An adaptive optics module for deep tissue multiphoton imaging in vivo

Cristina Rodríguez, Anderson Chen, José A. Rivera, Manuel A. Mohr, Yajie Liang, Wenzhi Sun, Daniel E. Milkie, Thomas G. Bifano, Xiaoke Chen, Na Ji

Posted on: 22 December 2020

Preprint posted on 26 November 2020

Article now published in Nature Methods at

Categories: biophysics


Imaging living organisms with subcellular resolution has been key for understanding different biological processes. Two photon (2P) fluorescence microscopy has been pivotal for observing cells and biological processes in vivo, in situ, deep inside tissues. Three photon (3P) fluorescence microscopy has allowed extending the imaging depth in opaque tissues. For 2P and 3P microscopy beyond relatively superficial depths, living biological tissues aberrate the wavefront of the excitation light, ultimately resulting in a limited performance. Adaptive optics have so far allowed imaging the mouse central nervous system at subcellular resolution. In their work, Rodriguez et al (1) report the development of a compact adaptive optics module that can be incorporated into 2P and 3P fluorescence microscopes, to measure and correct tissue-induced aberrations. This allowed resolving synaptic structures in deep cortical and subcortical areas of the mouse brain, and enabled obtaining high resolution imaging of neuronal structures and calcium responses in the spinal cord at unprecedented depths in vivo.

Key findings and developments

In their work, Rodriguez et al report a compact adaptive optics module for multiphoton microscopy composed of a high-speed segmented deformable mirror, two lenses and a field stop. In contrast to work where a slow deformable mirror or a digital micromirror device was used for frequency multiplexed modulation, and a spatial light modulator for aberration measurement and correction, using a single high-speed deformable mirror simplifies the module and enables faster measurement time, high power throughput, along with polarization and wavelength-independent operation, allowing its use in 2P and 3P fluorescence microscopy.

The authors then incorporated the AO module into a homebuilt 2P fluorescence microscope, placing it between the excitation laser and the microscope. Its performance for correcting artificial aberrations using signal from various features of different sizes, was then evaluated. They then applied the AO module to high resolution in vivo imaging.

Imaging zebrafish larvae. They were able to visualize myotomes in the mid-trunk of zebrafish larvae using a 920nm laser excitation wavelength, whereby aberration correction led to a 2-fold increase in 2P fluorescence signal, and 3.6-fold improvement in image contrast.

Imaging the mouse brain through a cranial window. The authors report significant improvements in image quality when imaging the brain through a cranial window using a 2P microscope. Aberration correction improved the image signal and contrast of labelled dendrites, and enabled identification of fine features. The authors report important improvements in signal, resolution, and contrast arising from adaptive optics correction of tissue-induced aberrations.

Incorporation of the AO module into a homebuilt 3P microscope. The authors were able to image neuronal structures through the mouse cerebral cortex in vivo. At 1300 nm excitation, a single correction pattern applied to the deformable mirror drastically improved the image quality of labeled neurons located 760 µm below the dura. The adaptive optics correction led to 3P signal increases ranging from 7-fold on the cell body to 19-fold on dendrites. Moreover, they report being able to observe previously invisible synaptic structures, due to the aberration correction. In all mouse brain examples, only 2-3 rounds of correction were used to obtain the final corrective wavefront, while additional rounds did not result in substantial improvement of the fluorescence signal. In different animals, and using different labels, aberration correction consistently improved the image signal and resolution, and allowed resolution of structures not identifiable without correction.

Imaging the spinal cord. Imaging beyond the mouse neocortex, into the hippocampus using conventional methods is challenging. The authors report that their adaptive optics module enabled imaging hippocampal structures with subcellular resolution without the need of invasive procedures other than the cranial window implantation. The positive results observed upon incorporating the adaptive optics module into a 3P microscope for imaging the brain, motivated the authors to image a more challenging structure, namely, the spinal cord. They were able to perform in vivo 3P imaging of labeled neuronal structures at depths exceeding 400µm below the dura through a dorsal laminectomy in adult mice, under 1300nm excitation. Aberration correction led to 2- to 5-fold signal improvements, and allowed a clearer visualization of fine neuronal structures. Finally, the authors report that the adaptive optics module enabled reliable recording of somatosensory-evoked calcium transients in the spinal cord dorsal horn at depths beyond 300 µm. Imaging at this depth had not been possible before, as it had been limited to relatively superficial imaging (at <100µm). This impossibility had prevented a comprehensive understanding of coding of somatosensory stimuli in the spinal cord circuitry. Besides the ability to achieve subcellular resolution at depth, the excitation power can be significantly reduced, minimizing out-of-focus background and decreasing the power delivered to the sample to levels below those where heating-related effects or photodamage would be problematic.

What I like about this paper

This is a fantastic development- intravital microscopy has allowed us to ask multiple questions regarding biological processes in vivo, and offers the opportunity to study those processes in living organisms within complex tissues. Still, while we have been able to answer many of those questions, intravital microscopy still faces limitations. This tool clearly allows overcoming important hindrances, while enabling us to answer questions previously impossible to answer, and ask new things.

Open questions

  1. This is a great development. In terms of depth, what are the limitations of your module, and do you envisage that these can be overcome in the future?
  2. What are the advantages of the module you present here, in comparison to GRIN lenses and microprisms that can be inserted into tissues to image at up to 1-2mm of depth?
  3. Are there any limitations arising from tissue auto-fluorescence?
  4. One of the main advantages of your work is the possibility to image deep into tissues without extremely invasive surgical procedures. You mention that operation with relatively low laser power allows for longitudinal imaging. Is your method compatible with chronic windows, and longitudinal imaging deep into tissues for say, several weeks?
  5. Are there dyes/tags more suitable for this method?


  1. Rodriguez et al. An adaptive optics module for deep tissue multiphoton imaging in vivo, bioRxiv, 2020.



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