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Assessment of Gate Width Size on Lifetime-Based F?rster Resonance Energy Transfer Parameter Estimation
摘要： F?rster Resonance Energy Transfer (FRET) enables the observation of interactions at the nanoscale level through the use of fluorescence optical imaging techniques. In FRET, fluorescence lifetime imaging can be used to quantify the fluorescence lifetime changes of the donor molecule, which are associated with proximity between acceptor and donor molecules. Among the FRET parameters derived from fluorescence lifetime imaging, the percentage of donor that interacts with the acceptor (in proximity) can be estimated via model-based fitting. However, estimation of the lifetime parameters can be affected by the acquisition parameters such as the temporal characteristics of the imaging system. Herein, we investigate the effect of various gate widths on the accuracy of estimation of FRET parameters with focus on the near-infrared spectral window. Experiments were performed in silico, in vitro, and in vivo with gate width sizes ranging from 300 ps to 1000 ps in intervals of 100 ps. For all cases, the FRET parameters were retrieved accurately and the imaging acquisition time was decreased three-fold. These results indicate that increasing the gate width up to 1000 ps still allows for accurate quantification of FRET interactions even in the case of short lifetimes such as those encountered with near-infrared FRET pairs.
关键词： fluorescence lifetime,F?rster Resonance Energy Transfer (FRET),gated ICCD,near infrared (NIR) dyes,time-resolved imaging,gate width,in vivo imaging
更新于2025-09-10 09:29:36
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[Progress in Molecular Biology and Translational Science] Volume 160 || Fluorescent Proteins as Sensors for Cellular Behavior in Mice
摘要： Imaging of cancer cells in mice expressing fluorescent proteins has allowed the real-time tracing of cancer growth and metastasis and determination of efficacy of candidate antitumor and antimetastatic agents, especially in mouse orthotopic models. The use of fluorescent proteins to differentially label cancer cells in the nucleus and cytoplasm can visualize the nuclear–cytoplasmic dynamics of cancer cells in vivo, including mitosis, apoptosis, cell-cycle position, and differential behavior of nucleus and cytoplasm that occurs during cancer cell deformation, migration, and extravasation. Recent applications of the technology described here include linking fluorescent proteins with cell cycle-specific proteins such that the cells change color from red to green as they transit from G1 to S phases. Any in vivo process can be imaged using fluorescent proteins, allowing molecular biology to advance from in vitro studies to studying molecular processes in the living animal.
关键词： cancer imaging,metastasis,RFP,in vivo imaging,GFP,fluorescent proteins
更新于2025-09-10 09:29:36
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Dynamic and non-contact 3D sample rotation for microscopy
摘要： Precise sample orientation is crucial for microscopy but is often performed with macroscopic tools and low accuracy. In vivo imaging of growing and developing samples even requires dynamic adaptation of the sample orientation to continuously achieve optimal imaging. Here, we present a method for freely positioning a sample in 3D by introducing magnetic beads and applying a magnetic field. We demonstrate magnetic orientation of fixed mouse embryos and artemia, and live zebrafish embryos and larvae on an epi-fluorescence microscope and on a light-sheet system for optimal imaging.
关键词： magnetic beads,microscopy,3D positioning,sample orientation,in vivo imaging
更新于2025-09-10 09:29:36
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<i>In vivo</i> monitoring of intracellular Ca <sup>2+</sup> dynamics in the pancreatic β-cells of zebrafish embryos
摘要： Assessing the response of pancreatic islet cells to glucose stimulation is important for understanding β-cell function. Zebrafish are a promising model for studies of metabolism in general, including stimulus-secretion coupling in the pancreas. We used transgenic zebrafish embryos expressing a genetically-encoded Ca2+ sensor in pancreatic β-cells to monitor a key step in glucose induced insulin secretion; the elevations of intracellular [Ca2+]i. In vivo and ex vivo analyses of [Ca2+]i demonstrate that β-cell responsiveness to glucose is well established in late embryogenesis and that embryonic β-cells also respond to free fatty acid and amino acid challenges. In vivo imaging of whole embryos further shows that indirect glucose administration, for example by yolk injection, results in a slow and asynchronous induction of β-cell [Ca2+]i responses, while intravenous glucose injections cause immediate and islet-wide synchronized [Ca2+]i fluctuations. Finally, we demonstrate that embryos with disrupted mutation of the CaV1.2 channel gene cacna1c are hyperglycemic and that this phenotype is associated with glucose-independent [Ca2+]i fluctuation in β-cells. The data reveal a novel central role of cacna1c in β-cell specific stimulus-secretion coupling in zebrafish and demonstrate that the novel approach we propose – to monitor the [Ca2+]i dynamics in embryonic β-cells in vivo – will help to expand the understanding of β-cell physiological functions in healthy and diseased states.
关键词： GCaMP6s,glucose-sensing of beta cells,early zebrafish development,cacna1c,in vivo imaging,Cav1.2 channel
更新于2025-09-09 09:28:46
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In vivo multiphoton microscopy imaging of melasma
摘要： Melasma is a skin disorder characterized by hyperpigmented patches due to increased melanin production and deposition. In this pilot study we evaluate the potential of multiphoton microscopy (MPM) to characterize non-invasively the melanin content, location, and distribution in melasma and assess the elastosis severity. We employed a clinical MPM tomograph to image in-vivo morphological features in melasma lesions and adjacent normal skin in 12 patients. We imaged dermal melanophages in most dermal melasma lesions and occasionally in epidermal melasma. The melanin volume fraction values measured in epidermal melasma (14±4%) were significantly higher (p<0.05) than the values measured in peri-lesional skin (11±3%). The basal keratinocytes of melasma and perilesions showed different melanin distribution. Elastosis was predominantly more severe in lesions than in perilesions and was associated with changes in melanin distribution of the basal keratinocytes. These results demonstrate that MPM may be a non-invasive imaging tool for characterizing melasma. Identifying the depth of excess pigment is critical for successful treatment of melasma. Multiphoton microscopy demonstrates the ability to visualize non-invasively the melanophages, a sign of a prior inflammatory response, key in the differential diagnosis of melasma. Patients with melasma may be diagnosed more accurately using a rapid, label-free, non-invasive microscopy technique.
关键词： in vivo imaging,melasma,microscopy
更新于2025-09-09 09:28:46
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In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish
摘要： Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.
关键词： sensory neuroscience,Issue 141,calcium imaging,GCaMP,Neuroscience,genetically encoded indicators,hair cells,in vivo imaging,Zebrafish,lateral line,confocal imaging
更新于2025-09-04 15:30:14
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AIEgen Nanoparticles of Arylamino Fumaronitrile Derivative with High Near-infrared emission for Two-photon Imaging and in vivo Cell Tracking
摘要： Developing of two-photon materials for live-cell imaging and in vivo analysis in-deep have been received great attention, and it is still urgent so that such microscopy technique could be promoted and advanced into new progress using the powerful probs. Herein, a new arylamino fumaronitrile derivative NPAPF was synthesized and transferred as aggregation-induced emission luminogen (AIEgen) fluorescent nanoparticles (AF-NPs) via assembly technique. This AF-NPs exhibited two-photon absorption cross-section at 2.6×106 GM with 19.5% of fluorescence quantum yield. Moreover, utilizing the great potential of AF-NPs, two-photon imaging of live cells with good cytocompatibility is realized upon two-photon microscopy. By in vivo long-term tracing studies of mesenchymal stem cells, we demonstrated the tremendous advantage of AF-NPs tracer in monitoring the stem cells transplant. Therefore, our unique AF-NPs provided an efficient two-photon-absorbing probe for investigating biological mechanism and behavior, and also opened a new avenue for spatiotemporal visualization of transplanted stem cells.
关键词： organic nanoparticles,aggregation-induced emission,two-photon,in vivo imaging,mesenchymal stem cell
更新于2025-09-04 15:30:14