mark hartnett software support engineer december 7, 2001 introduction to analysis and feature...

Mark HartnettSoftware Support EngineerDecember 7, 2001

Introduction to Analysis and Feature Extraction Software

Title of Presentation1 March, 2001

Agilent Restricted

Launching the Image Analysis Software• Double-click the Image

Analysis icon on the desktop

OR

• Click Start > Programs > Agilent Life-Sciences > Image Analysis


Agilent Restricted

Accessing the Image Analysis User Manual• Double-click the Agilent-

Life Sciences icon on the desktop

• OR

• Click Help > Help from the Image Analysis menu bar


Agilent Restricted

Accessing the Image Analysis Software Version• Click Help > About from

the Image Analysis menu bar

• The About Analysis dialog displays the software version


Agilent Restricted

How to Open a Scanned Image

• Click the Open icon on the Image Analysis toolbar

OR

• Click File > Open and select the image file in the Open dialog


Agilent Restricted

How to Save a Copy of a Scanned Image• Click the Save As icon on

the Image Analysis toolbar

OR

• Click File > Save As and name the image file in the Save As dialog


Agilent Restricted

How to Crop a Scanned Image

• Left-click and hold the mouse and then draw a box around the array

• Release the mouse button

• The cropped image appears in a new window


Agilent Restricted

How to Crop a Scanned Image (Continued)• Click the Save Cropped

View Icon in the new image window

OR

• Click File > Save Cropped View on the Image Analysis menu bar

• Complete the information in the Save Cropped View dialog


Agilent Restricted

How to Close an Image

• Click the Close icon on the scan image window

OR

• Click File > Close on the Image Analysis menu bar


Agilent Restricted

Changing the Color Range of the Image Display• Click the Set color range

icon in the toolbar of the scan image

OR

• Click Color > Set Data Range from the Image Analysis menu bar

• In the Set Data Range dialog, enter the minimum and maximum values or choose Auto Scale Image


Agilent Restricted

Setting the Data Range for the Color Scale• Changing the data range

only affects the visualization of the array

• Underlying pixel intensities - raw data - do not change

• Compressing the data range brightens the array

• Expanding the data range dims the array

expand scale -lower min

& raise max

compress scale -raise min &lower max

auto scale


Agilent Restricted

Changing between Log and Linear Color Scale • Click the Toggle Log Scale

Icon to switch between Log and Linear color scale

OR

• Click Color > Use Log Color Scale to toggle between Log and Linear color scale


Agilent Restricted

Viewing Images with Log or Linear Color Scales• Click Color > Use Log Color

Scale

• Toggle between log and linear color scale Log Scale

Linear Scale


Agilent Restricted

Changing the Color Palette

• Click on Color in the Image Analysis menu bar

• False Color and False Color with White BG are available for two-color images

• Gray Scale and Reverse Gray Scale are available for single-color images


Agilent Restricted

Histogram Graphs of Pixel Intensities• Histograms can be plotted for the entire view or a selected

region

• for entire view - click View > Entire Histogram

• for a selected region - right-click and draw the region

• Selected region can be rectangular or elliptical

• use the toggle button to switch between shapes

• For two color images - one or both colors can be plotted


Agilent Restricted

How to Select an Elliptical Area for a Histogram• Click the Toggle

Histogram Shape icon on the image toolbar

OR

• Click Options > Select Elliptical Area on the Image Analysis menu bar


Agilent Restricted

How to Select Graph Defaults for Histograms• Click Options > Set

Graph Defaults on the Image Analysis menu bar

• Specify either the Bin Size or the Maximum Number of Bins in the Set Bin Defaults dialog


Agilent Restricted

Histogram Plot - Sample

Elliptical histogramfor this feature


Agilent Restricted

Histogram Plot - Bin Size

• Bin size is set automatically by the Analysis software

• Users can change the bin size by two methods

• specifying the number of pixel intensity counts per bin

• specifying the maximum number of bins

• click Options > Set Graph Defaults -OR- Histogram > Change Bins


Agilent Restricted

How to Select Default Image Options• Click Options > Set

Image Defaults on the Image Analysis Toolbar

• Specify the desired default parameters in the Default Image Options dialog


Agilent Restricted

How to Set the Design File Search Path• Click Options > Set

Design File Search Path

• Click Browse in the Set XML Path dialog

• Locate the directory containing the design file in the Browse for Folder dialog

• Click Add in the Set XML Path dialog


Agilent Restricted

How to Check the Memory Status

• Click Help > Memory Status on the Image Analysis menu bar

• The Memory Status dialog is displayed


Agilent Restricted

Line Graphs

• Line graphs show a horizontal or vertical slice through the image

• To open the Select Row/Col dialog box

• click View > Select Row or Column

• for horizontal slice - click

• for vertical slice - click

• Choose Row -OR- Column -OR- All Rows or Columns


Agilent Restricted

Line Graph - Sample

Line graph for a row of pixelsthrough these features


Agilent Restricted

Exporting Images to other Applications• Scan images, histogram plots, and line graphs

can be exported to other applications

• Export methods

• high-resolution - click File >Save As and specify bitmap file type

• low-resolution - click the copy button to send the image to the clipboard


Agilent Restricted

Zooming In and Out

• Click View > Zoom from the menu

• select a magnification

• Mouse shortcuts

• To zoom in - Ctrl + left double click on the image

• To zoom out - Ctrl + right double click on the image

• Toolbar buttons

Zoom in Zoom out 100 percent


Agilent Restricted

Scan Image File Information

• Information regarding the scan image is stored within its TIF file

• chip barcode

• scan date and time

• scanner software versions

• scan region

• PMT voltage


Agilent Restricted

Viewing Scan Image File Information• Click View > FileInfo

• select General tab

• The scanner reads the barcode and generates the Array Identifier


Agilent Restricted


• select the General tab

• The scanner reads the barcode and generates the Array Identifier

• user can modify if necessary


Agilent Restricted


• select the Versions tab

• Scanner software versions are stored with the scan image


Agilent Restricted


• select the Image tab

• Scan regions and PMT Voltages are displayed


Agilent Restricted


• select the Image 2 tab

• Scan resolution is shown in the MicronsPerPixel field


Agilent Restricted

Feature Extraction Input Files

• Input Files - Required

• Scan image of Agilent microarray in 16-bit TIFF format

• 16-bit TIFF images are generated by the Agilent scanner. Scan images converted to other formats can not be feature extracted.

• Scan images of non-Agilent microarrays can be viewed - but not feature extracted - with the Agilent Image Analysis and Feature Extraction software.

• Array Design file - also called Pattern file - in GEML XML format

• Describes the layout and the probes on the microarray

• XML - Extensible Markup Language

• GEML - Gene Expression Markup Language


Agilent Restricted

Feature Extraction Input Files (Continued)• Input Files - Optional

• Printing File - applies to cDNA microarrays. This file contains information on the cDNA clones spotted on the microarray. Bad clones on the microarray are ignored and excluded from analysis.

• If no Printing File is found, the below dialog is displayed

• In-situ microarray customers should disregard this message

• cDNA microarray customers should place the appropriate Printing File in the Design File Search path


Agilent Restricted

Feature Extraction Output Files

• XML - Contains the gene expression profile for the microarray in GEML format. Expression data in the GEML format can be loaded directly into the Rosetta Resolver Gene Expression Analysis software.

• Tab-text - Contains the gene expression profile for the microarray in tab-delimited text format. This format is intended for customers who do not use Resolver for gene expression analysis. The data will load into other gene expression analsysis software with a minimum of manipulation.

• Visual Results - Contains the “shapes” annotation generated by the Feature Extraction software. This file allows for subsequent viewing of the “shapes” annotation without having to re-extract the scan image.

From theFeature Extraction UI


Agilent Restricted

How to Crop a Scanned Image

• Left-click and hold the mouse and then draw a box around the array

• Release the mouse button

• The cropped image appears in a new window


Agilent Restricted

How to Crop a Scanned Image (Continued)• Click the Save Cropped

View Icon in the new image window

OR

• Click File > Save Cropped View on the Image Analysis menu bar

• Complete the information in the Save Cropped View dialog


Agilent Restricted

How to Manually Input the Slide Barcode• The Agilent scanner

reads the barcode for each slide and stores that information inside the TIFF scan image

• In rare cases, the Agilent scanner does not read the slide barcode correctly

• The user must then manually enter the slide barcode when saving a cropped microarray scan image


Agilent Restricted

Agilent Slide Barcode Nomenclature• The Agilent microarray

barcode standard consists of 14 digits

• The six-digit design identifier is the same for all microarrays printed from the same pattern

• The six-digit serial number serves as a unique identifier for all microarrays printed from the same pattern

• The barcode uses Code 128C encoding 16NNNNNNNNNNNNV

ersi

on o

f th

e ba

rcod

e fo

rmat

Des

ign

iden

tifie

r Se

rial

num

ber

For

vers

ion

1: #

of di

gits

in the

des

ign

iden

tifie

r fie

ld


Agilent Restricted

Agilent Microarray Identifier Nomenclature• The Agilent microarray identifier is based on the slide

barcode

• Slides contain either one 22K-feature microarray or two 8.4K-feature microarrays

• 22K-feature microarray - one microarray per slide

• Suffix _A01 appended to the slide barcode

• 8.4K-feature microarray - two microarrays per slide

• Suffix _A01 appended to slide barcode - Left array

• Suffix _A02 appended to slide barcode - Right array

• Example: 16NNNNNNNNNNNN_A02barco

de


Agilent Restricted

Agilent Design and Printing File Nomenclature• Agilent design and printing

file naming nomenclature is based on the design ID and date

• Printing files are only generated for cDNA microarrays and contain information about bad clones on the array

• Do not rename the design or printing files!

• Deviating from the nomenclature prevents automatic association of microarray scan images with their design and printing files

NNNNNN_D_YYYYMMDD.xml

Des

ign

Iden

tifie

rD

indi

cate

s D

esig

n fil

e, P

indi

cate

s Pr

intin

g fil

e

Two-

digi

t M

onth

Four

-dig

it Y

ear

Two-

digi

t D

ay


Agilent Restricted

How to Set the Design File Search Path• Feature Extraction checks

the design file search path to find the microarray scan image’s design file

• Click Options > Set Design File Search Path

• Click Browse in the Set XML Path dialog

• Locate the directory containing the design file in the Browse for Folder dialog

• Click Add in the Set XML Path dialog


Agilent Restricted

How to Feature Extract a Scan Image• Make sure the design file is

in the design file search path

• Crop and save the microarray scan image (and manually enter the slide barcode if necessary)

• Click Feature Extraction > Feature Extractor

• Specify the directory path for the output files

• Click the Load All Defaults button and the Run button


Agilent Restricted

How the Feature Extraction Software Searches for Design Files• The design ID is part of the microarray identifier, which

is embedded in the microarray scan image

• The design ID is embedded in the filename of the design file

• The design ID is also embedded in the filename of printing files - applicable to cDNA microarray design files

• For all directories in the design file search path:

• the Feature Extraction software searches for design and printing files with embedded design IDs that match the design ID in the microarray scan image


Agilent Restricted

How to Manually Specify a Design File• If the microarray design file is not found in the

design file search path, the user is prompted to manually specify a design file

• Slide printing files can not be loaded using this method and therefore no clone information will be available

• The user must ensure that the manually-specified design file represents the microarray in the scan image

• If the manually-specified design file does not represent the microarray in the scan image, an erroneous gene expression profile will result


Agilent Restricted

How to Manually Specify a Design File

Warning 1

Warning 2

Design File Loading Logic


Agilent Restricted

Locating Corner Features in the Feature Extraction Software• The Feature Extraction software automatically

locates the corner features of the microarray and uses these as references to locate all other features

• In rare cases, the Feature Extraction software fails to locate the corner features of the microarray

• The user must then manually provide the locations of three corners of the microarray, to allow the software feature extract the microarray


Agilent Restricted

How to Manually Locate the Corner Features in a Microarray Scan Image• Zoom in on the top-left corner

feature on the microarray

• Place the mouse pointer over the center of the corner feature

• Left-click the mouse

• Click the enter button for Upper Left in the Find Spots tab of the Feature Extraction user interface

• The row and column coordinates are pasted into the user interface

• Repeat the process for the upper-right and lower-left corner features

top-leftcorner feature


Agilent Restricted

Viewing Feature Extraction Results

• Click View > Extraction Results

• Three on / off toggles

• View Results

• Hide Good

• Simple Colors


Agilent Restricted

Loading Feature Extraction Results

• Feature Extraction visual results can be saved

• makes re-extraction unnecessary

• click Feature Extraction > Load Results

mark hartnett software support engineer december 7, 2001 introduction to analysis and feature...

Documents

image analysis icon

image file

cropped image

image analysis toolbar

auto scale image slide

image display

image analysis menu

title of presentation