Zeiss Z.1 Lightsheet - sample preparation and basic imaging

Introduction

This tutorial provides a comprehensive guide on how to prepare and image samples using the Zeiss Z.1 Lightsheet system at Cardiff University's Bioimaging Research Hub. It covers the steps for preparing samples using LMP agarose and capillary tubes, as well as configuring the Lightsheet system for z-stacks, time series, and multiview imaging. This knowledge is essential for researchers looking to utilize advanced imaging techniques in their work.

Step 1: Preparing Samples with LMP Agarose

To prepare samples effectively for imaging with the Zeiss Z.1 Lightsheet, follow these steps:

1. Select Your Sample: Choose the biological sample you want to image, ensuring it is suitable for lightsheet imaging.
2. Mix LMP Agarose:
   • Use low-melting-point (LMP) agarose for embedding the sample.
   • Prepare a solution of LMP agarose according to the manufacturer’s instructions, typically around 1-2% concentration.
3. Pour the Agarose:
   • Place your sample in a suitable mold.
   • Pour the LMP agarose over the sample, ensuring it is fully submerged.
4. Allow to Set: Let the agarose set at room temperature until solidified.
5. Cut the Agarose Block: If necessary, trim the agarose block to fit into the imaging chamber of the Lightsheet system.

Step 2: Using Capillary Tubes for Sample Placement

Capillary tubes can be an alternative method for sample preparation. Follow these steps:

1. Prepare Capillary Tubes:
   • Select appropriate capillary tubes that can hold your sample.
   • Ensure the tubes are clean and free from contaminants.
2. Fill with Agarose:
   • Heat the LMP agarose until it becomes liquid.
   • Carefully fill the capillary tubes with the liquid agarose, ensuring no air bubbles are present.
3. Insert the Sample:
   • Place your sample into the capillary tube while the agarose is still liquid.
   • Allow the agarose to set, forming a solid structure around the sample.

Step 3: Configuring the Lightsheet System

Once your samples are prepared, configure the Zeiss Z.1 Lightsheet for imaging:

1. Set Up the Imaging System:
   • Power on the Lightsheet system and ensure all components are functioning properly.
   • Load your prepared sample into the imaging chamber.
2. Adjust Imaging Parameters:
   • Configure the system for z-stacks:
     • Select the appropriate slice thickness and number of slices based on your sample’s dimensions.
   • For time series imaging, set the time interval and total duration of the imaging session.
   • For multiview imaging, configure the system to capture images from multiple angles.
3. Calibrate the System:
   • Perform a calibration routine as per the manufacturer’s guidelines to ensure accurate imaging.
   • Focus the microscope on your sample and adjust the light settings to enhance visibility.

Conclusion

In this tutorial, you learned how to prepare samples using LMP agarose and capillary tubes, and how to configure the Zeiss Z.1 Lightsheet for effective imaging. Following these steps will help you utilize the Lightsheet system efficiently, allowing for detailed imaging of biological samples. For further exploration, consider experimenting with different sample types or advanced imaging settings to enhance your research findings. For more information, visit the Cardiff University Bioimaging Research Hub's website.