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<p>Biodistribution and sensitive tracking of immune cells with plasmonic gold nanostars</p>
摘要： Aim: To quantitatively and sensitively investigate the biodistribution of immune cells after systemic administration. Methods: Immune cells were loaded with plasmonic gold nanostars (GNS) tracking probes. Inductively coupled plasma mass spectrometry (ICP-MS) was used for quantitative gold mass measurement and two-photon photoluminescence (TPL) was used for high-resolution sensitive optical imaging. Results: GNS nanoparticles were loaded successfully into immune cells without negative effect on cellular vitality. Liver and spleen were identified to be the major organs for macrophage cells uptake after systematic administration. A small amount of macrophage cells were detected in the tumor site in our murine lymphoma animal model. Conclusion: GNS has great potential as a biocompatible marker for quantitative tracking and high-resolution imaging of immune cells at the cellular level.
关键词： biodistribution,immune cells,GNS,ICP-MS,two-photon microscopy
更新于2025-11-19 16:56:42
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Multiphoton imaging for morphometry of the sandwich-beam structure of the human stapedial annular ligament
摘要： Background: The annular ligament of the human stapes constitutes a compliant connection between the stapes footplate and the peripheral cochlear wall at the oval window. The cross section of the human annular ligament is characterized by a three-layered structure, which resembles a sandwich-shaped composite structure. As accurate and precise descriptions of the middle-ear behavior are constrained by lack of information on the complex geometry of the annular ligament, this study aims to obtain comprehensive geometrical data of the annular ligament via multiphoton imaging. Methods: The region of interest containing the stapes and annular ligament was harvested from a fresh-frozen human temporal bone of a 46-years old female. Multiphoton imaging of the unstained sample was performed by detecting the second-harmonic generation of collagen and the autofluorescence of elastin, which are constituents of the annular ligament. The multiphoton scans were conducted on the middle-ear side and cochlear side of the annular ligament to obtain accurate images of the face layers on both sides. The face layers of the annular ligament were manually segmented on both multiphoton scans, and then registered to high-resolution mCT images. Results: Multiphoton scans of the annular ligament revealed 1) relatively large thickness of the core layer compared to the face layers, 2) asymmetric geometry of the face layers between the middle-ear side and cochlear side, and variation of their thickness and width along the footplate boundary, 3) divergent relative alignment of the two face layers, and 4) different fiber composition of the face layers along the boundary with a collagen-reinforcement near the anterior pole on the middle-ear side. Conclusion and outlook: Multiphoton microscopy is a feasible approach to obtain the detailed three-dimensional features of the human stapedial annular ligament along its full boundary. The detailed description of the sandwich-shaped structures of the annular ligament is expected to contribute to modeling of the human middle ear for precise simulation of middle-ear behavior. Further, established methodology in this study may be applicable to imaging of other middle-ear structures.
关键词： Stapes,Two-photon microscopy,Multiphoton microscopy,Core layer,Face layer,Annular ligament
更新于2025-09-23 15:23:52
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Slide-free imaging of hematoxylin-eosin stained whole-mount tissues using combined third-harmonic generation and three-photon fluorescence microscopy
摘要： Intraoperative margin assessment of surgical tissues during cancer surgery is clinically important, especially in the case of tissue conserving surgery like Mohs micrographic surgery in which minimization of the surgical area is considered crucial. Frozen pathology is the gold standard of assessing excised tissues for signs of remaining cancerous lesions. The current protocol, however, is time-consuming and labor-intensive. Instead of the complex frozen sectioning, staining, and traditional white light microscopy imaging protocol, optically-sectioned histopathological imaging of hematoxylin-eosin stained whole-mount skin tissues with a sub-femtoliter resolution is demonstrated by using nonlinear microscopy in this study. With our proposed method, the reagents of staining and the contrast of imaging are fully consistent with the current clinical standard of frozen pathology, thus facilitating rapid intraoperative assessment of surgical tissues for future applications.
关键词： hematoxylin-eosin,three photon microscopy,third harmonic generation microscopy,surgical border,margin assessment
更新于2025-09-23 15:23:52
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Intracortical neural stimulation with untethered, ultrasmall carbon fiber electrodes mediated by the photoelectric effect
摘要： Objective: Neural stimulation with tethered, electrically activated probes is damaging to neural tissue and lacks good spatial selectivity and stable chronic performance. The photoelectric effect, which converts incident light into electric potential and heat, provides an opportunity for a tetherless stimulation method. We propose a novel stimulation paradigm that relies on the photoelectric effect to stimulate neurons around a free-floating, ultrasmall (7-8μm diameter) carbon fiber probe. Methods: A 2-photon microscope induced photo-stimulation with a laser. Chronoamperometry and chronopotentiometry were used to characterize the electrochemical properties of photo-stimulation, while the fluorescence of Rhodamine-B was used to quantify temperature changes. Results: Photo-stimulation caused a local cathodic potential pulse with minimal leakage current. Stimulation induced voltage deflections of 0.05 - 0.4V in vitro, varying linearly with the power of the laser source (5 – 40 mW). Temperature increases in the immediate vicinity of the electrode were limited to 2.5°C, suggesting that this stimulation modality can be used without inducing heat damage. Successful stimulation was supported in vivo by increased calcium fluorescence in local neurons at stimulation onset in a transgenic GCaMP-3 mouse model. Furthermore, cells activated by photo-stimulation were closer to the electrode than in electrical stimulation under similar conditions, indicating increased spatial precision. Conclusion: Our results support the hypothesis that the proposed photoelectric method is effective for neural stimulation. Significance: Photoelectric stimulation is precise and avoids the need for a potentially destructive tether, making it a promising alternative to electrical stimulation.
关键词： GCaMP,electrochemistry,neuromodulation,photovoltaic effect,2-photon microscopy,temperature-dependent,fluorescence
更新于2025-09-23 15:23:52
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Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy
摘要： Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying deeper cortical layers. Here, we developed a custom three-photon microscope optimized to image a vertical column of the cerebral cortex > 1 mm in depth in awake mice with low (<20 mW) average laser power. Our measurements of physiological responses and tissue-damage thresholds define pulse parameters and safety limits for damage-free three-photon imaging. We image functional visual responses of neurons expressing GCaMP6s across all layers of the primary visual cortex (V1) and in the subplate. These recordings reveal diverse visual selectivity in deep layers: layer 5 neurons are more broadly tuned to visual stimuli, whereas mean orientation selectivity of layer 6 neurons is slightly sharper, compared to neurons in other layers. Subplate neurons, located in the white matter below cortical layer 6 and characterized here for the first time, show low visual responsivity and broad orientation selectivity.
关键词： subplate neurons,deep brain imaging,visual cortex,neuronal activity,three-photon microscopy,GCaMP6s
更新于2025-09-23 15:23:52
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Ultrashort laser based two-photon phase-resolved fluorescence lifetime measurement method
摘要： This paper presents a two-photon phase-resolved fluorescence-lifetime measurement method based on the use of an ultrashort pulse laser. The proposed method also involves the use of a lock-in amplifier to control the phase difference between the reference and fluorescence signals, thereby facilitating the use of an alternative method for determining fluorescence lifetimes. Verification of the fluorescence lifetimes as measured in this study was performed using rhodamine B and a cellular thermoprobe as samples. In this study, we assume that the fluorescence decay was monoexponential in all cases. Rhodamine B was observed to exhibit an average fluorescence lifetime of 2.15 ns, whereas a temperature sensitivity of 1.39 ns/°C over a temperature range of 33.79–37.2 °C was demonstrated for the cellular thermoprobe. These results validate the feasibility of the proposed method for accurate measurement of fluorescence lifetimes using a simple laser configuration.
关键词： rhodamine B,ultrashort pulse laser,temperature sensor,phase-resolved fluorescence-lifetime,two-photon microscopy,cellular thermoprobe
更新于2025-09-19 17:13:59
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In Vivo Two-photon Calcium Imaging in Dendrites of Rabies Virus-labeled V1 Corticothalamic Neurons
摘要： Monitoring neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain. Two-photon Ca2? imaging in vivo using a cranial window and speci?c neuronal labeling enables real-time, in situ, and long-term imaging of the living brain. Here, we constructed a recombinant rabies virus containing the Ca2? indicator GCaMP6s along with the ?uorescent protein DsRed2 as a baseline reference to ensure GCaMP6s signal reliability. This functional tracer was applied to retrogradely label speci?c V1–thalamus circuits and detect spontaneous Ca2? activity in the dendrites of V1 corticothalamic neurons by in vivo two-photon Ca2? imaging. Notably, we were able to record single-spine spontaneous Ca2? activity in speci?c circuits. Distinct spontaneous Ca2? dynamics in dendrites of V1 corticothalamic neurons were found for different V1–thalamus circuits. Our method can be applied to monitor Ca2? dynamics in speci?c input circuits in vivo, and contribute to functional studies of de?ned neural circuits and the dissection of functional circuit connections.
关键词： Corticothalamic projection,In vivo Ca2? imaging,Two-photon microscopy,Primary visual cortex,Neural circuit tracing,Cranial window,Rabies virus,Dendrite
更新于2025-09-11 14:15:04
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Intravital imaging of adriamycin-induced renal pathology using two-photon microscopy and optical coherence tomography
摘要： Adriamycin (doxorubicin), a common cancer chemotherapeutic drug, can be used to induce a model of chronic progressive glomerular disease in rodents. In our studies, we evaluated renal changes in a rat model after Adriamycin injection using two-photon microscopy (TPM), optical coherence tomography (OCT) and Doppler OCT (DOCT). Taking advantage of deep penetration and fast scanning speed for three-dimensional (3D) label-free imaging, OCT/DOCT system was able to reveal glomerular and tubular pathology noninvasively and in real time. By imaging renal pathology following the infusion of °uorophore-labeled dextrans of di?erent molecular weights, TPM can provide direct views of glomerular and tubular °ow dynamics with the onset and progression of renal disease. Speciˉcally, glomerular permeability and ˉltration, proximal and distal tubular °ow dynamics can be revealed. 6–8 weeks after injection of Adriamycin, TPM and OCT/DOCT imaging revealed glomerular sclerosis, compromised °ow across the glomerular wall, tubular atrophy, tubular dilation, and variable intra-tubular °ow dynamics. Our results indicate that TPM and OCT/DOCT provide real-time imaging of renal pathology in vivo that has not been previously available using conventional microscopic procedures.
关键词： Two-photon Microscopy (TPM),Chronic kidney disease (CKD),Tubular Atrophy,glomerulosclerosis,Optical Coherence Tomography (OCT)
更新于2025-09-11 14:15:04
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Automatic Graph-based Modeling of Brain Microvessels Captured with Two-Photon Microscopy
摘要： Graph models of cerebral vasculature derived from 2-photon microscopy have shown to be relevant to study brain microphysiology. Automatic graphing of these microvessels remain problematic due to the vascular network complexity and 2-photon sensitivity limitations with depth. In this work, we propose a fully automatic processing pipeline to address this issue. The modeling scheme consists of a fully-convolution neural network to segment microvessels, a 3D surface model generator and a geometry contraction algorithm to produce graphical models with a single connected component. Quantitative assessment using NetMets metrics, at a tolerance of 60 μm, false negative and false positive geometric error rates are 3.8% and 4.2%, respectively, whereas false negative and false positive topological error rates are 6.1% and 4.5%, respectively. Our qualitative evaluation confirms the efficiency of our scheme in generating useful and accurate graphical models.
关键词： segmentation,graph,Cerebral microvasculature,deep learning,convolution neural networks,two-photon microscopy
更新于2025-09-09 09:28:46
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Endogenous Fluorophores Enable Two-Photon Imaging of the Primate Eye
摘要： Noninvasive two-photon imaging of a living mammalian eye can reveal details of molecular processes in the retina and RPE. Retinyl esters and all-trans-retinal condensation products are two types of retinoid fluorophores present in these tissues. We measured the content of these two types of retinoids in monkey and human eyes to validate the potential of two-photon imaging for monitoring retinoid changes in human eyes.
关键词： primate retina,retinoid cycle,two-photon microscopy,rod photoreceptors,cone photoreceptors
更新于2025-09-09 09:28:46