Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method

Area of Science:

• Biophysics
• Optical Microscopy
• Live-cell Imaging

Background:

• Multiconfocal microscopy offers a balance of speed and resolution but is limited by low fluorescence intensity and optical aberrations.
• Aberrations from refractive index mismatches distort the point spread function, reducing detected photons and image quality, hindering deep-sample imaging and molecular process analysis.

Purpose of the Study:

• To improve fluorescence detection and image quality in spinning disk confocal microscopy.
• To introduce an adaptive optics module that corrects aberrations without prior sample illumination, minimizing photobleaching and phototoxicity.

Main Methods:

• Utilized a compact adaptive optics module (adaptive optics box for sectioning optical microscopy) designed for spinning disk confocal microscopy.
• Developed a depth-dependent aberration model to correct aberrations during z-stack acquisition without pre-illumination.
• Applied the adaptive optics module and model to various biological samples.

Main Results:

• Successfully corrected aberrations in confocal imaging, overcoming limitations of low fluorescence intensity.
• The depth-dependent aberration model enabled aberration correction during z-stacking, minimizing sample photobleaching and phototoxicity.
• Achieved significant improvements in signal-to-background ratio and image contrast.

Conclusions:

• Adaptive optics, particularly with the developed depth-dependent model, enhances multiconfocal microscopy performance.
• This approach allows for improved imaging deep within biological samples with reduced phototoxicity.
• The technique facilitates more accurate analysis of molecular processes in live biological samples.