Research overview

Optical microscopy has fueled major discoveries in biology, from the cellular structures of organisms to the causes of infectious disease. Multiphoton microscopy stands out as a powerful method facilitating non-invasive three-dimensional imaging of cells and tissues.

However, structures such as blood vessels, cells, and subcellular components, render biological tissues highly distorting to light. With increasing imaging depth, these distortions impair both resolution and signal strength. This fundamental limitation, compounded by the brightness constraints of fluorescent probes, hinders examination of critical processes in their native biological context, such as synaptic plasticity in the brain, cellular dynamics in tumors, and neural circuits in the spinal cord.

The focus of the Rodríguez lab is to is to develop optical microscopy tools for visualizing critical biological processes deep within their native context, with a particular focus on applications in neuroscience. To this end, we combine diverse tools from physics and engineering, including multiphoton microscopy and wavefront shaping. Our technological innovations allow us to address unanswered questions in neuroscience using the mouse brain and spinal cord as model systems.

Going deep with 3-photon fluorescence microscopy

2-photon (2P) fluorescence microscopy (2PFM) has allowed researchers to observe biological structures and processes deep inside living organisms. For optically opaque tissues like the brain, however, the laser excitation power must be increased exponentially with imaging depth to compensate for the loss through scattering. Eventually, this leads to 2P excitation of out-of-focus fluorescent structures, generating background signals of similar brightness to the in-focus signal of interest, reaching the imaging depth limit. 3-photon (3P) fluorescence microscopy (3PFM) was developed to address this challenge and push beyond the limits of 2PFM. Here, a fluorescent molecule is excited via the simultaneous absorption of three photons, instead of two as in 2PFM, enabling the use of longer excitation wavelengths. Since longer wavelengths experience reduced light scattering, 3PFM enables better tissue penetration of the excitation light. In addition, the higher nonlinearity of 3P excitation leads to a stronger spatial confinement of the excitation, significantly reducing the out of focus fluorescence and improving the signal-to-background ratio at depth.

Our lab is using 3PFM to push the imaging depth limits in different model systems including the mouse brain and spinal cord.

Fig. 1: Energy diagram of 1-, 2-, and 3-photon excitation and cross-section view of the mouse brain showing optical accessibility of 1-, 2-, and 3-photon microscopy.

Adaptive optics: achieving subcellular resolution in deep brain layers

We work with adaptive optics – a method originally developed for astronomical telescopes – to actively shape the wavefront of the light used by our microscopes, and compensate for the aberrations introduced by tissues, making it possible to achieve subcellular resolution at depth.
 

Fig. 2: Principle of adaptive optics (AO) in a point scanning microscope. In an ideal case, a focusing beam forms a diffraction-limited focus (left). Refractive index inhomogeneities distort light (center). By pre-shaping the excitation light, AO recovers ideal performance (right). 

In our previous work, we used adaptive optics to image deep cortical and sub-cortical layers of the living mouse brain with drastically improved image quality. Adaptive optics enabled the visualization of synaptic structures otherwise invisible without aberration correction, including dendritic spines and axonal boutons, critical for cell communication and information processing (Rodríguez et al. 2021).
 

Fig. 3: 3-photon image of fluorescently labeled neuron in the mouse cortex, without and with adaptive optics (AO). 

Diving deep into the spinal cord with advanced optical imaging

Motivated by the excellent performance of our imaging technologies, we turned our attention to the spinal cord — a vastly understudied region of the nervous system, especially in the context of optical microscopy — critical for controlling movement, sensing touch, and supporting other vital functions. Despite its critical role, our understanding of the underlying mechanisms remains limited because optical microscopes have, until now, been unable to image beyond its surface. One major challenge is the superficial layer of densely packed nerve fibers (white matter), which significantly distorts light.
 
We demonstrated for the first time in vivo adaptive optical 3-photon imaging of neuronal structures and sensory-evoked calcium activity in the mouse spinal cord, at depths that far exceeded (2-3×) previous records. In particular, adaptive optics enabled the visualization of calcium signaling in temperature-sensitive neurons at tissue depths where such responses had not been previously studied. These findings open the door for investigating the processing and transmission of somatosensory information in deep neuronal layers. 

Fig. 4: First demonstration of adaptive optics (AO) for in vivo spinal cord 3-photon imaging. (left) Cross-section view of the mouse spinal cord showing optical accessibility of 2-photon and 3-photon AO microscopy. AO substantially improves 3-photon (center) structural and (right) functional imaging.

 
 

Our research is supported by: