3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy

Area of Science:

• Biophysics
• Optical Microscopy
• Cell Biology

Background:

• 3D live imaging is crucial for understanding biological processes but faces limitations with current techniques like spinning-disk confocal microscopy.
• Existing methods often struggle to balance speed, resolution, and minimal sample damage.
• Advanced microscopy techniques are needed to overcome these challenges in live-cell imaging.

Purpose of the Study:

• To provide a comprehensive guide to Bessel beam plane illumination microscopy (BPIM) for high-speed 3D live imaging.
• To explain the fundamental principles, operational modes, and optimization strategies for BPIM.
• To detail the construction, alignment, and operation of a BPIM system for researchers.

Main Methods:

• Explanation of the working principles of Bessel beam plane illumination microscopy.
• Discussion of the advantages and disadvantages of different operational modes.
• Demonstration of parameter optimization through practical examples.
• Step-by-step procedures for constructing and aligning a BPIM system.

Main Results:

• BPIM offers high-speed 3D live fluorescence imaging with near-isotropic resolution.
• The technique provides low photobleaching and photodamage, crucial for long-term live imaging.
• Different operational modes allow for tailored performance based on specific imaging needs.
• A detailed protocol and equipment list are provided for building a functional BPIM.

Conclusions:

• Bessel beam plane illumination microscopy is a powerful tool for advanced 3D live imaging of cellular and multicellular specimens.
• Optimizing operational modes and experimental settings is key to maximizing BPIM performance.
• The provided protocol facilitates the construction and implementation of BPIM systems for biological research.