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High dynamic range fluorescence imaging
摘要： Fluorescence acquisition and image display over a high dynamic range is highly desirable. However, the limited dynamic range of current photodetectors and imaging CCDs impose a limit on the fluorescence intensities that can be simultaneously captured during a single image acquisition. This is particularly troublesome when imaging biological samples, where protein expression fluctuates considerably. As a result, biological images will often contain regions with signal that is either saturated or hidden within background noise, causing information loss. In this manuscript we summarize recent work from our group and others, to extended conventional to high dynamic range fluorescence imaging. These strategies have many biological applications, such as mapping of neural connections, vascular imaging, bio-distribution studies or pharmacologic imaging at the single cell and organ level.
关键词： optical imaging,Intravital microscopy,drug imaging,in vivo imaging,high dynamic range
更新于2025-09-23 15:21:01
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Unraveling the Safety Profile of Nanoscale Particles and Materials - From Biomedical to Environmental Applications || Applications of Fluorescent Quantum Dots for Reproductive Medicine and Disease Detection
摘要： Understanding the mechanisms associated with fertility and disease management in animals remains challenging. Continuing advances in nanotechnology provide new tools and alternative approaches for the investigation of these mechanisms. Fluorescent quantum dot nanoparticles, for example, have unique physicochemical properties, which allow for in vivo and in vitro imaging in various areas of life sciences. Traditional quantum dots contain heavy metal semiconductor cores, which have raised concern over their potential for toxicity. The majority of available quantum dots today prevent heavy metal release with additional chemical and polymer layers for safe water solubility. In this chapter, the most widely used quantum dots made of cadmium selenide, which possess great potential for real-time imaging in disease detection and reproductive medicine, are discussed.
关键词： real-time imaging,spermatozoa,quantum dots,in vivo imaging,fertility,luminescence
更新于2025-09-23 15:21:01
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Probing Cell Mechanics with Bead-Free Optical Tweezers in the <em>Drosophila</em> Embryo
摘要： Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts' rheology.
关键词： Drosophila embryo,Developmental Biology,in vivo imaging,optical tweezers,Light sheet microscopy,force measurements,Issue 141,cell mechanics
更新于2025-09-23 15:21:01
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<em>In Vivo</em> Two-photon Imaging of Cortical Neurons in Neonatal Mice
摘要： Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.
关键词： Neuroscience,in vivo imaging,single-cell labeling,mouse,cerebral cortex,Newborn,two-photon
更新于2025-09-23 15:21:01
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Facile Synthesis of Noncytotoxic PEGylated Dendrimer Encapsulated Silver Sulfide Quantum Dots for NIR-II Biological Imaging
摘要： Near-Infrared-II (NIR-II, 1000-1700 nm) bioimaging features high penetration depth and high spatio-temporal resolution compared to traditional fluorescence imaging, but the key is to develop stable and biocompatible NIR-II fluorophores suitable for in vivo applications. Silver sulfide quantum dots (Ag2S QDs) have been demonstrated excellent for in vivo NIR-II imaging with unique optical properties and decent biocompatibility, but they often require complex post modifications for in vivo applications. Herein we demonstrate a facile one-pot strategy to synthesize the PEGylated dendrimer-encapsulated Ag2S QDs useful for in vivo NIR-II imaging. Silver ions were first loaded into the core of acylthiourea-functionalized dendrimer (PEG-PATU) through the coordination between silver ions and acylthiourea groups, followed by the addition of sodium sulfide to form Ag2S QDs in situ. The resulting PEG-PATU Ag2S QDs hold excellent NIR-II fluorescence signal, and thus could be executed for high efficiency labelling and tracking of A549 cancer cells mobility in vivo and real time visualization of vast circulatory network of a mouse.
关键词： in vivo imaging,PEGylated dendrimer,one-pot synthesis,Ag2S QDs,NIR-II bioimaging
更新于2025-09-23 15:19:57
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Polysaccharide enabled biogenic fabrication of pH??sensing fluorescent gold nanoclusters as a biocompatible tumor imaging probe
摘要： A biocompatible natural polysaccharide (PSP001) isolated from the fruit rind of Punica granatum was conjugated with L-cysteine (Y) to be used as a skeleton for the fabrication of fluorescent gold nanoclusters (AuNCs) represented as PSP-Y-AuNCs. With an average size of ~ 6 nm, PSP-Y-AuNCs demonstrated high quantum yield (31%), with a pH-sensitive fluorescence emission behavior. An emission maximum of 520 nm was obtained at acidic pH, which was blue shifted with increasing pH. This feature provides the possibilities for accurate ratiometric pH imaging. The PSP-Y-AuNCs not only demonstrated excellent biocompatibility with cancer cells and isolated peripheral lymphocytes and red blood cells but also demonstrated to be an active molecular imaging probe with appealing cellular uptake efficiency. The investigations with BALB/c mice further confirmed the non-toxic nature and in vivo imaging potential of the AuNCs. Estimation of the bio-distribution on solid tumor bearing syngeneic murine models revealed a tumor-targeted enhanced fluorescence emission pattern which is attributed to the pH responsive fluorescence behavior and the acidic microenvironment of the tumor. These findings were further confirmed with an impressive tumor accumulation pattern displayed in a xenograft of human cancer bearing nude mice. On account of their impressive biocompatibility and photophysical features, PSP-Y-AuNCs can exploited for the real-time fluorescence imaging of cancer tissues.
关键词： Polysaccharide,Tumor,pH ratiometry,Biocompatibility,In vivo imaging,Nanocluster,Fluorescence,Punica granatum,Gold nanoparticle
更新于2025-09-23 15:19:57
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Therapeutic Fluorescent Hybrid Nanoparticles for Traceable Delivery of Glucocorticoids to Inflammatory Sites
摘要： Treatment of inflammatory disorders with glucocorticoids (GCs) is often accompanied by severe adverse effects. Application of GCs via nanoparticles (NPs), especially those using simple formulations, could possibly improve their delivery to sites of inflammation and therefore their efficacy, minimising the required dose and thus reducing side effects. Here, we present the evaluation of NPs composed of GC betamethasone phosphate (BMP) and the fluorescent dye DY-647 (BMP-IOH-NPs) for improved treatment of inflammation with simultaneous in vivo monitoring of NP delivery. Methods: BMP-IOH-NP uptake by MH-S macrophages was analysed by fluorescence and electron microscopy. Lipopolysaccharide (LPS)-stimulated cells were treated for 48 h with BMP-IOH-NPs (1×10-5-1×10-9 M), BMP or dexamethasone (Dexa). Drug efficacy was assessed by measurement of interleukin 6. Mice with Zymosan-A-induced paw inflammation were intraperitoneally treated with BMP-IOH-NPs (10 mg/kg) and mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI) were treated intranasally with BMP-IOH-NPs, BMP or Dexa (each 2.5 mg/kg). Efficacy was assessed in vivo by paw volume measurements with μCT and ex vivo by measurement of paw weight for Zymosan-A-treated mice, or in the AAI model by in vivo x-ray-based lung function assessment and by cell counts in the bronchoalveolar lavage (BAL) fluid and histology. Delivery of BMP-IOH-NPs to the lungs of AAI mice was monitored by in vivo optical imaging and by fluorescence microscopy. Results: Uptake of BMP-IOH-NPs by MH-S cells was observed during the first 10 min of incubation, with the NP load increasing over time. The anti-inflammatory effect of BMP-IOH-NPs in vitro was dose dependent and higher than that of Dexa or free BMP, confirming efficient release of the drug. In vivo, Zymosan-A-induced paw inflammation was significantly reduced in mice treated with BMP-IOH-NPs. AAI mice that received BMP-IOH-NPs or Dexa but not BMP revealed significantly decreased eosinophil numbers in BALs and reduced immune cell infiltration in lungs. Correspondingly, lung function parameters, which were strongly affected in non-treated AAI mice, were unaffected in AAI mice treated with BMP-IOH-NPs and resembled those of healthy animals. Accumulation of BMP-IOH-NPs within the lungs of AAI mice was detectable by optical imaging for at least 4 h in vivo, where they were preferentially taken up by peribronchial and alveolar M2 macrophages. Conclusion: Our results show that BMP-IOH-NPs can effectively be applied in therapy of inflammatory diseases with at least equal efficacy as the gold standard Dexa, while their delivery can be simultaneously tracked in vivo by fluorescence imaging. BMP-IOH-NPs thus have the potential to reach clinical applications.
关键词： nanoparticle-based therapy,in vivo imaging,inflammatory disease,monitoring glucocorticoid delivery,hybrid nanoparticles
更新于2025-09-19 17:15:36
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Shell-free Copper Indium Sulfide Quantum Dots Induce Toxicity <i>In Vitro</i> and <i>In Vivo</i>
摘要： Semiconductor quantum dots (QDs) are attractive fluorescent contrast agents for in vivo imaging due to their superior photophysical properties, but traditional QDs comprise toxic materials such as cadmium or lead. Copper indium sulfide (CuInS2, CIS) QDs have been posited as a non-toxic and potentially clinically translatable alternative; however, previous in vivo studies utilized particles with a passivating zinc sulfide (ZnS) shell, limiting direct evidence of the biocompatibility of the underlying CIS. For the first time, we assess the biodistribution and toxicity of unshelled CIS and partially zinc-alloyed CISZ QDs in a murine model. We show that bare CIS QDs breakdown quickly, inducing significant toxicity as seen in organ weight, blood chemistry, and histology. CISZ demonstrate significant, but lower, toxicity compared to bare CIS, while our measurements of core/shell CIS/ZnS are consistent with literature reports of general biocompatibility. In vitro cytotoxicity is dose-dependent on the amount of metal released due to particle degradation, linking degradation to toxicity. These results challenge the assumption that removing heavy metals necessarily reduces toxicity: indeed, we find comparable in vitro cytotoxicity between CIS and CdSe QDs, while CIS caused severe toxicity in vivo compared to CdSe. In addition to highlighting the complexity of nanotoxicity and the differences between the in vitro and in vivo outcomes, these unexpected results serve as a reminder of the importance of assessing the biocompatibility of core QDs absent the protective ZnS shell when making specific claims of compositional biocompatibility.
关键词： in vivo imaging,biodegradable,CIS,nanotoxicity,nanomedicine,CuInS2,fluorescent contrast agent,QDs
更新于2025-09-19 17:13:59
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Noninvasive In Vivo Imaging in the Second Near-Infrared Window by Inorganic Nanoparticle-Based Fluorescent Probes
摘要： The fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) has emerged as a new method for in vivo imaging and attracted considerable attention in the past decade. Owing to the suppressed photon scattering and diminished autofluorescence, in vivo fluorescence imaging in NIR-II window can afford deep tissue penetration depth with high clarity. Inorganic nanoparticle-based fluorescent probes in NIR-II window have greatly prospered the field into a development stage because of their superior traits, including adjustable emission covering the whole NIR-II window, and abundant surface functional groups that facilitate chemical modification and bioconjugation, etc. In this feature article, we introduce the unique imaging performance of the NIR-II optical window, and highlight the latest development of noninvasive biological fluorescent imaging in NIR-II window using inorganic nanoparticle-based probes. A perspective on the challenge and future direction of inorganic nanoparticle-based NIR-II probes is also discussed.
关键词： noninvasive imaging,NIR-II,inorganic nanoparticles,fluorescence imaging,in vivo imaging
更新于2025-09-11 14:15:04
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Application of quantum dot‐based biosensor to the conjunctiva in Wistar albino rats
摘要： This study was conducted to get an idea about the distribution of the lymphatic fluid in conjunctiva throughout the body parts. For this purpose, Qdot655 (QD), fluorescence nanoparticles, spread onto the conjunctiva were used on Wistar albino rats. Drainage of QD particles from conjunctiva was followed up via fluorescence images at different hours on body parts such as eye, ears, forearms, hind legs and tails. The first fluorescence signals within the 30th minutes following administration of QD were observed in the nasal region and the anterior extremities. Whereas within 60th minutes following QD spread, fluorescent signals were obtained from the ears, forearms, hind legs and tail of the female and male rats.
关键词： rat,conjunctiva,quantum dot,biosensor,in vivo imaging,eyelid lymphatics
更新于2025-09-11 14:15:04