A practical guide to scanning light-field microscopy with digital adaptive optics

Area of Science:

• Biomedical Imaging
• Microscopy
• Optical Engineering

Background:

• Growing need for long-term, high-speed intravital fluorescence imaging to study cellular interactions.
• Limitations of traditional intravital microscopy in spatiotemporal resolution and phototoxicity.
• Scanning light-field microscopy (sLFM) offers a computational solution for volumetric imaging.

Purpose of the Study:

• To present a step-by-step protocol for implementing multicolor sLFM as an add-on to a wide-field fluorescence microscope.
• To provide a low-cost, accessible method using off-the-shelf components.
• To detail hardware and software implementation, including calibration and data processing.

Main Methods:

• Development of a multicolor sLFM system using readily available lenses and devices.
• Implementation of algorithmic post-processing for artifact correction and performance optimization.
• A one-step calibration method for robust imaging performance and an open-source GUI for synchronization and rendering.

Main Results:

• Successful implementation of a compact, low-cost multicolor sLFM system.
• Demonstration of improved spatiotemporal resolution and reduced phototoxicity compared to traditional methods.
• A comprehensive protocol enabling setup, calibration, and 3D reconstruction within 3-4 days.

Conclusions:

• The presented sLFM protocol provides an accessible and effective solution for advanced intravital fluorescence imaging.
• The method is adaptable to other light-field microscopy variants.
• This technique facilitates detailed observation of intercellular and intracellular dynamics in native biological states.