Using AutoQuant Deconvolution

Deconvolution is an image processing technique that improves the contrast and resolution of digital images captured by microscopes. 

Image-Pro can deconvolve both 2D and 3D data sets.

Deconvolution is located on the Process Ribbon.

With your dataset selected in the workspace, click on the Deconvolution button on the Process Ribbon.  The Deconvolution panel opens. Work your way through the 5 steps in the Deconvolution panel.

Step1: Confirm Metadata.

The Confirm Image Metadata step contains these fields:

  • Active Image
  • Volume Dimensions
  • Locations
  • Calibrations
  • Objective Lens
  • Lens Immersion RI
  • NA

Hover over any of these fields for a full definition in a tool tip.

Image-Pro will automatically populate as many of these fields as possible from meta data.  A warning is displayed for any fields that are required that cannot be automatically populated.  Manually add any required Image-Meta data.

Step 2: Confirm Channel Metadata

The Confirm Channel Metadata step consists of a table with these columns:

  • Channel
  • Modality
  • λ (nm)

Image-Pro will automatically populate as many cells in the table as possible from meta data. A warning is displayed for any cells for which data is required that cannot be automatically populated. Manually populate or edit any cells with warnings.

The checkbox in the Channel column indicates that a channel will be deconvolved. De-select this check box to pass a channel unprocessed into the new, deconvolved dataset. This ‘pass-through’ option is helpful for modalities that cannot be deconvolved, such as phase contrast or DIC.

Step 3: Choose Starting PSF

Select your starting PSF. For 3D datasets you can choose between either a Theoretical PSF or a Measured PSF. When working with 2D datasets you have the additional choice of a Derived PSF.

  • Theoretical PSF: The initial point-spread function estimate will be generated computationally from the supplied metadata (modality, calibration, numeric aperture, refractive index, and emission wavelength). It can be used with Fluorescence [Widefield], Laser Scanning Confocal, Spinning-Disk Scanning Confocal, Two-Photon Fluorescence, or STED.
  • Derived PSF: A PSF estimate which is made from the image data itself. It is not dependent on the metadata fields being correct.
  • Measured PSF: A dataset (which is open in Image-Pro) is used as the initial point-spread function estimate. This will be an image of a single sub-resolution bead collected under the same optical configuration as the sample being processed. By default, a loaded PSF dataset will be pre-processed to reduce noise and to center the peak intensity.

The spatial dimensions (width, height, and depth) of the PSF dataset do not have to match those of the sample. If the PSF consists of multiple volumes, each dimension length beyond XYZ ("hyperspatial dimensions") must either match that of the dataset being deconvolved or must be 1. The volumes of the PSF dataset are matched to the sample as follows:

If the hyperspatial dimensions of the PSF match those of the sample being processed, then each volume of the PSF will be matched to the corresponding volume in the sample.

If a hyperspatial dimension of the PSF has a length of 1, the lone element of that dimension will be applied to all points of the sample along the corresponding dimension; for example, if the time point count of the PSF is 1, but the sample has 10 time points, the first time point of the PSF will be applied to all time points of the sample.

Step 4: Choose Deconvolution Settings

Check the Use Defaults box to apply the recommended settings.

Selecting the Settings button gives access to further deconvolution settings where non-default values can be selected.

Click Deconvolve.

Your data is processed and a new, deconvolved version of the data set opens in the Image-Pro workspace.

You can monitor the progress of deconvolution jobs using the task manager.

Batch Deconvolution

To deconvolve multiple datasets using the settings in the AutoQuant Deconvolution panel, click on the drop-down menu on the right of the Deconvolution button and select the Run Batch... button.

A Select Files or Folders dialog opens. Navigate to, and select the data sets, or folder of datasets that you wish to deconvolve, and click Process Selection.

You can monitor the progress of your batch deconvolution using the task manager.

A new folder named Deconvolved is added to your source folder. The newly deconvolved datasets are saved in this folder. The datasets are saved with the name of the original file, prefixed with the number of iterations executed during the deconvolution.

 

Learn more about all features in The Process Tab.