Depth-enhanced molecular imaging with two-photon oblique plane microscopy.

Area of Science:

• Biophysics
• Microscopy
• Optical Imaging

Background:

• High-numerical-aperture (NA) oblique plane microscopy offers noninvasive subcellular imaging.
• Scattering and refractive-index variations limit deep tissue imaging performance.
• Out-of-focus background degrades resolution in multicellular specimens.

Purpose of the Study:

• To develop a two-photon oblique plane microscope for improved deep tissue imaging.
• To enhance resolution and reduce photodamage in scattering environments.
• To enable advanced live imaging of subcellular dynamics in vivo.

Main Methods:

• Combining high-NA single-objective detection with multiphoton plane illumination.
• Utilizing near-infrared nonlinear excitation for enhanced contrast and reduced photodamage.
• Achieving high-speed volumetric acquisition with single-molecule sensitivity.

Main Results:

• Achieved ~300 nm lateral and ~650 nm axial resolution in vivo.
• Demonstrated >5x faster volumetric acquisition (up to 3.25 x 10^6 voxels s-1) with reduced peak intensity compared to two-photon point scanning.
• Enhanced contrast by ~2x and restored volumetric resolving power by >2x in multicellular contexts using nonlinear excitation.

Conclusions:

• The developed two-photon oblique plane microscope significantly improves deep tissue imaging capabilities.
• It enables noninvasive, high-resolution, and fast live imaging of subcellular dynamics in complex biological samples.
• The technology facilitates advanced applications like multicolor transcription-factor dynamics and single-mRNA tracking.