FROM IMAGES TO ANSWERS July 2005 Silver Spring and San Diego Advanced Quantitative Image Analysis

Agenda Day One 9:00-10:00Advanced Microscopy Suite Setup 10:00-11:00Multi-Dimensional Acquisition 11:00-NoonMulti-Dimensional Analysis Noon-1:00Lunch (Catered) 1:00-2:00Simulated Live Cell Acquisition & Analysis 2:00-3:00Simulated Ratio Acquisition and Analysis 3:00-4:00Piezo Z-Axis Setup & Fast Acquisition 4:00-5:00Group Discussion 5:00PMAdjourn 6:00PMDinner at a Nearby Restaurant

Agenda Day Two 9:00-10:00Deconvolution of Widefield Images 10:00-Noon3D Rendering and Measurement of Image Stacks Noon-1:00Lunch (Catered) 1:00-2:003D Rendering and Measurement of Image Stacks 2:00-3:002D & 3D Tracking 3:00-4:00Using Workflow Toolbars to Automate Processes 4:00-5:00Group Discussion 5:00Adjourn 6:00Dinner at a Nearby Restaurant

Agenda Day Three 9:00-10:00Industry Best Practices with Macros 10:00-11:00Communicating with External Devices 11:00-NoonIntegrating Macros with Applications (Libraries and Resources) Noon-1:00PMLunch (Catered) 1:00-2:00Fun with Dimensioning Variables & Arrays; Building Loops 2:00-3:00Practical Examples of Macros in Practice 3:00-4:00Transitioning to Visual Basic for Applications in IQbase 4:00-5:00Group Discussion 5:00Adjourn

10 Things I Learned Doing IA Demos “Tell Me a Little About Your Research” (Stall) “It’s Never Done That Before” (Surprise) “Do You Have Administrator Privileges?” (Stall) “It Looks Like a Hardware Problem” (S/W Guy) “It Looks Like a Software Problem” (H/W Guy) “We’re Bringing in a Factory Expert” (We’re Clueless) “More Pixels are Better” (Pixel Envy) “Nobody Uses that Technology Any More” (Feed Ego) “All Your Colleagues are Using This Product” (Intimidation) “I’m Bringing in Our Imaging Specialist” (I’m Clueless)

Advanced Microscopy Setup FROM IMAGES TO ANSWERS

9-Step Career Program Follow the Following Steps for a Rewarding Career… Install Application Program(s) Install Capture Driver and Save Settings (VPF File) Calibrate Objective(s) Configure and Save Microscope Settings Configure AFA from Saved Settings Acquire Multi-Dimensional Images Get Bigger Grant to Buy More Stuff Win Nobel Prize Retire

Camera Setup Install Capture Driver from WEB Site (CDs are Always Out of Date)

Camera Setup Create Video Preference File (VPF)

Microscope Setup Set up Scope-Pro Configurator Select Microscope and Devices

Microscope Setup Set up Scope-Pro Configure Devices Save Common Settings Calibrate Objectives

Microscope Setup Calibrate Objectives Carefully Bad Calibrations = Bad Results!

Microscope Setup Set up Stage-Pro When in Doubt, Accept the Defaults (They’re Usually Right)

Microscope Setup Import Calibrations (Previously Created) When at Least One Objective is Calibrated, the Other Five Tabs Become Available

Microscope Setup Here Are the Next Four Tabs

Microscope Setup Set Z-Limits Just Above & Below the Focal Plane The Fifth Tab (Acquire) is Most Often Used

Microscope Setup Configure AFA using Saved Microscope & Capture Settings

FROM IMAGES TO ANSWERS Live Cell Imaging - Practical Issues Silver Spring & San Diego, June 2005

QED Setup Install Application Program* Select Capture Driver from Pull-Down List Calibrate Objective(s) Select Microscope Settings from Pull-Down List or Auto-Configure Acquire Multi-Dimensional Images * Check for Program Updates (Support, Update this Software)

QED Camera Setup

QED Spatial Calibration Setup

QED Parfocality & Paracentricty Setup

QED Microscope Setup

QED Fluoroescence Acquisition Setup

Live Cell Practical Considerations

When selecting which system to use for imaging living cells, one should consider three things: –Sensitivity of Detection –Speed of Acquisition –Viability of the Specimen See “Light Microscopy Techniques for Live Cell Imaging”, David J. Stephens et al, Science, Vol. 300, pp , 4 April 2003

What Do I Need to Control? –Camera (Gain, Binning & Averaging) –Speed of Acquisition (Exp. Time, Time Intervals, ROIs) –Dimensions of Acquisition (X,Y,Z, Time, Wavelength, Location) –Sample Environment (Temperature, Gases, etc.)

Controlling the Sensitivity of Detection

Controlling the Speed of Acquisition Region of Interest Frame Rate Binning Exposure Time

Control the Dimensions of Acquisition Time XY Color 3D Fast and Slow Hardware

Control the Specimen

Setup of Live Cell Imaging in 5 Dimensions

Long Time Lapse (24h single channel) Setup High Speed Time Lapse Define Acquisition Rate Calculate Number of Images Select “Start” to acquire Long Timelapse

High Speed Time Lapse (1000 I/30 sec) Setup High Speed Time Lapse Define Burst Rate Calculate Number of Images Select Burst Acquire at High Speeds

High Speed Ratio Time Lapse Setup High Speed Time Lapse Define Color Channels Check Color Channels Define Burst Rate Calculate Number of Images Select Burst Acquire at High Speeds

3D Time Lapse Multicolor Z or High-Speed Burst Acquisition XY multi point or area scans

Multiple Log-points Time Lapse

Multi Dimensional Live Cell Analysis

Ratio Analysis Intensity and Ratio Measurements with Fast Acquisition Advanced Ratiometric and Intensity based tracking Multi-Channel Ratio Experiments with Automated Microscopes and External Devices

Ion Channel and Fret Imaging

Tracking of objects Automatic Tracking Correlation Tracking on AOI Full or Phase Correlation for Tracking under Different Image Conditions >50 Morphological Measurements available including Intensity Tracking Smart Search Radius Detects Object Movement from Image Coherence Filter Adjusts for Directional Movement Static or Dynamic Intensity Tracking

3D Deconvolution No Neighbor, Nearest Neighbor Haze Removal and Inverse Filter Haze Restoration Algorithms 2D Blind and 3D Blind Deconvolution Algorithms These Algorithms are not Appropriate for Confocal Images

3D Reconstruction and Measurements

Image Pro 3D Suite Automatic Volumetric Measurements Over Time 4D Tracking of objects!!

Manage Images and Data with IQbase Manages Images and Data Drives Discovery Facilitates Collaboration Easy to Use Works the Way You Do Grows with Your Organization (Scalable)

Competitive Feature Comparison Image-Pro MC Most Comprehensive Package in the Industry; No Charge for Drivers MetaMorph Extra Fee for Camera drivers, Hardware Drivers, 2D & 3D Blind Deconvolution & 3D Simple PCI Extra Fee for Automated Image Capture, Image Processing and Analysis, Dynamic Intensity Analysis, Motion Tracking and Analysis IPLab Extra Fee for Ratio Plus, Fluorescence CV for Mac, MultiProbe for Win, Motion Control, Shutter & Filter Control OpenLab Extra Fee for 3D Module, Morphology Module, Ratio Module, FRET Module, Colocalization Module, Registration Module Intelligent Imaging Extra Fee for Ratio, TTL, 3D, 4D Tracking, Multiwell Plate Module, Statistics Package AxioVision Extra Fee for Z-Stack, Time Lapse, Tiling, Autofocus, Multichannel, Measurements, Imaging Plus

Competitive Feature Comparison Smart Camera Control YNNNNN Zoom & Pan Images YNNNNN Advanced Microscope Control YYYYYY Tiling, Mosaic, Slide and Well Scanning YYYYYY Multi-dimensional Acquisition YYYYYY Multi-Color Imaging YYYYYY Time Lapse Acquisition YYYYYY Color 3D with Piezo Focus Y, Extra $ YNYNN

Competitive Feature Comparison MCMMIPLab3ICompixAxioV Advanced Morphometric Measurment, Analysis & Classification YYYYYY Colocalization, Translocation FISH, and Fluorescence YYYYYY Ratio Imaging YYYYYY FRET Analysis YYYYYY Cell Tracking YYNYYN Vital Systems Control & Measurement YYNYNN Signals and Systems Control YYNYNN Data Analysis, Recording, and Reporting YYYYYY 3D rendering and measurements YNNYNN 3D Time Lapse Analysis YNNNNN Analog Digital signals support YYNYYN Trend chart analysis Intensity tracking YYYYYN

With Similar Features, How to Choose? Price (Highest to Lowest) MM > Zeiss > 3I > Compix > Scanalytics > MC Support (Best to Worst) MC > Compix > 3I > Zeiss > Scanalytics > MM Installed Base (Largest to Smallest) MC >> All Others Combined

With Similar Features, How to Choose? Zeiss Loyal Base due to Highty Integrated Systems and Direct Sales Few User Choices Molecular Devices (formerly UIC) Loyal (but Frustrated Base) Vulnerable due to Recent Changes in Personnel and Direction? Intelligent Imaging Innovations Lately Favored by Olympus; a Longtime Zeiss Collaborator (Strange Bedfellows) Reported to Have “More Mouse Clicks are Better” Syndrome Compix Released v. 4.0 this Spring with a Number of New Features and Improvements Considered the Low Price Leader for Basic Image Capture and Microscope Control Scanalytics Dealer Channel Remains Weak with Limited Representation Products Lag Behind Leaders; Typically Partners with Researchers for New Products

Updated Software Competitive Assessment Comparison Criteria: It is Impossible to Make an “Apples to Apples” Comparison (I wish There was) This Comparison Basis is Image Pro Plus MC versus Comparable Features in Other Packages The Data were Obtained from Respective Companies on their WEB Sites and Presumed to be Accurate as of June \My Documents\2005\Image Analysis Competitive Assessment 2005.xls

So What! What Do We Tell Customers? If You Have the Need for Speed: Choose QED for Fastest Acquisition when an External Device is Attached If You’re Prowling for Power: Choose Image Pro ®, the Most Powerful Analysis Package on the Planet If You Need Speed and Power: Choose a QED/IPA Bundle Combining the Speed of QED with the Horsepower of Image Pro

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