Imaging and Defining Emergent Behaviors of the Immune Response

Software Details

The GUI:

The graphical user interface (GUI) called ‘confocal.exe’ is an application to control devices and acquire data. This was written for us by Scott Hayden at Video Savant but is available for purchase along with their basic package . Again, Mike Sanderson is responsible for initially adopting the 4-channel acquisition capabilities of Video Savant and integrating their software. The GUI composed of a Main tab and three derivative tabs. Data can be acquired as a single timepoint, a z-series, a z-series over time or any of the above using multiple stage positions. Files are exported in multi-image .tif format that is accessible to Image J, MetamorphBitplane and the simpler Windows image viewers. If multiple stage positions are not used, the data is exported in a format:

When multiple stage positions are used, data is exported to files in the format:
BaseName_w#_s#_t#.tifMain Tab: 
The front page of the GUI looks like this:

Key features here are:

  • ‘Live’. The ability to see ‘Live’ images prior to acquisition. This is useful for adjusting focus, intensity of illumination, frame averaging, etc.
  • ‘Open Shutter’/’Close Shutter’. The ability to Open the hard shutter independently of image acquisition (for looking through the eyepieces or aligning beams, if necessary).
  • ‘Save Settings’/’Load Settings’. The ability to remember all of the data acquisitions settings from a given run and subsequently reload them into the GUI. This is useful, particularly with multiple users.
  • ‘Acquire’ which starts the process that you define in the other tabs (collection of data/timelapse/multiple stage positions/ multiple z-position)
  • The Export boxes allow you to choose which color channels you wish to export to .tif files. 

    Clicking the XY Stage button enables the XY stage tab functionality and permits the stage to ‘visit’ multiple positions at each timepoint.
    Clicking the Do Z series enables the Z tab and permits multiple Z planes to be collected per timepoint and/or stage position.

The Timelapse Tab:

In ‘Number of Time Points', you can enter the frequency that you wish to collect and the total duration of the experiment. Note that in the example shown below, only one timepoint is entered—this will result in a single image being collected (i.e. no timelapse). This would be useful if, for example, you just wanted to collect a z-scan or a series of xy stage positions. The delay between time points sets how long from start of timepoint n until the start of timepoint n+1.

Also under ‘Timelapse’, you can chose the details of frame averaging to boost the signal. This sets the number of 1/30th second scans that make up a frame.

The XY Tab:

Using either the Live tab to actively view real-time images or just viewing through the eyepiece, you can choose the positions you wish to visit at each timepoint and or the primary position of collection. You can rapidly add adjacent images by setting the step interval to a full-field width X (or height, Y) and using the right/left or up/down buttons respectively to add positions. Double click a saved position to move to this position. The buttons 3x3 Grid , 5x5 Grid, and 7x7 Grid create a series of stage positions centered around the current position. This is useful for taking images in a survey grid, particularly in a very big sample.

The Z Tab:

If you have chosen to do multiple z-position, then under ‘Z select the details of this z-series. If not, a single image is collected at the focus ‘Home’ Position (Usually the zero position). The Manual Positioning part of this GUI allows you to manually position the piezzo z-motor to find the ideal top and bottom of your scan.

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