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Multi-site XYZ

The Multi-site XYZ Scan allows images to be captured for display, archiving or processing, while scanning a two dimensional area with a motorized stage device. This can provide the ability to collect spatial information on static objects outside of a single field of view. To setup a Multi-site scan go to the Sequence pane and select XYZ Scan from the Select Scan Type drop-menu. Once the scan has been setup use the Save Scan icon to save scan settings for repeated use. Use the Open icon to load a saved scan.

Note: Calibration is Very Important!! Prior to adding the scan locations, load the correct spatial scale factor and calibrate the stage. For more information about calibration see "Spatial Calibration."

Progress

• Event Markers - Can be added to the data document file during acquisition as notable events occur in the experiment. Both the numbered buttons above and keyboard numbers may be used.
• Time Elapsed - The time from the start of the first scan.
• Delay Remaining - A reference to delay time. The right arrow button may be clicked to exit the delay.
• Frame Count - Total number of frames captured.
• FPS - Frames per second captured

Scan Settings

The Scan Settings panel is easy-to-use, simply set the speed, define the capture interval, enter the number of passes to capture and where to save the data.

Storage and Display

Use AutoSave to define where and how to store acquired data.

• CXD: Image data is organized and saved as a data document
• TIFF: Captured data is saved as individual image files
• MPTIFF: Captured data is saved as multipage tiffs

Determines how the data is structured when written to a data document.

• To a Single File: Store captured image data in successive fields.
• One File Per Pass: Store captured image data for each pass in a series of data documents called a Summary DocumentA Summary document (.cxs) is a collection of data documents (.cxd)..
• One File Per Position: Store captured image data on a positional basis. A Summary document (.cxs) is created with a Data Document (.cxd) for each position.

Determines if a live view is displayed in the image display or if acquired images may be reviewed during the acquisition.

• Update Image Display: During a sequence captured images are displayed in the image display
• Review Images: During a sequence captured images may be reviewed as newly captured images are added.

Speed

Select the maximum speed or a type of delay.

• Enable Maximum: Capture to memory at maximum speed. During maximum speed, items which slow down acquisition will be ignored (i.e., exposure protection and Z auto-focus).

• Field Delay: A pause between each frame of the scan. Enter a numerical value followed by units. (e.g., 5 seconds = 5s)

• Pass Delay: A pause between subsequent passes in a multi-pass scan. Enter a numerical value followed by units.
• Hours = h
• Minutes = t
• Seconds = s
• Milliseconds = m
• Microseconds = u

• Disk: Stream data directly to disk
• Memory: High speed acquisition stream to memory
• RAM: Sets the limits for the amount of RAM available for streaming

Control

Defines a specific Frame, Pass or Time point to stop the current scan.

• Continuous: The scan runs according settings without a preset endpoint. The scan will be stopped when resources run out or can be interrupted using the Finish button.
• Pass Count: Defines a specific number of passes to complete the current scan.

Refresh Z Focus (Auto-focus)

The Auto-focus is used to automatically find the right focus Z at a given X-Y position while acquiring an image sequence. One example is to update the Z position of a field in a time-lapse to allow for Z stage drift. Enter the number of fields to indicate how often to refocus the Z.  

XYZ Scan Setup

The XYZ Scan Setup panel provides a number controls for setting up simple line scan to complex multidimensional scans. Scan positions are displayed in a 2-dimensional grid view as a Scan Map and as ordered list of X, Y, Z coordinates called the Scan List.

Live Controls are used to navigating and adding positions to a scan, while other controls are used for defining scans and to provide location feedback.

• Live: Click the Live Stage Marker icon () to activate the live feedback mode that provides a live image and displays the current position of the stage. Navigate to points of interests using the stage joystick, the current location is displayed in the scan map and in the coordinates box described below.
• Add Position: Click the Add Position icon () while in the live feedback mode to add new positions to the scan. A green rectangle will be displayed as the position marker in the Scan Map. When not in the Live Feedback mode, new positions can be created at any location in the slide by using the right-click menu item Create View Field.
• Define Scan: Click the Define Scan icon () to launch the Define Scan Pattern dialog. Enter the scan parameters in the dialog based on the starting position and the number of fields in the X and Y directions to scan. Other settings include: Scan Direction (Raster or Meander) and XY Spacing/Overlap.
• Test: Click the Test Scan icon () to move the stage to each position in a defined scan. A defined scan has multiple positions that are connected by a scan path.
• Current Position: While in Live feedback mode, the X, Y, Z coordinates are continually updated providing the current stage position.

Scan Map

The Scan Map displays the coordinates of the current position as indicated by the white dot. The units are determined by the selected calibration. For more information about calibration see "Spatial Calibration." The green rectangles represent a selected location and the white dot indicates the current position. The XYZ coordinates for the current position are displayed above the grid. The red arrows trace the scan path to each of the selected locations. The XYZ Position Sliders allows the XYZ position to be changed by clicking and dragging the slider handle. The movement of the slider is relative, not absolute, so dragging the slider further up or down the scale will produce a faster movement in that direction. Click the Auto-focus icon to have HCImage determine the optimum Z position.

Icon	Name	Function
	Path	To create the path of travel between View Fields, select the Path button, then left-click on each View Field in turn to link the View Fields together. Click away from a View Field when finished. The path is shown as red connecting arrows. Deselect the Path button when done.
	Scan Region	Once all of the fields (locations) have been marked, view fields can be created as a group using Scan Region. See the Steps In this mode, click and drag to create a rectangle enclosing the grid intersections for the View Fields required, this creates a Group Box. Then right-click in the Group Box and select View Fields|Create from the right click menu. This will create a View Field at each intersection. To create a Path between the View Fields, right-click and select the Path > Create menu item. This will use the AutoTrace Properties to determine the route of the path and display it as a series of red arrows connecting the View Fields. Use the right-click menu item Path > AutoTrace Properties dialog to change the route selection.
	View Fields	View Fields can be modified when the Edit View Fields button is pressed . Left-click to select a View Field. The right-click menu contains several options. Also the selected View Field can be dragged to a new position on the slide; to see a live image at the same time in order to better align the View Field with the specimen, or to adjust the focus position. Select the View Field and use the right-click menu option Feedback. This will initiate the Feedback mode and drive the stage to the selected View Field position, displaying the live image in the Image Display. While in this mode the stage movements will move the View Field position and show these changes on the Scan Map. Unpress the Edit View Fields button and the Stage Marker Feedback button when done.
	Pan	Use Pan to change the field of view.
	Show All Scan Positions	Use the "Show All" button to fit all of the scan positions in the visible frame.

Scan List

The Scan List displays the coordinates for each of the scan positions. The list is interactive, click on a position in the list and the stage will move to that location. See the list below for more information on the interactive scan list.

• Go to Position: Click on a position in the list and the stage will move to that position.
• Update Position: Double-click on a position to update the position to the current stage coordinates.
• New Position: Click the New Position icon () to add the current stage position to the list. (Hot key = Insert)
• Delete Position: Click the Delete icon () to remove the selected position from the list. (Hot key = Delete)
• Move Position Order Up: Click the Move Item Up icon () to move the selected scan position up one level in the list. (Hot key = Alt + Up)
• Move Position Down: Click the Move Item Down icon () to move the selected scan position down one level in the list. (Hot key = Alt + Down)
• Add Offset to All Positions: Right-click and select Offset All Position, then enter an X, Y, Z, and/or Zf offset to all positions in the list.
• Update All Z Positions: Right-click and select Set All Z, updates the existing Z value with the current Z value for all of the positions in the list.

Auto-focus

The Auto-focus is used to automatically find the right focus Z at a given X-Y position prior to acquiring an image sequence. If you wish to perform an auto-focus while acquiring see "Refresh Z Focus (Auto-focus)."

Sub-Z Control

The Sub-Z Control is used to setup a Z scan at points along an XYZ scan. The Lambda ZIs a Z scan for each wavelength or channel; i.e. channel 1 Z scan, channel 2 Z scan, repeat up to 5 channels. option is available when acquiring up to five channels in mono. There are three Sub-Z control options:

• None: Sub-Z is disabled
• Relative: The start and end points are dependent upon the current Z position
• Absolute: The top and bottom points are defined

Note: What is the difference between a Raster and a Meander scan?