FROM IMAGES TO ANSWERS

Silver Spring, San Diego and Markham

Advanced Digital Automation & Analysis Destination Check…This is Not…American Dodgeball Association of America

Destination Check

This is Dodgeball

Agenda Day One

•9:00-10:00 Advanced Microscopy Suite Setup•10:00-11:00 Multi-Dimensional Acquisition•11:00-Noon Multi-Dimensional Analysis•Noon-1:00 Lunch (Catered)•1:00-2:00 Simulated Live Cell Acquisition & Analysis*•2:00-3:00 Simulated Ratio Acquisition and Analysis*•3:00-4:00 Piezo Z-Axis Setup & Fast Acquisition •4:00-5:00 Group Discussion•5:00PM Adjourn•6:00PM Dinner and a Movie in Markham!

*No Live Cells Were Harmed in the Acquisition of These Images!

Agenda Day Two

•9:00-10:00 Deconvolution of Widefield Images•10:00-Noon 3D Rendering and Measurement of

Image Stacks•Noon-1:00 Lunch (Catered)•1:00-2:00 3D Rendering and Measurement of

Image Stacks•2:00-3:00 3D & 2D Tracking•3:00-4:00 Using Workflow Toolbars to Automate

Processes•4:00-5:00 Group Discussion•5:00 Adjourn•6:00 Dinner at a Nearby Restaurant*

Many Brain Cells Will Be Harmed During This Dinner!

Agenda Day Three

•9:00-10:00 Industry Best Practices with Macros•10:00-11:00 Communicating with External

Devices•11:00-Noon Integrating Macros with

Applications (Libraries and Resources)

•Noon-1:00PM Lunch (Catered)•1:00-2:00 Fun with Dimensioning Variables &

Arrays; Building Loops•2:00-3:30 Practical Examples of Macros in

Practice•3:30-4:00 Transitioning to Visual Basic for

Applications in IQbase•4:00-5:00 Group Discussion•5:00 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)

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 http://www.solutions-zone.com

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

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

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 Time4D 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

Y N N N N N

Zoom & Pan Images

Y N N N N N

Advanced Microscope Control

Y Y Y Y Y Y

Tiling, Mosaic, Slide and Well Scanning

Y Y Y Y Y Y

Multi-dimensional Acquisition

Y Y Y Y Y Y

Multi-Color Imaging

Y Y Y Y Y Y

Time Lapse Acquisition

Y Y Y Y Y Y

Color 3D with Piezo Focus

Y, Extra $

Y N Y N N

Competitive Feature Comparison

MC MM BD Bio 3I Compix AxioV

Advanced Morphometric Measurment, Analysis & Classification

Y Y Y Y Y Y

Colocalization, Translocation FISH, and Fluorescence

Y Y Y Y Y Y

Ratio Imaging Y Y Y Y Y Y

FRET Analysis Y Y Y Y Y Y

Cell Tracking Y Y N Y Y N

Vital Systems Control & Measurement

Y Y N Y N N

Signals and Systems Control Y Y N Y N N

Data Analysis, Recording, and Reporting

Y Y Y Y Y Y

3D rendering and measurements

Y N N Y N N

3D Time Lapse Analysis Y N N N N N

Analog Digital signals support Y Y N Y Y N

Trend chart analysis Intensity tracking

Y Y Y Y Y N

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 2005

..\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 http://www.Solutions-Zone.com for Examples

Image-Pro and VB

Linked VB.Exe and Image-Pro

Often the Route of Choice for End User DevelopmentSimple 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

IQstudioAnalytical 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 ExamplesIQExplorer

IQstudio User Interface ExamplesDemo Projects – Metrology

IQstudio User Interface ExamplesDemo Projects – Cytology

IQstudio User Interface ExamplesDemo Projects

www.mediacy.comSilver 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…”