This page explains analyses available in VVDViewer, including segmentation and particle detection.

Analysis Tools

Open the Analyze dialog box by choosing Tools > Analysis Tools from the menu or by clicking the Analyze icon from the Main Tool Bar of the VVDViewer GUI.

Segmenting 3D Objects

Use the Paint Brush tab in the Analyze window to select individual objects (e.g., cells) in your rendered 3D volume.

Note: Segmentation in VVDViewer is optimized for use on VVDViewer pyramid files. It is recommended that you convert ExLLSM image volumes to this file type. Objects segmented in VVDViewer can then be saved as a TIFF series.

Creating a Mask

To segment out a cell, follow these steps to create a mask covering the cell:

1. If many cells are labeled, it might be helpful to use Clipping Planes to limit the view to a desired region.
2. Use the Select brush to select a small area, e.g., the soma.
3. Switch to the Diffuse brush to select regions thmain menu barat are connected to the original selection. Use the the Threshold slider to determine how similar in intensity two pixels have to be to be considered connected.
4. Inspect the mask to see if it covers all of the volume you want and nothing else.

• If there is a break in the cell mask, switch back to the Select brush to select the desired volume.
• If extraneous volumes are selected, use the Unselect brush to remove the selection,

Using Masks

Hide masked object or background

• After you have finished selecting the entire object, you can hide it or hide everything else by using Show Only Inside of Mask or Show Only Outside of Mask from the Pop-up menu in the Workspace panel.

The panels below show an example in which the a cell was segmented out from background fluorescence:

Particle Detection via EVE

Initial EVE Settings

The EVE (Exclusive Volume Embedding) function found on the Analysis tab of the Analyze window detects 3D particles by fitting non-overlapping 3D spheres, from large to small. We recommend segmenting objects to be counted using the Paint Brush tools to minimize calculation time.

To perform the analysis, First set the radius (in voxels) of the smallest (Min Radius) and largest (Max Radius) spheres to be fit, using the sliders. Note that the larger the Max Radius, the greater the calculation time required.

Next, set the Min Brightness slider to set the intensity threshold for spot detection. Signals dimmer than this value will be ignored.

Finally, click the Analyze button to run the spot detection analysis. After the analysis, VVDViewer will create a new channel in the Workspace panel and show the counting result on the Render View window. When the channel with the EVE results is selected, the parameters can be adjusted using the tools in the Properties panel.

EVE score adjustment

After the EVE spot-counting analysis, the spheres will be displayed in a new channel Workspace panel and in the Render View window. When this channel is selected, results of EVE analysis can be adjusted in the Properties panel.

The left side of the Properties panel lists the settings used for the current analysis.

The Transparency slider adjusts the transparency of the counting spheres.

The Min Score slider adjusts the minimum score that counting spheres must reach to be visible in the rendered view. The adjustments are made in real time.

The Number of Particles counted appears below these sliders. The number will change in real time as Min Score is adjusted.

Clicking Update will change the memo text in the left side of the panel.

Exporting EVE Results

To view a table of EVE results, select Tools > Measurement Tools... from the main menu bar

The table in the Measurement window lists the X, Y, Z coordinates of each particle detected and a score indicating the likelihood that the particle is signal, based on its brightness relative to the surrounding area. This table is updated in real time as you move the score adjustment slider in the Properties panel.

The table can be exported as .txt file when you click the disk icon on the toolbar in the Measurement window.

Spreadsheet