Glaucoma is characterized by the irreversible retinal ganglion cells (RGCs) loss and has received great research and clinical attention due to its complex mechanism and loss of effective treatment. Longitudinal in vivo study on glaucoma in mouse model can be a promising approach to unveil the mechanism of glaucoma. In this paper, we use a silicon oil (SO) induced glaucoma model on mouse and the adaptive optics two photon microscope system to evaluate whether the aberration from both mouse eye and SO can be corrected and then longitudinal imaging with subcellular resolution on the mouse retina can be achieved.
Imaging the brain in its natural state with high spatial resolution is a challenging task for visualization techniques. Two-photon microscopy offers a better approach to image deep brain tissue due to its inherent sectioning ability compared to single-photon confocal microscopy. Despite this, the optical heterogeneity of the skull severely compromises imaging contrast and spatial resolution, limiting imaging depth to the superficial layer. Recently, a new adaptive optics method called alpha-FSS has been applied to three-photon microscopy, enabling high-resolution imaging in deep brain tissue through the intact skull and demonstrating strong ability to correct large aberrations and scattering. In this study, we combined alpha-FSS adaptive optics with two-photon microscopy and demonstrated that this method works well in two-photon imaging system as well, which is much more widely used than three-photon microscopy. Using this system, we achieved subcellular resolution transcranial imaging of layer 5 pyramidal neurons up to 650um below pia in living mice. We also demonstrated the ability to perform functional calcium imaging with high sensitivity, as well as high-precision laser-mediated microsurgery through thinned skull. In summary, we applied alpha-FSS to two photon system and proved its ability to achieve near non-invasive high-resolution imaging in deep brain tissue.
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.
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