High-resolution structural and functional deep brain imaging using adaptive optics three-photon microscopy

Abstract

Multiphoton microscopy has become a powerful tool with which to visualize the morphology and function of neural cells and circuits in the intact mammalian brain. However, tissue scattering, optical aberrations and motion artifacts degrade the imaging performance at depth. Here we describe a minimally invasive intravital imaging methodology based on three-photon excitation, indirect adaptive optics (AO) and active electrocardiogram gating to advance deep-tissue imaging. Our modal-based, sensorless AO approach is robust to low signal-to-noise ratios as commonly encountered in deep scattering tissues such as the mouse brain, and permits AO correction over large axial fields of view. We demonstrate near-diffraction-limited imaging of deep cortical spines and (sub)cortical dendrites up to a depth of 1.4 mm (the edge of the mouse CA1 hippocampus). In addition, we show applications to deep-layer calcium imaging of astrocytes, including fibrous astrocytes that reside in the highly scattering corpus callosum.

Main

In scattering tissues such as the mammalian brain, two-photon excitation microscopy (2PM) is the gold standard for recording cellular structure and function in noninvasive and physiologically relevant conditions in vivo^1,2. However, the maximum penetration depth of two-photon microscopes is fundamentally limited by the onset of out-of-focus fluorescence near the surface with increasing excitation power, which for the mammalian brain prevents imaging beyond roughly 1 mm (ref. ³). Imaging approaches based on three-photon excitation microscopy (3PM) have shown potential for deep imaging beyond 1 mm with cellular resolution, owing to a substantially increased signal-to-background ratio (SBR) at depth and longer wavelength excitation^4,5,6,7.

As in 2PM, however, with increasing imaging depths optical aberrations due to tissue heterogeneities and refractive index mismatches, and subtle motion artifacts due to the animal’s heartbeat, degrade image resolution and overall performance, and result in a loss of subcellular details. This has so far prohibited the resolution of fine neuronal processes, synapses and subcellular Ca²⁺ transients in deep cortical and subcortical areas of the mouse brain in vivo without using highly invasive methods such as gradient index lens implantation⁸ or cortical aspiration⁹. While optical aberrations can be measured and compensated by using adaptive optics (AO) methods^10,11, previous implementations were predominantly based on 2PM and thus were limited in their effective imaging depth to at most 800 µm in the mouse brain^12,13,14,15. Alternative approaches based on wavefront shaping have the potential to image even further into highly scattering media, but limited fields of view (FOV) of only tens of micrometers and/or fast decorrelation times have prohibited their useful application in realistic intravital conditions^16,17,18.

A further challenge in in vivo deep brain imaging is that at large tissue depths, heart pulsation leads to intraframe motion artifacts that prevent frame averaging to enhance the signal-to-noise ratio (SNR), a standard technique that is essential to reliably resolve small structures such as dendrites and individual spines.

To address the above shortcomings, we developed a minimally invasive intravital imaging methodology based on 3PM, indirect AO correction and active electrocardiogram (ECG) gating to achieve aberration correction and near-diffraction-limited resolution up to a depth of over 1.4 mm in the mouse brain. This enabled us to resolve individual synapses down to roughly 900 µm in the cortex, and fine dendritic processes in the hippocampus at a depth of more than 1.4 mm. Furthermore, our noninvasive approach achieved in vivo functional characterization of fibrous astrocytes in the white matter and resolved Ca²⁺ transients in individual microdomains.

Results

3PM with ECG gating

Our intravital imaging method is based on a homebuilt 3P laser scanning microscope optimized for 1,300 nm excitation and the use of broad-bandwidth, low repetition-rate lasers (Fig. 1a and Extended Data Fig. 1). We optimized the 3P fluorescence signal by ensuring short-duration laser pulses (<50 fs) and efficient power delivery to the focal plane within the sample. Optimization of the in vivo fluorescence signal in 3PM is especially important to avoid laser-induced tissue heating or long-term tissue damage^7,19,20,21.