Nuclei Tracking

PARAMETER UPDATE TUTORIAL
Nuclei Tracking
The Nuclei Tracking recipe for CQ accurately detects nuclei and tracks their motion and
division in a fluorescence time-lapse image sequence. The recipe can be easily customized to
your needs through updates using the Recipe Console interface. In this tutorial, you will learn
how to update recipe parameters to optimize results using the Recipe Console interface.
(Left) Fluorescent Nuclei Tracking output with tracks and lineage overlay. (Right) Recipe Console with basic parameters displayed.
GETTING STARTED
Before starting this tutorial, please make sure you have the CQ module installed with the Nuclei Tracking recipe available. Please
contact your sales representative or visit www.cq-bioanalysis.com for more information about the CQ Recipe products.
We will demonstrate how to update recipe parameters in the Nuclei Tracking recipe
using the Recipe Console to optimize image analysis results. The Recipe Console is a
standard feature on CQ and we encourage you to explore its functionality and follow
along. The tutorial is split into two parts - update basic parameters only and update
basic and advanced parameters - with corresponding images described below.
lamin-RFP002-xy1_cropped_input.tif, on right, top: Nuclei stained with TRITC
is imaged and tracked over time. False detection can be removed by adjusting the
Minimum nuclei size basic parameter in Recipe Console.
Hela-H2B_40xoil_crop.tif, on right, bottom: Nuclei of HeLa cells expressing fusion
H2B-EGFP protein is imaged and tracked over time. H2B is a histone protein that
plays a role in the structure of chromatin within the nucleus. Nuclei detection can be
improved by modifying the Fill hole size parameter to close large holes in detection.
http://www.cq-bioanalysis.com
lamin-RFP002-xy1_cropped_input.tif
Hela-H2B_40xoil_crop.tif
SALES AND CONTACT INFORMATION
Sam Alworth, Director of Marketing
DRVision Technologies LLC
15921 NE 8th Street, Suite 200
Bellevue, WA 98008
Tel: (425)653-5589 | Fax: (425)746-0859
BASIC PARAMETER UPDATE TUTORIAL
1.
Load image
Go to File > Open and select the tutorial image file (lamin-RFP002-xy1_
cropped_input.tif ) and click Open to load image into CQ.
2.
Select recipe from Recipe Console
Choose the Nuclei Tracking recipe from the “Select recipe” pull-down menu
in the Recipe Console. Recipe steps will automatically populate the Recipe
Console panel.
3.
View available parameters in the Recipe Console
By default, parameter adjustment options are hidden in the Recipe Console.
Each step with available parameters will be indicated by the
icon. To view
the parameters in a single step, double-click on the header of the Recipe step.
You can view all the available parameters in the recipe by clicking on the
Expand all button below the recipe selection pulldown menu. To hide all
parameters, click on the Collapse all button.
4.
Recipe Console
The Recipe Console is designed to guide you through the image analysis
recipe. Image processing is broken down by steps and shown as a blue header
bar in the Console. There are four recipe step options.
Toggle parameter options | Toggle advanced/basic parameters
? Show description | Display step description dialog
Apply step | Apply recipe step to displayed image
Launch wizard | Launches procedure update wizard interface
http://www.cq-bioanalysis.com
SALES AND CONTACT INFORMATION
Sam Alworth, Director of Marketing
DRVision Technologies LLC
15921 NE 8th Street, Suite 200
Bellevue, WA 98008
Tel: (425)653-5589 | Fax: (425)746-0859
BASIC PARAMETER UPDATE TUTORIAL
5.
Apply recipe steps to image
Image background is removed and signal is enhanced in the Enhancement
step. The nuclei detection mask is generated in the Detection step. The nuclei
detection mask is partitioned in the Nuclei Separation step. Click the Apply
step button
to view initial processing result.
You can skip ahead to the Nuclei Separation step by clicking on the Apply
step button in the Nuclei Separation step header in the Recipe Console. All
previous recipe steps will be applied to the image.
6.
View initial processing result
When you apply a step to the image, the processing step is applied to the
entire sequence. The processing result is displayed in the “IndividualNuclei”
mask on the Downsampled channel.
Next we will apply the Subset Filtering step to the image.
http://www.cq-bioanalysis.com
7.
Apply Subset Filtering step to image
The Subset Filtering step provides several parameters to allow the user to
remove objects from the image based on certain characteristics. One of the
filtering parameter is minimum nuclei size, which removes false detections
that falls below the specified size threshold.
8.
View filtered result
Use the Play button below the image to view the entire sequence. On Frame
9, there is a large object mask between the two nuclei in the upper left
corner (pink rectangle) that was not removed using the minimum nuclei size
parameter. We can adjust the Min nuclei size parameter to remove that mask
and improve the detection accuracy.
SALES AND CONTACT INFORMATION
Sam Alworth, Director of Marketing
DRVision Technologies LLC
15921 NE 8th Street, Suite 200
Bellevue, WA 98008
Tel: (425)653-5589 | Fax: (425)746-0859
BASIC PARAMETER UPDATE TUTORIAL
9.
Modify parameter “Min Nuclei Size”
The false detection on Frame 9 could introduce error to the tracking
procedure as the algorithm will determine the object to be a valid “nuclei”.
Unhide the Subset Filtering parameter options by double-clicking on the step
header or clicking the Expand all button if the options are not displayed.
Click on the Min nuclei size parameter textbox and enter ‘120’ into the
textbox. You can also use the up and down arrow to fine-tune the parameter
value. To revert to original value, click on the Revert button to the right of
the parameter textbox.
10. Preview update result
Press Enter after you have input the updated parameter value. A preview of
the result is generated on the image. Compared to previous output, the false
detection between the two nuclei in the upper left corner is removed. Click
on the Apply step button in the header of the Subset Filtering step to
finalize the changes.
11. Run recipe
Click on the Run Recipe icon to apply the updated recipe to the image.
Please refer to the Data Analysis Tutorial for instructions on data viewing and
data export.
The track output are overlaid on the image. You can change the track
appearance in the pop-up dialog launched when the Track display options
button
on the toolbar is clicked.
12. Save changes to recipe in the Recipe Manager
To save the updated recipe, click on the Recipe Manager button below the
recipe selection pulldown menu to launch the Recipe Manager dialog. The
modified recipe is be selected automatically. Click Save to rename and save
the recipe to the recipe library. The updated recipe will be loaded into CQ
automatically when launched.
http://www.cq-bioanalysis.com
SALES AND CONTACT INFORMATION
Sam Alworth, Director of Marketing
DRVision Technologies LLC
15921 NE 8th Street, Suite 200
Bellevue, WA 98008
Tel: (425)653-5589 | Fax: (425)746-0859
ADVANCED PARAMETER UPDATE TUTORIAL
1.
Load image
Go to File > Open and select the tutorial image file (Hela-H2B_40xoil_crop.
tif ) and click Open to load image into CQ.
2.
Select recipe from Recipe Console
Choose the Nuclei Tracking recipe from the “Select recipe” pull-down menu
in the Recipe Console. Recipe steps will automatically populate the Recipe
Console panel.
3.
Apply Detection step to image
The Detection step generates a nuclei region detection mask to detect bright
nuclei in the image. The detection is output to a mask on the original channel
image. Click the Apply step button on the Detection step header to view
results.
4.
View Detection result
Click on the “Downsample” channel tab located below the image to switch
view to the downsampled channel. To view the detection mask, click on the
“NucleiRegion” mask tab located above the image.
Click on the Play button below the image sequence to view detection result
on the image sequence. On the first frame, parts of several nuclei are not
detected (pink rectangle). We can increase detection by reducing the Contrast
threshold parameter in the Detection step.
http://www.cq-bioanalysis.com
SALES AND CONTACT INFORMATION
Sam Alworth, Director of Marketing
DRVision Technologies LLC
15921 NE 8th Street, Suite 200
Bellevue, WA 98008
Tel: (425)653-5589 | Fax: (425)746-0859
ADVANCED PARAMETER UPDATE TUTORIAL
5.
Modify “Contrast Threshold”
As shown on the mask overlay image on Step 4, parts of some nuclei were not
detected. This may be due to the high contrast threshold value applied to the
image.
Click on the Contrast threshold parameter textbox and enter ‘1’ into the
textbox, you can also use the up and down arrow to fine-tune the parameter
value. You can try a few different values to find the optimum result. To revert
to original value, click on the Revert button
to the right of the textbox.
6.
Preview update result
Press Enter after you have input the updated parameter value. A preview of
the result is generated on the image. Compared to previous output, only
one of the nuclei remains not fully detected, while there are quite a few false
detections on the image background. (Do not worry, we can take care of the
false detections in the Subset Filtering step as we have shown in the Basic
Parameter update tutorial.)
The missing detection may be fixed by adjusting the Fill hole size parameter.
http://www.cq-bioanalysis.com
7.
Show advanced parameters
In the Detection step header, click on the Show more options icon to show
additional parameters for the step. Click on the textbox next to Fill hole size
and enter the value ‘150’ and press Enter to show preview.
8.
Apply Nuclei Separation step to image
The Nuclei Separation step partitions the NucleiRegion mask into individual
nuclei components for tracking. Click on the Apply step button on the
Nuclei Separation step header to view results.
SALES AND CONTACT INFORMATION
Sam Alworth, Director of Marketing
DRVision Technologies LLC
15921 NE 8th Street, Suite 200
Bellevue, WA 98008
Tel: (425)653-5589 | Fax: (425)746-0859
ADVANCED PARAMETER UPDATE TUTORIAL
9.
Select alternate partition method
In this image, even though the H2B-EGFP protein is expressed throughout
the nuclei, there are regions of high intensity and low intensity within a single
nucleus. As a result, an intensity-based partition method could introduce
unwanted cuts on the nuclei.
We can click on the Show advanced options button
in the Nuclei
Separation header to show additional parameters. Here, we will select
“Shape based partition” from the pulldown menu. Enter the value ‘2’ for the
parameter Shape sensitivity to reduce cut sensitivity.
10. Preview update result
Press Enter after you have input the updated parameter value. A preview
of the result is generated on the image. Click on the Play button below the
image sequence to view partition results over whole sequence.
11. Tracking large nuclei
The Fluorescence Nuclei Tracking recipe is optimized for the most common
input image settings. For uncommon image settings, the Nuclei Tracking
recipe provides alternate tracking operations that are based on nuclei size in
pixels. Here we want to use the tracking option optimized for large nuclei.
Click on the Show advanced options button
in the Nuclei Tracking
step header and select the “Track Large Nuclei” option from the switchable
operations dropdown menu. Note that preview will not be generated. Click
on the Apply step button in the header to view results.
12. Finalize results
Click the Run Recipe button at the top of the Recipe Console to run the
recipe on the entire image. You can view and export tracking results, or
perform track and mask editing on the image as needed.
To save the updated recipe, click on the Recipe Manager and select the
updated recipe in the Recipe Manager dialog.
http://www.cq-bioanalysis.com
SALES AND CONTACT INFORMATION
Sam Alworth, Director of Marketing
DRVision Technologies LLC
15921 NE 8th Street, Suite 200
Bellevue, WA 98008
Tel: (425)653-5589 | Fax: (425)746-0859
TROUBLESHOOTING
This section contains information about common problems and methods to resolve the problems while using CQ image analysis.
For usage instructions, please refer to the in the previous section. For specific inquiry about using CQ Recipes on your image
data, please contact your sales representative or email us at [email protected]
I cannot see the basic parameters in the recipe.
If the recipe step has basic parameters, a
icon will be visible in the recipe step header. To display the basic parameters in a
single step, double-click on the recipe header. A gray panel will be displayed below the blue recipe step header. To display all the
basic parameters in the recipe, click on the Expand all button below the recipe selection pulldown menu.
I want to hide the basic parameters in the recipe.
To hide the basic parameters in a single step, double-click on the recipe header of an expanded step. To hide all basic parameters
in the recipe, click on the Collapse all button below the recipe selection pulldown menu.
I see a lot of parameters in the recipe step.
First, verify that you are in basic parameter display mode by looking at the Toggle parameter display icon in the recipe step header.
If you see a
icon, that means you are in basic parameter view mode; please refer to the troubleshooting solution above on
hiding the basic parameters. If you see a
icon, that means you are in advanced parameter view mode; click on the icon to
toggle back to basic parameter view.
I want to revert parameter value to the recipe default values.
You can revert a parameter value back to its original value by clicking on the Revert button
textbox.
to the right of the parameter value
Where can I find additional help on what the parameters mean?
You can click on the Show Description button ? on the recipe step header to display the Description dialog. The Description
dialog provides a brief description of the step and explanation of the parameters. You can also find the information in the
Parameter Update Guideline document available on our website at www.cq-bioanalysis.com.
I updated a parameter/applied a step but I did not apply the steps before it.
When you update a parameter or applied a step, CQ will automatically apply the steps before it. You do not have to apply every
step when using the Recipe Console for updates.
I returned to an earlier step to change a parameter and my previous changes to the later steps are no longer shown.
This is expected as CQ will automatically apply all the steps in the recipes up to your last changed step. You can go to the later
step and click Apply step to apply the changes made in the earlier step and the later step to the image.
I am not sure how to set the recipe parameters.
Please refer to the Parameter Update Guideline for explanation of the parameters and parameter usage; and to the Image
Troubleshooter for how to best adjust parameters based on your image.
http://www.cq-bioanalysis.com
SALES AND CONTACT INFORMATION
Sam Alworth, Director of Marketing
DRVision Technologies LLC
15921 NE 8th Street, Suite 200
Bellevue, WA 98008
Tel: (425)653-5589 | Fax: (425)746-0859