Intravital imaging of Bacillus thuringiensis Cry1A toxin binding sites in the midgut of silkworm

Identification of the resistance mechanism of insects against Bacillus thuringiensis Cry1A toxin is becoming an increasingly challenging task. This fact highlights the need for establishing new methods to further explore the molecular interactions of Cry1A toxin with insects and the receptor-binding region of Cry1A toxins for their wider application as biopesticides and a gene source for gene-modified crops. In this contribution, a quantum dot-based near-infrared fluorescence imaging method has been applied for direct dynamic tracking of the specific binding of Cry1A toxins, CrylAa and CrylAc, to the midgut tissue of silkworm. The in vitro fluorescence imaging displayed the higher binding specificity of CrylAa–QD probes compared to CrylAc–QD to the brush border membrane vesicles of midgut from silkworm. The in vivo imaging demonstrated that more CrylAa–QDs binding to silkworm midgut could be effectively and distinctly monitored in living silkworms. Furthermore, frozen section analysis clearly indicated the broader receptor-binding region of Cry1Aa compared to that of Cry1Ac in the midgut part. These observations suggest that the insecticidal activity of Cry toxins may depend on the receptor-binding sites, and this scatheless and visual near-infrared fluorescence imaging could provide a new avenue to study the resistance mechanism to maintain the insecticidal activity of B. thuringiensis toxins.     

In Vivo image Analysis Using iRFP Transgenic Mice

Fluorescent proteins with light wavelengths within the optical window are one of the improvements in in vivo imaging techniques. Near-infrared (NIR) fluorescent protein (iRFP) is a stable, nontoxic protein that emits fluorescence within the NIR optical window without the addition of exogenous substrate. However, studies utilizing an in vivo iRFP model have not yet been published. Here, we report the generation of transgenic iRFP mice with ubiquitous NIR fluorescence expression. iRFP expression was observed in approximately 50% of the offspring from a matings between iRFP transgenic and WT mice. The serum and blood cell indices and body weights of iRFP mice were similar to those of WT mice. Red fluorescence with an excitation wavelength of 690 nm and an emission wavelength of 713 nm was detected in both newborn and adult iRFP mice. We also detected fluorescence emission in whole organs of the iRFP mice, including the brain, heart, liver, kidney, spleen, lung, pancreas, bone, testis, thymus, and adipose tissue. Therefore, iRFP transgenic mice may therefore be a useful tool for various types of in vivo imaging.     

量子点在癌症研究中的应用

半导体量子点具有长时间、多目标和灵敏度高等独特的光化学性质,这些特性使量子点成为细胞标记和生物应用中得到了广泛的应用。利用量子点目标定位癌细胞,对于寻找癌变部位具有指导的作用。近年来,利用量子点作为光动力学治疗癌症的能量供体也得到了一定的研究。简单地介绍了量子点独特的光学性质,并从量子点标记癌细胞、可视化癌细胞表面功能和在光动力学治疗癌症等方面综述了量子点在癌症诊断和治疗中的应用。     

Lead sulfide near-infrared quantum dot bioconjugates for targeted molecular imaging

In this paper, we report the use of lead sulfide quantum dot (PbS QD) bioconjugates as near infrared (NIR) contrast agents for targeted molecular imaging with expanded emission wavelengths beyond 1000 nm. The red-shifted emission band, coupled with the small particle size, which will facilitate clearance, both afford PbS QDs unique properties for noninvasive, high resolution in vivo NIR imaging applications. We have performed imaging experiments at the molecular level using surface-modified PbS NIR QDs, together with our lab-built NIR imaging system. This novel instrumentation and fluorescent contrast agent have enabled us to study the relatively unexplored NIR biomedical imaging spectral region of 900-1200 nm. Preliminary experimental results indicate that PbS-QD/antibody bioconjugates are promising candidates for targeted NIR molecular imaging and future in vivo NIR tissue imaging applications.     

Endolithic chlorophyll d-containing phototrophs

Cyanobacteria in the genus Acaryochloris are the only known oxyphototrophs that have exchanged chlorophyll a (Chl a) with Chl d as their primary photopigment, facilitating oxygenic photosynthesis with near infrared (NIR) light. Yet their ecology and natural habitats are largely unknown. We used hyperspectral and variable chlorophyll fluorescence imaging, scanning electron microscopy, photopigment analysis and DNA sequencing to show that Acaryochloris-like cyanobacteria thrive underneath crustose coralline algae in a widespread endolithic habitat on coral reefs. This finding suggests an important role of Chl d-containing cyanobacteria in a range of hitherto unexplored endolithic habitats, where NIR light-driven oxygenic photosynthesis may be significant.     

Microbial diversity of biofilm communities in microniches associated with the didemnid ascidian Lissoclinum patella

We assessed the microbial diversity and microenvironmental niche characteristics in the didemnid ascidian Lissoclinum patella using 16S rRNA gene sequencing, microsensor and imaging techniques. L. patella harbors three distinct microbial communities spatially separated by few millimeters of tunic tissue: (i) a biofilm on its upper surface exposed to high irradiance and O₂ levels, (ii) a cloacal cavity dominated by the prochlorophyte Prochloron spp. characterized by strong depletion of visible light and a dynamic chemical microenvironment ranging from hyperoxia in light to anoxia in darkness and (iii) a biofilm covering the underside of the animal, where light is depleted of visible wavelengths and enriched in near-infrared radiation (NIR). Variable chlorophyll fluorescence imaging demonstrated photosynthetic activity, and hyperspectral imaging revealed a diversity of photopigments in all microhabitats. Amplicon sequencing revealed the dominance of cyanobacteria in all three layers. Sequences representing the chlorophyll d containing cyanobacterium Acaryochloris marina and anoxygenic phototrophs were abundant on the underside of the ascidian in shallow waters but declined in deeper waters. This depth dependency was supported by a negative correlation between A. marina abundance and collection depth, explained by the increased attenuation of NIR as a function of water depth. The combination of microenvironmental analysis and fine-scale sampling techniques used in this investigation gives valuable first insights into the distribution, abundance and diversity of bacterial communities associated with tropical ascidians. In particular, we show that microenvironments and microbial diversity can vary significantly over scales of a few millimeters in such habitats; which is information easily lost by bulk sampling.     

Design and development of high bioluminescent resonance energy transfer efficiency hybrid-imaging constructs

Here we describe the design and construction of an imaging construct with high bioluminescent resonance energy transfer (BRET) efficiency that is composed of multiple quantum dots (QDs; λ_em = 655 nm) self-assembled onto a bioluminescent protein, Renilla luciferase (Rluc). This is facilitated by the streptavidin–biotin interaction, allowing the facile formation of a hybrid-imaging construct (HIC) comprising up to six QDs (acceptor) grafted onto a light-emitting Rluc (donor) core. The resulting assembly of multiple acceptors surrounding a donor permits this construct to exhibit high resonance energy transfer efficiency (∼64.8%). The HIC was characterized using fluorescence excitation anisotropy measurements and high-resolution transmission electron microscopy. To demonstrate the application of our construct, a generation-5 (G5) polyamidoamine dendrimer (PAMAM) nanocarrier was loaded with our HIC for in vitro and in vivo imaging. We envision that this design of multiple acceptors and bioluminescent donor will lead to the development of new BRET-based systems useful in sensing, imaging, and other bioanalytical applications.     

Microwave‐assisted aqueous synthesis of new quaternary‐alloyed CdSeTeS quantum dots; and their bioapplications in targeted imaging of cancer cells

In this study, we report for the first time a one‐pot approach for the synthesis of new CdSeTeS quaternary‐alloyed quantum dots (QDs) in aqueous phase by microwave irradiation. CdCl₂ was used as a Cd precursor during synthesis, NaHTe and NaHSe were used as Te and Se precursors and mercaptopropionic acid (MPA) was used as a stabilizer and source of sulfur. A series of quaternary‐alloyed QDs of different sizes were prepared. CdSeTeS QDs exhibited a wide emission range from 549 to 709 nm and high quantum yield (QY) up to 57.7 %. Most importantly, the quaternary‐alloyed QDs possessed significantly long fluorescence lifetimes > 100 ns as well as excellent photostability. Results of high‐resolution transmission electron microscopy (HRTEM), energy dispersive X‐ray spectroscopy (EDX) and powder X‐ray diffraction (XRD) spectroscopy showed that the nanocrystals possessed a quaternary alloy structure with good crystallinity. Fluorescence correlation spectroscopy (FCS) showed that QDs possessed good water solubility and monodispersity in aqueous solution. Furthermore, CdSeTeS QDs were modified with alpha‐thio‐omega‐carboxy poly(ethylene glycol) (HS‐PEG‐COOH) and the modified QDs were linked to anti‐epidermal growth factor receptor (EGFR) antibodies. QDs with the EGFR antibodies as labeling probes were successfully applied to targeted imaging for EGFR on the surface of SiHa cervical cancer cells. We believe that CdSeTeS QDs can become useful probes for in vivo targeted imaging and clinical diagnosis. Copyright © 2012 John Wiley & Sons, Ltd.     

Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping

Intraoperative near-infrared (NIR) fluorescence imaging provides the surgeon with real-time image guidance during cancer and other surgeries. We have previously reported the use of NIR fluorescent quantum dots (QDs) for sentinel lymph node (SLN) mapping. However, because of concerns over potential toxicity, organic alternatives to QDs will be required for initial clinical studies. We describe a family of 800 nm organic heptamethine indocyanine-based contrast agents for SLN mapping spanning a spectrum from 775 Da small molecules to 7 MDa nanocolloids. We provide a detailed characterization of the optical and physical properties of these contrast agents and discuss the advantages and disadvantages of each. We present robust methods for the covalent conjugation, purification, and characterization of proteins with tetra-sulfonated heptamethine indocyanines, including mass spectroscopic site mapping of highly substituted molecules. One contrast agent, NIR fluorescent human serum albumin (HSA800), emerged as the molecule with the best overall performance with respect to entry to lymphatics, flow to the SLN, retention in the SLN, fluorescence yield and reproducibility. This preclinical study, performed on large animals approaching the size of humans, should serve as a foundation for future clinical studies.     

Near‐Infrared,Heavy Metal‐Free Colloidal “Giant” Core/Shell Quantum Dots

“Giant” core/shell quantum dots (g‐QDs) are a promising class of materials for future optoelectronic technologies due to their superior chemical‐ and photostability compared to bare QDs and core/thin shell QDs. However, inadequate light absorption in the visible and near‐infrared (NIR) region and frequent use of toxic heavy metals (e.g., Cd and Pb) are still major challenges for most g‐QDs (e.g., CdSe/CdS) synthesized to date. The synthesis of NIR, heavy metal‐free, Zn‐treated spherical CuInSe₂/CuInS₂ g‐QDs is reported using the sequential cation exchange method. These g‐QDs exhibit tunable NIR optical absorption and photoluminescence (PL) properties. Transient fluorescence spectroscopy shows prolonged lifetime with increasing shell thickness, indicating the formation of quasi type‐II band alignment, which is further confirmed by simulations. As a proof‐of‐concept, as‐synthesized g‐QDs are used to sensitize TiO₂ as a photoanode in a photoelectrochemical (PEC) cell, demonstrating an efficient and stable PEC system. These results pave the way toward synthesizing NIR heavy metal‐free g‐QDs, which are very promising components of future optoelectronic technologies.     

综述——基于纳米材料的生物检测与医学诊断技术：功能化量子点在肿瘤活体成像中的应用

量子点表面经生物分子或药物分子修饰而具有生物功能．功能化量子点具有独特的光学性质和生物相容性,在生物医学光学诊断和治疗领域具有广泛的应用．本文简要介绍了功能化量子点制备及修饰方法,综合评述了量子点在肿瘤活体诊断和治疗中的应用,包括活体淋巴结成像、血管动态成像、肿瘤成像和抗肿瘤药物示踪等,讨论了功能化量子点在肿瘤活体诊断和治疗中的应用前景以及面临的挑战．     

Characterization and cancer cell targeted imaging properties of human antivascular endothelial growth factor monoclonal antibody conjugated CdTe/ZnS quantum dots

High luminescence quantum yield water‐soluble CdTe/ZnS core/shell quantum dots (QDs) stabilized with thioglycolic acid were synthesized. QDs were chemically coupled to fully humanized antivascular endothelial growth factor₁₆₅ monoclonal antibodies to produce fluorescent probes. These probes can be used to assay the biological affinity of the antibody. The properties of QDs conjugated to an antibody were characterized by ultraviolet and visible spectrophotometry, fluorescent spectrophotometry, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transmission electron microscopy and fluorescence microscopy. Cell‐targeted imaging was performed in human breast cancer cell lines. The cytotoxicity of bare QDs and fluorescent probes was evaluated in the MCF‐7 cells with an MTT viability assay. The results proved that CdTe/ZnS QD–monoclonal antibody nanoprobes had been successfully prepared with excellent spectral properties in target detections. Surface modification by ZnS shell could mitigate the cytotoxicity of cadmium‐based QDs. The therapeutic effects of antivascular endothelial growth factor antibodies towards cultured human cancer cells were confirmed by MTT assay. Copyright © 2014 John Wiley & Sons, Ltd.     

Recent advances in synthesis and surface modification of lanthanide-doped upconversion nanoparticles for biomedical applications

Lanthanide (Ln)-doped upconversion nanoparticles (UCNPs) with appropriate surface modification can be used for a wide range of biomedical applications such as bio-detection, cancer therapy, bio-labeling, fluorescence imaging, magnetic resonance imaging and drug delivery. The upconversion phenomenon exhibited by Ln-doped UCNPs renders them tremendous advantages in biological applications over other types of fluorescent materials (e.g., organic dyes, fluorescent proteins, gold nanoparticles, quantum dots, and luminescent transition metal complexes) for: (i) enhanced tissue penetration depths achieved by near-infrared (NIR) excitation; (ii) improved stability against photobleaching, photoblinking and photochemical degradation; (iii) non-photodamaging to DNA/RNA due to lower excitation light energy; (iv) lower cytotoxicity; and (v) higher detection sensitivity. Ln-doped UCNPs are therefore attracting increasing attentions in recent years. In this review, we present recent advances in the synthesis of Ln-doped UCNPs and their surface modification, as well as their emerging applications in biomedicine. The future prospects of Ln-doped UCNPs for biomedical applications are also discussed.     

Cadmium-containing quantum dots: properties,applications, and toxicity

The marriage of biology with nanomaterials has significantly accelerated advancement of biological techniques, profoundly facilitating practical applications in biomedical fields. With unique optical properties (e.g., tunable broad excitation, narrow emission spectra, robust photostability, and high quantum yield), fluorescent quantum dots (QDs) have been reasonably functionalized with controllable interfaces and extensively used as a new class of optical probe in biological researches. In this review, we summarize the recent progress in synthesis and properties of QDs. Moreover, we provide an overview of the outstanding potential of QDs for biomedical research and innovative methods of drug delivery. Specifically, the applications of QDs as novel fluorescent nanomaterials for biomedical sensing and imaging have been detailedly highlighted and discussed. In addition, recent concerns on potential toxicity of QDs are also introduced, ranging from cell researches to animal models.

     

Gold nanoclusters as novel optical probes for in vitro and in vivo fluorescence imaging

Fluorescent probes play an important role in the development of fluorescence-based imaging techniques for life sciences research. Gold nanoclusters (AuNCs) are a novel type of fluorescent nanomaterials which have attracted great interest in recent years. Composed of only a few atoms, these ultrasmall AuNCs exhibit quantum confinement effects and molecule-like properties. Fluorescent AuNCs have an attractive set of features including ultrasmall size, good biocompatibility and photostability, and tunable emission in the red to near-infrared spectral region, which make them promising as fluorescent labels for biological imaging. Examples of their application include live cell labeling, cancer cell targeting, cellular apoptosis monitoring, and in vivo tumor imaging. Here, we present a brief overview of recent advances in utilizing these emissive ultrasmall AuNCs as optical probes for in vitro and in vivo fluorescence imaging.     

量子点在生物学中的研究进展

量子点作为一种新型的荧光标记物近年来已在生物学中获得广泛应用。本文总结了量子点的主要光学特性,其中包括荧光激发和发射光谱特性、量子产额、光漂白特性和荧光寿命等。重点综述了量子点在细胞标记、活体和组织成像、组合标记和光动力学治疗等生物学中的应用及其最新研究进展。同时讨论了量子点在应用中可能存在的细胞毒性等主要问题,最后对量子点在生物学中的应用前景作了展望。     

Synthesis of near‐infrared‐emitting CdTeSe and CdZnTeSe quantum dots

We exploited the synthesis of near‐infrared (NIR) emitting ternary‐alloyed CdTeSe and quaternary‐alloyed CdZnTeSe quantum dots (QDs) with rod and tetrapod morphologies, which have tunable emission in the NIR electromagnetic spectrum. The morphologies of the QDs depended strongly on their growth kinetics, probably due to the coordinating ligands used in the preparation. Using oleic acid, stearic acid and hexadecylamine as ligands and keeping the same reaction parameters, QDs with tetrapod and rod morphologies were created. Not only had the capping ligands influenced the morphologies of QDs, but also they influenced the optical properties of QDs. The molar ratios of Cd/Zn and Te/Se upon preparation were adjusted for investigating the effect of composition on the properties of resulting QDs. By varying the composition of QDs, the photoluminescence (PL) wavelength of QDs was tuned from 650 nm to 800 nm. To enhance PL efficiency and stability, QDs were coated with a CdZnS shell. As NIR PL has numerous advantages in biological imaging detection, these QDs hold great potential for application. Copyright © 2012 John Wiley & Sons, Ltd.     

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2^E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from α-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.     

Aqueous synthesis of highly luminescent surface Mn2+‐doped CdTe quantum dots as a potential multimodal agent

Mn²⁺‐doped CdTe quantum dots (QDs) were synthesized directly via a facile surface doping strategy in aqueous solution. The best optical property emerged when the added amount of Mn²⁺ was 5% compared to Cd²⁺ in the CdTe nanoparticles and the reaction temperature was 60 °C. The fluorescence and magnetic properties of the QDs were studied. The as‐prepared Mn²⁺‐doped CdTe QDs have high quantum yield (48.13%) and a narrow distribution with an average diameter of 3.7 nm. The utility of biological imaging was also studied. Depending on the high quantum yield, cells in culture were illuminated and made more distinct from each other compared to results obtained with normal QDs. They also have a prominent longitudinal relaxivity value (r₁ = 4.2 mM^?1s^?1), which could indicate that the Mn²⁺‐doped CdTe QDs can be used as a potential multimodal agent for fluorescence and magnetic resonance imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

Synchrotron radiation based STXM analysis and micro-XRF mapping of differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on Fe and S

The differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on substrates Fe^2 + and S⁰ was investigated by using synchrotron radiation based scanning transmission X-ray microscopy (STXM) imaging and microbeam X-ray fluorescence (μ-XRF) mapping. The extracellular thiol groups (SH) were first alkylated by iodoacetic acid forming Protein-SCH₂COOH and then the P-SCH₂COOH was marked by calcium ions forming P-SCH₂COOCa. The STXM imaging and μ-XRF mapping of SH were based on analysis of SCH₂COO-bonded Ca^2 +. The results indicated that the thiol group content of A. ferrooxidans grown on S⁰ is 3.88 times to that on Fe^2 +. Combined with selective labeling of SH by Ca^2 +, the STXM imaging and μ-XRF mapping provided an in situ and rapid analysis of differential expression of extracellular thiol groups.