Two-photon microscopy has become the method of choice for in vivo brain imaging in neuroscience research during the past decades owing to its inherent sectioning capability and large penetration depth in scattering tissues. By integrating with a gradient refractive index (GRIN) lens that implanted into the brain, two-photon microendoscopy further extends the imaging depth down to subcortical regions. However, the imaging resolution and field of view (FOV) are compromised due to the intrinsic aberrations of the inserted GRIN lens. Here, we developed an adaptive optics (AO) two-photon microendoscopy based on direct wavefront sensing that can measure and correct the aberrations of GRIN lens during in vivo brain imaging. Using our system, the diffraction-limited resolution was restored and the fine structures such as dendritic spines of hippocampal CA1 neurons can be clearly resolved over a much extended FOV.
Two-photon microscopy is a powerful tool for in vivo brain imaging that has greatly facilitated the neuroscience research in the past few decades. However, it still remains a challenge to image deep inside the brain with near diffraction-limited resolution due to the optical aberrations induced by the biological tissue and the cranial window. Here, we used an adaptive optics approach based on direct wavefront sensing to correct the aberration induced by the thinned skull window and achieved minimally invasive imaging of cerebral cortex with near-diffraction-limit resolution. Besides, by compensating the intrinsic aberration of a miniature gradient-index lens that implanted into the brain, two-photon imaging of hippocampal dendritic spines was realized over an extended field of view. The improvement in fluorescence intensity and imaging resolution enabled us to resolve the fine structures in live mouse brain such as dendritic spines that were invisible without the help of adaptive optics.
KEYWORDS: Brain, Luminescence, Neuroimaging, Two photon imaging, Alzheimer's disease, Near infrared, In vivo imaging, Skull, Green fluorescent protein, Spectroscopy
Amyloid depositions in the brain represent the characteristic hallmarks of Alzheimer’s disease (AD) pathology. The abnormal accumulation of extracellular amyloid-beta (Aβ) and resulting toxic amyloid plaques are considered to be responsible for the clinical deficits including cognitive decline and memory loss. In vivo two-photon fluorescence imaging of amyloid plaques in live AD mouse model through a chronic imaging window (thinned skull or craniotomy) provides a mean to greatly facilitate the study of the pathological mechanism of AD owing to its high spatial resolution and long-term continuous monitoring. However, the imaging depth for amyloid plaques is largely limited to upper cortical layers due to the short-wavelength fluorescence emission of commonly used amyloid probes. In this work, we reported that CRANAD-3, a near-infrared (NIR) probe for amyloid species with excitation wavelength at 900 nm and emission wavelength around 650 nm, has great advantages over conventionally used probes and is well suited for twophoton deep imaging of amyloid plaques in AD mouse brain. Compared with a commonly used MeO-X04 probe, the imaging depth of CRANAD-3 is largely extended for open skull cranial window. Furthermore, by using two-photon excited fluorescence spectroscopic imaging, we characterized the intrinsic fluorescence of the “aging pigment” lipofuscin in vivo, which has distinct spectra from CRANAD-3 labeled plaques. This study reveals the unique potential of NIR probes for in vivo, high-resolution and deep imaging of brain amyloid in Alzheimer’s disease.
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