Chronic in vivo optical imaging of the spinal cord is an effective way to study the biological processes during and after spinal cord injury (SCI) in mouse models. It normally relies on an implanted spinal chamber to provide continuous optical access to the spinal cord. However, the chronic window consists of multiple layers of transparent materials with various optical properties and irregular thickness, which induce large optical aberration. Therefore, the image quality of multiphoton microscopy as well as the precision of femtosecond laser axotomy were dramatically degraded. In this work, we developed an adaptive optics (AO) microscope system integrating stimulated Raman scattering (SRS) and twophoton excited fluorescence (TPEF). Using our system, the aberrations induced by the spinal cord window were measured and compensated accordingly, enabling both high-resolution imaging and precise laser axotomy of the mouse spinal cord.
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.
Non-invasive retinal imaging has greatly facilitated the research of eye disease and neurodegenerative disorders in the central nervous system (CNS). Two-photon microscopy is a powerful tool for in vivo imaging of mouse retina because it provides intrinsic optical sectioning capability and the infrared laser is less likely to excite the photoreceptors. However, the dilated mouse eye has large optical aberrations, which must be corrected to achieve high-resolution or even diffraction-limited imaging. Here, we developed an adaptive optics (AO) two-photon microscope for in vivo imaging of retinal neurons through the eyeball of living mouse. We used the two-photon excited fluorescence signal of retina as the guide star to measure and correct the aberration of mouse eye. After AO correction, the fluorescence signal was increased by at least fivefold and the fine structures such as axons of retinal ganglion cells (RGC) were clearly resolved. To take advantage of the non-invasive high-resolution imaging, we demonstrated functional calcium imaging of RGC responding to the light stimulations.
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.
To understand the mechanisms of important lipid-related biological processes and diseases, it is highly demanded to study the dynamics of lipids in living biological system with high spatiotemporal resolution. However, in vivo quantitative analysis of lipid synthesis and lipolysis has been technically difficult to achieve by conventional lipid extraction and fluorescent staining methods. Recently, SRS microscopy has emerged as a powerful tool to probe small molecules with alkyne (C≡C) or deuterium (C-D) labeling in cell-silent region. The Raman tags have been used for the quantitative study of lipids in cells. In this study, we investigated metabolic dynamics of lipid droplets (LDs) by tracing the alkyne-tagged fatty acid 17-ODYA and deuterium-labeled saturated and unsaturated fatty acids PA-D31 & OA-D34 in living C. elegans. Specifically, we developed a hyperspectral SRS microscope system for LDs characterization. The system can sequentially excite and probe the stimulated Raman scattering-induced CH2 stretching of endogenous lipids information (2863 cm-1), C≡C stretching from 17-ODYA (2125 cm-1) and C-D stretching from deuterium-labeled fatty acids (2117 cm-1). We first examined the concentration levels of fatty acids in E. coli OP50. Two major lipid metabolic processes, namely uptake and turnover, were further studied in adult C. elegans. We imaged alkyne-tagged and deuterated fatty acids using SRS and traced their uptake, transportation, incorporation and turnover over time. Additionally, several other treatments including starvation were also conducted to study their effects on metabolic dynamics of pulse labeled 17-ODYA, PA-D31 and OA-D34.
Macrophages are essential for the regeneration of skeletal muscle after injury. It has been demonstrated that depletion of macrophages results in delay of necrotic fiber phagocytosis and decreased size of regenerated myofibers. In this work, we developed a multi-modal two-photon microscope system for in vivo study of macrophage activities in the regenerative and fibrotic healing process of injured skeletal muscles. The system is capable to image the muscles based on the second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals simultaneously. The dynamic activities of macrophages and muscle satellite cells are recorded in different time windows post the muscle injury. Moreover, we found that infiltrating macrophages emitted strong autofluorescence in the injured skeletal muscle of mouse model, which has not been reported previously. The macrophage autofluorescence was characterized in both spectral and temporal domains. The information extracted from the autofluorescence signals may facilitate the understanding on the formation mechanisms and possible applications in biological research related to skeletal muscle regeneration.
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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