Volocity Tutorial Creating a Measurement Protocol in Volocity Software

Transcription

1 TUTORIAL NOTE Cellular Imaging and Analysis Volocity Tutorial Creating a Measurement Protocol in Volocity Software This tutorial will illustrate the process of creating a Measurement Protocol in Volocity Quantitation. 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.

2 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. Image preview shows feedback as measurements are made Drag measurement protocol tasks to this pane to make protocols Measurement protocol tasks Measurements are shown here as a table or histogram 2/7

3 To start a protocol for finding objects in the red channel, drag a finding task such as Find Objects 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 label 02. Give the population a name, which will allow you to identify these objects later. You may also wish to change the color. Click on the cog icon on the task to adjust the selection. The dialog shows a histogram of the intensity values present within the dataset and selected values are shown as a shaded red area on the histogram. The automatic setting, shown by the green vertical line, has selected significant intensities and avoided background; however the selection may be adjusted if required. 3/7

4 The Find Objects task also allows the exclusion of objects below a size threshold. The default value in this field will exclude most noise in biological images, but can be adjusted. Volume measurements for all objects are automatically made, and shown in the measurements table, to aid in this decision. 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. Change the measurements that are made by clicking the word Measure at the bottom of the series. Refine the protocol by adding tasks, dragging them into the existing series for a population to extend it. Continue until the preview shows the required selection. 4/7

5 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. A progress bar may be shown on the 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. 5/7

6 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... 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 (µm 3 ), summarize by Sum and organize by the Item Name. This organization by item name gives one value for the whole dataset. 6/7

7 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. PerkinElmer, Inc. 940 Winter Street Waltham, MA USA P: (800) or (+1) For a complete listing of our global offices, visit Copyright 2010, PerkinElmer, Inc. All rights reserved. PerkinElmer is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 7/7