The LaVision BioTec UltraMicroscope II is a light-sheet microscope ideally suited for imaging of large, fixed, cleared, fluorescent samples, with cellular resolution. This guide describes the basic principles of this system’s operation, the main configurations available, how to minimize risks when using it, tips for sample preparation and mounting and an extensive discussion of how to optimize imaging parameters. It also provides advice for how to handle data transfers from the microscope, as well as how to configure a workstation for data visualization and basic analysis. Finally, it provides standard operating procedures that can be used with this system in other labs or core facilities. This guide aims to provide a baseline of practical information and recommended workflows for new users of this system.

Contents

• Title Page and Copyright Notice
• 1. Administrivia
  • 1.1. Who is this for?
  • 1.2. Why should you listen to me?
  • 1.3. Why did I write this?
  • 1.4. What this is not
• 2. System Basics
  • 2.1. Basic principles of system operation
  • 2.2. What is this system good for?
• 3. System Configuration
  • 3.1. What are the main configuration options?
  • 3.2. Acquisition computer specifications
  • 3.3. What else will you need?
• 4. Risks
  • 4.1. Risks to users
  • 4.2. Risks to equipment
• 5. Workflow Overview
• 6. Sample Preparation Tips
  • 6.1. Trimming to the biological question
  • 6.2. Clearing and labeling
• 7. Sample Mounting Tips
• 8. Optimizing Imaging Parameters
  • 8.1. Introduction
  • 8.2. Light sheet parameters and how to adjust them optimally
  • 8.3. Additional imaging considerations
  • 8.4. Workflow summary and examples
• 9. Alignment Tips
• 10. Deconvolution
• 11. Data Transfer
  • 11.1. Defining the problem
  • 11.2. Defining other problems that the UNC Core cannot solve quickly, cheaply or at all
  • 11.3. Constraints
  • 11.4. Narrowing the problem
  • 11.5. Current configuration in the UNC Core
  • 11.6. Thoughts about even bigger datasets
• 12. Visualization/Analysis Workstation
• 13. Acknowledgements
• ANNEX - Standard Operating Procedures UNC
• References