FROM IMAGES TO ANSWERS Silver Spring, San Diego and Markham Advanced Digital Automation & AnalysisDestination Check… This is Not… American Dodgeball Association of America

Destination Check This is DodgeballDodgeball

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 and a Movie in Markham! *No Live Cells Were Harmed in the Acquisition of These Images!

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 2:00-3:003D & 2D Tracking 3:00-4:00Using Workflow Toolbars to Automate Processes 4:00-5:00Group Discussion 5:00Adjourn 6:00Dinner at a Nearby Restaurant* Many Brain Cells Will Be Harmed During This Dinner!

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 2:00-3:30Practical Examples of Macros in Practice 3:30-4:00Transitioning 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Camera Setup Install Capture Driver from WEB Site (CDs are Always Out of Date) Solutions-Zone is

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) New Automatic Calibration Macro Now Available (as of last week!)

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 Focal Limits at Extents of Volume The Fifth Tab (Acquire) is Used Most Often

Microscope Setup Configure AFA using Saved Microscope & Capture Settings

FROM IMAGES TO ANSWERS Live Cell Imaging - Practical Software 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 Fluorescence 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 2003David J. Stephens

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 Use a Combination of Gain, Binning and Averaging to control the Sensitivity of Detection.

Controlling the Speed of Acquisition Region of Interest Use a Combination of ROI, Binning, Exposure Time and Frame Rate to control the Speed of Acquisition.

Control the Dimensions of Acquisition Use a Combination of XY, Z, Color to control the Dimensions of the Live Cell Acquisition. 3D XY Color

Control the Viability of the Specimen Use a Combination of Trend Charting, Signals & Actions and Vital Systems Control to control the Viability of the Specimen. Trend Charting Vital Systems Control Signals & Actions

Setup for Live Cell Imaging in 5 Dimensions

Long Time Lapse (24h single channel) 1.Define Sensitivity of Detection 2.Define Speed of Acquisition 3.Select “Start” to acquire Long Time Lapse

High Speed Time Lapse (1000 I/30 sec) 1.Define Sensitivity of Detection 2.Define Speed of Acquisition 3.Calculate # of Images 4.Select “Burst” to acquire at High Speeds

High Speed Ratio Time Lapse 1.Define Sensitivity of Detection 2.Define Speed of Acquisition 3.Define Ratio Channels 4.Calculate # of Images 5.Select “Burst” to acquire at High Speeds

3D Time Lapse 1.Define Sensitivity of Detection 2.Define Speed of Acquisition 3.Define Number of Planes 4.Define Z Spacing 5.Select 3D Time Lapse 6.Select “Start” to acquire 3D Time Lapse

Multiple Stage Positions & Time Lapse 1.Define Sensitivity of Detection 2.Define Speed of Acquisition 3.Define Number of Log Points 4.Select Time Lapse 5.Select “Start Multi Acquire” to acquire Multiple Log-points Time Lapse

Multi Dimensional Live Cell Analysis

Real-Time Ratiometric Analysis 1.Define Sensitivity of Detection 2.Define Speed of Acquisition 3.Select Time Lapse 4.Define Multiple ROIs 5.Select Trend Feature 6.Display Live or Compute on Movie

Ion Channel Imaging 1.Define Background Correction 2.Define Intensity Calibration 3.Define Psuedo Color Display 4.Define Multiple ROIs

Tracking of objects 1.Define Tracking Measurements 2.Define Tracking Options 3.Define Multiple ROIs 4.Filter Data

3D Deconvolution 1.Verify Imported Deconvolution Parameters 2.Define Deconvolution Options 3.Deconvolve

3D Reconstruction and Measurements 1.Verify Imported 3D Parameters 2.Define 3D Options 3.Measure 3D Colocalization

3D Reconstruction and Measurements Automatic Volumetric Measurements Over Time 4D Tracking of objects!! 1.Verify Imported 3D and Time Parameters 2.Define 3D Options 3.Define Tracking Options 4.Track 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)

Group Discussion

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 MCMMBD Bio3ICompixAxioV 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 > BD Bio > MC Support (Best to Worst) MC > Compix > 3I > Zeiss > BD Bio > MM Installed Base (Largest to Smallest) MC >> All Others Combined (>60,000)

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 (div. of Hamamatsu Photonics, Inc.) 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 (div. of BD BioSciences) 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 is Based on 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

Deconvolution & 3D Rendering/Measurement

Deblurring vs Deconvolution We License these Algorithms from Auto-Quant® The No Neighbor & Nearest Neighbor Algorithms are Deblurring Tools (Intensities are Removed) The Inverse Filter Algorithm is a Haze Restoration Tool (Intensities are Re-Assigned) The Optional 2D Blind and 3D Blind Tools are Deconvolution Algorithms (Intensities are Re- Assigned) These Algorithms are not Appropriate for Confocal Images Because They Use Widefield PSFs

Deblurring vs Deconvolution

3D User Interface

3D User Tools Pointer to Toggle to Measurements Pan Tool to Adjust Volume Home Position Set New Home Position Change Perspective (View) Display Box Around Volume Change View Y,Z or Z,X or Y,X Use Control Click for LR or UD, Click for UD, Alt Click to Toggle Between Pan and Pointer

3D User Options

3D Rendering with 3D Filters

3D Volume Measurements

3D Volume Measurements Over Time

3D Branch / End Point Measurements

IPbasic – A Short Course

Industry Best Practice with Macros There is No Single Solution That Works for All Applications Complex Processing Batch Operations Generating Reports Consistent, Repeatable Algorithms Simple User Interfaces

Communicating with External Devices Examples of External Devices Cameras Microscope controllers: – IpStageRead( ) and IpStageWrite( ) Serial Interfaces: – Solution 2119, COMM class Analog and Digital Devices – IO-Pro

Integrating Macros with Applications Excel, Word, and other apps –Report Generation, Numerical Processing –Graphs and Charts Controlling Image-Pro from VB –Customer Developed Interfaces –VB Controls and Simple Dialogs Plug-ins using the IPP SDK –Tightly Integrated Applications –Custom Applications like Gel-Pro

Working with Excel (and Word, and…) Control Excel Through OLE –Add a Reference to Excel to View Excel Help –In Macro, Obtain a Handle to Excel –Use Excel commands: Prefix with “Handle” or Use in “With” block –Close Handle as Soon as Possible to Avoid Hanging References See for Exampleshttp://

Image-Pro and VB Linked VB.Exe and Image-Pro Often the Route of Choice for End User Development Simple to Write, Limited Functionality –IpBasic is a Subset of VB Any Macro Code can be Run in VB The Major Difference is Dialog Handling –Add Ipc32.bas Module to VB project –Set Project Target and Compile to IPP Folder –Call IpBasic Functions

Image-Pro Plug-Ins Create New Modules with the SDK Receive Events Use Lower Level Functions for Speed IpBasic Code Can be Used Directly Fully Integrated with Image-Pro Gel-Pro, Array-Pro, and 3D-Constructor are all Examples

Fun with Calls, Arrays & Loops! Generalizing recorded macros with variables –Allows a Macro to Apply to other Images –One of the Most Important Macro Calls is IpDocGet(GETACTDOC, …) Looping over Arrays of Data –For and Do Loops Decision Trees in Your Code –If Then and Select Statements

Practical Examples of Macros White_Balance –Generalized Tool Written as a Macro Halves, Mosaic_Images –Display Tools Filter_Peaks, MinMaxScale –Complex Processing Hardness_Testing –Task Specific Number Crunching

IQstudio Analytical Imaging Development Kit Rapid Development of Image Analysis Applications Develop your own applications using our tools Add image analysis to existing products Standardize image analysis within your organization Redistribute your code through run-time licensing

Tour of IQstudio Features Some Highlights: –Interface for choosing projects –Cytology project example –Metrology –Help for programming example projects

IQstudio User Interface Examples IQExplorer

IQstudio User Interface Examples Demo Projects – Metrology

IQstudio User Interface Examples Demo Projects – Cytology

IQstudio User Interface Examples Demo Projects

Silver Spring, MD (Headquarters) Worldwide Offices in: Boston, MA, Pittsburgh, PA & San Diego, CA Leiden, The Netherlands Goettingen, Germany Wokingham, United Kingdom Singapore, Republic of Singapore Tokyo, Japan Shanghai, China

“Elvis Has Left the Building…”