To acquire a 3D Z projection:

Z Sweep Acquisition, or Optical Axis Integration (OAI), is useful for acquiring 2D projections of live specimens. Instead of collecting an individual image at each focal plane, OAI collects and integrates one continuous image through an extended Z movement. OAI has several advantages over optical sectioning: it is much faster, it reduces cell exposure to harmful light, and it has less total camera read noise. You can use the SnapShot tool to acquire a 3D Z projection or you can set up and run an experiment to acquire one or more of these projections.

Note:  Optical Axis Integration provides 3D Z projections that are equivalent to those obtained from numerous optical sections when you are acquiring images of cells or other live specimens on a coverslip. Optical sectioning may be a better option when the sample contains a large amount of fluorescence throughout 3D space (e.g, a tumor spheroid that has large amount of fluorescence.)

 

To acquire a 3D Z Projection with the Snapshot Tool:

  1. From the Resolve3D window, choose File | Snapshot (or click the Snapshot Icon_Snapshot.jpg icon) to open the Snapshot window.

  2. Select the thickness of the section to acquire (in microns) and then select the DO OAI Scan option.

  3. To deconvolve the image, select Deconvolve 2D Images. (This option does not perform advanced image restoration. Instead, it provides 2D image previews that closely resemble images processed with advanced 3D image restoration techniques.)

  4.  Enter the file name for your image and select the channels (filters) that you would like to use. (Snapshot uses the exposure conditions that were last used for those channels.)

    5. softWoRx adds the _R3D.dv extension to the file name. If you enter a file name without a directory path, the file will be saved in the current data folder. You can specify to save the file in another directory by including the path in the file name (e.g., /tmp/myfile).

  5. Click Do It.

    6. Snapshot acquires a projection of data for the thickness that you specified (the stage position at the start of the acquisition is the mid point of the section). The Image window opens and displays the new image.

 

To set up and experiment that acquires a 3D Z Projection:

  1. Manually acquire an image and mark the top and bottom of the sample.

  2. Click the Experiment Icon_Experiment.jpg icon to open the Design/Run Experiment window.

  3. Click the Design tab and then click the Sectioning tab.

  4. Select the Z Sectioning and Enable OAI Scan options.

  5. Click the Run tab and click Start Scan Run_icon_sm.JPG to start the experiment.