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

FROM IMAGES TO ANSWERS

July 2005 Silver Spring and San Diego

Advanced Quantitative Image Analysis

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 at a Nearby Restaurant

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•Install Capture Driver from WEB Site (CDs are Always Out of Date)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

•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


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

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 of 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)



3. Calculate # of Images

4. Select “Burst” to acquire at High Speeds

High Speed Ratio Time Lapse



3. Define Ratio Channels

4. Calculate # of Images

5. Select “Burst” to acquire at High Speeds

3D Time Lapse



3. Define # of Planes

4. Define Z Spacing

5. Select 3D Time Lapse

6. Select “Start” to acquire 3D Time Lapse

Multiple Log-points Time Lapse



3. Define # of Log Points

4. Select Time Lapse

5. Select “Start Multi Acquire” to acquire Multiple Log-points Time Lapse

Multi Dimensional Live Cell Analysis

Live Ratio Analysis



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


FRET Imaging

1. Define Background Correction

2. Define Channels

3. Calculate FRET Image

Tracking of objects

1. Define Tracking Measurements

2. Define Tracking Options


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

Image Pro 3D Suite 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)

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

http://www.improvision.com/products/openlab/

http://www.image1.com/products/metamorph/


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



MC MM IPLab 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 > 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 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

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

http://www.mediacy.com/

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