1.1. What is My Dongle Number and SMA Expiry Date?

What is My Dongle Number and SMA Expiry Date?

The Volocity dongle/serial number is an 8-digit number programmed into the Volocity or Imaging License Server (ILS) dongle. Each dongle is also programmed with a Software Maintenance Agreement (SMA) expiry date.

Locating the Dongle Number:

1. Physically Check the Dongle:

   • The dongle number is printed on the tag attached to the dongle.
   • For standalone dongles: It will be plugged into the workstation where Volocity is installed.
   • For License Server dongles (ILS dongles): It is usually plugged into a server on your local network.

2. Verify Using Volocity:

   • Start Volocity.
   • Go to the Window menu and select Show Session Log.

The session log will display the dongle/serial number and the validity of your SMA.

1.2. How to Transfer Volocity Preferences Between Different User Accounts in Windows

How to Transfer Volocity Preferences Between Different User Accounts in Windows

On Windows, transferring Volocity preferences involves exporting the user-level preferences from the Registry and importing them into the target user account.

Steps to Transfer Volocity Preferences:

1. Export Preferences from the Donor Account:

   • Log in to the donor account (usually an administrator).
   • Open the Registry Editor:
     • Press Win + R, type regedit, and press Enter.
   • Navigate to the following location:
     HKEY_CURRENT_USER > Software > Improvision > Volocity
   • To export preferences:
     • Right-click the Volocity folder and select Export.
     • Save the exported file as a .reg file in an accessible location, such as a shared folder or a USB drive.

2. Log in to the Target Account:

   • Log out of the donor account and log in to the new user account.
   • Access the shared folder or portable drive containing the exported .reg file.
   • Copy the .reg file to the desktop.

3. Import Preferences to the Target Account:

   • Double-click the .reg file on the desktop.
   • Confirm the prompt to allow changes to the Registry.
   • The preferences will be imported automatically to the correct location for the new user account.

4. Verify Preferences in Volocity:

   • Start Volocity in the new account. The preferences should match those from the donor account.

1.3. How to Transfer Volocity Preferences Between Different User Accounts on macOS

How to Transfer Volocity Preferences Between Different User Accounts on macOS

Volocity user-level preferences include light paths, acquisition protocols, current analysis settings, and measurement protocols. Be cautious when copying and pasting preferences or registry folders, as they contain extensive configuration information. Ensure that you are targeting the correct Volocity preferences folder.

Steps to Transfer Volocity Preferences:

1. Copy Preferences from the Donor Account:

   • Log in to the administrator account (or the account you want to use as the "donor").
   • Navigate to:
     Macintosh HD > Users > [Account Home Directory] > Library > Preferences
   • Locate the folder named Volocity Preferences.
   • Select the folder, go to the Edit menu, and choose Copy.

2. Paste Preferences in a Shared Location:

   • Navigate to:
     Macintosh HD > Users > Shared
   • Paste the Volocity Preferences folder in this location using Edit > Paste.

3. Copy Preferences to the Target Account:

   • Log in to the new user account.
   • Navigate to:
     Macintosh HD > Users > Shared
   • Copy the Volocity Preferences folder using Edit > Copy.
   • Navigate to:
     Macintosh HD > Users > [Account Home Directory] > Library > Preferences
   • Paste the preferences folder here using Edit > Paste.

4. Verify Preferences in Volocity:

   • Start Volocity in the new account. The preferences should initially match those in the donor account.

Note on Compatibility:

Apple removed support for 32-bit applications starting with macOS versions after High Sierra. Volocity will not function on operating systems later than macOS High Sierra.

2.1. Changing the Imaging License Server (ILS) Computer – Key Considerations

Changing the Imaging License Server (ILS) Computer – Key Considerations

1. Workstation Requirements

• The workstation must meet the minimum specifications outlined in the Volocity System Requirements.

2. Static IP Address or DNS

• The ILS workstation must have a static IP address or a DNS.

• This IP address/DNS must be shared with users connecting to Volocity via client computers.

3. Port Configuration

• By default, the server uses:

  • Port 15002 for TCP/IP communication with clients.

  • Port 15003 for HTTP communication with the administration tool.

• Ensure the following:

  • Windows Installations: Add firewall exceptions for these ports.

  • Internal Networks: Confirm that network firewalls also allow communication through these ports. Consult your IT staff if needed.

4. Administration Tool

• The ILS administration tool runs via a web browser.

• Refer to Page 7 of the Imaging License Server User Guide for a list of supported browsers.

5. Power Settings

• Configure the ILS workstation's power settings to prevent sleep or hibernation.

• If the server workstation is turned off or set to sleep, the ILS daemon/service will stop, preventing client machines from connecting to the server.

6. Backup and Transfer of Preferences

• Create a backup copy of the Improvision License Server preference folder on the original ILS server computer and transfer it to the new server.

• This folder contains two critical files:

  • Users and Groups.mk

  • Server log.mk

• These files contain all configuration information for the ILS (this data is not stored on the dongle).

File Locations:

• Windows XP:
  C:\Documents and Settings\All Users\Application Data\Improvision License Server

• Windows 7, 10, 11:
  C:\Program Data\Improvision License Server

• Legacy Mac OS X:
  Mac HD \ Library \ Application Support \ Improvision License Server

• Once copied, place the folder in the same location on the new ILS computer.

Installation Process

1. Unplug the Dongle: If the ILS dongle is already plugged in, unplug it.

2. Download and Install: Download the Imaging License Server (ILS) software from the ILS Downloads Page and install it.

3. Shutdown the Computer: Once installed, shut down the computer.

4. Reconnect the Dongle: Plug the ILS dongle into an available USB port.

5. Restart the Computer:

   • Upon restart, the LED on the dongle should light up (indicating power).

6. Verify Setup: Access the ILS admin tool by entering localhost:15003 into the browser's address bar.

   • Verify that the ILS is running and the correct preference files are loaded.

   • Confirm that Users, Groups, and Configurations mirror the setup on the original ILS machine.

2.2. Troubleshooting ILS Admin Tool Connection – Running a Traceroute

Troubleshooting ILS Admin Tool Connection – Running a Traceroute

Running a Traceroute on Windows

1. Open the Command Prompt:
   • Go to Start > Run.
   • Type cmd and press Enter.
   • A command prompt window will open with a blinking cursor, e.g., C:\Documents and Settings\yourname>_.
2. Run the traceroute command:
   • Type: tracert [hostname]
     (Replace [hostname] with the IP address or name of the server you are testing.)
3. Wait for the test to complete:
   • The process may take up to a minute or more.
   • It will generate a list of the connections between the client and the server’s IP address.

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Running a Traceroute on macOS

1. Use Built-in Network Tools:
   • Open Finder and navigate to Macintosh HD > Applications > Utilities.
   • Open one of the following tools:
     • Network Utility:
       • Click the Traceroute tab.
       • Enter the hostname of the server you are testing.
     • Terminal:
       • Type: traceroute [hostname]
         (Replace [hostname] with the name or IP address of the server you are testing.)
2. Wait for the test to complete:
   • The process may take up to a minute or more.
   • It will generate a list of the connections between the client and the server’s IP address.

2.3. Deleting Imaging License Server (ILS) Settings in the Registry

Deleting Imaging License Server (ILS) Settings in the Registry

1. Deleting Registry Entries (Windows)

1. Open the Registry Editor:
   • Press the Windows Start icon and select Run.
   • Type regedit and hit Enter.
2. Navigate to the following registry path:
   • HKEY_CURRENT_USER\SOFTWARE\IMPROVISION
3. Delete the Improvision folder:
   • Right-click on the Improvision folder and select Delete.

2. Clearing ILS Configuration Files

Like Volocity, the ILS stores its configurations in a separate file. To start with a fresh ILS installation, you’ll need to clear out the users&groups.mk file from the following locations:

• Windows 7/10/11:
  C:\Program Data\Imaging License Server
  (Note: This folder is hidden by default. You’ll need to enable "Show hidden files, folders, and drives" to reveal it.)

• macOS:

  • macOS 10.7 and above:
    /Users/Shared/Library/Application Support/Imaging License Server
  • macOS 10.6 and earlier:
    /Library/Application Support/Imaging License Server

2.4. Uninstalling the License Server on macOS

3.1. Volocity ILS & Windows Defender

Volocity ILS & Windows Defender

Windows Defender is a preinstalled antivirus program in Windows 8.1 and Windows 10. Follow these steps to allow an application through Windows Defender:

Step 1: Open Security and Maintenance

1. Open the Windows Control Panel.
2. Select Security and Maintenance.

Step 2: Allow an App Through Windows Defender

1. Click Allow an app or feature through Windows Defender Firewall.

Step 3: Select the Application

1. Scroll through the list of programs to locate the application you want to allow through Windows Defender.
2. Check the box next to the application to enable it.

3.2. Restoring a corrupt Library in Volocity

Restoring a corrupt Library in Volocity

A Volocity library is an essential database that combines image data and metadata. If a library becomes corrupt, it may be difficult or impossible to fully restore the data without a proper backup. For this reason, frequent backups of critical data are highly recommended. Both the .mvd2 file and its corresponding Data folder must remain intact for the library to function properly.

Why Volocity Libraries Are Difficult to Restore:

• The files in the Data folder do not merely store raw image data; they are interlinked with metadata in the .mvd2 file to create a functional database.
• Without both the .mvd2 file and the complete Data folder, the library cannot function, and it is not possible to extract and import the files into another library.

Steps to Attempt Restoring a Corrupt Library:

If the library is corrupted but still recognized by Volocity, you can try the following procedure:

1. Open the Corrupt Library in Volocity:

   • Launch Volocity and attempt to open the corrupted library.

2. Compact the Library:

   • Go to the File menu and select Compact Library.
   • In the Compact Library dialog box, check the option Also remove cached data and preferences.

3. Start the Compacting Process:

   • Click Compact to initiate the process.
   • This operation will clean up the library by removing unnecessary cached data and attempting to optimize the library's database structure.

4. Assess the Restored Data:

   • Once the process is complete, review the library to see if the corruption has been resolved and if your data is accessible.

Best Practices for Preventing Corruption:

1. Backup Regularly:

   • Always maintain regular backups of your Volocity libraries.
   • Store backups in a secure, redundant location (e.g., external drives, cloud storage).

2. Avoid System Interruptions:

   • Do not shut down the workstation or interrupt Volocity while saving or compacting a library.

3. Monitor Disk Health:

   • Use tools like CrystalDiskInfo or your system's built-in diagnostics to monitor the health of your storage drives.
   • Replace failing drives promptly to prevent data loss.

4. Limit Concurrent Access:

   • If libraries are stored on a shared network drive, limit simultaneous access by multiple users, as this can increase the risk of corruption.

Final Note:

If the above steps do not resolve the issue, and no backup exists, the corrupted library may be irrecoverable. In such cases, consider reaching out to Volocity support or IT professionals with experience in data recovery for further assistance.

3.3. Connecting a Leica DMi8 Microscope to Volocity

Connecting a Leica DMi8 Microscope to Volocity

To connect a Leica DMi8 microscope to Volocity, you need to configure the microscope's Virtual COM Port (VCP). The Leica SDK, which is included with the DMi8 upon delivery, installs the necessary driver to establish communication between the microscope and the computer via USB. While the Leica SDK does not require a COM Port, Volocity requires one to communicate with the microscope.

Follow these steps to enable and configure the VCP:

Steps to Enable the Virtual COM Port (VCP):

1. Install the Leica SDK and Driver:

   • Ensure that the Leica SDK and associated drivers are installed on your computer. These drivers are typically included with the DMi8 microscope upon delivery.

2. Access Device Manager:

   • Open the Start Menu and search for Device Manager.
   • Click to open the Device Manager window.

3. Locate the DMi8 Device:

   • Expand the Universal Serial Bus (USB) Controllers section.
   • Locate the DMi8, which should appear as a USB Serial Controller.

4. Open Device Properties:

   • Double-click the USB Serial Controller or right-click it and select Properties.

5. Enable the VCP:

   • In the Properties window, navigate to the Advanced tab.
   • Check the box for Load VCP.

6. Verify the COM Port Configuration:

   • Once the VCP is enabled, the DMi8 will now appear under the Ports (COM & LPT) section in Device Manager.
   • Note the COM Port assigned to the DMi8 (e.g., COM3, COM4, etc.).

Configure the Microscope in Volocity:

1. Launch Volocity:

   • Open the Volocity software on your workstation.

2. Set Up the Serial Device:

   • Go to the Preferences menu (Windows: Edit > Preferences; macOS: Volocity > Preferences).
   • Navigate to the Devices or Serial Devices tab.

3. Select the Appropriate COM Port:

   • Locate the COM Port assigned to the DMi8 in the previous step (e.g., COM3).
   • Configure Volocity to use this COM Port for communicating with the Leica DMi8 microscope.

4. Test the Connection:

   • Ensure that Volocity can successfully communicate with the DMi8 by verifying its control options (e.g., stage movement, light path settings).

Notes and Tips:

• Driver Issues:

  • If the DMi8 does not appear in Device Manager or under USB Controllers, ensure that the Leica SDK and drivers are correctly installed.
  • Reinstall the SDK if necessary and restart your computer.

• COM Port Conflicts:

  • If the assigned COM Port is already in use by another device, you may need to change it.
  • In the Device Manager, right-click on the COM Port, select Properties, and manually assign an unused COM Port.

• Cable and Connection:

  • Ensure that the USB cable is securely connected between the DMi8 and the computer. Use a high-quality USB cable for reliable communication.

• Supported Operating Systems:

  • Verify that the operating system on your workstation is supported by the Leica SDK and Volocity.

By following these steps, you can establish a reliable connection between the Leica DMi8 microscope and Volocity, enabling precise control and data acquisition.

3.4. Transferring Volocity Configurations Between Workstations or Users

Transferring Volocity Configurations Between Workstations or Users

If you have purchased a new workstation or need to standardize settings across multiple user accounts on a single workstation, Volocity configurations can be transferred. Follow the steps below to safely transfer both user-level settings and system-level configurations.

On Windows Workstations

1. Export User-Level Settings

• Path:
  The user-level settings for Volocity are stored in the following registry key:
  HKEY_CURRENT_USER > Software > Improvision > Volocity
  These settings include lightpaths, import preferences, and other user-specific configurations.

• Steps to Export:

  1. Log in using an Admin account.
  2. Go to the Start menu, search for regedit, and open the Registry Editor.
  3. Navigate to the path:
     HKEY_CURRENT_USER > Software > Improvision > Volocity.
  4. Right-click on the Volocity key and select Export.
  5. Save the exported file as a .reg file in a safe location.

• Steps to Import:

  1. Log into the desired user account.
  2. Double-click the exported .reg file to import the settings into the registry.

2. Export System-Level Settings

• Path:
  System-level configurations for Volocity (e.g., serial port setup, objective calibrations) are stored in the following registry key:
  HKEY_LOCAL_MACHINE > Software > Improvision > Volocity.

• Steps to Export:

  1. In the Registry Editor, navigate to:
     HKEY_LOCAL_MACHINE > Software > Improvision > Volocity.
  2. Right-click on the Volocity key and select Export.
  3. Save the file as a .reg file.

• Steps to Import:

  1. Double-click the exported .reg file on the target workstation to import the system configurations.

3. Important Notes for Windows:

• If transferring both user-level and system-level settings, you must export and import both .reg files.
• Caution: Only export the specific Volocity keys. Accidentally exporting and importing the entire registry (e.g., all keys under HKEY_LOCAL_MACHINE) can severely disrupt the system, potentially requiring a complete OS reinstallation.
• Lightpath Settings Issue:
  • After transferring configurations to a new workstation, lightpaths may appear missing because the camera ID in the registry has changed.
  • For DCAM cameras (e.g., Hamamatsu), you may need to update the DCAM IDs in the registry to restore lightpaths. See detailed instructions here.

On Macintosh Workstations (version 6.3 and older)

1. Export User-Level Settings

• Path:
  User-level settings are stored in:
  Hard Disk > Users > Home Directory (House Icon) > Library > Preferences > Volocity Preferences.

• Steps to Export:

  1. Navigate to the path above.
  2. Copy the Volocity Preferences folder to a safe location.

• Steps to Import:

  1. Move the Volocity Preferences folder to the same path in the target user account’s home directory.

2. Export System-Level Settings

• Path:
  System-level settings are stored in:
  Hard Disk > Library > Preferences > Volocity Preferences.

• Steps to Export:

  1. Navigate to the path above.
  2. Copy the Volocity Preferences folder to a safe location.

• Steps to Import:

  1. Copy the folder into the same path on the target workstation.

3. Important Notes for macOS:

• Avoid Mixing Files: Both the user-level and system-level folders have the same name ("Volocity Preferences"). To avoid confusion, create parent folders (e.g., "User Settings" and "System Settings") before exporting.
• Ensure the files are placed in the exact corresponding paths on the target workstation.

Final Notes on Configuration Transfers

• Lightpath and Camera Settings:

  • On Windows systems, lightpath settings tied to specific cameras may not transfer correctly due to changes in camera IDs. For Hamamatsu DCAM cameras, you can manually adjust the DCAM IDs in the registry to restore these settings.

• Backup and Test:

  • Always create backups of the original settings before making changes.
  • After transferring settings, test Volocity to ensure all configurations have been successfully applied.

By following these steps, you can efficiently transfer Volocity configurations across workstations or user accounts while minimizing potential issues.

3.5. Calculating PSFs with Intermediate Magnification from Optivars

In Volocity, Point Spread Functions (PSFs) are calculated in object space, also referred to as the back-projected plane or specimen plane. This approach ensures that PSFs remain independent of magnification, as they are applied based on the physical dimensions of the images being deconvolved. This eliminates the need to manually input the magnification parameter when calculating the PSF, provided the image calibration is correctly set.

PSF Considerations for Spinning Disk Confocal Systems

For spinning disk confocal systems, the calculation of PSFs must account for the interference pattern caused by the multiplicity of pinholes in the spinning disk. The following key factors are involved:

1. Back-Projected Pinhole Size and Spacing:

   • The physical size and spacing of the pinholes in the spinning disk must be projected back to the sample plane, considering all magnification components between the spinning disk and the sample.
   • Typical parameters for a spinning disk include:
     • Disk spacing: 250 µm
     • Pinhole size: 50 µm
   • For a 100X objective lens, these values project to:
     • Distance between pinholes: 2.5 µm
     • Pinhole size: 0.5 µm

2. Intermediate Magnification (Optivar):

   • If an Optivar is positioned between the spinning disk and the physical sample, its magnification must be included in the calculation of the back-projected pinhole size and spacing.
   • For example, if a 1.6x Optivar is used with a 100X objective lens, the total magnification becomes:
     • Total Magnification = Objective Magnification × Optivar Magnification
     • Total Magnification = 100 × 1.6 = 160X
   • However, intermediate magnification components that lie outside the optical path between the spinning disk and the sample should not be included in this calculation.

3. Numerical Aperture (NA) Considerations:

   • When an Optivar is added, it effectively creates a new lens system composed of the objective and the Optivar.
   • The focal length (f) of this new system is reduced by the Optivar magnification (M), while the size of the entrance aperture remains unchanged.
   • At small angles, the Numerical Aperture (NA) is calculated as:
     NA = f / Radius of the Entrance Aperture
   • Adding the Optivar reduces the focal length without changing the entrance aperture, causing the NA to decrease.
   • For example, if a 1.6x Optivar is used, the NA must also be divided by 1.6.

Calculating PSFs in Volocity

If you choose the Calculated PSF option in Volocity, you must manually account for the additional magnification introduced by the Optivar and adjust the NA accordingly:

1. Total Magnification:

   • Multiply the objective magnification by the Optivar magnification.
   • Example:
     Total Magnification = 100 × 1.6 = 160X

2. Adjusted NA:

   • Divide the original NA of the objective lens by the Optivar magnification.
   • Example:
     Adjusted NA = Original NA ÷ 1.6

Verifying the Pinhole Size and Spacing

To physically verify the pinhole size and spacing:

1. Stop the spinning disk and measure the pattern in the image plane.
2. For a 100X objective lens:
   • Disk spacing: 250 µm back-projects to 2.5 µm.
   • Pinhole size: 50 µm back-projects to 0.5 µm.

Conservation of Etendue and NA Limitations

Adding an Optivar introduces a physical limitation due to the conservation of etendue, which governs the trade-off between magnification, aperture size, and light throughput. For more information on this topic, consult relevant optical theory resources.

By carefully accounting for intermediate magnification and the adjusted NA, you can ensure accurate PSF calculations for deconvolving data acquired with spinning disk confocal systems.

3.6. Playback Speed Factors for Multi-Z Plane Image Sequences

Playback Speed Factors for Multi-Z Plane Image Sequences

When playing through a time-series dataset in Volocity, you may notice that playback is smoother in the XY plane mode, but significantly slower in XYZ, XZ, or YZ viewing modes. This difference arises from how image data is stored and processed, as well as the hardware performance of the system.

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Why Is Playback Slower in XYZ, XZ, and YZ Views?

1. Memory Caching and Resource Usage

• On first playback, the entire image volume is loaded from the storage device into memory.
• Subsequent playback for the same timepoints benefits from cached memory, improving performance.
• However, when playing through many timepoints, the memory cache may reach its limit, causing older data to be pushed out and reloaded from storage, reducing the cache's effectiveness.

2. Memory Layout of Z Planes

• XY planes are stored sequentially in memory for efficient access. For example:

  • A simple 3x3 XY plane filled with zeros is stored in memory as:
    0 0 0 0 0 0 0 0 0.

• 3D Volumes (multiple XY planes) are stored as a linear block:

  • 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2.

• To display XZ or YZ planes, Volocity must extract specific voxels from the 3D block and rearrange them into a format suitable for display (e.g., an XZ plane appears in memory as 0 1 2 0 1 2 0 1 2).

  • This extraction and rearrangement process is computationally intensive, especially for large datasets.
  • For datasets with many Z planes (e.g., 100 or more), extracting the XZ or YZ planes is significantly slower than displaying a single XY plane.

3. Dataset Size and Disk Throughput

• Large datasets require high-speed storage to minimize delays in loading data. For example:
  • A 22GB dataset with timepoints sized at 700MB will take longer to load if stored on older storage systems.
  • The first playback may take longer as the dataset is loaded into memory from disk.

Example Calculation:

For a 700MB timepoint stored on modern NVMe SSDs with a read speed of 3,500MB/s:

• Time to load each timepoint = Size of timepoint ÷ Disk speed
  • 700MB ÷ 3,500MB/s ≈ 0.2 seconds per timepoint

In comparison, older storage systems (e.g., HDDs or SATA SSDs) with slower read speeds (e.g., 100MB/s) would take significantly longer (700MB ÷ 100MB/s = 7 seconds per timepoint).

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Modern Hardware Recommendations for 2025

To achieve optimal playback speeds, consider upgrading your system with modern hardware:

1. Use NVMe SSDs:

   • NVMe SSDs provide sequential read speeds of 3,000–7,000MB/s, far outperforming older RAID or SATA SSD setups.
   • Example drives: Samsung 980 Pro, Western Digital SN850X, or enterprise-grade NVMe drives for heavy workloads.

2. Increase System RAM:

   • More RAM allows larger datasets to be cached in memory, reducing the need to reload data from disk.
   • For large datasets (e.g., 22GB or larger), consider at least 64GB or 128GB of RAM.

3. Free Up Disk Space:

   • Keep storage drives at least 30-40% free to prevent latency from disk fragmentation or overuse.

4. Upgrade to PCIe 5.0 or 4.0 Storage Interfaces:

   • PCIe 5.0 and 4.0 NVMe drives offer higher bandwidth, reducing data transfer times for large datasets.

5. GPU Acceleration:

   • Modern GPUs with CUDA support (e.g., NVIDIA RTX 4000/5000 series or A-series professional GPUs) can offload some computational tasks, improving processing times for rendering XYZ, XZ, and YZ planes.

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How to Optimize Your Workflow:

1. Optimize Cache Usage:

   • Play through smaller ranges of timepoints at a time to make the most of cached memory.

2. Minimize AV Interference:

   • If using Windows Defender or institutional antivirus, exclude Volocity directories and data files from real-time scanning. This can improve performance during initial playback.

3. Check Disk Throughput:

   • Use modern benchmarking tools (e.g., CrystalDiskMark) to verify disk read/write speeds.
   • Ensure your storage setup is meeting expected performance levels.

4. Monitor Hardware Resources:

   • Use system monitoring tools (e.g., Task Manager, HWInfo) to ensure no bottlenecks in CPU, GPU, or RAM usage during playback.

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Key Takeaways:

• Playback performance is heavily influenced by storage speed, RAM availability, and dataset size.
• Upgrading to NVMe SSDs, increasing RAM, and optimizing antivirus settings can significantly improve playback performance.
• For very large datasets, consider systems with high-speed storage interfaces (e.g., PCIe 5.0 NVMe drives) and professional GPUs to reduce computational overhead.

By leveraging modern hardware and optimizing system configurations, you can achieve smoother playback and improved performance in Volocity, even with large multi-Z plane datasets.

3.7. How to Identify Objects Inside Other Objects in Volocity

3.8. How to Produce and Export a Multi-Channel Merged Image or Image Sequence for Publication

How to Produce and Export a Multi-Channel Merged Image or Image Sequence for Publication

Volocity allows you to create high-quality merged images or image sequences suitable for publication. This can be done by capturing snapshots or using the "View as..." export options. Below are detailed instructions for each method.

Method 1: Capture Snapshot (Single 2D Image)

This method captures a single 2D image from the current view and saves it in the millions of colors format. Snapshots can incorporate display options such as scales, color reference tables, or time overlays.

1. Open the Image Sequence:

   • In the Volocity Library, highlight the desired image sequence.
   • Double-click on the thumbnail to open the image sequence in its own window.

2. Select the Viewing Mode:

   • Switch to the Image Tab.
   • Use the dropdown menu to select the viewing mode (e.g., Extended Focus, XYZ, or other options).

3. Select the Correct Timepoint (if applicable):

   • For time series datasets, ensure the correct timepoint is selected.
   • Use the Time Navigation Bar below the view to switch between timepoints.
   • If the navigation bar is not visible, enable it by clicking on Image > Show Time Navigation.

4. Show or Hide Channels:

   • Use the Channel Controls on the right-hand side of the image window to display or hide specific channels.
   • If the channel controls are not visible, enable them via Image > Show Channels.

5. Edit the Display:

   • Add elements such as a scale bar, color reference table, or time display from the Image > Display menu.
   • Ensure the Z slice is correctly selected if viewing in XYZ, YZ, XZ, or XY mode.

6. Capture the Snapshot:

   • Go to Image > Capture Snapshot.
   • A new thumbnail for the snapshot will appear at the bottom of the library.
   • The snapshot incorporates all displayed options (e.g., scales, color tables, etc.) and is stored in the millions of colors format.

7. Export the Snapshot:

   • Highlight the snapshot in the library.
   • Go to File > Export.
   • Choose an export format suitable for publication:
     • Item as TIFF
     • Item as TIFF for Publication
   • Save the file to your desired location.

Method 2: Export Using the "View as..." Option (Merged Image or Movie)

This method is commonly used to quickly produce merged images or movies that appear exactly as they are shown in the Image View.

1. Open the Image Sequence:

   • In the Volocity Library, highlight the desired image sequence.
   • Double-click on the thumbnail to open it in its own window.

2. Select the Viewing Mode:

   • Switch to the Image Tab.
   • Use the dropdown menu to select the viewing mode (e.g., Extended Focus, XYZ, etc.).

3. Show or Hide Channels:

   • Use the Channel Controls on the right-hand side of the image window to display or hide specific channels.
   • If the channel controls are not visible, enable them via Image > Show Channels.

4. Edit the Display:

   • Add elements such as a scale bar, color reference table, or time display from the Image > Display menu.

5. Export the View:

   • Go to File > Export.
   • Select one of the View as... export options based on your requirements:
     • View as TIFF: Exports a single 2D image.
     • View as AVI Movie: Exports a time series dataset as a multi-channel merged movie.
     • Other formats, such as PNG or JPEG, are also available for single 2D images.

Key Considerations:

• For publication purposes, it is recommended to use the Item as TIFF for Publication option, as it provides high-quality output suitable for scientific figures.
• When exporting time series datasets as movies (e.g., AVI), ensure that all relevant channels and display options are configured before exporting.
• Always review the exported file to confirm it meets your publication or presentation needs.

By following these steps, you can produce and export merged images or sequences that are ready for use in scientific publications or professional presentations.

3.9. Avoiding Conflicts Between Volocity and Antivirus Software

3.10. How to Change the Spatial Calibration of a Dataset in Volocity

How to Change the Spatial Calibration of a Dataset in Volocity

Proper spatial calibration is essential for accurate quantitative analysis in Volocity. Each objective lens on the acquisition system should be calibrated using a stage graticule, which ensures that the measurements correspond to real-world dimensions. For detailed instructions on performing this calibration with Volocity Acquisition, refer to page 151 of the Volocity User Guide.

However, if the correct calibration values (microns per pixel) are already known, these can also be manually entered.

Steps to Change the Spatial Calibration:

1. Select the Image Sequence:

   • Open the dataset for which you want to adjust the spatial calibration.

2. Access Image Properties:

   • Go to the Edit menu and select Properties.

3. Modify Calibration Values:

   • Adjust the following fields as needed:
     • µm/pixel (X): Represents the pixel size in the X-axis.
     • µm/pixel (Y): Represents the pixel size in the Y-axis.
     • µm/pixel (Z): By default, this is set to the physical step size during acquisition but can be modified to account for spherical aberration.

Calculating µm/pixel for X and Y:

If the spatial calibration has not been performed using a stage graticule, you can calculate the µm/pixel value manually using the following equation:

µm/pixel = Camera Pixel Size (µm) / (Lens Magnification × Intermediate Magnification)

Example Calculation:

Scenario:

• Camera: Hamamatsu Orca AG
  • Pixel size: 6.45 µm (X and Y)
• Objective Lens: 63x magnification
• Intermediate Magnification: 0.63x reduction C-mount

Calculation:
µm/pixel = 6.45 / (63 × 0.63)
µm/pixel = 0.163

Result:
The spatial calibration values for X and Y would be 0.163 µm/pixel, which can be entered into the µm/pixel (X) and µm/pixel (Y) fields in the Properties dialog.

Best Practice: Calibrate Using a Stage Graticule

While manual calculations can provide approximate calibration values, it is best practice to use a stage graticule for calibration. This ensures the calibration values are as accurate as possible, accounting for all variations in optical components and camera setup.

3.11. How to Create a Measurement Item in Volocity

How to Create a Measurement Item in Volocity

A Measurement Item allows you to save and analyze measurements performed on points, lines, or regions of interest in your image data. Follow these steps to create and store a Measurement Item in Volocity.

Steps to Create a Measurement Item:

1. Make Measurements:

   • In the Measurements View, perform measurements on points, lines, and/or regions of interest.
   • Use the analysis tools available in the protocol to track objects or calculate properties of interest, such as volume, intensity, or shape.
   • Example: For a time-series dataset, you can construct a protocol to track objects over time.

   Note:

   • You do not need to include the Measure Objects task in your protocol to create a Measurement Item.
   • Include the Measure Objects task only if you want to measure objects at specific points within the protocol.

2. Use the "Make Measurement Item" Command:
   • Go to the Measurements Menu and select Make Measurement Item.
   • The Make Measurement Item Dialog will appear, allowing you to save your results as a new Measurement Item or append them to an existing one.

3. Configure Timepoint Options:
   • In the Make Measurement Item Dialog, choose which timepoints to measure:
     • Current Timepoint: Measure only the currently selected timepoint in the timeline.
     • All Timepoints: Analyze all timepoints in the time-series dataset.
     • Selected Timepoint(s): Measure specific timepoints that you have selected in the image sequence timeline or the time navigation bar.
   • If no timepoints are selected, the current timepoint is measured by default.

4. Save the Measurement Item:
   • Once created, the Measurement Item will be stored in the Volocity Library.
   • It will appear as a new library entry, allowing you to access, analyze, or export the data as needed.

By following these steps, you can efficiently create and manage Measurement Items in Volocity, ensuring your data is accurately recorded and ready for analysis.

3.12. How to Enter an SMA Code in Volocity

How to Enter an SMA Code in Volocity

To activate your Software Maintenance Agreement (SMA), follow these steps to enter your SMA code.

On macOS:

1. Start Volocity.
2. Go to the Volocity menu and select Unlock Codes.
3. Enter the code exactly as provided in your release code letter.
4. Confirm the entry and complete the process.

On Windows:

1. Start Volocity.
2. Go to the Help menu and select Unlock Codes.
3. Enter the code exactly as provided in your release code letter.
4. Confirm the entry and complete the process.

3.13. Exporting Charts for Use in Publications and Presentations

3.14. How to Import a Measurement Protocol in Volocity

How to Import a Measurement Protocol in Volocity

Importing a measurement protocol allows you to apply pre-configured analysis settings to your image data, saving time and ensuring consistent measurements across datasets. Follow these steps to import and restore a measurement protocol in Volocity:

Steps to Import a Measurement Protocol:

1. Start Volocity:

   • Launch Volocity and ensure you are on the correct workstation or user account.

2. Open an Image Sequence:

   • Load the image sequence you want to analyze.
   • Make sure the image sequence is correctly calibrated for measurement purposes.

3. Switch to the Measurements View:

   • Navigate to the Measurements View by selecting it from the view options in Volocity.
   • This view enables access to tools and options for analyzing your dataset.

4. Restore the Measurement Protocol:

   • Go to the Measurement menu and select Restore Protocol.
   • A dialog box will appear prompting you to locate and import the protocol file.

5. Import the Protocol:

   • Click the Import button in the Restore Protocol dialog box.
   • Browse to the location of the protocol file you wish to import.
   • Select the protocol file, which must be in .assf format, and click Open.

6. Save and Use the Protocol:

   • Once imported, the protocol will appear in the list of saved settings within the Restore Protocol window.
   • To apply the protocol, double-click on it or select it and click the appropriate option to restore the settings.
   • Volocity will automatically apply the imported measurement protocol to the active image sequence.

7. Verify and Customize (Optional):

   • Review the imported protocol settings to ensure they match your analysis requirements.
   • You can modify the protocol if necessary and save it as a new protocol for future use.

Benefits of Importing Measurement Protocols:

• Consistency: Ensures consistent analysis settings across multiple datasets.
• Efficiency: Saves time by avoiding the need to manually configure measurement settings for each dataset.
• Customizability: Imported protocols can be edited and saved as new templates, adapting them to evolving experimental needs.

By following these steps, you can streamline your measurement workflow and ensure accurate, reproducible results in your analyses.

3.15. How to Install the IEEE1394 OHCI Compliant Host Controller

How to Install the IEEE1394 OHCI Compliant Host Controller

This process applies to Windows 7 and Windows 7 x64 platforms using Hamamatsu Firewire (Dcam) cameras.

Steps to Install the IEEE1394 OHCI Compliant Host Controller (Legacy):

1. Open the Device Manager:

   • Go to the Windows menu.
   • Right-click on My Computer and select Properties.
   • Click on Device Manager.

2. Locate the IEEE1394 Bus Host Controllers:

   • Expand the IEEE1394 Bus Host Controllers section by clicking the dropdown arrow.
   • If you have multiple Firewire cards/controllers, identify the one connected to the Hamamatsu camera.

3. Update the Driver Software:

   • Right-click on the 1394 OHCI Compliant Host Controller and select Update Driver Software.
   • Choose Browse my computer for driver software.
   • Select Let me pick from a list of device drivers on my computer.

4. Install the Legacy Driver:

   • From the list, select the 1394 OHCI Compliant Host Controller (Legacy) option.
   • Click Next to install the driver.

5. Verify Installation:

   • If the installation is successful, you will see the message:
     "Windows has successfully updated your driver software".
   • In the Device Manager, confirm that the Legacy version of the OHCI Compliant Host Controller is installed.

6. Restart the System:

   • Turn off the Hamamatsu camera and shut down the workstation.
   • Power on the workstation first, then turn on the camera.

Following these steps ensures that the legacy driver is properly installed, resolving compatibility issues with Hamamatsu Firewire (Dcam) cameras.

3.16. Moving Items Between Libraries in Volocity

Moving Items Between Libraries in Volocity

The most convenient way to move items between libraries in Volocity is by using the Library Clipping export format. This method allows you to export and import items seamlessly between libraries.

Steps to Move Items Between Libraries:

1. Export Items from the Source Library:

   • Select the item(s) in the open library that you wish to move.
   • Go to the File menu and select Export.
   • Enter a name for the file and choose a location to save it.
   • From the Save as type dropdown menu, select Library Clipping (*.acff) as the export format.
   • If multiple items are selected, the Export dialog will display a Naming... button.
     • Use this button to define how individual files will be named.
   • Click the Export button to save the library clipping file.

2. Import Items into the Target Library:

   • Open the target library where you want to import the items.
   • Drag and drop the Library Clipping (.acff) file into the library view.

The selected items will now be added to the new library, maintaining their integrity and structure.

3.17. Understanding Software Autofocus Functionality

Software autofocus in Volocity is designed to find the optimal focal point by maximizing the signal-to-noise ratio (SNR) within the autofocus range. This means the algorithm identifies the point where the brightest point in the image divided by the darkest point in the image is greatest. By focusing on this ratio, the system ensures sharp and clear imaging.

Method Used: Golden Section Search

• Volocity uses the golden section search method to determine the optimal focus point.
• This mathematical optimization algorithm minimizes the number of sample exposures required, making the autofocus process faster and more efficient.
• More details on the golden section search can be found here.

Key Considerations:

1. Autofocus Limits:

   • It is crucial to set the autofocus range limits appropriately.
   • Incorrect or overly broad limits can cause the autofocus to be "thrown off" by local maxima—bright areas that may not represent the actual focal plane.

2. Region of Interest (ROI):

   • If a region of interest (ROI) is drawn on the live preview, the autofocus algorithm will restrict its calculations to this specific area.
   • This ensures that the autofocus is applied to the most relevant part of the image and avoids distractions from less important areas.

Advantages of Software Autofocus:

• It reduces manual adjustments, saving time during image acquisition.
• By using a mathematical approach to maximize SNR, the process is highly repeatable and precise, ideal for quantitative imaging tasks.
• The use of the golden section method minimizes unnecessary exposures, preserving the sample and improving workflow efficiency.

By setting the autofocus limits carefully and utilizing the ROI feature when necessary, Volocity's software autofocus can deliver highly accurate results, ensuring optimal imaging quality.

3.18. Generating Intensity-Modulated Display Images

Generating Intensity-Modulated Display Images

An intensity modulated display image is created by combining information from a ratio image (e.g., a FRET image) and a modulator image to accurately represent both intensity and ratio data in a single display. This process ensures that variations in intensity and the spatial distribution of the ratio values are visually preserved.

Steps to Generate an Intensity Modulated Display Image:

1. Convert the Ratio Image to an RGB Image:

   • The ratio image, often derived from fluorescence measurements (such as FRET), is first converted into an RGB image.
   • This step assigns color values to the ratio data, which represent the relative intensity or interaction levels in the original dataset.

2. Multiply by the Modulator Image:

   • The modulator image, which contains the intensity information, is then applied to the RGB ratio image through pixel-wise multiplication.
   • This step combines the ratio and intensity data, ensuring that both components contribute to the final visualization.
   • Areas with higher intensity values will appear brighter, while areas with lower intensity will appear dimmer, preserving the relationship between the ratio and intensity.

3. Normalize the Result:

   • The resulting image from the multiplication step is divided by 256.
   • This normalization step ensures that the resulting RGB values fall within the appropriate range for display (0-255 for each color channel).
   • Without this step, the resulting image might appear overly bright or contain artifacts due to out-of-range pixel values.

4. Display the Intensity Modulated Image:

   • The normalized RGB image is displayed as the intensity modulated image, where:
     • The color represents the ratio values (e.g., FRET efficiency).
     • The brightness represents the intensity or signal strength from the modulator image.

Why Use Intensity Modulated Images?

• Intensity modulated images are particularly useful for visualizing FRET data or other ratio-based measurements where both ratio and intensity information need to be presented simultaneously.
• This method avoids misinterpretation that could arise from viewing ratio data alone, as areas with low intensity are appropriately dimmed in the final display.

This process combines complex data into an easy-to-interpret format that maintains both spatial and quantitative information.

3.19. How to Derive Calibration Properties (µm/pixel) for Quantitative Analysis

How to Derive Calibration Properties (µm/pixel) for Quantitative Analysis

If you are performing quantitative analysis on image data, it is most accurate to retrieve the calibration properties from the acquisition system or re-acquire the data after calibrating with the specific objective lens.

Equation for Calibration Properties:

To calculate the spatial calibration (µm/pixel) for your image, use the following equation:

µm/pixel = Camera Cell Size / Total Magnification

Example Calculation:

For an image acquired using a Hamamatsu C9100-13 camera and a 40X objective lens (no intermediate magnification):

• Camera Cell Size (from the Hamamatsu C9100-13 specification sheet): 16 µm x 16 µm
• Magnification: 40
• Calculation:
  16 µm ÷ 40 = 0.4 µm/pixel

Notes:

• Magnification includes the objective lens magnification, but may also factor in:
  • C-mount magnification
  • Magnification introduced by other intermediate lenses (e.g., an Optovar).

Accurate calibration ensures reliable quantitative analysis of your image data.

3.20. How to Create a Debug Shortcut in Volocity

How to Create a Debug Shortcut in Volocity

Creating a debug shortcut in Volocity can be very useful for troubleshooting issues, especially in the event of a crash. The debug log provides detailed insight into specific problems.

Steps to Create a Debug Shortcut:

1. Create a Shortcut:

   • Navigate to: C:\Program Files\Volocity.
   • Locate the Volocity (x64).exe file.
   • Right-click on the .exe file and select Create Shortcut.
   • When prompted, place the shortcut on the desktop.
   • Rename the shortcut to Volocity Debug Shortcut.

1. Edit the Shortcut Properties:

   • Right-click on the newly created shortcut and select Properties.
   • In the Shortcut tab, locate the Target field.
   • Add the following text to the end of the target path:
     /d /logtofile
     (Note: Ensure there is a space before /d and /logtofile.)
   • The full Target field should read:
     "C:\Program Files\Volocity\Volocity (x64).exe" /d /logtofile
   • Click OK to save your changes.

1. Launch Volocity in Debug Mode:

   • Use the debug shortcut to launch Volocity when troubleshooting.
   • For conventional operation, launch Volocity as usual.

2. Retrieve the Debug Log:

   • After quitting Volocity (or if it crashes), retrieve the debug log from the following location:
     C:\Users\[Current User]\AppData\Local\Volocity7.0.0
   • Use this log to gain insights into the issues.

3.21. How to Check the Time Remaining in Your Acquisition

How to Check the Time Remaining in Your Acquisition

After starting a Volocity acquisition by pressing the Record button, you can track the time remaining in your acquisition.

Steps to Check the Time Remaining:

1. Locate the green box labeled Elapsed in the top-right corner of the screen.
2. Click on the Elapsed box.
3. You may need to click the box 2 or 3 times to toggle the display from Elapsed to Remaining.

This will show the remaining time for your acquisition.

3.22. Lost Lightpaths After Downgrading from Volocity 5.3 or Higher to an Older Version

Lost Lightpaths After Downgrading from Volocity 5.3 or Higher to an Older Version

If you experience lost lightpaths after moving from Volocity 5.3 or higher to an older version, this issue may be related to the HASP dongle device drivers included with the Volocity installer package. When the drivers are installed, any active processes using the dongle may be suspended.

Resolution:

1. Restart the License Server Computer:
   • Restarting the License Server will reactivate the dongle and resolve the issue with lightpaths.

This should restore functionality and resolve the lightpath issue in the older version of Volocity.

3.23. Lost Lightpaths After Moving Configuration Files to a Different Workstation – How to Reassign DCAM ID

Lost Lightpaths After Moving Configuration Files to a Different Workstation – How to Reassign DCAM ID

When moving configuration files to a different workstation, the DCAM ID for connected cameras may need to be reassigned. Follow these steps on a Windows workstation to resolve this issue:

Steps to Reassign DCAM ID:

1. Open the Windows Registry Editor:

   • Select the Start menu and search for regedit.exe.
   • Launch regedit.exe.

2. Export the Volocity Registry Settings:

   • Expand the data tree to locate the Volocity user-level settings.
   • Right-click on the Volocity folder and select Export.
   • Save the exported registry key to a local directory, such as the Desktop.

3. Edit the Registry Key:

   • Navigate to the saved registry key file and open it in a text editor (e.g., Notepad).
   • Search for the phrase "DCAM" within the text document.
   • Locate the hex key for each camera's DCAM ID.

4. Reassign DCAM IDs:

   • Modify the first two digits of the hex key to reassign the DCAM ID.
   • For example, if swapping the DCAM IDs of two cameras, adjust the first two digits of their respective hex keys accordingly.

5. Save Changes:

   • Save the changes to the registry file and close the text editor.

6. Apply the New Settings in Volocity:

   • Double-click the amended registry key file.
   • When prompted, select Yes to add the updated key to the Windows registry.

Your DCAM IDs will now be reassigned, and the lightpaths should work correctly in Volocity.

3.24. Why Do the Loop and Shuttle Options Work Under the Image Menu but Not Under the Movie Menu?

Why Do the Loop and Shuttle Options Work Under the Image Menu but Not Under the Movie Menu?

The behavior of the Loop and Shuttle commands depends on whether you are working with an Image Sequence or a Movie in Volocity.

Image Menu:

• The Loop and Shuttle commands in the Image menu are specifically designed for image sequences.
• These commands will not work if applied to movies.

Movie Menu:

• The Loop and Shuttle commands in the Movie menu are intended for movies created in Volocity.
• To use these commands, you must be viewing a movie, not an image sequence.

Ensure you are working with the correct file type (image sequence or movie) when using these commands in Volocity.

3.25. The Size of the Video Preview Has Changed – How Do I Restore It?

The Size of the Video Preview Has Changed – How Do I Restore It?

If you and your colleagues use the same user account, it’s possible that one of the following settings has been changed. Follow the steps below to identify and correct the issue.

1. Binning:

• Location: Found in the Device Controls when using the Video Preview.
• Explanation: Binning combines 2 or more camera pixels. If the same area of your sample is visible but the image size has changed, the binning level may have been altered.
• Solution: Ensure you are using the same binning level as before. Increasing the binning level will reduce the size of the Video Preview.

2. Crop Video:

• Location: Found in the Video menu under Crop Video.
• Explanation: Cropping reduces the video size to a specific region of the CCD. Smaller crop sizes will reduce the Video Preview size.
• Solution:
  • To undo the crop, select Full Frame in the Crop Video options.
  • Alternatively, select the Crop Tool, then click anywhere outside the crop boundary to restore the full frame.

3. CCD Active Area:

• Location: Found in the Video menu under CCD Active Area.
• Explanation: Margins may have been set by another user, reducing the overall image size. The CCD Active Area feature excludes unwanted regions of the field of view.
• Solution:
  • Open the CCD Active Area window.
  • Click the Default button to reset all margins to 0.

By following these steps, you can restore the Video Preview to its original size.

3.26. Tools Missing or Moved in the Toolbar

Tools Missing or Moved in the Toolbar

In Volocity 5 or higher on macOS, the toolbar can be customized to include only the tools you need. If all users share the same macOS account, customizations might confuse some users. To move or restore a missing tool, follow these steps:

Steps to Customize the Toolbar:

1. Start Volocity.
2. Navigate to the view where the tool is missing or misplaced.
3. Hold the Control key and click on the toolbar.
4. From the pop-up menu, select Customize Toolbar.
5. A window will appear with the prompt to "Drag your favorite items into the toolbar."
6. Drag the missing or moved tool to the desired location in the toolbar.

Note:

• This feature is only available on macOS.
• Customizing the toolbar is not supported on Windows.

3.27. What Is the Difference Between Z Step and µm/pixel (Z) in the Properties?

What Is the Difference Between Z Step and µm/pixel (Z) in the Properties?

• Z Step (µm):

  • Refers to the physical step used to capture the original images.
  • Changing this value does not affect how the data is represented in the image—it is purely for reference purposes.

• µm/pixel (Z):

  • Refers to the Z calibration, which is the optical step corrected for spherical aberration.
  • This value may be the same as the physical step but can be adjusted to account for spherical aberration, providing more accurate calibration.

Understanding these differences ensures accurate representation and interpretation of your imaging data.

3.28. What Is the Easiest Way to Capture Fluorescent and Brightfield Images Without Merging Them Manually?

What Is the Easiest Way to Capture Fluorescent and Brightfield Images Without Merging Them Manually?

A more efficient method is to acquire two channels while using Manual Intervention in the Brightfield light path. This allows you to capture both fluorescent and brightfield images without acquiring and merging them separately.

Steps to Enable Manual Intervention:

1. Double-click the Light Path Manager button.
2. In the Light Path Properties dialog, check the box for Requires Manual Intervention if the light path requires manual adjustments.
3. When this light path is called during acquisition, a message will prompt you to make the necessary manual changes.

This process streamlines the acquisition of both fluorescent and brightfield images in a single workflow.

3.29. Why Are Focus Positions Not Stored When Creating XY Points?

Why Are Focus Positions Not Stored When Creating XY Points?

If the focus position is not being stored when creating or reviewing XY points, it is likely because a Focus Device has not been set in the X-Y Stage preferences. Without a Focus Device configured, Volocity cannot store the Z position for each point.

How to Set the Focus Device:

On macOS:

1. Go to the Volocity menu and select Preferences.
2. Select X-Y Stage.
3. Choose the Focus Device from the pop-up menu.

On Windows:

1. Go to the Edit menu and select Preferences.
2. Select X-Y Stage.
3. Choose the Focus Device from the pop-up menu.

Once the Focus Device is set, the Z positions will be stored correctly when creating or reviewing points.

3.30. Why Can't I Change the Filter Turret Position with My Leica Microscope Using Volocity?

Why Can't I Change the Filter Turret Position with My Leica Microscope Using Volocity?

If the filter turret does not move to the transmitted light position when requested in Volocity, this is likely due to the Contrast Mode setting on Leica microscopes. Leica microscopes have contrast modes such as "Fluorescence," "DIC," or "DIC and Fluorescence," which can limit software control over the filter turret position.

Steps to Resolve the Issue:

1. Check and Adjust the Contrast Mode:

   • Ensure that the Contrast Mode is set to the desired setting (e.g., "DIC and Fluorescence" for compatibility with both transmitted and fluorescent light paths).

2. Include the Contrast Mode in the Lightpath:

   • Double-click on the desired lightpath button in the Video Preview.
   • In the Device Checklist, ensure that Contrast Mode is checked.
   • Click Save to update the lightpath settings.

If necessary, you can manually move the filter turret to the transmitted light position using the buttons on the microscope.

3.31. Why Can't I Make Changes to My Data After Importing It from a Portable Hard Drive or Another Workstation?

Why Can't I Make Changes to My Data After Importing It from a Portable Hard Drive or Another Workstation?

If your dataset is still linked to a source file located outside the Volocity library (e.g., on a portable hard drive), you will not be able to perform permanent changes, such as creating an image sequence or cropping data. The dataset must be adopted by Volocity to enable these changes.

Steps to Adopt Data in Volocity:

1. Select the dataset thumbnail in the Library window.
2. Go to the Actions menu and select Adopt Items.
3. Volocity will locate the source file(s) and adopt the data, incorporating it into the Volocity library.

Once the dataset is adopted, you will be able to make permanent changes within Volocity.

3.32. Why Do My Objects Appear Elongated in the Z Axis?

Why Do My Objects Appear Elongated in the Z Axis?

Elongation in the Z axis is a common issue in optical microscopy and is often caused by a mismatch between the physical and optical step size between Z layers during imaging.

Steps to Address Z-Axis Elongation:

1. Verify Spatial Calibration:

   • Ensure that the spatial calibration of the objective lenses has been performed correctly.
   • Detailed instructions are available in the Volocity User Guide (pages 120-121).

2. Understand Optical Aberrations:

   • Elongation in Z can result from axial aberrations inherent to optical microscopy.
   • These factors are discussed in the Volocity User Guide (pages 355-356) and in Technical Note 382.

Additional References on Z-Axis Aberrations:

• Kam, Z. et al. (2001). Computational adaptive optics for live three-dimensional biological imaging. PNAS, 98, 3790-3795.
• Scalettar, B. A. et al. (1996). Dispersion, aberration, and deconvolution in multi-wavelength fluorescence images. Journal of Microscopy, 182, 50-60.
• Sherman, J. et al. (2002). Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror. Journal of Microscopy, 206, 65-71.

3.33. Why Is the Length of the 'Skeleton' Not Always Equal to the Skeletal Length Measurement in the Measurement Item?

Why Is the Length of the 'Skeleton' Not Always Equal to the Skeletal Length Measurement in the Measurement Item?

The difference arises due to how Volocity calculates skeletal length and how the skeleton is visually represented.

Definition of Skeletal Length

As described in the Volocity Help menu:

• Skeletal length is calculated by thinning or skeletonizing the object from all sides until only a single voxel-wide line remains.
• This measurement is best suited for elongated shapes.
• The line's direction is elongated to meet the edges of the object, and the length of this line is the skeletal length.
• For objects with branching skeletons, skeletal length is measured as the longest path across the object without crossing any point more than once.

Skeletal Diameter

The skeletal diameter is the diameter of a hypothetical cylinder that has:

• A length equal to the skeletal length of the object.
• A volume equal to the object's measured volume.

The skeletal diameter is derived using the equation for the volume of a cylinder:
V = πr²l

Skeleton Feedback

• When the skeletal length is measured, Volocity populates the measurement columns with skeletal length and skeletal diameter for each measured object.
• A measurement row is added, visually representing the skeletal length with a line (the skeleton).
• The skeleton serves as feedback, with the parent ID column reflecting the ID of the source object.

Sub-Sampling and Visual Discrepancies

• To create the skeleton, Volocity uses a sub-sampling method, selecting a subset of the total number of voxels (nodes) in the skeletonized object.
• This process can cause the skeleton to visually "cut corners," as the path of the yellow line (skeleton feedback) does not necessarily align with the red object (original shape).

Key Consideration

• The skeleton shown in the image is intended only as a visual representation of the path taken through the object after skeletonization.
• The measured skeletal length is calculated using the algorithm and is accurate, but it may not match the distance along the visual skeleton line.

3.34. Calibrate Stage Option Greyed Out

Why Is the "Calibrate Stage" Option Greyed Out Even Though My X-Y Stage Is Recognized?

The preferences for the X-Y stage are stored in two locations within Volocity:

1. Device Preferences:

   • Accessible via Edit > Preferences > Devices (or Volocity > Preferences > Devices on macOS).

2. X-Y Stage Preferences:

   • Accessible via Edit > Preferences > X-Y Stage (or Volocity > Preferences > X-Y Stage on macOS).

Troubleshooting Steps:

1. Verify that the stage appears in the Device Preferences and that the X-Y Stage option is selected.
2. If the "Calibrate Stage" function in the Stage menu is still greyed out, ensure that an appropriate X-Y stage device is selected in the X-Y Stage Preferences.

This ensures that Volocity recognizes the correct stage settings and enables the calibration functionality.

3.35. Configuring the Serial port of the DMi8 for Volocity

Configuring the Serial Port of the DMi8 for Volocity

When you install the Leica SDK (delivered with the DMi8 microscope), it includes a driver that allows the computer to connect to the microscope via USB. This driver sets up a "Virtual Com Port" (VCP) for serial port communication. While the Leica SDK does not require the VCP for communication, the Volocity software does.

Steps to Enable the VCP for the DMi8:

1. Open Device Manager:

   • Press Win + X and select Device Manager.

2. Locate the DMi8 Device:

   • The DMi8 will appear under USB Controllers as a USB Serial Controller.

3. Access Device Properties:

   • Double-click the device, or right-click and select Properties.

4. Enable the Virtual Com Port (VCP):

   • Navigate to the Advanced tab.
   • Check the box for Load VCP.

5. Verify the Device in "Ports":

   • After enabling the VCP, the device will now appear under Ports in Device Manager.
   • Note the assigned COM Port number.

6. Configure Volocity:

   • In Volocity, set up your serial devices and select the appropriate COM Port for the DMi8.

3.36. Volocity ILS & Windows Defender

Technical Guide: Configuring Windows Defender Firewall Exceptions

Overview

Windows Defender (integrated into Windows 8.1, 10, and 11) includes a built-in firewall designed to monitor and restrict network traffic. For networked applications like Volocity ILS, the firewall may block the connection between the client and the license server. Manually "allowing" the application creates an exception rule, permitting necessary data packets to pass through without compromising the system's overall security.

Configuration Procedure

1. Access Security Settings

Navigate to the Control Panel. The most direct route to firewall management is through the Security and Maintenance category. This hub provides a high-level overview of the system’s protective status, including antivirus and network filtering.

2. Enter Firewall Permissions

Within the Security and Maintenance interface, locate and select:

Allow an app or feature through Windows Defender Firewall

Note: You may be prompted for Administrator credentials at this stage to modify system-level permissions.

3. Define the Exception

A list of installed applications and features will appear. To enable traffic for a specific tool (e.g., Volocity):

• Locate the Application: Scroll to find the specific executable or service name.

• Assign Network Profiles: Check the box next to the application name.

• Determine Scope: Select Private (for secure office/home networks) or Public (for open networks) depending on your IT environment. For license servers, "Private" is the standard recommendation.

Troubleshooting Tip

If the application is not listed, click the "Allow another app..." button to manually browse for the .exe file in its installation directory.

3.37. Volocity Supported File Formats

Volocity Supported File Formats

File Formats for Import

Volocity supports the following file formats for reading:

• BioRad PIC
• Windows BMP
• Volocity Library Clipping
• DeltaVision
• ICS/IDS (Image Cytometry Format)
• JPEG
• Leica LIF (generated by Leica confocals)
• Neurolucida Tracing XML format
• Openlab LIFF
• PerkinElmer AIC
• PerkinElmer Harmony "Index and Images" and "Index with Referenced Images" (generated by PerkinElmer Operetta High Content imaging systems)
• Apple PICT
• QuickTime Images (e.g., Photoshop PSD and other still image formats supported by QuickTime)
• TIFF, including:
  • Improvision TIFF extensions (metadata in the ImageDescription tag)
  • Leica TIFF (generated by Leica confocals)
  • Olympus TIFF (generated by Olympus FluoView confocals, xcellence, and cell^R systems)
  • PerkinElmer TIFF (generated by PerkinElmer UltraVIEW systems)
  • GE TIFF (generated by GE Healthcare INCell 1000 systems)
  • OME TIFF (as described by the Open Microscopy Environment: openmicroscopy.org)
  • Zeiss LSM (a TIFF variant)
  • Zeiss ZVI
  • MetaMorph STK (a TIFF variant)
  • Olympus OIF (a TIFF variant)
  • TILLVision

File Formats for Export

Volocity supports writing the following file formats:

• Windows BMP
• Volocity Library Clipping
• ICS/IDS (Image Cytometry Format)
• JPEG
• Openlab LIFF
• MATLAB v5 image format (compatible with MATLAB versions 5 and 6)
• TIFF (with Improvision extensions)
• OME-TIFF (2007 schema, as described by the Open Microscopy Environment: openmicroscopy.org)
• TIFF for Publication
• QuickTime Movies
• AVI Movies (Windows only)
• WMV Movies (Windows only)

Volocity also supports writing views as QuickTime, AVI, and WMV movies where these formats are available.