Modeling bidirectional transport of quantum dot nanoparticles in membrane nanotubes

This paper develops a model of transport of quantum dot (QD) nanoparticles in membrane nanotubes (MNTs). It is assumed that QDs are transported inside intracellular organelles (called here nanoparticle-loaded vesicles, NLVs) that are propelled by either kinesin or dynein molecular motors while moving on microtubules (MTs). A vesicle may have both types of motors attached to it, but the motors are assumed to work in a cooperative fashion, meaning that at a given time the vesicle is moved by either kinesin or dynein motors. The motors are assumed not to work against each other, when one type of motors is pulling the vesicle, the other type is inactive. From time to time the motors may switch their roles: passive motors can become active motors and vice versa, resulting in the change of the vesicle’s direction of motion. It is further assumed that QDs can escape NLVs and become free QDs, which are then transported by diffusion. Free QDs can be internalized by NLVs. The effects of two possible types of MT orientation in MNTs are investigated: when all MTs have a uniform polarity orientation, with their plus-ends directed toward one of the cells connected by an MNT, and when MTs have a mixed polarity orientation, with half of MTs having their plus-ends directed toward one of the cells and the other half having their plus-ends directed toward the other cell. Computational results are presented for three cases. The first case is when organelles are as likely to be transported by kinesin motors as by dynein motors. The second case is when organelles are more likely to be transported by kinesin motors than by dynein motors, and the third case is when NLVs do not associate with dynein motors at all.     

Microvessel diameter estimation: error bias correction of serial measurements

The assessment of vessel patency can be substantially improved by serial microvessel diameter measurements taken successively along an extensive length of the vessel. It is possible to avoid making the a priori assumptions about the existence or location of local constriction sites implicit in single diameter measurements. The problem then becomes one of making sense of tens or hundreds of measurements for each vessel. Equivalent diameter is defined here as as the diameter of a uniform circular cylinder of the same length as the original vessel, and having the same total resistance. Direct computation of the equivalent diameter, without taking measurement errors into account, leads to an underestimation of the true equivalent diameter even if the individual diameter measurements were not biased. We have developed a method for effectively eliminating this bias. It has been applied to serial microvessel diameter measurements of the guinea pig cochlea, automatically measured using an image analysis system. In this report, the results were developed for diameter estimates with an approximate gaussian distribution; however the method is readily extended to other error distributions. Convergence of the bias compensation was rapid. Use of the new method is advisable with as few as three diameter estimates per vessel.     

k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics

We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or "blinking". Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities that agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of alpha5-integrin/enhanced green fluorescent protein constructs in a transfected CHO cell imaged on a total internal reflection fluorescence microscope using charge-coupled device area detection.     

Detection, identification, and correction of a bias in an epidemiological study

The relative lack of epidemiological studies of natural populations is partly due to the difficulty of obtaining samples that are both large enough and representative of the population. Here, we present the result of an epidemiological study (December 1992-August 1995) of feline immunodeficiency virus (FIV) in a free-roaming population of domestic cats (Felis catus), with a special emphasis on sample bias. Over five trapping periods, the prevalence of FIV in sampled cats steadily declined. Across these samples we consistently achieved a very large sampling fraction (approximately 60% of the population), the sex ratio, age and weight distributions remained stable with time in the samples, and the sex ratio was similar in the samples and the population. These indices would normally indicate that our samples were representative, suggesting the decline in FIV prevalence to be real. However, a concomitant ecological study of the whole population revealed an important bias in the samples, with an initial high probability of capturing a few individuals, which appeared significantly more likely to be FIV-infected, and then a lower probability of recapturing them. Since our protocol resulted in a non-random sampling, subsequent trappings were designed to avoid this bias, by also capturing individuals who had previously learned to escape capture. This modified capture regime revealed that FIV prevalence was in fact constant in the population. This study shows how samples of large size, which are stable and appear representative of the population, can still be biased. These results may have major implications for other studies based on trapping.     

A three-dimensional digital image correlation technique for strain measurements in microstructures

A three-dimensional digital image correlation technique is presented for strain measurements in open-cell structures such as trabecular bone. The technique uses high-resolution computed tomography images for displacement measurements in the solid structure. In order to determine the local strain-state within single trabeculae, a tetrahedronization method is used to fill the solid structure with tetrahedrae. Displacements are calculated at the nodes of the tetrahedrae. The displacement data is subsequently converted to a deformation tensor in each of the tetrahedral element centers with a least-squares estimation method. Because the trabeculae are represented by a mesh, it is possible to deform this mesh according to the deformation tensor and, at the same time, visualize the calculated local strain in the deformed mesh with a finite element post-processing tool. In this way, the deformation of a single trabecula from an aluminum foam sample was determined and validated with rendered images of the three-dimensional sample. A precision analysis showed that a rigid translation or rotation does not affect the accuracy. Typical values for the standard deviation in the displacement and strain components are 2.0 microm and 0.01, respectively. Presently, the precision limits the technique to strain measurements beyond the yield strain.     

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.     

Maximizing specificity and yield of PCR by the quantum dot itself rather than property of the quantum dot surface

We found that semiconductor quantum dots (QDs) dramatically improved both product yield and specificity of PCR. The concentration of QDs is important for improving PCR amplification. In the presence of appropriate concentration of mercaptoacetic acid (MAA)-coated QDs, specificity and yield of PCR were enhanced. Also, strong nonspecific bands and weaker smeared bands were eliminated. At lower annealing temperatures (25–45 °C), addition of MAA-coated QDs into the PCR reagent produced specific PCR products without nonspecific sequence amplification. MAA alone did not improve PCR amplification. Streptavidin (SA) surface modified QDs with different size also effectively improved the specificity of PCR, demonstrating that the observed effect was not due to property of the QD surface but instead due to the QD itself. Bovine Serum Albumin (BSA) could relieve Taq polymerase from MAA-coated QDs in PCR by interaction with QDs and therefore imply that QDs improve specificity of PCR by interaction with Taq polymerase. These results demonstrate that QDs, added to reaction mixes at appropriate concentrations, can increase PCR yield and improve PCR specificity, even at low annealing temperatures. We assume that many different surface modified polymeric nanoparticles might have similar effects.     

量子点荧光光谱学与生命科学

近年来,量子点(半导体纳米微晶体)的研究引起国内外研究者的广泛兴趣,其研究内容涉及物理学、化学、材料等多学科,已成为一门新兴的交叉学科。虽然量子点在生物学中的应用才刚刚起步,但是已经取得了有意义的进展,成为人们极为关注的一个热点。现就量子点的光学特性、制备方法,以及在生物学中的研究进展和应用前景作一简要综述。     

Dynamics and mechanisms of quantum dot nanoparticle cellular uptake

Background  

The rapid growth of the nanotechnology industry and the wide application of various nanomaterials have raised concerns over their impact on the environment and human health. Yet little is known about the mechanism of cellular uptake and cytotoxicity of nanoparticles. An array of nanomaterials has recently been introduced into cancer research promising for remarkable improvements in diagnosis and treatment of the disease. Among them, quantum dots (QDs) distinguish themselves in offering many intrinsic photophysical properties that are desirable for targeted imaging and drug delivery.     

Tendon strain measurements with dynamic ultrasound images: evaluation of digital image correlation

Strain is an essential metric in tissue mechanics. Strains and strain distributions during functional loads can help identify damaged and pathologic regions as well as quantify functional compromise. Noninvasive strain measurement in vivo is difficult to perform. The goal of this in vitro study is to determine the efficacy of digital image correlation (DIC) methods to measure strain in B-mode ultrasound images. The Achilles tendons of eight male Wistar rats were removed and mechanically cycled between 0 and 1% strain. Three cine video images were captured for each specimen: (1) optical video for manual tracking of optical markers; (2) optical video for DIC tracking of optical surface markers; and (3) ultrasound video for DIC tracking of image texture within the tissue. All three imaging modalities were similarly able to measure tendon strain during cyclic testing. Manual/ImageJ-based strain values linearly correlated with DIC (optical marker)-based strain values for all eight tendons with a slope of 0.970. DIC (optical marker)-based strain values linearly correlated with DIC (ultrasound texture)-based strain values for all eight tendons with a slope of 1.003. Strain measurement using DIC was as accurate as manual image tracking methods, and DIC tracking was equally accurate when tracking ultrasound texture as when tracking optical markers. This study supports the use of DIC to calculate strains directly from the texture present in standard B-mode ultrasound images and supports the use of DIC for in vivo strain measurement using ultrasound images without additional markers, either artificially placed (for optical tracking) or anatomically in view (i.e., bony landmarks and/or muscle-tendon junctions).     

Intracellular transport of sulfated macromolecules in parotid acinar cells

Intracellular transport of sulfated macromolecules in parotid acinar cells was investigated by electron microscopic radioautography after injection of 35S-sulfate. Ten minutes after injection radiosulfate was concentrated in the Golgi region. By 1 hr, much of the radioactive material had been transported to condensing vacuoles. These vacuoles were subsequently transformed into zymogen granules which contained almost 70% of the radioactivity 4 hrs after injection. These results indicate that, in addition to its packaging function, the Golgi apparatus in parotid acinar cells is capable of utilizing inorganic sulfate for the production of sulfated macromolecules. These molecules, following an intracellular route similar to that taken by digestive enzymes, become an integral component of zymogen granules. The possibility that sulfated macromolecules play a role in exocrine secretion by aiding in the packaging of exportable proteins is discussed.     

Selection of quantum dot wavelengths for biomedical assays and imaging

Fluorescent semiconductor nanocrystals (quantum dots [QDs]) are hypothesized to be excellent contrast agents for biomedical assays and imaging. A unique property of QDs is that their absorbance increases with increasing separation between excitation and emission wavelengths. Much of the enthusiasm for using QDs in vivo stems from this property, since photon yield should be proportional to the integral of the broadband absorption. In this study, we demonstrate that tissue scatter and absorbance can sometimes offset increasing QD absorption at bluer wavelengths, and counteract this potential advantage. By using a previously validated mathematical model, we explored the effects of tissue absorbance, tissue scatter, wavelength dependence of the scatter, water-to-hemoglobin ratio, and tissue thickness on QD performance. We conclude that when embedded in biological fluids and tissues, QD excitation wavelengths will often be quite constrained, and that excitation and emission wavelengths should be selected carefully based on the particular application. Based on our results, we produced near-infrared QDs optimized for imaging surface vasculature with white light excitation and a silicon CCD camera, and used them to image the coronary vasculature in vivo. Taken together, our data should prove useful in designing fluorescent QD contrast agents optimized for specific biomedical applications.     

Mortalin imaging in normal and cancer cells with quantum dot immuno-conjugates

Quantum dots are the nanoparticles that are recently emerging as an alternative to organic fluorescence probes in cell biology and biomedicine, and have several predictive advantages. These include their ⑴broad absorption spectra allowing visualization with single light source, ⑵exceptional photo-stability allowing long term studies and ⑶narrow and symmetrical emission spectrum that is controlled by their size and material composition. These unique properties allow simultaneous excitation of different size of quantum dots with a single excitation light source, their simultaneous resolution and visualization as different colors. At present there are only a few studies that have tested quantum dots in cellular imaging. We describe here the use of quantum dots in mortalin imaging of normal and cancer cells. Mortalin staining pattern with quantum dots in both normal and cancer cells mimicked those obtained with organic florescence probes and were considerably stable.     

ZnS量子点的生物合成及对阿萨尔基亚芽胞杆菌的标记

通过微生物合成金属量子点是目前研究的热点。本研究通过白色念珠菌合成ZnS量子点,对合成的量子点用高分辨透射电镜(HR-TEM)和X射线衍射(XRD)进行表征,用生物合成的ZnS量子点标记生防菌阿萨尔基亚芽胞杆菌,为建立生防菌阿萨尔基亚芽胞杆菌的荧光探针标记提供科学依据。实验结果表明,ZnSO4浓度为20 mmol/L时白色念珠细胞内合成ZnS量子点,通过反复冻融破细胞壁提出胞内ZnS量子点,紫外-可见光谱(UV)检测ZnS量子点在348 nm处显示有吸收带,HR-TEM测其粒径约7. 06 nm,荧光分光光谱分析量子点激发波在280 nm和363 nm,XRD显示ZnS量子点特征峰。ZnS量子点中加入1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)偶联剂的芽胞杆菌样本的标记效率明显高于不加偶联剂的样本。白色念珠菌合成的ZnS量子点可以用于微生物标记。     

Capillary transport of macromolecules: pores and other endothelial pathways

Can a pore or pore-equivalent model account for transport of macromolecules across microvascular endothelium, or are alternate nonpore pathways necessary? Pores may be defined as aqueous channels of any shape or configuration, including those through a fiber matrix. Such pathways exhibit selective restriction to passage of macromolecules depending on their size, shape, and electrical charge. At least two pore pathways (small and large), differing in both sieve-element spacing and in hydraulic conductivity by an order of magnitude, are required to account for observed size selectivity for plasma proteins of similar shapes and charges. For the two organs examined critically in this review (cat ileum and dog paw), transport of macromolecules through small and large pore pathways is predominately convective. Total transport through small and large pores (alternatively, narrow and wide slits or fine and coarse fiber matrices) is insufficient to account for observed transport rates at low-to-moderate levels of volume flow. Either the estimated pore sizes and hydraulic conductivities derived from measurements of high volume-flow sieving are incorrect or other nonconvective transport pathways contribute substantially to macromolecular transport at low (normal) volume flow.     

Plasmodesmata: intercellular tunnels facilitating transport of macromolecules in plants

The major structural and enzymatically active protein in spicules from siliceous sponges, e.g., for Suberites domuncula studied here, is silicatein. Silicatein has been established to be the key enzyme that catalyzes the formation of biosilica, a polymer that represents the inorganic scaffold for the spicule. In the present study, it is shown, by application of high-resolution transmission and scanning transmission electron microscopy that, during the initial phase of spicule synthesis, nanofibrils with a diameter of around 10 nm are formed that comprise bundles of between 10 and 20 nanofibrils. In intracellular vacuoles, silicasomes, the nanofibrils form polar structures with a pointed tip and a blunt end. In a time-dependent manner, these nanofibrillar bundles become embedded into a Si-rich matrix, indicative for the formation of biosilica via silicatein molecules that form the nanofibrils. These biosilicified nanofibrillar bundles become extruded from the intracellular space, where they are located in the silicasomes, to the extracellular environment by an evagination process, during which a cellular protrusion forms the axial canal in the growing spicule. The nanofibrillar bundles condense and progressively form the axial filament that becomes localized in the extracellular space. It is concluded that the silicatein-composing nanofibrils act not only as enzymatic silica bio-condensing platforms but also as a structure-giving guidance for the growing spicule.     

Multidimensional spectroscopic data correlation in the conformation transition of biological macromolecules

A computerized spectrophotometer system which is capable of simultaneously obtaining three spectral dimensions, that is, the absorbance, the circular dichroism and the fluorescence intensity, from one sample solution has been developed for the purpose of attaining a higher resolving power in the study of conformational transitions of biological macromolecules. Measurement conditions, such as the wavelength, the temperature, pH or the concentration of reagents in the sample solution, can be scanned according to a sequence that is set just prior to the measurement. A computer-driven micro-injector and a pH electrode directly immersed in the sample solution make it possible to obtain a titration curve in parallel to the optical measurement. All the data taken are stored on a magnetic disk for later retrieval. They can be processed and displayed in any required form. The helix-coil transitions of a polynucleotide caused by temperature and those of a polypeptide caused by pH, and the denaturation of proteins caused by guanidine hydrochloride, were studied by this measuring system. The continuous plotting of transition profiles and the correlation diagrams among different spectral dimensions has proved to be a good way of demonstrating the existence of different modes of transition.     

Estimation and correction of seed recovery bias from moist-soil cores

Scientists estimate seed abundances to calculate seasonal carrying capacities and assess wetland management actions for waterfowl and other wildlife using soil core samples. We evaluated recovery of known quantities of moist-soil seeds from whole and subsampled experimental core samples containing 12 seed taxa representing small, medium, and large size classes. We recovered 86.3% (SE = 1.8) of all seeds added to experimental cores; 8.3% (SE = 1.2) of seeds were destroyed during the sieving process and 5.4% (SE = 1.2) were not recovered by observers. Recovery rates varied by seed size, but not seed quantity or disproportionate ratios of seed-size classes. Overall seed recovery rates were similar between subsampled ( = 81.2%, SE = 3.6) and whole–processed core samples ( = 86.3%, SE = 1.8). We used recovery rates to generate size-specific, taxon-specific, and constant correction factors and applied each to actual core sample data. Size-specific correction factors increased seed mass estimates in the Mississippi Alluvial Valley ( = 10.1%, SE = 0.32), upper Midwest ( = 21.2%, SE = 0.61), and both regions combined ( = 15.7%, SE = 0.51) differently, as seed composition in core samples varied regionally. We suggest scientists consider using size-specific correction factors to account for seed recovery bias in core samples because these factors may be applied to a variety of taxa and produced similar mass estimates as taxon-specific correction factors. However, if data from core samples are unavailable at the resolution of seed size classes, we suggest increasing seed mass estimates by 16% to account for seed recovery bias. © 2011 The Wildlife Society.