Michael Hooker Microscopy Facility (MHMF.ORG)

Using Volocity

These are terse notes on using Volocity are (more) specific to version 4.4.

1.  Using Volocity

• Start Volocity
• Create or open a library

2.  Loading Images from microscope scans

• Zeiss database:
  • File -> Open -> New Library
  •  In the white area of the Library window
    • Right-click
    • Select Create -> Image Sequence
    • A blank Image Sequence window will appear
  • Drag and drop the desired .lsm file into the Image Sequence window
    • Indicate the number of color channels and time/channel naming order (usually "Channels, then Z-series...") when prompted and press OK
    • You should see one thumbnail for each color channel in the Image Sequence window.  You will probably have to expand the window to see them all if there are more than one.
• Leica  series:
  • File -> New Library  (ctrl + N)    or    File -> Open  (ctrl + O)
  • Load image sequences by either:
    • Dragging the .lei file or .lif file (see *Note below) into the white space
  • or
    • In the white area of the Library window
    • Right-click
    • Select Create -> Image Sequence
    • Double click on the Image Sequence Entry
    • A blank Image Sequence window will appear
    • In a Windows folder window, highlight all images from .tif series images
    • Drag and drop the group of images into the Image Sequence window
      When prompted,
    • Indicate the number of color channels and time/channel naming order (usually usually "Channels, then Z-series, then time) and press OK
  • You should see one thumbnail for each color channel in the Image Sequence window.  You will probably have to expand the window to see them all if there are more than one
  • *Note: .lif files will not import if the .lif image database contrains xz scans or xy scans with large dimensions (8192 pixels will not work).  Importing will cause Volocity to either give an error message or crash.  z-series and time lapses should import cortrectly.
• Olympus confocal series in .oif format:
  • If images are in .oib format make an .oif version using the Olympus FV10 software on \\Sutton (or install and use the free FV10-asw 2.0c viewer, or later, on your PC computer)
  • File -> Open  (or  File -> New Library, ^N)
  • File -> Import  the .oif file (or drag the .oif file from Windows Explorer into the Volocity Library window pane)
  • May need to change channel colors
  • May need to check that image dimensions are correct (right click on image and hit properties)
• Importing Olympus 3D time series stacks (4D image data) e.g. MATL data (multi area time lapse z-stacks)
  • After a MATL has completed just dragging the append folder to the Library windows pane will provide a fairly well organized 4-D series.
    • Color for channels will need to be manually set.  This is best done after all the tracks are imported, then selecting all the volumes so that color is changed on all track volumes at once.
      • Choose one or more spliced sequences
      • Tool -> Change Colors - select appropriate color for one channel at a time
  • - or -
  • If volumes need to be manually assembled or the MATL has not completed or an append series has not been generated:
    • Drag .oif files into gray Library area  (or File -> Import - select .oif files)
    • Select library items there are part of the same image series (i.e. have same dimensions, e.g. 3-D stacks at different times, but not different positions)
    • Right click on selection and choose Splice Sequences
    • Choose one or more spliced sequences
    • Tool -> Change Colors - select appropriate color for one channel at a time
• C-Imaging series -e.g. Spinning Disk Confocal Stacks, Fluorescence Dissecting/Macro-scope
  • Talk to facility personnel
• Biorad PIC format
  • Contact Michael
  • Or just click and drag the .pic files into an image sequence window.
• Free standing tiff files
  • Contact Michael

3.  Organizing Images in Volocity Database

• 3.1  Copying
• 3.2  Renaming
• 3.3  Combining
  • Single times points into a time series
    • In the library panel select images which are to be combined (Left mouse clip on first, hold Shift, left mouse click on last;  Individual images can be selected of deselected by holding down crotrol key and pressing left mouse button)
    • Right click on slected images and choose "Splice sequences"
    • New combined image is created and will appear at bottom of library
    • Can move, e.g. closer to individual images, by left clicking inage icon and dragging
  • Combining individual channels into an overlay
  • Select images of the different channels to be combined
  • Right click on slection ---> Create New ---> Image Sequence from selection
  • Edit dimensions if necessary
• 3.4  Changing displayed colors
• 3.5  Hiding channels
• 3.6  Removing
• 3.7 

4.  Displaying

4.1  Orthogonal view (Image tab) [V6]

• Click or double click on the an entry in the Library
  • 
  • Click on "Image" tab
  • Right click on image and choose viewing mode
  • Scrolling tools - Inspection Tool -> can use mouse to scroll through slices
  • Use CHS1 & CHS2 & etc. sliders to adjust relative intensities
  • Hit the slow/hide channel square button to turn on/of each channel
  • The square button with a red target selects the channel for operations.
• Cropping
  • Select the "Rectangular ROI tool"
  • Resize with mouse
    • Actions ---> Crop to Selection (Cropped image becomes a new entry in the Library)
  • Select "Selection Tool" and place cross hairs inside selected region.  Since we are dealing with 3-D space so this is worth doing in case thins are strange. 
• Changing channel colors - Tools ---> Change Colors
  • Select channel then choose color button for each channel
• Selecting/Deselecting
  • Selection shapes - Geometric
    • Rectangle  |  Freehand ROI tool |  Circular Freehand Tool |
  • Selection based on intensity
    • Lasso
    • Magic Wand
      • Double click - only/all objects | Brighter than, Darker Than | Similar with tolerance levels  | Filter out noise. ( makes selection smoother) .
  • Click inside shape and press deleted (object 2 will deleted by pressing the "Delete" key on the keyboard)
  • ^E (control - E ) deselects all  or  2 clicks in a non-selected area also deselects all

3.  Measurements

• Select "Measurements" tab
• Things that may help with selections:
  • Zoom tool
  • Changing viewing mode - e.g. Extended focus
  • Changing brightness
• Select channel on which selection is to be based

4.  Rendering

• Modes - Opacity
• Brightness
• Density (Opacity)
• Black level

5.  Create Movie Sequence

• Right-click in the white space of the Library window
  • Create a new Movie Sequence
• A window “Untitled Sequence” will open
• Insert actions (rotating, zooming, etc), durations, etc.
  • IMPORTANT NOTE:  The orientation of the image as it is displayed in the Image Window is the starting point for the Movie Sequence.
    • To ensure consistent rotation, orientation, etc. in subsequent plays of the movie, always reset the image view to zero rotation. 
    • To reset image view, right-click on the image and select Reset View.
• When movie sequence is complete, select newly created sequence rectangle in the image database
• Go to menu bar item Sequence -> Make Movie
  • NOTE:  This will apply the highlighted Movie Sequence to ALL of the Image Sequence files open from the image database library!
  • The movie will be added to the Library with a folder icon
• Export Movies from created folder
  • Right-click on the Movie file (in the Library Window)
  • Choose Export
    • Select options and choose codec.  On Windows XP 64 bit only the "Microsoft Indeo 1" or "Intel IYUV" codecs are available.  Save in this format and use another program (e.g. QuickTimePro) on another computer to convert to, preferably, uncompressed or the Cinepak codec in order to be compatible with Power Point on a Windows XP (standard 32 bit) computer.  For Apple users convert to a QuickTime format.

6. Colocalization Analysis

• 
• Ensure have started Volocity with the Quant(itation) Option selected
• Create a Volocity library and drag in files (or open an existing library)
• Click on image to analyze
• 
• Select colocalization tab
• Select channels to compare
• Channel brightness sliders may be adjusted to get a good bright and balanced view.  Nte: This has no effect on quantization.
• Set background levels for thresholding
  • Select a shape tool
  • Outline dark area with no features in the image panel
  • Press the blue threshold dropper for each channel
  • 
  • Click outside the selected image to clear the selected region
  • Scattergram will be for entire area where pixels are above the threshold level (don't want colocalization statistics for dark area, i.e. background)

		Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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