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