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Highly Sensitive Shack-Hartmann Wavefront Sensor: Application to Non-Transparent Tissue Mimic Imaging with Adaptive
Javier Morgado Brajones1, Gregory Clouvel2, Guillaume Dovillaire2
1ITAV, Université de Toulouse, CNRS, 31106 Toulouse, France.
Methods and Protocols
|July 25, 2019
Summary
We developed a wavefront sensor adaptive optics SPIM (WAO-SPIM) to improve deep-tissue imaging. This advanced microscopy technique corrects optical aberrations in thick samples, enhancing visualization of cellular structures.
Area of Science:
- Biomedical Imaging
- Optical Microscopy
- Adaptive Optics
Background:
- High-quality 3D imaging in thick biological samples is challenging due to scattering and aberrations.
- Selective Plane Illumination Microscopy (SPIM) offers subcellular resolution but is limited in imaging depth.
- Adaptive Optics (AO) corrects optical aberrations to improve microscope performance.
Purpose of the Study:
- To incorporate adaptive optics into SPIM (WAO-SPIM) for correcting aberrations in optically-thick samples.
- To develop a high-sensitivity Shack-Hartmann wavefront sensor (SHWS) for use with faint non-linear guide stars (NGS).
- To demonstrate the effectiveness of WAO-SPIM in imaging thick, inhomogeneous samples like multi-cellular tumor spheroids (MCTS).
Main Methods:
- Integration of a wavefront sensor adaptive optics scheme into SPIM.
- Utilizing two-photon fluorescence to generate a non-linear guide star (NGS) within the sample.
- Development and application of a high-sensitivity Shack-Hartmann wavefront sensor (SHWS).
Main Results:
- Successful correction of aberrations in thick, inhomogeneous samples like MCTS using WAO-SPIM.
- Demonstrated significant improvements in spatial frequencies relevant to cellular and subcellular features (up to 79%).
- The developed SHWS effectively detected and corrected aberrations using faint NGS.
Conclusions:
- WAO-SPIM with a novel SHWS enables high-quality, in-depth imaging of challenging biological samples.
- This technique overcomes depth limitations in SPIM caused by optical aberrations.
- Improved imaging resolution facilitates better visualization of cellular and subcellular structures in thick tissues.
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