FROM IMAGES TO ANSWERS Advanced Quantitative Image Analysis

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

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

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

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

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)

FROM IMAGES TO ANSWERS Advanced Microscopy Setup

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Camera Setup • Install Capture Driver from WEB Site (CDs are Always Out of Date)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 • The Fifth Tab (Acquire) is Most Often Used • Set Z-Limits Just Above & Below the Focal Plane

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

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. 82 -86, 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 Frame Rate Region of Interest Binning Exposure Time

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

Control the Specimen

Setup of Live Cell Imaging in 5 Dimensions

Long Time Lapse (24 h 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 Timelapse Setup High Speed Time Lapse • Define Color Channels • Check Color Channels • Define burst rate • Calculate Number of Images • Select Burst acquire at high speeds

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

Multiple Log-points Timelapse

Multi Dimensional Live Cell Analysis

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

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

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

3 D Reconstruction and Measurements

Image Pro 3 D Suite Automatic Volumetric Measurements Over Time 4 D 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 In. Vivo/ Analyzer 3 D Most Comprehensive Package in the Industry; Only 2 Upgrades Possible (Sharpstack Plus & IO Pro); No Charge for Drivers Meta. Morph Extra Fee for Camera drivers, Hardware Drivers, 2 D & 3 D Blind Deconvolution & 3 D 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, Multi. Probe for Win, Motion Control, Shutter & Filter Control Open. Lab Extra Fee for 3 D Module, Morphology Module, Ratio Module, FRET Module, Colocalization Module, Registration Module Intelligent Imaging Extra Fee for Ratio, TTL, 3 D, 4 D Tracking, Multiwell Plate Module, Statistics Package Axio. Vision Extra Fee for Z-Stack, Time Lapse, Tiling, Autofocus, Multichannel, Measurements, Imaging Plus

Competitive Feature Comparison Smart Camera Control Y N N N Advanced Zoom Navigator Y N N N Advanced Microscope Control Y Y Y Tiling, Mosaic, Slide and Well Scanning Y Y Y Multi-dimensional Acquisition Y Y Y Multi-Color Imaging Y Y Y Time Lapse Imaging and Burst Acquisition Y Y Y Burst Color 3 D with Piezo Focus Y Y N N

Competitive Feature Comparison MC MM IPLab 3 I Compix Axio. V Advanced Morphometric Measurment, Analysis & Classification Y Y Y Colocalization, Translocation FISH, and Fluorescence Y Y Y Ratio Imaging Y Y Y FRET Analysis Y Y Y Cell Tracking Y Y N Vital Systems Control & Measurement Y Y N N Signals and Systems Control Y Y N N Data Analysis, Recording, and Reporting Y Y Y 3 D rendering and measurements Y N N 3 D Time Lapse Analysis Y N N N Analog Digital signals support Y Y N Trend chart analysis Intensity tracking Y Y Y N

With Similar Features, How to Choose? • Price (Highest to Lowest) • MM > Zeiss > 3 I > MC > Compix > Scanalytics • Support (Best to Worst) • MC > 3 I > Compix > 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 • Generally Considered the High Price Leader • 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 be good-to-Excellent on Acquisition, Fair-to-Good on Analysis • 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 Features in Other Packages • The Data were Obtained from Respective Companies on their WEB Sites and Presumed to be Accurate as of June 2005 . . My Documents2005Image 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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