distribution of motion information for each cell in an image can be displayed in a 2D map. In addition, users can gate a cell group to obtain a motion profile. A B A B A. The software can automatically recognize cells to evaluate and analyze multiple cells simultaneously. B. In addition users can freehand draw a ROI around a cell shape and the software will identify cells of interest. A. Cells with identified by ROI (ight blue). B. Cells gated in 2D map (pink). 9

10 Cell Tracking Detects Trajectory and Supplies Quantitative Data The tracking function detects cell trajectory and can calculate quantitative data such as trajectory (XY chart), distance and speed. This makes the system effective at detecting and measuring for example, the trajectories of cell migration and sperm motion. The tracking function can also analyze parameters such as area, perimeter, and roundness which enables the software to follow shape changes of, for example, ex-vivo cardiomyocytes and colony formations. The software can detect the trajectory of cell movement as seen in this example of sperm movement. The Nucleus tracking feature can be used to analyze cancer cell migration. When a ROI is identified the software follows shape changes of target area. In this example, ex-vivo cardiomyocytes are tracked and the software calculates changes in long axis length due to shrinkage. Mophology tracking allows analysis of changes in cell proliferation such as colony formation. 10

11 Cardio Model Applications Label-free visualization and quantification of contraction propagation in cardiomyocyte monolayer Magnitude of all motion vectors detected for the whole image ( pixels) is shown with a heat map. Upper panels represent contraction wave and lower panels are relaxation wave. The interval of each picture is 13ms. The field of view is 4mm 4mm. This type of quantification makes the SI8000 an ideal system for measuring and quantifying torsadogenic risk in drugs or compounds offering a much deeper understanding of cardiovascular risk. Contraction wave Relaxation wave 2mm 0ms 13ms 26ms 39ms 52ms 65ms 78ms 91ms 104ms Propagation of contraction Isochrone map The SI8000 accurately quantifies the propagation of the contraction wave (top panel) and relaxation wave (middle panel). Analyzing the contraction and propagation wave allows for a precise quantification of the propagation of the contraction (bottom panel). 11

12 Contractile motion of single ventricular cardiomyocyte Ventricular cardiomyocytes were paced via an external stimulator and their beat profile was modified as expected via the application of the beta-adrenergic agonist, Isoproterenol, which is known to increase heart rate. The application of Isoproterenol increased maximum contraction velocity, maximum relaxation velocity, and average deformation distance while decreasing beat duration (analogous to field potential duration). These results demonstrate the power of the SI8000 to detect pharmacological effects of compounds label free with high spatial and temporal resolution providing deeper data quality. Control Isoproterenol SPEED [μm/s] SPEED [μm/s] Time[s] Time[s] The image on the left shows isolated primary ventricular cardiomyocytes. The beat profile is obtained under control conditions (center) and in response to Isoproterenol (right). Contractile motion of single ventricular cardiomyocyte Ventricular cardiomyocytes were paced via an external stimulator and their beat profile was modified as expected via the application of the calcium channel blocker, Verapamil, which is known to slow heart rate. The application of Verapamil decreased maximum contraction velocity, maximum relaxation velocity, and average deformation distance while decreasing beat duration (analogous to field potential duration). Results demonstrate how the SI8000 can detect pharmacological effects of compounds label free with high spatial and temporal resolution providing precise data. SPEED [μm/s] Control Verapamil SPEED [μm/s] Time[s] Time[s] The image on the left shows isolated primary ventricular cardiomyocytes. The beat profile is obtained under control conditions (center) and in response to Verapamil (right). 12

13 Research Model Applications Cell viability assay with human ips cell derived neural cell The SI8000 can measure the motion of neurons. The Power Spectral Density (PSD) of a neuron s motion can be used to accurately predict cell death. The PSD represents the strength of a cell s motion in the frequency domain. The figures below show how the PSD of a neuron culture can be used to predict cell death much more precisely than traditional survivability measures. Time after addition 1 h 2 h 4 h 6 h 8 h PSDLow Control A23187 [10µM] Trypan Blue intensity Elapsed Time [h] Elapsed Time [h] *p<0.01 n=20 PSDLow * * Trypan Blue intensity ips derived neural cells (icell Neuron) Cell death was induced by adding 10 μmol/l of Ca 2+ ionophore A Cell death was analyzed by adding 0.01% of Trypan blue (top). Graph (bottom) a comparison of the Trypan blue cell death analysis (line) and the PSD (bars). The graph on the bottom right shows a comparison of Trypan blue cell death analysis (line) and PSD (bars) 1 8 hours after applying Ca 2+ ionophore. The PSD analysis of the SI8000 more reliably predicts cell death than the more traditional measure of Trypan blue cell death analysis, offering a deeper understanding of the data. 13

14 Migration Assay with human cancer cell The SI8000 is suited for cellular migration assays using time lapse imaging. By measuring migration speed and migration distance, a deep understanding of cellular motion is possible. HeLa cells (human cervical cancer cells) migration is demonstrated in the images below. Control Migration speed Migration distance Cytochalasin D Migration speed Migration distance Control Paclitaxel 10nM GM601 20uM Cytochalasin D Time [min] Control Paclitaxel 10nM GM601 20uM Cytochalasin HeLa cells demonstrate the migration assay capabilities of the SI8000. The speed of migration under control conditions (top) and heat map of cellular motion where relatively fast speeds are represented by red and slow speeds by blue (right). Cellular migration slows in response to Cytochalasin D (anti-cancer agent) (bottom). The bar graph represents migration speed under control conditions and in response to the anti-cancer agents Paclitaxel, GM601, and Cytochalasin. Migration speed significantly reduced in response to all anti-cancer agents. This data indicates that the SI8000 offers precise data analysis of cellular migration. The line graph represents migration distance under control conditions and in response to the anti-cancer agents Paclitaxel, GM601, and Cytochalasin. Migration distance significantly decreased over time in response to all anti-cancer agents. This data indicates that the SI8000 offers a deep understanding of data for cellular migration. Monitoring blood flow in Zebrafish Larva Using a high speed video camera, the SI8000 tracked the development of blood flow in Zebra Fish Larvae. Below shows the velocity of motion of blood flow (color) from very little (early day 1) to nearly mature (third day of larval stage.) The color in the images above represent the speed of blood flow in four stages of Zebra Fish larvae development. The red color represents relatively fast blood flow and the blue represents slow. Blood flow increases as a function of development. The ability to quantify subtle differences in blood flow demonstrates the precision and depth of data that is possible with the SI

15 Models and Specifications The system is available in Cardio and Research models. The Cardio model enables quantification of cardiomyocytes using contraction and relaxation speed, average deformation distance, as well as contraction, and relaxation duration. The Research model enables observation of live cell behaviors for a broad spectrum of applications including, for example, viability, and migration assays. Each model is available in basic or full configurations. Basic configurations include high performance workstation with large memory capacity and 24 monitor, specialized acquisition and analysis software, and high speed camera (500 FPS for Cardio model, 2752x2200 for Research model). Full configurations include plus confocal microscope (4x/10x/20x), light source and rack in addition to components of Basic configuration. An optional CO2 incubator reliably manages an optimal environmental for in-vitro cell observation. Focus and light conditions are automated through the SI8000 software. Video imaging captures activity at a high frame rate to deliver deep data-rich results. Displacement and magnitude of cellular motion are calculated using a Motion Vector algorithm developed by Sony for HD television. This accurately detects movement of single cell or cell clusters such as monolayers continuously for extended periods of time. In full configurations the scanning stage controls the X, Y, and Z position of the imaging system for precise and repeatable positioning and repositioning, supporting high throughput analysis. Phase-contrast Microscope Stage Top Incubator (option) Fluorescence Observation Unit (option) Video Camera Full configuration of the SI

16 Models Cardio Model Basic System Full System Research Model Basic System Full System Workstation: GPU with large capacity memory Workstation: GPU with large capacity memory Monitor: 24 inch Cardio camera Acquisition software - SI8000 View software - Real-time HQ mode - Auto focus Cardio analysis software Microscope 4x/10x/20x Auto stage Auto measurement Light source Auto light control Rack Incubator optional optional Monitor: 24 inch Research camera Acquisition software - SI8000 View software - Real-time HQ mode - Auto focus Research analysis software Microscope 4x/10x/20x Auto stage Auto measurement Light source Auto light control Rack Incubator optional optional Specifications Imaging Unit Cardio model (SI8000C) Research model (SI8000R) Microscope 4x, 10x, 20x Phase-contrast Microscope (auto-focus) Light* Video camera* White-color LED Frame Rate: fps Frame Rate: 1-72 (frame rate depends on the pixel size selected) Pixel size: max x 2048 Pixel size: 2704 x 3384 Required environment Temperature: 5-35 C change : ±5 C) Humidity: 20-80%RH (no condensation) Avoid direct sunlight Work station* Hi-Performance GPU large capacity memory SI8000C View Software SI8000R View Software Software Microscope & Video Control SI8000C Analyzer Software SI8000R Analyzer Software Data Analysis for Cardio model Data Analysis for Research model Data format Video: AVI/Still: BMP/Graph: PNG/Analysis Data: CSV or Open XML * SI8000 Cell Motion Imaging System are system-integration products which include Sony products and non-sony products.

17 North America/International Japan Europe 1730 North First Street San Jose, CA U.S.A. Voice: FAX: , Konan, Minato-Ku, Tokyo, Japan Tel: Fax: The Heights, Brooklands. Weybridge, Surrey, KT13 0XW, UK Sony Biotechnology Inc. All rights reserved. Sony and the Sony logo are trademarks of Sony Corporation. For non-clinical research use only. Not for use in diagnostic or therapeutic procedures, or for any other clinical purpose. Specifications subject to change without notice

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