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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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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
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Bio‐orthogonal AIE Dots Based on Polyyne‐Bridged Red‐emissive AIEgen for Tumor Metabolic Labeling and Targeted Imaging
摘要： In this work, we aim to develop cancer cell-targeting AIE dots based on the polyyne-bridged red-emissive AIEgen, 2TPE-4E, through the combination of metabolic engineering and bio-orthogonal reactions. Azide groups on tumor were efficiently produced by intravenous injection of Ac4ManNAz and glycol-metabolic engineering. These bio-orthogonal azide groups could facilitate the specific targeting of DBCO-AIE dots to the tumor cells undergoing metal-free click reaction in vivo. The efficiency of this targeting strategy could be improved along with further development of new bio-orthogonal chemical groups with higher reactivity and sufficient AIEgens could be delivered to tumor for diagnosis.
关键词： in vivo,metabolic labeling,aggregation-induced emission,tumor,bio-orthogonal chemistry
更新于2025-09-04 15:30:14
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Estimate of Nonuniformity of Dose Distribution in Regime of Total Therapeutic Irradiation with In Vivo Dosimetry Using Semiconductor Detectors
摘要： We have estimated the nonuniformity of the dose distribution along the body of a patient using in vivo dosimetry by semiconductor detectors with total therapeutic irradiation of cancer patients before bone marrow transplantation. The method of in vivo dosimetry was developed and used at the Blokhin Oncology Center since 2007. Based on the results of the investigation of 35 patients, we determined a mean nonuniformity of 10–15% of the dose distribution along the body of the patient, which conforms to international recommendations. We show a linear dependence of the accumulated dose on the mass of the patient.
关键词： total irradiation,in vivo dosimetry,semiconductor detectors,radiation therapy
更新于2025-09-04 15:30:14
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Comprehensive Biomedical Physics || Bioluminescence Imaging
摘要： Bioluminescence imaging is a research technique that uses light-producing enzymes from naturally occurring species or modified versions of these enzymes to follow biological processes inside cells or animals. The luciferase enzyme is often encoded in a reporter gene that is introduced into the cells or animals of interest. The amount of light that is detected by a light-sensitive camera represents changes in the chosen biological process.
关键词： Bioluminescence imaging,In vitro,Reporter gene,In vivo,Luciferase
更新于2025-09-04 15:30:14
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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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Evaluation of the corneal epithelium in non-Sj?gren’s and Sj?gren’s dry eyes: an in vivo confocal microscopy study using HRT III RCM
摘要： Background: The corneal epithelium is directly affected in dry eye syndrome. Thus, we attempted to describe the morphological features and evaluate the cellular density within the corneal epithelial layers in patients with non-Sj?gren’s (NSDE) and Sj?gren’s syndrome dry eyes (SSDE) by in vivo confocal microscopy (IVCM). Methods: Central cornea was prospectively imaged by IVCM in 68 clinically diagnosed aqueous tear-deficient dry eyes and 10 healthy age-matched control eyes. Morphological characteristics of corneal epithelial layers and cellular densities were evaluated by four trained graders from the Doheny Eye Institute. Results: Corneal epithelium in dry eyes presents morphological changes such as areas of enlarged and irregular shaped cells. In comparison with controls, the density of superficial epithelial cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01); the density of the outer layer of wing cells was smaller but not significantly different in NSDE (P > 0.05), but was lower in the SSDE group (P < 0.01); the density of the inner layer of wing cells was decreased in both the NSDE (P < 0.05) and SSDE groups (P < 0.01) and the density of basal epithelial cells was lower in both the NSDE (P < 0.01) and SSDE groups (P = 0.01). For all cell counts, the interclass correlation coefficient showed good agreement between graders (ICC =0.75 to 0.93). Conclusions: IVCM represents a reliable technique for examining the corneal epithelial microstructural changes associated with dry eyes, as well as for objectively and reproducibly quantifying cell densities within all corneal epithelial layers.
关键词： Dry eye syndrome,corneal epithelium,Sj?gren’s syndrome,in vivo laser scanning confocal microscopy
更新于2025-09-04 15:30:14
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Ex Vivo Confocal Microscopy Of Basal Cell Carcinoma On A 3-Color Scale
摘要： Ex vivo confocal microscopy (CM) is capable of visualizing freshly excised tissue in real-time with cellular resolution without routine processing. Depending on the laser wavelength, either reflectance (RCM) or fluorescence (FCM) is utilized. Ex vivo CM is useful for the rapid evaluation of tumor margins during Mohs micrographic surgery (MMS). Initially, ex vivo RCM studies used acetic acid as contrast agent to enhance basal cell carcinoma (BCC) cell nuclei. However, thin strands of BCC were frequently missed. The use of fluorophores improves contrast, so that even small strands of BCC can be spotted in FCM mosaics. Acridine orange (AO) is the dye most widely used. AO binds specifically to DNA and emits fluorescence, so images of living cell nuclei can be enhanced and displayed as bright structures in FCM mosaics. Even though good resolution and morphological correlation are achieved with this standard technique, nowadays confocal mosaics are displayed in a grey scale format. However, dermato-pathologists are often neither familiar with nor comfortable assessing these black-and-white images. We herein report a new technique for obtaining 3-color scale confocal mosaics (3CS-FCM) with the simultaneous use of AO and ethidium bromide (EB) as fluorescent dyes. In this technique, the excised skin sample is first soaked with liquid nitrogen. The sample is then sectioned into 20-30 μm-thick slices using a cryostat and stained with the dye mixture (AO 0.1 mM + EB 0.25 mM) for about one minute. The sample is then placed in the confocal microscope plate for imaging (Nikon A1R+, NIKON CORPORATION?, Japan). The tissue is scanned simultaneously with two different wavelength lasers (405 and 488 nm) and the collected fluorescence displayed on the screen as a 3-color-scale mosaic. Around 10 to 15 minutes are required for completion of the tissue processing and for final mosaics to be developed. Unlike AO, EB binds specifically to the DNA of BCC cells that are damaged due to freezing. As a result, BCC nests are stained by EB and emit red fluorescence after laser stimulation; in contrast, the epidermis and dermis are stained by AO and emit green fluorescence. Blue color corresponds to the background tissue autofluorescence. All fluorescence is collected by the microscope displaying the final images in a 3-color scale format. AO and EB staining do not affect additional fixation or staining of the sample. Figure 1 shows completed BCC mosaics displayed with this new technique. Each color represents a different skin structure, making the mosaics easier to read. In this way, 3CS confocal mosaics are more user-friendly and can be interpreted by healthcare professionals without previous experience with FCM. Moreover, with frozen sample processing, the tissue is completely flattened and the entire sample can be displayed on the screen. These developments represent important advantages over previously described images obtained with CM. In conclusion, 3CS-FCM is an innovative technique that provides colored images, expanding significantly the applicability of FCM. Larger studies are nevertheless required to validate the technique for MMS and other applications.
关键词： fluorescence confocal microscopy,3-color scale mosaics,basal cell carcinoma,ethidium bromide,acridine orange,ex vivo confocal microscopy,Mohs micrographic surgery
更新于2025-09-04 15:30:14
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Confinement of Singlet Oxygen Generated from Ruthenium Complex-Based Oxygen Sensor in the Pores of Mesoporous Silica Nanoparticles
摘要： We synthesized mesoporous silica nanoparticles bearing ruthenium complexes in their pores (MSN-Ru) and characterized their photochemical properties. The ruthenium complexes that were immobilized in the pores showed oxygen-dependent phosphorescence, similar to the complexes that were not tethered to nanoparticles. Cellular imaging and in vivo experiments revealed that hypoxic cells and tissues could be visualized by monitoring the phosphorescence of MSN-Ru. Our most important finding was that the toxic effect of singlet oxygen (1O2), which was generated by excitation of the complexes, was effectively suppressed by the deactivation before leaking out from the pores. In addition, we observed a negligible toxic effect of the ruthenium complexes themselves due to the blockage of their direct interaction with intracellular biomolecules. Thus, MSN-Ru is a promising molecular probe of oxygen levels in living cells and tissues.
关键词： ruthenium complexes,singlet oxygen,in vivo experiments,cellular imaging,oxygen sensor,mesoporous silica nanoparticles
更新于2025-09-04 15:30:14
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Sensitive, Real-Time and In-Vivo Oxygen Monitoring for Photodynamic Therapy by Multifunctional Mesoporous Nanosensors
摘要： Real-time monitoring of oxygen consumption is beneficial to predict treatment response and optimize therapeutic protocols for photodynamic therapy (PDT). In this work, we first demonstrate that deformable hollow mesoporous organosilica nanoparticles (HMONs) can be used to load [(Ru(dpp)3)]Cl2 for detecting oxygen (denoted as HMON-[(Ru(dpp)3)]Cl2). This nanoprobe shows significantly improved biocompatibility and high cellular uptake. In-vitro eperiments demonstrate that the HMON-[(Ru(dpp)3)]Cl2 can sensitively detect oxygen changes between 1 %-20 %. On this basis, photosensitizer chlorin e6 (Ce6) and [(Ru(dpp)3)]Cl2 are simultaneously loaded in the HOMNs (denoted as HMON-Ce6-[(Ru(dpp)3)]Cl2) for real-time oxygen monitoring during photodynamic therapy. The HMON-Ce6-[(Ru(dpp)3)]Cl2 can reflects oxygen consumption in solution and cells in photodynamic therapy. Furthermore, the ability of HMON-Ce6-[(Ru(dpp)3)]Cl2 nanosensor to monitor oxygen changes is demonstrated in tumor-bearing nude mice.
关键词： oxygen detection,mesoporous organosilica,photodynamic therapy,in-vivo,real-time
更新于2025-09-04 15:30:14