Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking...

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Single-molecule localization microscopy (SMLM) enables nanoscale imaging but faces challenges with photobleaching and photon budget in thick specimens.
• Multiphoton excitation (MPE) offers advantages for deep tissue imaging but requires optimization for SMLM.

Purpose of the Study:

• To develop and validate a single-wavelength temporal-focusing multiphoton excitation (TFMPE) SMLM technique for improved 3D imaging.
• To optimize TPE wavelength for enhanced photon detection from blinking fluorophores.
• To visualize subcellular structures and pathological features in thick biological samples.

Main Methods:

• Implemented single-wavelength TFMPE for wide-field and axially confined two-photon excitation (TPE) of blinking fluorophores.
• Performed TPE spectral measurements to determine optimal excitation wavelengths.
• Combined TFMPE-SMLM with astigmatic imaging for 3D reconstructions.

Main Results:

• Achieved nanoscale spatial resolution (approx. 51 nm) and high localization precision (18±6 nm) in 2D SMLM of cancer cell microtubules.
• Demonstrated 3D TFMPE-SMLM imaging of amyloid-beta deposits in Alzheimer's disease mouse brain tissue.
• Successfully reduced photobleaching effects in out-of-focus regions, improving photon budget utilization.

Conclusions:

• TFMPE-SMLM is a powerful tool for high-resolution 3D imaging of subcellular structures and disease pathology in thick specimens.
• The optimized TPE wavelength significantly enhances photon detection efficiency for SMLM.
• This technique offers a promising approach for studying complex biological systems at the nanoscale.