Odyssey Infrared Imaging System Tutorial Manual v 2.1 - LI

ODYSSEY
Infrared Imaging System
Tutorial
Manual
Version 2.1
®
®
ii
Notice
Printing History
The information contained in this document is
subject to change without notice.
Publication Number 984-08465
Printed June, 2006
LI-COR MAKES NO WARRANTY OF ANY
KIND WITH REGARD TO THIS MATERIAL,
INCLUDING, BUT NOT LIMITED TO THE
IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR
PURPOSE. LI-COR shall not be liable for
errors contained herein or for incidental or
consequential damages in connection with the
furnishing, performance, or use of this
material.
This document contains proprietary information which is protected by copyright. All
rights are reserved. No part of this document
may be photocopied, reproduced, or translated
to another language without prior written
consent of LI-COR, Inc.
LI-COR is an ISO9001 registered company. © 20012006 LI-COR Inc. Specifications subject to change.
LI-COR, Odyssey, MousePOD and IRDye are trademarks or registered trademarks of LI-COR, inc. Alexa
Fluor® 680 is a registered trademark of Molecular
Probes. Adobe and Acrobat are registered trademarks of
Adobe Systems Inc. Windows and Microsoft are registered trademarks of Microsoft Corporation. The Odyssey
Infrared Imaging System and IRDye 800CW are covered
by U.S. patents 6,495,812, 6,995,274, foreign equivalents, and patents pending.
iii
Table Of Contents
Chapter 1. Getting Started
Chapter 4: Quantification
How to Get Started...................................... 1
Access Privileges...................................... 1
Odyssey Overview ...................................... 2
The Infrared Advantage ............................ 2
Two Infrared Dyes.................................... 2
Odyssey Detection System ....................... 3
Network Connectivity .............................. 3
The Project Model.................................... 3
Starting Scans ........................................... 4
Analyzing Your Images............................. 4
What’s Next? ............................................... 4
Checking Settings.......................................25
Identifying Concentration Standards........... 27
Centering Features .................................. 28
Resizing Features .................................... 30
Naming the Standard .............................. 30
Entering the Concentration of the
Standard .................................................31
Quality Control Using the Standards Plot ... 33
Quantifying Unknown Concentrations ....... 34
Quantifying the 700 Channel Image........... 35
Using Details View for Data Comparison ... 38
Printing Quantification Reports .................. 39
Report Templates .................................... 39
Printing a Report ..................................... 39
Saving the Project ...................................... 41
Chapter 2: Starting Scans
Projects, Scans, and Analyses ..................... 5
Starting Scans .............................................. 5
Scan Procedure ........................................... 6
Stopping a Scan......................................... 11
Closing Odyssey Software During a Scan . 11
Completing a Scan in the New Analysis
Window .................................................... 12
Other Scanning Methods ........................... 14
Chapter 3: Creating a New Analysis
Importing Images Into the Tutorial
Project....................................................... 15
Creating a New Analysis............................ 17
Changing the Appearance of the
Images....................................................... 19
Rotating an Image................................... 19
Cropping an Image ................................. 21
Opening an Image View............................ 24
Chapter 5: Quantification Using
Grids
Importing the Microplate Tutorial Images .... 43
Opening the Grid Analysis ......................... 44
Applying a Grid Template .......................... 46
Viewing Data in the Grid Sheet.................. 51
Quantification Using Grids ........................ 51
In-Cell Western Assays............................... 52
What’s Next ...............................................52
Chapter 6: Sizing Bands
Overview ...................................................53
Importing Images Into the Tutorial
Project .......................................................54
Checking the Settings ................................. 55
Starting a New Analysis.............................. 56
iv
Adding Lanes............................................. 58
Moving and Resizing Lanes.................... 60
Adding Multiple Lanes ........................... 62
Editing Band Markers................................. 65
Band Finding Threshold ......................... 65
Adding and Resizing Band Markers........ 65
Deleting Band Markers .......................... 70
Entering the Size of Each MW Marker........ 73
Linking MW Lines to MW Marker Bands ... 74
Reshaping and Moving MW Lines.......... 76
Applying MW Lines to Both Images ........... 76
Plotting Size Standards............................... 78
Generating Lane Reports............................ 79
Outputting Lane Data to a File ............... 80
Saving the Project ...................................... 82
Continuing the Tutorial.............................. 82
Chapter 7: Starting Scans From the
Odyssey Front Panel
Starting Scans ............................................ 83
Scan Procedure ...................................... 83
Scan Dimensions ................................... 84
File Naming Conventions....................... 84
Reviewing Scan Status............................ 84
Stopping a Scan ..................................... 85
Downloading Files for Analysis.................. 85
1
Chapter 1: Getting Started
How to Get Started
Information on Odyssey Software can be found both in this manual
and in the Odyssey User Guide. In addition, software functions related
to the Odyssey MousePOD™ Accessory are discussed in the Odyssey
In vivo Imaging Guide.
A good place to start is to read the overview of Odyssey given later in
this chapter. If you need to install Odyssey or Odyssey Software,
instructions are given in the Odyssey Installation Manual. Any instructions more recent than the printed manual will be included in a
“Readme” file on the Odyssey CD.
The User Guide is a reference manual with detailed descriptions of
all the functions of Odyssey Software. This Tutorial Manual is more
task oriented and shows each step necessary to perform common
tasks such as scanning, sizing, and quantification. Each chapter
builds on the previous chapter, so it is best to do the tutorial from
beginning to end, though you don’t have to do it all at once.
Access Privileges
Before you get started with the tutorial, make sure you have Control
access rights to Odyssey. A user with Administrator access rights can
change your access rights, if necessary. Control rights are required to
start new scans, download data, and analyze images.
2 CHAPTER 1
Getting Started
Odyssey Overview
The Infrared Advantage
In the past, visible wavelength fluorescence technology has been
widely used for protein and nucleic acid imaging applications.
However, most visible systems have limited capabilities for imaging
membranes due to strong background fluorescence at visible
wavelengths. The Odyssey Infrared Imager eliminates these detection
barriers by using near-infrared fluorophores.
Low background fluorescence at infrared (IR) wavelengths provides
a much higher signal-to-noise ratio than fluorescence detection at
visible wavelengths. The sensitivity of the Odyssey system is comparable to chemiluminescence on film, but chemiluminescent
substrates and film are not required.
Two Infrared Dyes
The Odyssey system typically uses two IR dyes for simultaneous twocolor detection. Two-color detection adds many new detection capabilities. For example, two different antibodies can be used as probes on the
same blot by using antibodies labeled with different IR dyes.
Use of IR-labeled antibodies and compatible dyes and stains are
described in the Odyssey Application Protocols manual and pack inserts
included with the reagents. The latest Odyssey protocols are posted on
LI-COR’s support web site at http://biosupport.licor.com/support. Files
are in Adobe® Acrobat® format (*.PDF), requiring a copy of Adobe®
Reader® to open the files. Adobe Reader is available at no charge at
http://www.adobe.com.
3
Odyssey Detection System
The Odyssey detection system uses two completely independent
detection channels – one for each IR dye. Two diode lasers provide
excitation light at 685 and 785 nm. Two avalanche photodiodes are
filtered to detect fluorescence at 720 and 820 nm. Scans can be
performed at resolutions ranging from 21 - 337 µm. A more complete
description of the detection system can be found in the Odyssey
Operator’s Manual.
Network Connectivity
It is easiest to think of Odyssey as a combination of a scanner and a
file server. A computer running Windows® (XP/NT/2000/95/98) can
connect directly to the Odyssey Imager or both the computer and
Odyssey can be connected to your network. Connection to a
network adds greater flexibility since scans can be accessed
anywhere on the network. Security protocols assure that users can
access only the files that they are permitted to access. Changes to
access rights can be made only by a user with Administrator access
rights.
The Project Model
Odyssey uses projects to manage your workflow. All scans created in
Odyssey become part of a project. Projects are simply containers that
provide a logical way to group related scans. The analysis data for
each scan is also part of the project structure. Once a project is
opened, new scans can be added to the open project.
4 CHAPTER 1
Getting Started
Starting Scans
Scans can be started from the Odyssey front panel, from Odyssey
Software, or an Internet browser. When a scan is initiated, scan
parameters like scan area and resolution are specified. The scan
parameters are sent to the Odyssey Imager, which starts the scan and
controls data collection. A separate image is collected for each IR
dye. Scan data are stored on an internal hard disk in the Odyssey
Imager. If Odyssey Software is used to start the scan, scan data are
automatically transferred to the computer in real time.
Analyzing Your Images
After a scan is complete, sizing or quantification is started by creating
an analysis that becomes part of the project. When an analysis is
opened, the two images from the scan are displayed in different
colors. An overlay mode makes it easy to compare samples since
areas of fluorescence overlap are displayed in a third color. Fast,
simple tools allow images to be analyzed quickly. Software tools for
both band sizing and quantification are provided. Optional software
modules for In-Cell Western analysis and the MousePOD Accessory
provide additional functionality if you have purchased those modules.
After analysis is complete, a variety of printed reports are available.
Analysis data can also be exported in a file format compatible with
external databases.
What’s Next?
In the next chapter you will learn how to start a new scan using
Odyssey Software. All readers will benefit by reading Chapters 2, 3,
and 7. Depending on your application, you will also want to read the
quantification tutorials in Chapters 4 and 5, or the band sizing
tutorial in Chapter 6.
5
Chapter 2: Starting Scans
Projects, Scans, and Analyses
All files in Odyssey are contained within projects. Within each
project there can be many scans. Each scan contains one or two TIFF
images from the Odyssey Imager, depending on whether probes for
one or both dyes were imaged. An analysis holds all the sizing or
quantification data created when a scan is analyzed.
After each new scan, a New Analysis window is opened to create the
first analysis on the new scan files. Additional analyses within a scan
might be created for a variety of reasons. For example, if the scanned
images include multiple membranes for different users, each user
may want to independently analyze a portion of the image in a
separate analysis. If the original image is flipped or rotated in some
way, a new analysis can be created to correct the orientation of the
image. When an analysis is created, the image can be flipped,
rotated, cropped, filtered, or have its background fluorescence
subtracted.
Starting Scans
There are three ways to start scans with the Odyssey Imager:
•
From the instrument front panel
•
From an Internet browser
•
From Odyssey Software
6 CHAPTER 2
Starting Scans
Odyssey Software operation is discussed in this chapter. "One button
scanning" from the Odyssey front panel is described in Chapter 7 and
the use of an Internet browser for scanning is discussed in the
Odyssey Operator’s Manual.
Scan Procedure
1) Start the Odyssey Software and choose File > New to start a new
project.
Note: Scans are usually added to existing projects rather than starting a new one.
Existing projects are opened by choosing File > Open.
C:\Documents and Settings\user name\My Documents\licor\Odyssey\Projects\
2) Set the Path to the path shown above but
substitute your Windows users name for
“user name” in the path. Enter Tutorial in the
Name field to name the new project.
(Location of the Licor directory may vary.
Use the Browse button to find the directory,
if needed.)
3) Click Scan to create the new project and
start a scan that will become part of the
Tutorial project.
4) In the Scanner Login window, enter your
User Name and Password (case sensitive).
Note: User accounts (names and passwords) must
be added to both the Odyssey Imager and Odyssey
Software. See the Odyssey Operator’s Manual if you
are denied access.
5) Click OK to open the
Scanner Console window.
7
The Scanner Console window is used to name the scan and specify
important scan parameters like size and resolution.
6) Enter FirstScan in the
Name field to name
the scan.
7) Choose the scan
Group that matches
your user name.
Scan groups are like directories on the Odyssey Imager,
except that access can be
restricted to specific users.
8) Choose Membrane
from the Preset
drop-down list.
Presets are sets of scan
parameters that have been
saved. For repetitive
scanning, Presets are more
efficient than entering each
parameter individually.
9) Enter First Tutorial Scan in the Description field.
Descriptions can be included in analysis reports.
Choosing a Preset automatically loads all of the scan parameters. A
brief description of each scan parameter is given below and
complete information can be found in the Odyssey User Guide. After
the Preset is loaded, any scan parameter can be edited as needed. In
fact, the next step is to set the Scan Area.
• Resolution can be set to 21, 42, 84, 169, or 337 µm. 169 µm is used for
typical scans of membranes, gels, or microplates.
8 CHAPTER 2
Starting Scans
• Quality determines how much detector signal is processed for a given area
on a membrane to form a pixel on the image. Medium is typical.
• Focus Offset is always zero for membranes. For microplates the Focus
Offset is 3 mm and for gels Focus Offset is equal to half the thickness of
gels in millimeters (4 mm maximum).
• Select Microplate (flip image) when scanning microplates since micro-
plates are scanned through the bottom of the plate.
• The Channels check boxes are used specify whether to detect the 700
channel dye, 800 channel dye, or both.
• Intensity is typically set to 5.0 for membranes, 8.0 for DNA gels, and 5.0
for protein gels or microplates.
• Scan Area is drawn by clicking and dragging a rectangle on the scan grid.
The Origin and scan Size fields are automatically entered based on the
size and location where the rectangle is drawn. Scan area can also be
entered in the Origin and Size fields.
10)Double-click the scan grid to
erase the initial scan area. Click
in the lower left corner of the
scan grid and hold down the left
mouse button. Drag the mouse to
draw a rectangle on the scan grid
that is 5 cm high by 20 cm wide
and release the mouse button.
The lower left corner of the scan grid is
the origin (0,0) and corresponds to the
front-left corner of the scan surface on the
Odyssey Imager. The scan rectangle can
be drawn anywhere on the scan grid. It
does not have to start at the origin.
Important: Always make the scan area larger than the membrane or
gel. Adding an extra centimeter on each side will assure that labels
placed on the image will be displayed properly.
9
11) After setting the scan area, note the file size.
File sizes should be kept below 7 MB per image if possible, but can
be as large as 14 MB. See Chapter 2 of the Odyssey User Guide for
details about file sizes.
12) Since you probably do not have a membrane prepared, place the
scanning ruler that comes with Odyssey on the lower left corner
of the scan surface in the orientation shown below.
Note: Techniques for placing membranes, microplates, and gels are given in the
Odyssey Operator’s Manual.
Left border
of scan area
Lower border
of scan area
13) Close the lid on Odyssey.
The tip of the arrow on the lower left corner
of the scan surface is the origin (0,0).
Tip: Rectangular membranes (etc.) will
scan faster if the long dimension is
oriented horizontally along the lower
border of the scan area. Placement in a
vertical orientation requires the laser
microscope to travel further and increases
scan time.
10 CHAPTER 2
Starting Scans
14) Click Start Scan in the Scanner Console to send the scan parameters to the Odyssey Imager and start the scan.
The image is displayed in
real time in the Scanner
Console window.
15)After some of the
image is displayed,
click Alter Image
Display, select
Linear Manual and
increase the Sensitivity on both
image channels
until you can see
the ruler.
Note: The Alter Image
Display controls only
change how image data
are displayed and are
not the same as the
Intensity parameters in
the Scanner Console,
which change how
fluorescence is
detected.
At the bottom of the Scanner Console window, the
status line indicates how much time is required to
finish the scan and a progress bar indicates how
much of the area has been scanned.
11
When scanning membranes, if bands or dots are dim, use the
brightness and contrast controls in the Alter Image Display window.
If there is no fluorescence showing, increase the sensitivity using the
Linear Manual Sensitivity slider.
By default, the 700 and 800 channel images are shown overlaid. In
the default red/green color scheme, areas that are yellow have
intense fluorescence in both channels. The Show This Image check
boxes in the Alter Image Display window can be used to display only
one channel at a time if you prefer to look at each channel separately.
Note: The Adjust Image Curves button can also be used to change the appearance of
the image, as described in Chapter 11 of the Odyssey User Guide.
16) Click OK to close the Alter Image Display window.
Stopping a Scan
Odyssey automatically completes the scan and saves the image files
after the entire scan area has been scanned. To finish a scan before
automatic completion, click the Stop button in the Scanner Console
window or press the Stop key twice on the Odyssey front-panel
keypad. When a scan is stopped, the image files are closed and
saved, allowing the files to be analyzed. If you want to abandon a
scan and not save the image files, click Cancel rather than Stop in the
Scanner Console window.
Closing Odyssey Software During a Scan
If you do high resolution scans with long scanning times, Odyssey
Software can be closed while the scan is being completed. To close
the software, just choose File > Exit. Do not close the Scanner
Console window by clicking Stop or Cancel. When the project is
opened again, Odyssey Software will automatically download the
finished image files from the Odyssey Imager.
12 CHAPTER 2
Starting Scans
Completing a Scan in the New Analysis Window
When Odyssey finishes scanning, a New Analysis window is opened
for the TIFF image files that were just scanned. At this stage, the TIFF
image files are now stored on the computer in a directory with the
same name as the project (transfer is automatic).
17)Name the analysis
Original Analysis.
18)Click OK to save the
unaltered original
images.
In the next chapter, you will learn that you can also crop, flip, rotate,
change brightness and contrast, and perform background
subtraction before clicking OK. Any changes to the images are
permanent, so if you want to keep unaltered copies of the images in
your project, it is best to just click OK without making changes.
13
After the analysis is saved, it is displayed in the navigation tree in the
Odyssey window.
19)Click First Scan in the
navigation tree to reveal the
analysis saved at the end of
the scan.
20)Double-click Original Analysis in the navigation tree to display
the images from the scan.
In the next chapter, you will learn how to create a new analysis that
uses copies of these original images.
14 CHAPTER 2
Starting Scans
Other Scanning Methods
This chapter has introduced a scanning method that can be used for
membranes. The same basic method can also be used for DNA gels
and microplates by just choosing the appropriate preset in the
Scanner Console window. Scanning microplates also requires use of
a scanning guide (included) that places the microplate at a known
location that approximately matches the Microplate2 preset. For
higher throughput, up to six microplates can be scanned by choosing
File > Scan Multiple Plates. Chapter 2 in the User Guide and Chapter 3
in the Operator’s Manual contain additional information on scanning
single and multiple microplates. Chapter 2 of the optional In vivo
Imaging Guide illustrates how to scan up to three mice using the
Odyssey MousePOD™ Accessory.
15
Chapter 3: Creating a New Analysis
New scans are listed under the designated project in the navigation
tree on the left side of the Odyssey window. Clicking the plus symbol
[+] next to the scan reveals the first analysis added at the end of the
scan.
In the practice scan for the Tutorial project, real samples were not
scanned. The first step in this chapter will be to import a set of images
into the tutorial project and create a new analysis.
Importing Images Into the Tutorial Project
The images needed for this tutorial are located on the Odyssey CD in
a directory called Tutorial Images. The file names are Q700.tif and
Q800.tif. After importing these images into the Tutorial project and
creating a new analysis for the images, you will be ready for the
quantification tutorial in Chapter 4.
1) If necessary, open the Tutorial project created in Chapter 2 by
choosing File > Open and selecting the project.
16 CHAPTER 3
Creating A New Analysis
2) Choose File > Import Images.
Note: The Import Images choice on the File menu is normally
used to import TIFF files if the original scan file has been lost
or deleted. A scan file (*.scn) is one of the files generated
during a scan and contains information like the location of the
TIFF image files, scan resolution, etc.
3) Enter QuantScan as the new scan name and
Original Analysis as the analysis name.
4) Click Browse for the
700 channel to
select an image.
6) Click OK to import the
two images.
5) In the File Selection
window, select a file
named Q700.TIF in
the folder named
Tutorial Images on
your CD drive (insert
the Odyssey software
CD if necessary).
Click Open to select
the file.
The path to the 800 channel
image is always entered
automatically as long as it is
in the same directory as the
700 channel image and
ends with the suffix 800.tif.
17
Creating a New Analysis
The images are now stored in the Tutorial project. Rather than alter
these original images, a new analysis should be started that uses
copies of the original images.
1) Click QuantScan in the list of scans for the Tutorial project.
2) Create a new analysis in QuantScan by clicking the New Analysis
button on the toolbar.
A new analysis can also
be created by choosing
File > New Analysis.
18 CHAPTER 3
Creating A New Analysis
3) Enter Quant1 as the Name
for the new analysis.
Note: Never use slashes,
colons, commas, or
periods in analysis names.
4) Enter "Quantification of
Q700 and Q800 Tutorial
Images" as the Description.
5) Make sure Original Analysis
is selected.
The analysis selected in the
Available Analyses list is the
analysis that the image files will be
copied from. Only analyses in the
current scan are shown in the list.
6) Both the 700 and 800 check boxes should
be selected so images from both image
channels are imported.
In the Analysis Image section of the window, a thumbnail of the
images is shown with the two image channels overlaid. Fluorescence
in the 700 channel image is red. Fluorescence in the 800 channel is
green. If there were any locations where fluorescence from the two
channels overlap, the images would be some shade of yellow. On the
tutorial images there is no fluorescence overlap.
19
Changing the Appearance of the Images
The buttons in the Analysis Image section of the New Analysis
window are used to change the appearance of the image. Flip and
Rotate let you change image orientation. Subtract and Filter let you
change the image by performing background subtraction, sharpening, etc. Crop is used to choose a smaller segment of the whole
image to analyze. Alter Image Display changes the brightness and
contrast of the images. Note that Rotate, Subtract, and Filter can
change quantification results if used incorrectly (see Chapter 4 of the
Odyssey User Guide).
Although the tutorial images require no changes, let’s try a few to see
how they work.
Rotating an Image
7) Click the Rotate button to
rotate the image.
Note: The New Analysis window
is the only window in Odyssey
where images can be cropped,
rotated, filtered etc. Once you
click OK to close the New
Analysis window, changes are
permanent so it is important to
change the image the way you
want it before closing the
window.
20 CHAPTER 3
Creating A New Analysis
8) Click the Counter-Clockwise button, set
the degrees to 90, and click OK.
For images that are only slightly rotated, a rotation less than
90° can be specified in the Free field, but note that rotation
at angles that are not a multiple of 90° can change quantification results due to image interpolation.
9) Since the images do not need to be rotated for
this analysis, click the Undo button to return
the images to their previous orientation.
Odyssey has multiple Undo capability. Continuing to click
Undo will sequentially undo each image manipulation
made since the New Analysis window was opened.
21
Cropping an Image
In some cases you may scan several membranes at once. The Crop
tool is very useful for these types of images since you can crop out a
portion of the image and analyze each membrane in a separate
analysis.
Let’s assume that you want to crop out the first seven dots on both
rows of dots. On these images, there are dots that are not displayed
due to the brightness and contrast settings. Before starting the crop,
the Alter Image Display button can be used to change image
brightness, contrast, and sensitivity settings to find out where all the
dots are.
10)Click Alter Image Display
to open the Alter Image
Display window.
22 CHAPTER 3
Creating A New Analysis
On the image, green dots
correspond to the 800
channel and red dots are
in the 700 channel image.
A separate group of
image controls are
provided for each image.
Tip: Use the Show This
Image check boxes
when you want to display
only one image at a time.
11)Increase the
Linear Manual
Sensitivity sliders
until all dots are
visible.
12)Click OK.
The Linear Manual Sensitivity sliders change the way LI-COR 16-bit
TIFF images are mapped to the display. Eight different sensitivities are
available by moving the Linear Manual slider. The range of intensity
values on the original image determines which sensitivity might be
most appropriate.
In the case of our tutorial images, increasing the sensitivity on each
image reveals many dots that could not be seen at lower sensitivity.
In general, if bands or dots are missing on an image, increase the
sensitivity using the Linear Manual Sensitivity slider. If bands are just
dim, use the Brightness and Contrast sliders.
Now that all the dots are revealed on the tutorial images, let’s crop
out the first seven dots of each channel to include in the new
analysis.
23
13)Draw a selection rectangle
around the first seven dots in
both the 800 (green) channel
and 700 (red) channel. To
draw the selection rectangle,
click in the upper left corner,
hold down the mouse button,
and drag to the bottom of the
images – just to the right of
the seventh column of dots –
and release the mouse button.
Tip: Always leave empty
background around the edges of
the image when cropping an
image. Odyssey uses a variety of
labels that may not display
properly if the image is cropped
too closely.
14)Click Crop to crop out
the portion of the image
in the selection
rectangle.
15)Click OK to create the
new analysis using a
cropped portion of the
original images.
24 CHAPTER 3
Creating A New Analysis
Opening an Image View
When the new analysis is created it is added to the navigation tree
under the scan to which it belongs.
16)Double-click the Quant1 analysis in the
navigation tree to open the images.
17)Choose File > Save to save the Tutorial project.
When the images in a
new analysis are
opened for the first
time, both images are
displayed in the image
view with image
channels overlaid.
When the image view
is opened, many of the
tools on the toolbar are
activated so analysis
can be started.
With the image view open, you are ready to begin identifying
concentration standards. The next chapter describes how to quantify
the dots on the tutorial images. If you want to stop, you can resume
the tutorial later by opening the Tutorial project and double clicking
the Quant1 analysis again.
25
Chapter 4: Quantification
The quantification tutorial in this chapter begins where the New
Analysis Tutorial left off in the last chapter. The Tutorial project and
the Quant1 analysis should be open. If necessary, the project can be
reopened by choosing File > Open and selecting the Tutorial project.
The Quant1 analysis can be opened by double-clicking its icon in
the navigation tree.
Checking Settings
Before starting quantification, there are two settings to check.
1) Choose Settings > Application
to open the Application
Settings.
2) Select Display
Values from the
Settings List.
3) Select Concentration to display
calculated concentration values
when image objects are quantified.
26 CHAPTER 4
Quantification
4) Select Image View
Features from the
Settings List.
Image View settings
control the appearance of
annotations on the image.
5) Select the Quantification,
and Boundary check
boxes.
6) Select the Name, Quantification, and Boundary
check boxes.
When features are drawn around
dots or bands on the image,
different labels can be displayed
depending on whether the
features are selected or not.
Showing names for only selected
features reduces “screen clutter”.
These settings, as well as font size, can
be changed at any time during analysis
to improve how information is
displayed.
7) Select Save to close the
Image View settings.
27
Identifying Concentration Standards
Important: For scans with both 700 and 800 channel images,
concentration standards on one image cannot be used to quantify
bands on the other image. Concentration standards for both dyes
must be loaded and each image must be analyzed separately.
8) Begin by clicking
on the toolbar so only one image is
displayed in Single Channel mode. If the red dots of the 700
channel are displayed, click
to display the green dots of the
800 channel.
The scan, analysis, and image
name are all displayed in the
title bar.
On the tutorial image, the first and seventh dots are concentration
standards. The first dot is 10 pmole and the seventh dot is 0.5 pmole.
Two standards are required for quantification, but the more standards
you have the higher the accuracy will be.
Concentration standards are identified using a shape tool (rectangle,
circle, oval, or freeform shape) to draw a feature on the image that
encloses the standard. After the feature is drawn, it is selected and the
Concentration Standards window is used to enter the size.
Note: For images that have bands in lanes, the next chapter and the User Guide show
how to define lanes and use band markers in lanes for quantification, rather than
features drawn with shape tools.
28 CHAPTER 4
Quantification
Odyssey requires that concentration standards be added in order
(either lowest-to-highest or highest-to-lowest). On your own images,
you will want to start by visually identifying all the concentration
standards on the image. This will help you to add them in the correct
order.
9) Click the Circle tool on the left-side toolbar. A circle is the best
match for the fluorescence dots on the tutorial image (rectangle,
oval, and freeform tools are also available).
10)Draw a circle around the first dot on the left as shown below.
Imagine a bounding
rectangle surrounding
the dot and place the
cursor in the upper left
corner.
Click and hold down
the mouse button.
Drag downward
and to the right until
the circle encloses
the dot.
Release the mouse
button. If the circle is not
centered, it can be moved
as described below. ID:1
is an object name
assigned by Odyssey. n/a
means the concentration
is not assigned.
Centering Features
It is easiest to center features with the Details View open. The Details
View is a window that shows an enlarged view of the dot or band,
and a variety of statistics that will be used later in the tutorial.
29
11)Click
Cross hairs indicate the center
of the feature. The bounding
rectangle indicates the
borders of the feature. All
green pixels should be inside
the circle. The pixels on the
outside perimeter of the blue
rectangle are used to
calculate background. Before
quantifying your own samples,
read the section on using
Details View to verify the
background calculation
method in Chapter 8 of the
User Guide.
on the toolbar to open Details View.
Intensity
curves show
the intensity of
the pixels
underneath the
vertical and
horizontal
crosshairs.
12) After examining the cross hairs in the Details View, return to the
image view window. Center the circle, if necessary, by moving
the cursor into the center of the circle on the image until the
cursor has arrows in all four directions as shown below.
13) Move the circle by clicking and dragging it, or by using the arrow
keys on the keyboard to move the circle one pixel at a time. (The
arrow keys on the keyboard work only when the cursor is inside
the circle and the “all arrows” cursor is displayed.)
30 CHAPTER 4
Quantification
Resizing Features
If a feature (circle, etc.) is too large or too small, move the cursor
toward a corner until the cursor turns to a diagonal arrow as shown
below. With the diagonal arrow cursor displayed, the feature can be
enlarged or reduced by clicking and dragging.
Deleting Features: If you make a mistake and want to delete
the feature, select the feature and press the Delete key on the
key board or click
on the toolbar.
Naming the Standard
Odyssey automatically assigns an ID number to each feature, but on
reports it may be useful to enter your own name and description.
14) Make sure the circle is still selected and click
to open the Properties for the circle.
on the toolbar
15) Enter Std1 as the Name, 10 pmole as the Description and click
OK.
The name is now shown on the image. The description is
not shown because we did not enable Descriptions in
the Image View settings at the beginning of this chapter.
Tip: Short names and descriptions are preferable since bands and dots are often close
together on the images.
31
Entering the Concentration of the Standard
16) With the circle still selected, choose Analyze > Concentration
Standards or click
on the toolbar.
17)Make sure the
Channel is set to
800 to match the
image you are
analyzing.
18)Enter 10 as the
concentration
value and select
picomoles as the
units.
19)Click Add New to add the standard and
then OK to close the Concentration
Standards window.
32 CHAPTER 4
Quantification
20) Repeat steps 10-16 for the second concentration standard
(seventh dot from the left). In the Properties, name the standard
Std2 and enter a description of 0.5 pmole.
The image should
appear something
like this after the
second standard is
added.
21) Select Std2 and then choose Analyze > Concentration Standards.
22) Make sure the Channel is set to 800.
23) Enter 0.5 as the concentration value and leave the units set to
picomoles.
24) Click Add New to add the second standard.
After at two standards
have been added, the
integrated intensities of
the standards (X-axis)
are plotted against
concentration (Y-axis).
The interpolation
method used to fit the
data points was Linear.
The standards plot can
be used as a quality
control as described
below.
33
25) Click OK to close the Concentration Standards window.
The concentration values
are now displayed for
both standards. Any new
circles drawn around
dots of unknown concentration will be quantified
automatically, as you will
soon see.
Quality Control Using the Standards Plot
The purpose of the concentration standards plot is to look for
anomalous standards. Since the standards plot for the tutorial only
has two standards, there are not enough points on the plot to detect
errors.
Suppose, however, that there are four standards that plot as shown
below.
These standards plot in a fairly straight line if they have been
accurately assigned. Any standard that is out of position on the plot
may need editing. For this set of standards, if the straight line were
broken by a standard that is too high or too low, as shown below, you
may want to review the standard.
34 CHAPTER 4
Quantification
Standard #2 in the standards plot above appears to have a concentration value assigned to it that is too high. When reviewing an
anomalous standard, make sure the standard is fully enclosed by the
feature surrounding it and that the feature is centered over the
standard. Also make sure that the correct concentration value has
been assigned to the correct band or dot.
Quantifying Unknown Concentrations
Now that standards are defined, each of the remaining five dots can
be quantified by drawing circles around them. Quantification is
automatic and values are displayed immediately after the feature is
drawn.
26) Click the Circle tool and draw a circle around the second dot
from the left. Center the circle and resize it as needed.
35
27) To save time, press F5 on the keyboard to repeat the Circle tool
selection and draw another circle around the third dot from the
left.
Note: Selected features (circles, etc.) can also be copied and pasted.
28) Continue to use F5 and the Circle tool to draw circles around the
rest of the dots on the 800 channel image until it looks like the
image below.
Quantifying the 700 Channel Image
The dots on the 700 channel image have to be quantified just like the
800 channel image. One strategy is to choose Edit > Select All, copy
all the circles, switch to the 700 channel image, move the cursor to
the left most dot, and paste the circles (features paste starting at the
cursor position). In this case however, we will use the Add Multiple
Features tool to add seven circles with equidistant spacing.
29)Click
on the toolbar to switch to the 700 channel (red) image.
30)Select the Circle tool and draw a circle around the dot on the left.
The first feature is one end of the
line (straight or curved) along
which copies of the first feature
will be placed.
36 CHAPTER 4
Quantification
31)Click the Add Multiple Features tool (
) on the left toolbar.
Six features will be
added that are equally
spaced horizontally.
Features will be
named automatically
from left to right
starting with Spot_1
and ending with
Spot_7.
32)Six more circles need to be added to the one already on the
image, so set Number of Features to 6.
33)Select Horizontally so that all features are equally spaced in the
horizontal direction.
34)Click Continue to return to the image and move the cross hair
cursor until it is centered in the dot on the right side of the image.
37
35)Double click and six more circles will be added at equally
spaced distances.
36)The new circles are very near the fluorescence that they should
enclose, so the circles can be moved automatically into final
position by clicking
on the toolbar or choosing Analyze >
Adjust Feature Location.
The Adjust Feature Location
function finds fluorescence
near the feature and moves
the feature over it. Spot
finding accuracy can be
increased using the Application Settings as
described in Chapter 7 of
the User Guide.
37)Adjust the location of any circles as needed and choose Edit >
Deselect All.
38)Click the circle around the first dot on the left, choose Analyze >
Concentration Standards, add a new standard with 10 picomole
concentration, and click OK.
39)Click the circle around the seventh dot. Repeat step 38 and add
the second standard with a concentration of 0.5 picomole.
The concentration of the other five dots will be calculated automatically after the second 700 channel standard is entered. Quantification of both images is now complete. In the last part of this chapter
you will learn how to compare quantification data for various image
features and how to prepare reports.
38 CHAPTER 4
Quantification
Using Details View for Data Comparison
40)Click
on the toolbar so both images are displayed again with
channels overlaid.
41)If the Details View is not open, choose Analyze > Details View or
click
on the toolbar.
42)Click Clear in Details View to clear all the entries in the data
table.
43)Click one of the dots from the 800 channel (green) and one from
the 700 channel (red).
Each time an object is clicked on the
image, specifications for that object
are added to the table in Details
View. When channels are overlaid,
objects from both channels can be
compared.
The scroll bar underneath the table
can be used to view all the statistics.
Columns in the Details View can be
rearranged by dragging the column
headers to new positions.
44)Click Close to close the Details View.
39
Printing Quantification Reports
Now that you have seen how to compare quantification data on
screen, it’s time to learn how to output data in a report. Reports can
be printed to a printer or the data can written to a text file that can be
imported by most spreadsheet, database, or analysis programs.
Report Templates
All Odyssey reports use report templates. Report templates provide a
means of generating standardized reports. Templates also make it
easy to quickly print a report without having to designate which data
to include in the report. Two report templates for quantified image
features are included with Odyssey. The two are similar except one
prints data to a printer and the other saves data to a file. To learn how
to modify these templates or create your own, see Chapter 10 in the
Odyssey User Guide.
Printing a Report
45)Assuming the Quant1 analysis is
still open, choose Edit > Select All
to select all the circles on the
image.
Note: Only selected features are included in
reports.
40 CHAPTER 4
Quantification
46)Choose Report > Features.
47)Select Feature_Print
as the report
template.
The title for this report
automatically enters the
project name.
The report format is for
printing.
The Path field is not used
for printed reports.
48)Click Preview to display a
preview of the report.
49)If you are connected
to a printer click
Print All to print the
entire report, otherwise click Close.
For multi-page reports, the
Previous and Next buttons
can be used to page
through the report.
50)Click Cancel in the Report Features window to close the window.
41
Saving the Project
Projects should be saved periodically and after completing an
analysis.
51)To save the Tutorial project, choose File > Save.
Changes to the project will be abandoned if you don’t save the
project and ignore the warning messages when you close the
Odyssey program.
iii
43
Chapter 5: Quantification Using
Grids
In the last chapter, you learned two methods for quantifying dots on
dot blots with relatively low numbers of dots. Drawing features
around fluorescence on an image is practical when the required
number of features is low. It becomes less practical for scans of
microplates with 96 or 384 wells.
For scans where fluorescent dots are evenly spaced in a grid pattern,
Odyssey’s grid tool can be used to rapidly apply an array of circles or
rectangles to an image. The grid tools make it easy to scan microplates. A microplate alignment guide is included with Odyssey that
places a microplate in known scan position on the scan surface to
simplify repetitive scanning (see Operator’s Manual). The Microplate2
scan preset has scan dimensions and X,Y offsets that match the
alignment guide and many standard microplates.
In this chapter you will see how to apply a grid to a scan of a 96-well
microplate. First task is to import the tutorial images. Next, a grid
template that specifies the grid is applied to the images. Finally, the
the GridSheet tool can be used to examine the data in a table format.
Importing the Microplate Tutorial Images
The images needed for this tutorial are located on the Odyssey CD in
the Tutorial Images directory. The file names are T700.tif and T800.tif.
1) If necessary, open the Tutorial project created in Chapter 2 by
choosing File > Open and selecting the project.
44 CHAPTER 5
Quantification Using Grids
2) Choose File > Import Images.
3) Enter PlateScan as the new scan name and
GridAnalysis as the analysis name.
4) Click Browse for the 700
channel.
5) In the file selection
window, select a file
named T700.TIF in the
folder named Tutorial
Images of the Odyssey
CD. Click Open to select
the file.
6) Click OK to import the
two images.
The path to the 800 channel image
should be entered automatically.
Opening the Grid Analysis
Copies of the images are now stored in the Tutorial project. It is
usually a good idea to start another new analysis and analyze copies
of the original images, but for the tutorial we will analyze the
imported images.
45
7) Double-click GridAnalysis in
TiterScan to open the tutorial
images.
46 CHAPTER 5
Quantification Using Grids
Applying a Grid Template
The tutorial image is a scan of a 96-well microplate. There is a
standard grid template for 96-well microplates included with
Odyssey. Let’s begin by applying the standard 96-well template.
8) Click the grid tool (
) in the toolbar on the left side.
9) Select the 96_Well_Plate grid template from the drop-down list
and click OK.
47
The grid template controls where the grid is placed, the size of the
grid, whether the grid is composed of circles or squares, and the size
of the circles or squares. When the grid template is applied to new
scans of microplates, the initial grid placement and size will match
the microplate image closely, but may require some adjustment for
your particular instrument. The images for the tutorial are intentionally offset so you can learn how to change templates and resize
grids.
Since the default 96-well grid does not match the image very well,
let’s delete the grid, modify the template, and apply a new grid that
more closely matches the image.
10) Click one of the grid lines to select the grid. The entire grid should
turn yellow.
11) Click
to delete the grid.
12) Click the grid tool (
) again. Select the 96_Well_Plate template
from the drop-down list and click Modify.
You can also modify grid
templates by choosing Settings >
Grid Templates.
48 CHAPTER 5
Quantification Using Grids
Our image has eight rows and twelve columns, so the Grid Size
fields do not need to be modified.
Since typical 96-well microplates have 9.0 mm well spacing, the
vertical and horizontal grid spacing should match the tutorial image.
To make the grid fit the tutorial image, the X,Y offset needs to be
adjusted so the grid is properly placed over the wells, and the size of
the wells should be increased.
13) Change the X Offset to 20 mm and the Y Offset to 13.
14) Change the Quantify Well Diameter and Physical Well Diameter
to 7.0 mm.
49
15) Click Save As to save the changes under a new name. Name the
template TutorialGrid.
Note: The Odyssey User Guide explains how grids can be moved, enlarged, reduced,
and rotated using the mouse.
16) Select TutorialGrid from the drop-down list and click OK.
The grid is still slightly
offset. Final positioning
can be accomplished
using the mouse or
arrow keys.
50 CHAPTER 5
Quantification Using Grids
17) Click the grid to select it. Then move the cursor over one of the
lines on the interior of the grid. The cursor should change to the
“all arrows” cursor.
The arrow keys can be
used to move the grid
one pixel at a time as
long as the “all arrows
cursor is displayed and
the grid is selected.
18) With the “all arrows” cursor displayed, click and drag the grid so
it is in the best possible alignment with the image.
Now that the grid is in place, any individual circles that are still out
of position can be moved to complete the grid placement.
19) Click a circle that is misplaced to select it (turns yellow).
Tip: Multiple features can be selected by
holding down the Control key and clicking
additional features, or by dragging a
selection rectangle around multiple
features.
20) Move the cursor to the center of the circle until the “all arrows”
cursor is displayed.
21) With the “all arrows” cursor displayed, click and drag the circle
until it surrounds all the fluorescence on the image.
The arrow keys can also be used to move a selected circle one pixel at a time as long
as the “all arrows” cursor is displayed.
51
Viewing Data in the Grid Sheet
Grid features are usually closely spaced, so labels showing data
values are not displayed. A “Grid Sheet” is available to view
integrated intensity values from each feature in the grid.
22) Click
to open the Grid Sheet.
The rows and columns of
integrated intensity
values are arranged in the
same order as the rows
and columns of features
in the grid (A1 = A1, etc.).
The Channel drop-down
list can be used to view
data for a different image.
23) After examining the data, click the close box on the window
frame to close the Grid Sheet.
Quantification Using Grids
Now that all features in the grid are properly positioned, quantification can proceed as described in Chapter 4. The individual steps
are not shown here, but you may want to quantify the image on your
own by assigning some concentration standards (pick any wells).
Concentration standards are assigned by selecting the individual
features and choosing Analyze > Concentration Standards (steps 16-19
in Chapter 4). After the concentration standards are assigned,
concentration values are automatically calculated for all other
52 CHAPTER 5
Quantification Using Grids
features in the grid. Concentration values can be viewed in the Grid
Sheet or by generating a feature report (Chapter 10, Odyssey User
Guide) that lists the concentrations for each feature.
In-Cell Western Assays
The In-Cell Western (ICW) Module for Odyssey Software has another
option for applying grids. Grids can be applied automatically by
choosing In-Cell Western > Align Grid.
When a grid is applied, the ICW calculations are performed automatically using the ICW template that was last used. ICW data can be
displayed in table format by choosing In-Cell Western > View ICW
Analysis (the data are meaningless for the tutorial images). ICW
templates can be created or changed by choosing In-Cell Western>
Change ICW Parameters.
If you have the ICW Module for Odyssey Software and would like to
learn more about configuring Odyssey for In-Cell Western assays,
Chapter 9 in the Odyssey User Guide describes the software and the
Odyssey Applications Manual contains protocol examples.
What’s Next...
In the next chapter you will import another set of images and use
those images for band sizing. If you are not interested in band sizing
applications, you may want to read Chapter 7 to learn how to start
scans from the Odyssey front panel keypad.
53
Chapter 6: Sizing Bands
Overview
For band sizing applications, Odyssey Software allows two samples
labeled with different IR dyes to be loaded into the same lane and
imaged separately. Unlike concentration standards, size standards
need to be run in only one of the two image channels. Odyssey has
a unique method for applying size standard information from one
image channel to the other.
Sizing begins by adding lanes to the image with both image channels
overlaid. With channels overlaid, lanes are added to both images
simultaneously and in identical positions. Band finding is automatic
as soon as lanes are found.
Identification of size standard bands is performed separately on each
image while viewing only one of the two image channels. Size
standard lanes are identified and the sizes of all standards in the lanes
are entered. After size standards are identified, all other bands are
sized automatically. The last step is to apply the size standard information from one image to the other. As soon as the standards are
applied to the second image, all the bands are automatically sized.
Though band sizing and quantification are not always performed on
the same image, they can be in Odyssey. If size standards and
concentration standards are both run in the same gel, bands can be
both sized and quantified in the same analysis. Once lanes are
defined for an image, Odyssey allows the band markers to be
selected and used as shape objects for quantification in the same way
circles were used in the quantification tutorial (Chapter 4).
54 CHAPTER 6
Sizing Bands
Importing Images Into the Tutorial Project
The images for this chapter are located on the Odyssey CD in the
Tutorial Images directory. The file names are S700.tif and S800.tif.
First, you will import the images into the Tutorial project and then
analyze them in a new analysis.
1) If necessary, open the Tutorial project created in Chapter 2 by
choosing File > Open. (The most recently used projects are also
listed at the bottom of the File menu.)
2) Choose File > Import Images.
3) Enter SizingScan as the new scan name and Original_Analysis as
the analysis name.
4) Click Browse for the 700
channel.
5) In the File Selection
window, select the file
named S700.TIF in the
Tutorial Images folder of
the Odyssey CD. Click
Open to select the file.
6) Click OK to import the
two images.
55
Checking the Settings
Before starting band sizing, choose which labels will be displayed
using the Application Settings.
7) Choose Settings > Application and select Image View Features
from the Settings List.
8) Select the Name and
Boundary check boxes.
9) Select the Name,
Molecular Weight, and
Boundary check boxes.
For each lane and band marker that
will be created, various annotations
can be displayed depending on
whether the lane or band marker is
selected or not. Controlling which
annotations are displayed, as well as
the font size, can decrease “screen
clutter”.
10)Select Save to close the
Application Settings.
56 CHAPTER 6
Sizing Bands
Starting a New Analysis
Images can be displayed individually in single channel mode or
overlaid as a composite image. When an analysis with two images is
opened for the first time, the images are overlaid so lanes can be
added. Adding lanes with images overlaid is more efficient since
lanes are added to both images at once. After adding lanes, each
image must be analyzed separately in single channel mode.
11)Click SizingScan in the list
of scans for the tutorial
project or Original Analysis
within SizingScan.
12)Create a new analysis in
SizingScan by clicking
on the toolbar or choosing
File > New Analysis.
57
13)Enter Sizing as the Name
for the new analysis.
14)Make sure Original Analysis
is selected (it contains the
images we want to copy).
15)Make sure both the 700
and 800 check boxes are
selected.
16)Click OK.
17)Double click the Sizing
analysis in SizingScan.
58 CHAPTER 6
Sizing Bands
Now that the images are displayed at full size, the image intensity
can be accurately changed.
18) Click
on the toolbar to open the Alter Image Display window.
19)Adjust the Linear
Manual Sensitivity for
each image until the
bands are clearly
visible.
Sensitivity values of 4 (700
channel) and 7 (800 channel)
should be about right for these
images.
20)Click OK.
Adding Lanes
On the gel that was used to create the tutorial images, the first and
last lanes were loaded with molecular weight (MW) markers. The five
lanes in the middle are loaded with samples. At the top of the gel,
some lanes have fluorescence that is an artifact of electrophoresis
and not part of the sample or MW marker lanes. These artifacts can
be excluded from analysis by starting the lanes below the artifacts.
59
21) Scroll the image so all bands are displayed as shown below.
22) Click the
(add lane) tool in the left toolbar.
23) Move the cursor above the highest MW marker at the top of the
first lane. Center the cursor in the lane and click as shown below.
Click Here
60 CHAPTER 6
Sizing Bands
24) Double-click at the bottom-center of the lane, as shown below.
Double-clicking finishes the
lane finding operation.
Straight lanes (vertical or
slanted) need only two points
to define the shape of the lane.
For lanes that are curved,
additional points can be
added as described in the
Odyssey User Guide.
After the lane is created, lane
boundaries are displayed and
band markers are automatically place around all bands
that have been found.
Double-Click Here
If you make a mistake and need to delete the lane, click the lane to
select it and click
on the toolbar.
After a lane is created, it is important to check both the lane finding
and band finding.
Moving and Resizing Lanes
Always check each lane to make sure it is centered over the lane on
the image and that the lane boundary is wide enough to enclose all
the bands in the lane.
61
Moving Lanes
25)Move the lane that was just added by moving the
cursor over the center line until the “all arrows” cursor
is displayed and the center line changes to a white
dashed line. Click and drag the lane until the center
line of the lane is centered over the lane on the image.
Tip: If the lane is slanted compared to the lane on the image, either
of the two end-points of the lane can be moved by clicking and
dragging the point.
Changing Lane Width
The band markers should fully enclose the bands on the image, so
the lane should be slightly wider than the bands. The new lane may
be too wide or narrow because the lane settings do not match the
lane width on the image. If you use a certain size comb on your gels,
you can change the default lane width to match the comb (Application Settings). Even if the lane is correctly sized, try the steps below
to learn how to resize lanes.
26)Move the cursor over the lane boundary on the right
side until the cursor changes to a right-left arrow
cursor. Click and drag the lane boundary to the right
until all the bands on the right side of the lane are
within the lane border.
27)Using the same technique, move the left boundary to
the left until all the bands on the left side of the lane
are within the lane border.
Tip: Both boundaries can be widened at once by selecting the lane,
clicking the Properties button (
), and selecting the Symmetric Left
and Right Boundaries check box.
62 CHAPTER 6
Sizing Bands
The other lanes could be added by repeating the steps above,
however, when all lanes have similar shapes it is more efficient to
copy and paste the existing lane. On gels with distorted lanes (smiles,
etc.) each lane may need to be created individually.
28) If necessary, click the first lane to select it and press Control + C
on the keyboard to copy the lane.
29) Move the mouse pointer to the top-center of the second lane as
shown below.
30) Press Control + V on the keyboard to paste a new lane at the
position of the mouse pointer. Bands are found automatically on
both 700 and 800 channel images.
Adding Multiple Lanes
You could continue to paste the other five lanes, but the Add Multiple
Lanes tool (
) can be used to add all five lanes at once. The Add
Multiple Lanes tool works best with images that have straight, vertical
lanes. In our tutorial example, all seven lanes could have been added
using the Add Multiple Lanes tool, but we added lanes manually as
an exercise.
31) Start by clicking (
) in the left toolbar.
63
32) Enter 5 as the number of lanes to create and click OK.
33)Move the cursor above the third lane and center it in the lane as
shown below.
34)Click and drag until the cursor is at the bottom of the last lane on
the right with the cursor centered in the lane as shown below.
64 CHAPTER 6
Sizing Bands
35)Release the mouse button and five vertical lanes will be created
at evenly spaced intervals. If necessary adjust the width of each
lane individually.
All lanes should now be marked and bands should be enclosed by a
rectangular band marker. In some cases, however, there are too many
or two few band markers, or they may not be placed in optimal
positions. The next step is to edit the band markers.
65
Editing Band Markers
Band Finding Threshold
In some cases new sample lanes may have too many bands (e.g.
lanes 2 through 6 on the tutorial image). In other lanes, there may be
too few bands (the weak band at the bottom is not marked). This is
due to the default setting for band finding threshold. Later in this
chapter you will see how band finding threshold controls the number
of bands that are found in a lane.
Band finding threshold will vary when scanning membranes or gels
from different manufacturers. The amount of background fluorescence influences the threshold settings. As you gain experience, you
will empirically determine a band finding threshold that is optimal
for your membranes and samples. The default value of the band
finding threshold can be changed by choosing Settings > Application
and then selecting Lane from the Settings List.
Adding and Resizing Band Markers
Band markers can only be edited in single channel mode, so before
editing you must switch to single channel mode.
36)Click
to switch to single channel mode and then click
to
switch from the 700 channel image (red) to the 800 channel
image (green image with the MW marker bands on it).
66 CHAPTER 6
Sizing Bands
In each of the two MW marker lanes, there are nine marker bands,
but the faint band of the lowest marker was not found during
automatic lane finding. There are two techniques in Odyssey for
adding band markers. To see how they both work, each lane will be
corrected using a different technique.
37)Click the add band marker tool (
) in
the tool bar and click in the center of the
faint band at the bottom of Lane 1.
If you cannot see the band, use the brightness and
contrast controls (
) to make the band more
prominent.
Odyssey has a lane profile tool that allows you to view fluorescence
curves for a lane that is selected. The next step is to use the lane
profile to check the new band marker that was added and to review
the other eight bands.
38)Click the boundary of lane 1 to select the lane and then choose
Analyze > Lane Profile.
See Chapter 5, Odyssey User Guide for a thorough discussion of the
Lane Profile window.
67
39)Examine each peak to make sure
band markers are centered. The
'+' symbol should be at the apex
of the peak if the band marker is
properly centered.
The vertical magenta lines
are the boundaries of the
band and are in the same
location as the band markers
on the image.
Notice that the new
band marker (band 9)
does not cover the
entire peak. The
band marker should
be enlarged as
shown below.
The left side is the
top of the lane and
the right side is the
bottom.
40)Click OK.
If you need to move a band marker to center it, move the cursor to
the middle of the band marker until it turns to an all-arrows cursor.
Click and drag the band marker until it is centered.
68 CHAPTER 6
Sizing Bands
41)Click on Band 9 to select it.
42)Move the cursor over the bottom
boundary of the band marker until it
changes to an up-down arrow cursor.
43)Click and drag the lower boundary of
the band marker until it is enlarged
enough to enclose the entire band.
44)Repeat steps 34 and 35 for the upper
boundary of the band marker.
45)Click on Lane 1 to select it and choose
Analyze > Lane Profile again. The band
markers on the lane profile and the
image should appear like those shown
to the right.
46)Click OK to close the Lane Profile
window.
The second technique for adding (or deleting) bands is to use the lane
profile window to interactively change the band finding threshold
until the correct number of bands are displayed. This technique is
particularly useful when you are trying to find the optimum threshold
for a particular brand of membrane or gel formulation. After
changing threshold for lanes on a number of membranes you will
eventually determine a threshold value that produces good results for
most scans. This number can be entered as the default band finding
threshold by choosing Settings > Application and then selecting Lane
from the Settings List.
47)Click Lane 7 to select it.
69
48)Choose Analyze > Lane Profile.
49)Move the Threshold slider from the default value of 10 to
about 17. Watch the last peak on the profile. As the threshold
nears 17, the ninth band should be found.
Increasing the Threshold
value will find more
bands and decreasing
the threshold finds fewer
bands.
50)Click Apply to see the new band on the image. (The new band
is also added to the image if you click OK.)
51)Click OK to close the lane profile.
70 CHAPTER 6
Sizing Bands
Deleting Band Markers
In Lane 6, too many bands were found. The upper band marker is
placed over some minor background fluorescence due to an electrophoresis artifact. If you want to verify the lack of fluorescence at this
location, select the lane and open the lane profile window. Since
Band 1 in Lane 6 is a false band it should be deleted.
52)Click on Band 1 in Lane 6 to select it.
53)Click
on the toolbar to delete the band.
TIP: The band can also be deleted by pressing the Delete key on the keyboard or
by using the Lane Profile window to decrease band finding threshold for Lane 6.
54)Edit the remaining four sample lanes as needed.
Depending on the default threshold settings, you may have lanes
with bands that need to be deleted or others that need to have bands
added.
71
When you are finished editing the 800 channel image, there should
be one band in lanes 3-6, no bands in lane 2, and nine bands in both
lanes 1 and 7.
Now that the 800 channel image has been edited, the 700 channel
image should also be edited.
55)Click
to switch from the 800 channel image to the 700
channel image (red).
72 CHAPTER 6
Sizing Bands
On the 700 channel image, there are only three bands, located in
lanes 2, 4, and 6.
56)Delete any extra bands or add any missing bands. Use the lane
profile window, if necessary to verify which band markers should
be deleted. Do not delete any empty lanes.
57)Click
to switch from the 700 channel image back to the 800
channel image so the molecular weight markers can be
identified.
73
Entering the Size of Each MW Marker
The nine molecular weight marker bands in lanes 1 and 7 have been
found, but the size of each marker still needs to be entered. Marker
sizes can be entered individually or loaded as a set. The Odyssey
User Guide describes how to save sets of markers, which are efficient
for repetitive scanning because all markers can be loaded at once.
For this tutorial, all nine marker sizes will be entered individually.
58)Choose Analyze > Edit Size Standards to begin adding MW
markers.
59)The size of the lowest standard is 154 bp, so
enter 154 in the New MW Value field.
60)Click Add MW Line.
A MW line is displayed on
the image that will connect
all the MW standard bands
of a particular weight (154 bp
in our example). The
placement of the line is not
important, yet. In later steps
the line will be snapped into
position over the molecular
weight bands it represents.
74 CHAPTER 6
Sizing Bands
61)Repeat steps 59 and 60 for each of the following sizes: 234, 298,
453, 653, 1033, 1230, 1766, and 2176.
Important: The total number of standards you define must exactly
match the number of bands in the MW standards lanes.
After all the standards are added, all the MW lines will be displayed
at the top of the image as shown below.
Now the lines need to be linked to specific MW bands in standards
lanes. The first step is to select the lanes that contain the MW marker
bands.
Linking MW Lines to MW Marker Bands
62)Click the Select Lanes radio button so the MW marker lanes can
be selected.
75
63)Select Lane 1 on the 800 channel image by clicking it.
TIP: Lanes are selected by clicking the center line, but the center line is often covered
by band markers. Clicking near the top or bottom is usually easier. When the lane is
selected, the dashed center line turns to the highlight color (yellow).
64)Hold down the Control key and click Lane 7 so both MW
standard lanes are selected.
Important: On images with more than two MW standards lanes, all MW standard lanes
must be selected.
65)Click the Snap To Lane button in the Edit Size Stds window.
76 CHAPTER 6
Sizing Bands
Reshaping and Moving MW Lines
The MW lines should now be properly positioned over the MW
standard bands. A movable control point (square) is added over each
standard band in the middle of the MW standard lanes. The control
points should be placed in the middle of each MW standard band.
When control points are out of position, they can be moved by
clicking Modify Points in the Edit Size Stds window and then clicking
and dragging points that need to be moved. Whole lines can be
moved by clicking Modify Lines in the Edit Size Stds window and
then clicking and dragging a MW line into position. (Multiple lines
can be selected by holding down the Control key while clicking
additional lines.)
In the tutorial images, MW lanes are straight and nearly horizontal.
On large gels with smiles, the lines may not follow the contour of the
gel very well because there are too few control points. It is important
that the MW lines follow the contour of the gel. Control points can
be added and moved by clicking Add Points in the Edit Size Stds
window as described in Chapter 6 of the Odyssey User Guide.
Applying MW Lines to Both Images
At this stage of analysis, the new MW lines are shown on the image,
but have not been permanently applied. You have the choice to apply
the MW lines to only the current image or to both images using the
Apply or Apply to Both buttons in the Edit Size Stds window (respectively). Apply is used when you are only analyzing one image or
77
when each image has its own MW markers. Apply to Both is used
when you want to use MW markers on one image to size bands on
both images, as is the case with the tutorial images.
66)Click Apply to Both to apply the MW lines to both the 700 and
800 channel images.
67)Click OK in the warning window to proceed.
When Apply to Both is clicked, all the bands on both images are
automatically assigned a size.
68)Click on any band marker to display the size of the band.
At the beginning of the chapter, you changed the Application Settings
to display MW only on selected shapes. If you would like to see the
MW of all bands at once, choose Settings > Application, select
Image View Features from the Settings List, select the Molecular
Weight check box under "Show for Unselected Shapes", and click
Save.
78 CHAPTER 6
Sizing Bands
Plotting Size Standards
Plotting the standards gives you a means of checking the standards in
each lane. Using the size standards plot, you can set the interpolation
method for the plot and verify that the standards are accurately
assigned.
69)Click on any lane to select it.
70)Choose Analyze > Size Standards to open a plot of the sizing
standards.
The graph plots MW on the
Y-axis vs. scan line where the
band center is located on the
X-axis. (A scan line is one row
of image pixels. Scan line
number one is at the bottom of
the image.)
71)Select Std1 Channel
700 (if necessary) to
review the standards
in the first lane on the
700 channel image.
72)Set the Units for
the standards to
basepairs, if
necessary.
73)Set the Interpolation
Method to Reciprocal
Fit, which provides the
most accurate fit for
these standards. (See
User Guide for details).
Note: Units and Interpolation Method apply to all lanes and don’t have to be
set individually for each lane.
79
74)Select each of the remaining three standards lanes from the Lane
list and examine the standards plot.
Look for anomalous bands. For example, in the standards plot for the
tutorial images, if the smooth curve were broken by a band that is too
high or too low, the position of that band on the image should be
reviewed. Check any standard that is out of position to make sure the
band marker is centered and that the correct MW has been assigned
to the standard.
75)Select each sample lane from the Lane drop-down list and
examine the standards plots for each lane.
For sample lanes, molecular weight standards are located where the
MW lines cross the center lines of lanes. For gels with even band
migration (straight molecular weight lines), the plots of the sample
lanes will be very similar to the standards lanes. On the tutorial
images, all sample lanes have similar plots and there should not be
any misplaced standards.
For gels with smiles or other gel artifacts, extra control points are
added to bend the MW lines in a particular direction which can
cause standards to be out of position. Close examination of the
standards plot for each sample lane is needed on images with smiles.
(See the Odyssey User Guide for complete details.)
76)Click OK to close the Size Standards window.
Sizing is now complete. A report can now be generated for the MW
data from each lane.
Generating Lane Reports
Lane reports are similar to the feature reports described in Chapter 4.
They can be printed or output to a data file. Lane reports also rely on
templates to create standardized reports.
80 CHAPTER 6
Sizing Bands
Two report templates for lanes are included with Odyssey – one
prints data to a printer and the other saves data to a file. See Chapter
10 in the Odyssey User Guide for additional details.
Outputting Lane Data to a File
Suppose you have a database that is used to import lane data from
tab delimited text files. In this example, you will make some minor
modifications to the Lane_Data template and then output the lane
data to a file that is database and spreadsheet compatible.
77)Click
to display both images again.
78)Choose Edit > Select All to select all the lanes and band markers
on the image.
Note: Only selected lanes and bands are included in reports.
79)Choose Report > Lanes.
80)Select Lane_Data as
the report template.
The report format is for file
generation.
81)Click Edit to display
the Lane_Data report
template.
81
82)Select File Export, if
necessary, to export report
data to a file.
83)Change the default
file name to
LaneData.txt.
84)Verify the Field
Separator is Tab for
tab delimited data.
85)Select Sort Output
and Ascending.
86)Change the By Field
to Lane Name to
group all bands for a
given lane when
sorted.
If you would like to view all the
fields that will be included in
the report, scroll through the
Fields In Use list.
87)Click Save to save
changes to the report
template.
82 CHAPTER 6
Sizing Bands
88)Create the report file by
clicking OK in the Report
Lanes window.
Saving the Project
Sizing is now complete and all the data have been exported to a
report file. The data can be viewed with any text editor. Before going
any further, now would be a good time to save the Tutorial project by
choosing File > Save.
Continuing the Tutorial
The next chapter concludes the Odyssey tutorials by showing you
another way to start scans using the buttons on the Odyssey front
panel.
83
Chapter 7: Starting Scans From the
Odyssey Front Panel
Starting Scans
At times it may be easier to start scans from the Odyssey front panel
rather than from Odyssey Software. Front panel scans use Preset
parameters and automatic file naming conventions to minimize the
steps needed to start a scan.
Scan Procedure
1) Press the Start key on the Odyssey front panel.
2) Press the Next key until the Membrane Preset is shown on the
top line of the Odyssey front panel display.
Note: There are two groups of Presets – those stored in the Odyssey Imager and those
stored on the computer with Odyssey Software. Only Presets stored in the Odyssey
Imager are displayed when starting a scan from the front panel. See the Odyssey
Operator’s Manual for information on saving Presets in the Odyssey Imager.
3) With the Membrane Preset displayed, press the Start key.
The scan is started in a paused state so that you can place the
membrane or gel on the scanning surface. (Techniques for placing
membranes and gels are given in the Odyssey Operator’s manual.)
While the word Paused is displayed on the front panel, the instrument
is reserved for your scan and no other user can start a scan.
84 CHAPTER 7
Starting Scans From The Odyssey Front Panel
4) Place the membrane on the scan surface within the area specified
in the Preset.
Scan Dimensions
The scan dimensions are part of the Preset. The scan dimensions are
10 x 10 cm for Membrane, DNAGel, and ProteinGel presets. The
Microplate preset uses a scan area of 13 x 9 cm. See the Odyssey
Operator’s Manual for how to change preset scan dimensions.
5) Press Start to begin the scan. If you need to abort the scan, press
the Stop key instead.
File Naming Conventions
All scans started from the Odyssey front panel are stored in the public
scan group on Odyssey’s internal hard disk (see the Odyssey
Operator’s Manual for an explanation of scan groups). Unlike your
own scan group, scans in the public scan group are accessible by any
user.
Scan files are automatically named when the scan is started. The
format of the scan name is XXXXXXX-MM-DD_N, where XXXXXXX
is the first seven characters of the Preset name, MM is the month, DD
is the day, and N is a number that increments for every scan on a
given day and resets at the end of the day.
Reviewing Scan Status
6) While a scan is in progress, information about the scan can be
reviewed on the front panel by repeatedly pressing the Next
key.
85
Five different parameters can be displayed, including time remaining
in the scan, percent complete, the scan group that the file is being
saved into, IP address of the scanner, and the address of the network
card in the scanner.
Stopping a Scan
When the scan area specified in the preset has been scanned, the
scan will terminate automatically. If you need to stop a scan before
automatic completion, press the Stop key. Pressing Stop returns
you to the Paused state where you can press Stop again to end the
scan. When the scan is stopped, all files are closed and saved. Any
scan data collected before Stop was pressed are saved.
Downloading Files for Analysis
In order to analyze scans started on the Odyssey front panel, the scan
files stored in the Odyssey Imager must be imported into an existing
project using the Download Scan function.
1) Open the Tutorial project created in Chapter 2 (if necessary).
2) Choose File > Download Scan.
3) Select the Scanner (if
necessary).
4) Enter your user name
and password (case
sensitive).
5) Click OK.
86 CHAPTER 7
Starting Scans From The Odyssey Front Panel
6) Select public from the
Scan Groups drop-down
list).
7) Select the file that was
just scanned. The name
starts with Membran
followed by today’s date.
8) Click OK.
After selecting a scan and clicking OK, the selected scan will be
downloaded into the open project. The scan will be listed in the
navigation tree on the left side of the Odyssey window. Analysis can
proceed normally after the scan is downloaded.
Download PDF
Similar pages