Achieving intravital optical imaging with diffraction-limited spatial resolution of deep-brain structures represents an important step toward the goal of understanding the mammalian central nervous system1–4. Advances in wavefront-shaping methods and computational power have recently allowed for a novel approach to high-resolution imaging, utilizing deterministic light propagation through optically complex media and, of particular importance for this work, multimode optical fibers (MMFs)5–7. We report a compact and highly optimized approach for minimally invasive in vivo brain imaging applications. The volume of tissue lesion was reduced by more than 100-fold, while preserving diffraction-limited imaging performance utilizing wavefront control of light propagation through a single 50-μm-core MMF. Here, we demonstrated high-resolution fluorescence imaging of subcellular neuronal structures, dendrites and synaptic specializations, in deep-brain regions of living mice, as well as monitored stimulus-driven functional Ca2+ responses. These results represent a major breakthrough in the compromise between high-resolution imaging and tissue damage, heralding new possibilities for deep-brain imaging in vivo.

Topographic representation of the receptor surface is a fundamental feature of sensory cortical organization. This is imparted by the thalamus, which relays information from the periphery to the cortex. To better understand the rules governing thalamocortical connectivity and the origin of cortical maps, we used in vivo two-photon calcium imaging to characterize the properties of thalamic axons innervating different layers of mouse auditory cortex. Although tonotopically organized at a global level, we found that the frequency selectivity of individual thalamocortical axons is surprisingly heterogeneous, even in layers 3b/4 of the primary cortical areas, where the thalamic input is dominated by the lemniscal projection. We also show that thalamocortical input to layer 1 includes collaterals from axons innervating layers 3b/4 and is largely in register with the main input targeting those layers. Such locally varied thalamocortical projections may be useful in enabling rapid contextual modulation of cortical frequency representations.

Achieving optical access to deep-brain structures represents an important step towards the goal of understanding the mammalian central nervous system. The complex refractive index distribution within brain tissue introduces severe aberrations to long-distance light propagation thereby prohibiting image reconstruction using currently available non-invasive techniques. In an attempt to overcome this challenge endoscopic approaches have been adopted, principally in the form of fibre bundles or GRIN-lens based endoscopes. Unfortunately, these approaches create substantial mechanical lesions of the tissue precipitating neuropathological responses that include inflammation and gliosis. Together, lesions and the associated neuropathology may compromise neural circuit performance. By replacing Fourier-based image relay with a holographic approach, we have been able to reduce the volume of tissue lesion by more than 100-fold, while preserving diffraction-limited imaging performance. Here we demonstrate high-resolution fluorescence imaging of neuronal structures, dendrites and synaptic specialisations, in deep-brain regions of living mice. These results represent a major breakthrough in the compromise between high-resolution imaging and tissue damage, heralding new possibilities for deep-brain imaging in vivo. Keywordsmicroendoscopy, in vivo imaging, dendritic spines, calcium imaging, multimode fibre, holography IntroductionAt present, non-invasive (surface) high-resolution imaging of brain tissue can achieve micrometre resolution up to penetration depths of about 1 mm 1 . Even in small mammals, such a mice, this severely restricts optical access to almost all subcortical structures many of which are implicated in important neuronal processes such as memory formation and gating of sensory and motor information, as well as neurological diseases, for example Alzheimer's disease 2-4 . Attempts to gain access to these brain regions have precipitated the use of invasive strategies, including the removal of overlying cortical structures 5 , the insertion of fibre bundles 6 and graded index (GRIN) lenses 7,8 . However, where these techniques are uniformly problematic is the considerable extent to which they damage the brain 9 . The degree to which this damage is consequential for the behaviour of the animal and the physiology of networks is not fully understood 10,11 , nonetheless, it is clear that were it possible to cause little or no damage, then such an approach would be wholly desirable as a method with which to explore the nervous system in vivo.Advances in holographic methods and computational power have recently allowed for a novel approach to high-resolution imaging, utilising deterministic light propagation through optically complex media and, of particular importance for this work, multimode optical fibres (MMF) 12-14 .MMF probes offer the advantage that they are many times thinner than other types of microendoscopes. To exploit this advantage we have developed a compact and highly optimised approach for minimally inva...

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