Three-dimensional stimulation and imaging-based functional optical microscopy of biological cells

Area of Science:

• Biophotonics
• Optical Microscopy
• Cellular Imaging

Background:

• Functional optical microscopy is crucial for observing cellular dynamics.
• Existing methods often lack simultaneous 3D stimulation and volumetric imaging capabilities.
• Precise control over cellular states is essential for research, particularly in optogenetics.

Purpose of the Study:

• To introduce a novel optical microscope system, three-dimensional (3D) stimulation and imaging-based functional optical microscopy (SIFOM).
• To demonstrate SIFOM's ability for precise, simultaneous 3D stimulation and volumetric fluorescence recording.
• To validate the system's effectiveness in biological experiments.

Main Methods:

• SIFOM utilizes digital holograms on a phase-mode spatial light modulator to generate multiple 3D spots for precise cellular stimulation.
• Common-path off-axis incoherent digital holographic microscopy, with a diffraction grating and focusing lens on another spatial light modulator, enables single-shot 3D fluorescence acquisition.
• The system integrates stimulation and imaging into a single acquisition process.

Main Results:

• The system successfully achieved precise, simultaneous stimulation of user-defined biological cells.
• Volumetric fluorescence distribution was recorded in a single acquisition.
• Experiments with fluorescent microbeads and human lung cancer cells confirmed the system's effectiveness at various defocused positions.

Conclusions:

• SIFOM represents a novel advancement in functional optical microscopy.
• The system's capability for precise 3D stimulation and simultaneous volumetric imaging is validated.
• SIFOM holds significant potential for applications in optogenetics and cellular state manipulation.