Breaking the 100-nm resolution barrier with multiphoton microscopy using image scanning microscopy and optical...

Area of Science:

• Optical microscopy
• Super-resolution imaging
• Biophotonics

Background:

• Multiphoton fluorescence microscopy excels with thick, scattering samples but offers lower resolution than single-photon super-resolution techniques.
• Conventional laser scanning multiphoton microscopy suffers from reduced spatial resolution compared to confocal microscopy.
• Achieving high resolution in deep tissue imaging remains a significant challenge in biological sciences.

Purpose of the Study:

• To enhance the spatial resolving power of multiphoton microscopy by integrating super-resolution methods.
• To overcome the inherent resolution limitations of standard laser scanning multiphoton microscopy.
• To enable sub-100 nm imaging in thick, scattering biological specimens.

Main Methods:

• Combined two-photon microscopy with image scanning microscopy and super-resolution optical fluctuation imaging.
• Detected descanned fluorescence signals using a 23-element single photon avalanche detector.
• Analyzed temporal signal fluctuations via higher-order cumulant calculations and image deconvolution.

Main Results:

• Achieved a lateral resolution better than 100 nm, specifically 75 nm for quantum dots emitting at 625 nm.
• Demonstrated a 5-fold resolution improvement over standard two-photon microscopy.
• Validated the method's applicability on biological samples, including fixed mouse neurons labeled with quantum dots.

Conclusions:

• The novel combination of two-photon microscopy, image scanning microscopy, and optical fluctuation imaging significantly boosts resolution.
• This technique provides a 3.5-fold improvement over conventional widefield imaging for the same fluorophores.
• Represents a breakthrough in achieving sub-100 nm resolution with multiphoton laser scanning microscopy.