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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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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
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[IEEE 2018 40th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) - Honolulu, HI (2018.7.18-2018.7.21)] 2018 40th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) - Fully Convolutional DenseNets for Segmentation of Microvessels in Two-photon Microscopy*
摘要： Segmentation of microvessels measured using two-photon microscopy has been studied in the literature with limited success due to uneven intensities associated with optical imaging and shadowing effects. In this work, we address this problem using a customized version of a recently developed fully convolutional neural network, namely, FC-DensNets. To train and validate the network, manual annotations of 8 angiograms from two-photon microscopy was used. Segmentation results are then compared with that of a state-of-the-art scheme that was developed for the same purpose and also based on deep learning. Experimental results show improved performance of used FC-DenseNet in providing accurate and yet end-to-end segmentation of microvessels in two-photon microscopy.
关键词： deep learning,two-photon microscopy,FC-DenseNets,microvessels,Segmentation
更新于2025-09-09 09:28:46
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A Two-Photon Ratiometric Probe for Hydrogen Polysulfide (H2Sn): Increase in Mitochondrial H2Sn Production in a Parkinson’s Disease Model
摘要： Hydrogen polysulfide (H2Sn, n>1), which is primarily generated during the crosstalk between H2S and reactive species (ROS and RNS), is receiving increasing attention in biochemical research. H2Sn is mostly generated in the mitochondria, and abnormal mitochondrial function and oxidative stress are directly related to many disorders including Parkinson's disease (PD). We now report a two-photon fluorescent probe (SPS-M1) for in situ detection of H2Sn and its application to a PD model to account the H2Sn levels. The probe exhibited selective and fast response to H2Sn along with a marked blue-to-green color change. SPS-M1 is sensitive enough to quantitative detection of endogenous H2Sn content in mitochondria using two-photon microscopy (TPM). Ratiometric TPM imaging of live neurons and brain slices using SPS-M1 revealed that H2Sn production is increased to a greater extent in the A53T α-synuclein (α-syn) overexpressing model than in the wild-type control. These findings suggest that the interactions of H2S and the increased ROS caused by α-syn overexpression may generate more H2Sn. By employing our recently published TP probe for mitochondrial H2S, we also found the relationship between the H2Sn and H2S; increased H2Sn and decreased H2S levels, indicating that H2S and H2Sn may play a significant role in the pathogenesis of PD. This result may be useful to biomedical studies, including PD.
关键词： two-photon microscopy,fluorescent probe,Parkinson’s disease,Hydrogen polysulfide,mitochondria
更新于2025-09-04 15:30:14
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A stage-scanning two-photon microscope equipped with a temporal and a spatial pulse shaper: Enhance fluorescence signal by phase shaping
摘要： Here, we present a stage-scanning two-photon microscope (2PM) equipped with a temporal pulse shaper and a spatial light modulator enabling full control over spectral and spatial phases of the exciting laser pulse. We demonstrate the capability of correcting wavefronts and temporal pulse distortions without cross-dependencies induced by optical elements at the same time enhancing the fluorescence signal. We implemented phase resolved interferometric spectral modulation for temporal pulse shaping and the iterative feedback adaptive compensation technique for spatial pulse modulation as iterative techniques. Sample distortions were simulated by cover glass plates in the optical path and by chirping the exciting laser pulses. Optimization of the spectral and spatial phases results in a signal increase of 30% and nearly complete recovery of the losses. Applying a measured spatial compensation phase within a real leaf sample shows the enhancement in contrast due to wavefront shaping with local fluorescence increase up to 75%. The setup allows full independent control over spatial and spectral phases keeping or improving the spatial resolution of our microscope and provides the optimal tool for sensitive non-linear and coherent control microscopy.
关键词： two-photon microscopy,wavefront shaping,spatial light modulator,fluorescence signal enhancement,temporal pulse shaping
更新于2025-09-04 15:30:14