Photoimprint photoacoustic microscopy for three-dimensional label-free subdiffraction imaging

Area of Science:

• Optical microscopy
• Biomedical imaging
• Nanotechnology

Background:

• Conventional optical microscopy is limited by the diffraction limit, restricting the resolution of cellular and subcellular imaging.
• Photoacoustic microscopy (PAM) offers unique contrast mechanisms but typically lacks optical sectioning capabilities.
• Subdiffraction imaging techniques aim to overcome the diffraction limit for enhanced resolution.

Purpose of the Study:

• To develop a simple method for subdiffraction photoacoustic imaging (SPAI) applicable to both fluorescent and nonfluorescent samples.
• To enhance lateral resolution beyond the diffraction limit using a novel double-excitation process.
• To introduce inherent optical sectioning capability into photoacoustic microscopy.

Main Methods:

• Combined absorption-based photoacoustic effect with intensity-dependent photobleaching.
• Employed a double-excitation process: a first pulse inhomogeneously bleaches molecules, biasing signal contributions from a second pulse.
• Utilized the differential signal between excitations to isolate signal from the excitation center, sharpening resolution.

Main Results:

• Achieved subdiffraction resolution in photoacoustic imaging.
• Demonstrated three-dimensional imaging of fluorescent and nonfluorescent species by scanning the excitation beam.
• The nonlinear signal generation provided inherent optical sectioning, improving depth discrimination.

Conclusions:

• The developed method enables subdiffraction photoacoustic imaging with enhanced lateral resolution and optical sectioning.
• The technique's reliance on molecular absorption suggests potential for label-free subdiffraction imaging.
• This approach is versatile and can be adapted for other optical imaging modalities or combined with existing subdiffraction methods.