Single particle tracking of fluorescent nanodiamonds in cells and organisms

Ever since the discovery of fullerenes in 1985, nanocarbon has demonstrated a wide range of applications in various areas of science and engineering. Compared with metal, oxide, and semiconductor nanoparticles, the carbon-based nanomaterials have distinct advantages in both biotechnological and biomedical applications due to their inherent biocompatibility. Fluorescent nanodiamond (FND) joined the nanocarbon family in 2005. It was initially developed as a contrast agent for bioimaging because it can emit bright red photoluminescence from negatively charged nitrogen-vacancy centers built in the diamond matrix. A notable application of this technology is to study the cytoplasmic dynamics of living cells by tracking single bioconjugated FNDs in intracellular medium. This article provides a critical review on recent advances and developments of such single particle tracking (SPT) research. It summarizes SPT and related studies of FNDs in cells (such as cancer cell lines) and organisms (including zebrafish embryos, fruit fly embryos, whole nematodes, and mice) using assorted imaging techniques.     

Mass production and dynamic imaging of fluorescent nanodiamonds

Fluorescent nanodiamond is a new nanomaterial that possesses several useful properties, including good biocompatibility, excellent photostability and facile surface functionalizability. Moreover, when excited by a laser, defect centres within the nanodiamond emit photons that are capable of penetrating tissue, making them well suited for biological imaging applications. Here, we show that bright fluorescent nanodiamonds can be produced in large quantities by irradiating synthetic diamond nanocrystallites with helium ions. The fluorescence is sufficiently bright and stable to allow three-dimensional tracking of a single particle within the cell by means of either one- or two-photon-excited fluorescence microscopy. The excellent photophysical characteristics are maintained for particles as small as 25 nm, suggesting that fluorescent nanodiamond is an ideal probe for long-term tracking and imaging in vivo, with good temporal and spatial resolution.     

Real-time monitoring of intracellular mRNA hybridization inside single living cells

A molecular beacon, an oligonucleotide probe with inherent signal transduction mechanisms, is an optimal tool for visualizing real-time mRNA hybridization in single living cells. Each molecular beacon (MB) consists of a single-stranded DNA molecule in a stem-loop conformation with a fluorophore linked to the 5' end and a quencher at the 3' end. In this study, we demonstrate real-time monitoring of mRNA-DNA hybridization inside living cells using molecular beacons. A MB specific for beta-actin mRNA has been designed and synthesized. After microinjection into the cytoplasm of single living kangaroo rat kidney cells (PtK2 cells), the MB hybridizes with beta-actin mRNA as shown by fluorescence measurements over time. Hybridization dynamics have been followed. Strict control experiments have been carried out to confirm that the fluorescence signal increase is indeed due to the hybridization of mRNA inside single living cells. Variation in the MB/mRNA hybridization fluorescent signal has been observed for different PtK2 cells, which indicates the amount of mRNA in different cells is different. We have also monitored the beta-1 andrenergic receptor mRNA inside the PtK2 cells. These studies demonstrate the feasibility of using MBs and the ultrasensitivity achieved in our fluorescence imaging system for real-time detection of mRNA hybridization and for the visualization of oligonucleotide/mRNA interactions inside single living cells.     

Particle tracking microrheology of cancer cells in living subjects

Cumulative evidence shows that microenvironmental conditions play a significant role in the regulation of cell functions, and how cells respond to these conditions are of central importance to regenerative medicine and cancer cell response to therapeutics. Here, we develop a new method to examine cell mechanical properties by analyzing the motion of nanoparticles in living in mice, combining particle tracking with intravital microscopy. This method directly examines the mechanical response of breast carcinoma cells and normal breast epithelial cells under intravital microenvironments. Our results show both carcinoma and normal cells display significantly reduced compliance (less deformability) in vivo compared to the same cells cultured in 2D, in both sparse and confluent conditions. While the compliance of the normal cells remains steady over time, the compliance of carcinoma cells decreases further as they form tumor-like architectures. Integrating the cancer cells into spheroids embedded in 3D collagen matrices in part redirected the mechanical response to a state closer to the in vivo setting. Overall, our study demonstrates that the microenvironment is a crucial regulator of cell mechanics and the intravital particle tracking method can provide novel insights into the role of cell mechanics in vivo.     

Subcellular tracking of drug release from carbon nanotube vehicles in living cells

The direct observation of drug release from carbon nanotube vehicles in living cells is realized through a unique two-dye labeling approach. Single-walled carbon nanotubes (SWNTs) are firstly marked with fluorescein isothiocyanate (FITC) to track their location and movement inside the cell. Then a fluorescent anticancer drug doxorubicin (DOX) is attached by means of π-stacking onto SWNTs. Delivered by SWNTs into cells, DOX will detach from the vehicle in an acidic environment due to the pH-dependent π-π stacking interaction between DOX and SWNTs. From observation of the two different kinds of fluorescence (green and red) that respectively represent the carrier SWNTs and drug DOX, the process of drug release inside the living cell can be monitored under a confocal microscope. Results show that the drug DOX detaches from SWNTs inside the lysosomes to yield free molecules and escape into the cytoplasm and finally into the nucleus, while the vehicle SWNTs are trapped inside the lysosomes, without entering the nucleus. The current observations confirm previously proposed mechanisms for drug/DOX release inside cells. The experimental establishment of drug-release mechanisms in living cells here might provide important insights for future design of new drug-delivery and release systems.     

Real-time measurements of dissolved oxygen inside live cells by organically modified silicate fluorescent nanosensors

Optical PEBBLE (probes encapsulated by biologically localized embedding) nanosensors have been developed for dissolved oxygen using organically modified silicate (ormosil) nanoparticles as a matrix. The ormosil nanoparticles are prepared via a sol-gel-based process, which includes the formation of core particles with phenyltrimethoxysilane as a precursor followed by the formation of a coating layer with methyltrimethoxysilane as a precursor. The average diameter of the resultant particles is 120 nm. These sensors incorporate the oxygen-sensitive platinum porphyrin dye as an indicator and an oxygen-insensitive dye as a reference for ratiometric intensity measurement. Two pairs of indicator dye and reference dye, respectively, platinum(II) octaethylporphine and 3,3'-dioctadecyloxacarbocyanine perchlorate, and platinum(II) octaethylporphine ketone and octaethylporphine, were used. The sensors have excellent sensitivity with an overall quenching response of 97%, as well as excellent linearity of the Stern-Volmer plot (r(2) = 0.999) over the whole range of dissolved oxygen concentrations (0-43 ppm). In vitro intracellular changes of dissolved oxygen due to cell respiration were monitored, with gene gun injected PEBBLEs, in rat C6 glioma cells. A significant change was observed with a fluorescence ratio increase of up to 500% after 1 h, for nine different sets of cells, which corresponds to a 90% reduction in terms of dissolved oxygen concentration. These results clearly show the validity of the delivery method for intracellular studies of PEBBLE sensors, as well as the high sensitivity, which is needed to achieve real-time measurements of intracellular dissolved oxygen concentration.     

Visualization of nonengineered single mRNAs in living cells using genetically encoded fluorescent probes

Single mRNA imaging in live cells is a useful technique to elucidate its precise localization and dynamics. We developed a method for visualizing endogenous mRNAs in living cells with single molecule sensitivity using genetically encoded probes. An RNA-binding protein of human PUMILIO1 (PUM-HD) was used for recognizing base sequences of a target mRNA, β-actin mRNA. Two PUM-HDs were modified by amino acid mutations to bind specifically to tandem 8-base sequences of the target mRNA. Because each PUM-HD was connected with amino- and carboxyl-terminal fragments of enhanced green fluorescent protein (EGFP), the probes emit fluorescence by reconstitution of EGFP fragments upon binding to β-actin mRNAs. The EGFP reconstituted on the mRNAs was monitored with a total internal reflection fluorescence microscope. Results show that each fluorescent spot in live cells represented a single β-actin mRNA and that distinct spatial and temporal movement of the individual β-actin mRNAs was visualized. We also estimated the average velocity of the movement of the single mRNAs along microtubules in live cells. This method is widely applicable to tracking various mRNAs of interest in the native state of living cells with single-mRNA sensitivity.     

Fingerprint orientation alignment and similarity measurement

Abstract

In this paper, an algorithm for fingerprint orientation alignment is proposed in which a fingerprint is aligned to a particular direction according to its ridge flows. The algorithm works equally well for different types of fingerprints, including the type with no reference points. Aligned fingerprint images provide a lot of useful information for various applications. To demonstrate the use of such information, a fingerprint similarity measurement system is developed in which fingerprints are expressed by vectors with different elements derived from aligned images. When given two fingerprints, this system can help determine which of the two is more similar to a third fingerprint and which one is less similar by calculating distances among vectors. The alignment algorithm has been tested on 165 images in the NIST-4 fingerprint database. By rotating a test image in different degrees, 20 images are created. The alignment precision is calculated by an equivalent Euclidean norm, E(I), of the 20 rotated images to the original image. A smaller E(I) indicates a higher accuracy of the algorithm. The experimental results show that the average E(I) of these 165 samples is 1.234, and the spread of these 165 E(I)s is 1.1.     

Flower-shaped gold nanoparticles: synthesis, characterization and their application as SERS-active tags inside living cells

The detection of Raman signals inside living cells is a topic of great interest in the study of cell biology mechanisms and for diagnostic and therapeutic applications. This work presents the synthesis and characterization of flower-shaped gold nanoparticles and demonstrates their applicability as SERS-active tags for cellular spectral detection. The particles were synthesized by a facile, rapid new route that uses ascorbic acid as a reducing agent of gold salt. Two triarylmethane dyes which are widely used as biological stains, namely malachite green oxalate and basic fuchsin, were used as Raman-active molecules and the polymer mPEG-SH as capping material. The as-prepared SERS-active nanoparticles were tested on a human retinal pigment epithelial cell line and found to present a low level of cytotoxicity and high chemical stability together with SERS sensitivity down to picomolar particle concentrations.     

Quantum theory of preparation and measurement

The conventional postulate for the probabilistic interpretation of quantum mechanics is asymmetric in preparation and measurement, making retrodiction reliant on inference by use of Bayes' theorem. Here, a more fundamental symmetric postulate is presented, from which both predictive and retrodictive probabilities emerge immediately, even where measurement devices more general than those usually considered are involved. It is shown that the new postulate is perfectly consistent with the conventional postulate.     

A genetically encoded fluorescent indicator capable of discriminating estrogen agonists from antagonists in living cells

A genetically encoded fluorescent indicator was developed for the detection and characterization of estrogen agonists and antagonists. Two different color mutants of green fluorescent protein were joined by a tandem fusion domain composed of LXXLL (L = leucine, X = any amino acid) motif from the nuclear receptor-box II of steroid receptor coactivator 1, a flexible linker sequence, and the estrogen receptor alpha ligand binding domain (ERalpha LBD). Monitoring real-time ligand-induced conformational change in the ERalpha LBD to recruit the LXXLL motif allowed screening of natural and synthetic estrogens in single living cells using fluorescence resonance energy-transfer technique. The indicator was named SCCoR (single cell-coactivator recruitment). The high sensitivity of the present indicator made it possible to distinguish between estrogen strong and weak agonists in a dose-dependent fashion, immediately after adding ligand to live cells. Discrimination of agonists from antagonists was efficiently achieved using the present study. The approach described here can be applied to develop biosensors for other hormone receptors as well.     

Quantum analysis of measurement results

It is shown that a stochastic matrix operator (quantum measurement matrix) may be considered in some cases as a density matrix. Results of measurements in the form of figure numbers, interconnected with a matrix of quantum measurements, are similar to quantum observable quantities determined in quantum statistical theory. __________ Translated from Izmeritel’naya Tekhnika, No. 12, pp. 3–8, December, 2006.     

Highly sensitive fluorescent analysis of dynamic glycan expression on living cells using glyconanoparticles and functionalized quantum dots

A double signal amplification strategy was designed for highly sensitive and selective in situ monitoring of carbohydrate on living cells. The double signal amplification included the multiplex sandwich binding of functionalized quantum dots (QDs) to both glycan groups on the cell surface and glyconanoparticles and a cadmium cation sensitized fluorescence emission of Rhod-5N. Using the sialic acid-phenylboronic acid recognition system as a model, the 3-aminophenylboronic acid functionalized QDs (APBA-QDs) were synthesized by covalently binding APBA to mercaptopropionic acid capped CdS QDs, and the glyconanoparticles, polysialic acid stabilized gold nanoparticles (PSA-AuNPs), were prepared by a one-pot procedure. The APBA-QDs first recognized the sialic acid (SA) groups on BGC-823 human gastric carcinoma (BGC) cells and then the PSA on AuNPs, which were further used to bind more APBA-QDs on the cell surface for signal amplification. After the bound QDs were dissolved to release the Cd(2+), a Cd(2+)-sensitized fluorescence method was developed for the detection of BGC cells in a linear range from 5.0 × 10(2) to 1.0 × 10(7) cells mL(-1) with a limit of detection down to 210 cells mL(-1) (8 cells in 40 μL of solution) and the dynamic monitoring of SA expression variation on the cell surface. The monitoring result was identical with that from flow cytometric analysis. This approach showed high specificity and acceptable reproducibility. This strategy provided a promising platform for highly sensitive cytosensing and cytobiologic study.     

Influence of hydrogen bonds on the colloidal and fluorescent properties of detonation nanodiamonds in water,methanol and ethanol

This paper presents the results of research of influence of hydrogen bonds with different strength on fluorescence and colloidal properties of detonation nanodiamonds with surface carboxylic groups in the solvents. It is established that the colloidal properties of detonation nanodiamonds are almost independent on hydrogen bonds strength in water, methanol and ethanol. The fluorescent properties of detonation nanodiamonds are dependent on the type of solvent: the more intensive fluorescent properties correspond to weaker hydrogen bonds in solvents.     

Thermo/moisture responsive shape-memory polymer for possible surgery/operation inside living cells in future

The recoverable strain of shape-memory polymers (SMPs) is an order higher than their metal counterpart, i.e., shape-memory alloys. The recent finding of the influence of moisture on the glass transition temperature of a polyurethane SMP, which is traditionally known for its thermo-responsive nature, enables us to realize not only water-driven for shape recovery, but also recovery following a pre-determined sequence. Utilizing these features of this SMP, we propose the concept of delivering a tiny device which is made of this material into a pore through a very small hole. The feasibility of the concept is demonstrated experimentally. This concept could eventually be used to deliver a micro/nano device into living cells for surgery or operation inside of them.     

Quantum dots with phenylboronic acid tags for specific labeling of sialic acids on living cells

Sialic acids with a nine-carbon backbone are commonly found at the terminal position of the glycans structures on cell membranes. The unique distribution and ubiquitous existence of sialic acid on the cell membrane make them important mediators in various biological and pathological processes. We report a new class of imaging probes based on semiconductor quantum dots with small molecular phenylboronic acid tags for highly specific and efficient labeling of sialic acid on living cells. Our results have shown that the use of these probes enables one-step labeling and continuous tracking of the cell surface sialic acid moieties without any pretreatment of living cells. The one-step procedure with fast binding kinetics and the biocompatibility of these probes make it an ideal noninvasive technology for living cell imaging. We also find that the labeled sialic acids undergo quick internalization shortly after surface binding via endocytosis and eventually distribute in the perinuclear region. This distribution pattern is consistent with the notion that sialylated glycoproteins are populated on cell membranes and recycled through the vesicular exocytotic and endocytic pathways. The superior photostability and brightness of quantum dots enable quantitative analysis of the diffusion dynamics of sialic acids, which has been a significant challenge for glycan imaging.     

新型碟式太阳能跟踪平台设计和姿态工作空间分析

传统的碟式太阳能热发电系统采用回转支承和螺旋升降机来实现太阳方位角和高度角的跟踪,不足之处在于无法自动调整聚光器镜面单元的位姿以应对外界扰动。采用并联机构来实现太阳跟踪,不仅具有刚度高、跟踪误差小等内在优势,还可以自动调整聚光器镜面单元位姿。若采用传统球铰,由于其物理限制,跟踪机构的偏转能力不能精确满足太阳跟踪角度范围大的技术要求。针对上述问题,采用新型被动球铰,设计了一种基于3-RPS并联机构的新型碟式太阳能跟踪平台,并计算了其跟踪角度的范围;同时,通过建立通用3-RPS并联机构的逆运动学方程,结合球坐标搜索法计算了3-RPS并联跟踪机构的姿态工作空间,并结合结构参数对其姿态工作空间的影响规律,确定了各结构参数的最优值。结果表明：当新型碟式太阳能跟踪平台选用新型被动球铰,动、定平台形状为等腰三角形,转动副轴线共面且呈三角形布置,动、定平台的半径比为2以及支链长度为动平台半径的2倍时,其结构符合设计要求且调整后的姿态工作空间满足太阳跟踪所需的角度范围。研究结果可为后续的太阳跟踪平台结构设计和参数优化提供参考。     

Genetically encoded fluorescent indicators to visualize protein phosphorylation by extracellular signal-regulated kinase in single living cells

Extracellular signal-regulated kinase (ERK) is a serine/threonine protein kinase that regulates a wide variety of cell functions, such as cell growth and differentiation. To study the spatiotemporal dynamics of protein phosphor-ylation by activated ERK in living cells, we have developed genetically encoded fluorescent indicators for ERK. The present indicators are based on fluorescence resonance energy transfer (FRET) between two green fluorescent protein mutants. Phosphorylation of the indicators by activated ERK changes the FRET efficiency due to their conformational alterations. We visualized the cytosolic and nuclear activity of ERK using the present indicators. We thus found that the activation duration of ERK is considerably different between the cytosol and nucleus in living cells. The subcellular difference in the ERK activity may be fundamental to the regulation of cell functions by ERK. The present fluorescent indicators provide a powerful tool to reveal the spatiotemporal dynamics of protein phosphorylation by ERK in single living cells.