Data

Alzheimer’s disease plaque (red channel – disease plaque; green channel – microglial cells)

Dataset courtesy of Professor Elizabeth Head, Department of Molecular and Biomedical Pharmacology, Sanders-Brown Center on Aging, Lexington, KY, USA.

Aim

To find the total volume of all objects labeled in the red channel.

Workflow

The process has three stages:

1. Creation of a Measurement Protocol to locate significant intensities in the red channel. The protocol is used to threshold data to select areas of interest and, from that selection, include or exclude objects based on a range of criteria.

2. Creation of a Measurement Item.

3. Analysis of the contents of the Measurement Item to find the total labelled volume of all objects in the red channel.

Cellular Imaging and Analysis

Volocity Tutorial Creating a Measurement Protocol

This tutorial will illustrate the process of creating a Measurement Protocol in Volocity® Quantitation.

TUTORIAL NOTE

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1) Creation of a Measurement Protocol to locate significant intensities in the red channel Open the required dataset in the library. To obtain correct values for calibrated spatial measurements, it is important to ensure that the data is calibrated correctly by setting the X, Y and Z pixel dimensions in the Properties dialog. From the Edit menu, select Properties… Most file formats supported by Volocity will enter this information automatically, but it is advisable to check and adjust if necessary.

View the Measurements tab. The Measurements View contains all the tools and information needed for selecting objects.

Measurements are shown here as a table or histogram

Measurement protocol tasks

Drag measurement protocol tasks to this pane to make protocols

Image preview shows feedback as measurements are made

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To start a protocol for finding objects in the red channel, drag a ‘Find’ task such as ‘Find Objects Using Intensity’ to the protocol pane above. In the task dialogue, select the channel which you are interested in, in this case the red channel, which has the name ‘02’. The task shows a histogram of the intensity values present within the dataset and selected values are shown as a shaded red area on the histogram. Drag the vertical, red lines to adjust the selected intensity range. Here the red line indicating the lowest intensity to be selected has been moved to the right to avoid selecting background. The red line indicating the highest intensity to be selected has been left at the maximum so that all intensities above background are selected.

The selected objects will be shown as a colored overlay in the image preview and standard morphological and intensity measurements will be displayed in the measurements table.

Lowest selected intensity

Highest selected intensity

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Next, noise is removed from the selection by adding the ‘Remove Noise From Objects’ task to the protocol. Adjust the protocol, as required, until the preview shows the required selection.

2) Creation of a Measurement Item

Select Make Measurement Item… from the Measurements menu. Choose the option to make a new Measurement Item and enter a name for it. Alternatively, you can add these measurements to an existing Measurement Item, which may be made from the same data or from a different dataset in the library. Measurement Items may therefore combine measurements from different sources. Click on OK.

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A progress bar is shown on screen whilst the measurements are being made and a new item will be added to the library.

Click the Measurement Item in the library to open the Raw View showing the table of numbers.

3) Analyzing the contents of the Measurement Item to find the total labeled volume

Click to View the Analysis tab. From the Analysis menu select Analyze...

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In this example, we are interested in finding the total, or sum, of the volume found in this table. In the Edit Analysis dialogue box, select to analyze the data by Volume (µm3), summarize by Sum and organize by the Item Name. This organization by item name gives one value for the whole dataset.

From here we may wish to export some of this information into a format which can be viewed in other software packages. In the file menu, choose Export… and the required format. Any table in a Measurements View or Measurement Item may be exported as tab delimited or comma delimited text. These are the standard formats for exporting numerical data to other spreadsheet packages, should you wish to perform further analysis.

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