Nonbleaching fluorescence of gold nanoparticles and its applications in cancer cell imaging

In this paper, we investigated the fluorescent properties of gold nanoparticles (GNPs) with several tens of nanometers by ensemble fluorescence spectrometry, fluorescence correlation spectroscopy (FCS), and fluorescence microscopy. We observed that GNPs synthesized by the citrate reduction of chloroauric acid possessed certain fluorescence, narrow full width at half-maximum (17 nm), and with an increase of particle sizes, the emission intensity showed a gradual increase while the emission wavelength remained almost constant (at 610 nm). Especially, the fluorescence of GNPs possessed the excellent behavior of antiphotobleaching under strong light illumination. Despite their low quantum yields, GNPs exhibited strong native fluorescence under relatively high excitation power. The fluorescence of GNPs could be characterized by fluorescence imaging and FCS at the single particle level. On the basis of this excellent antiphotobleaching of GNPs and easy photobleaching of cellular autofluorescence, we developed a new method for imaging of cells using GNPs as fluorescent probes. The principle of this method is that after cells stained with GNPs or GNPs bioconjugates are illuminated by strong light, the cellular autofluorescence are photobleached and the fluorescence of GNPs on cell membrane or inside cells can be collected for cell imaging. On the basis of this principle, we imaged living HeLa cells using GNPs as fluorescent probes and obtained good cell images by photobleaching of cellular autofluorescence. Furthermore, anti-EGFR/GNPs were successfully used as targeted probes for fluorescence imaging of cancer cells. Our preliminary results demonstrated that GNPs possessed excellent behaviors of antiphotobleaching and were good fluorescent probes in cell imaging. Our cellular imaging method described has potential applications in cancer diagnostics, studies, and immunoassays.     

Dynamic SERS imaging of cellular transport pathways with endocytosed gold nanoparticles

Dynamic SERS imaging inside a living cell is demonstrated with the use of a gold nanoparticle, which travels through the intracellular space to probe local molecular information over time. Simultaneous tracking of particle motion and SERS spectroscopy allows us to detect intracellular molecules at 65 nm spatial resolution and 50 ms temporal resolution, providing molecular maps of organelle transport and lisosomal accumulation. Multiplex spectral and trajectory imaging will enable imaging of specific dynamic biological functions such as membrane protein diffusion, nuclear entry, and rearrangement of cellular cytoskeleton.     

Lysosome‐Targeted Bioprobes for Sequential Cell Tracking from Macroscopic to Microscopic Scales

Longitudinal tracking of living cells is crucial to understanding the mechanism of action and toxicity of cell‐based therapeutics. To quantify the presence of administered cells in the host tissue without sacrifice of animals, labeling of the target cells with a nontoxic and stable contrast agent is a prerequisite. However, such long‐term live cell tracking is currently limited by the lack of fluorophores with steady optical and physicochemical properties in the near‐infrared (NIR) window. Herein, for the first time, the design of fixable cell‐tracking NIR fluorophores (CTNFs) with high optical properties, excellent cell permeation and retention, and high stability against chemical treatments is reported. Efficient cellular labeling and tracking of CTNFs using intraoperative optical fluorescence imaging by following the fate of NIR‐labeled cells from the time of injection into animals to ex vivo cellular analysis after resection of the target tissue is demonstrated. Due to the lipophilic cationicity and primary amine docking group, CTNF126 outperforms the other tested fluorophores with rapid diffusion into the cytoplasmic membrane and sequestration inside the lysosomes, which prevents cellular efflux and improves cellular retention. Thus, CTNF126 will be useful to track cells in living organisms for the mechanism of action at the single cell level.     

Single-particle tracking of hepatitis B virus-like vesicle entry into cells

HBsAg, the surface antigen of the hepatitis B virus (HBV), is used as a model to study the mechanisms and dynamics of a single-enveloped virus infecting living cells by imaging and tracking at the single-particle level. By monitoring the fluorescent indicator of HBsAg particles, it is found that HBsAg enters cells via a caveolin-mediated endocytic pathway. Tracking of individual HBsAg particles in living cells reveals the anomalously actin-dependent but not microtubule-dependent motility of the internalized HBsAg particle. The motility of HBsAg particles in living cells is also analyzed quantitatively. These results may settle the long-lasting debate of whether HBV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell, and how the virus moves in the cell.     

Chrominance to dimension: a real-time method for measuring the size of single gold nanoparticles

Noble metal nanoparticles have excellent optical and chemical properties and are widely used in optics, sensors, and biomedicines. The inherent characteristics of metal nanoparticles, particularly their size, play important roles in their applications. The ability to readily measure the size of single nanomaterials on-site is crucial to the rapid development of single-particle sensors. In this study, we developed a facile and real-time method for estimating the diameter of single gold nanoparticles (GNPs) that range from 35 to 110 nm in diameter; this technique uses the chrominance of the GNP's plasmon resonance scattering light that is captured by a dark-field microscope (DFM). The RGB (three primary colors, red, green, and blue) chrominance information from the dark-field image can be directly converted into the diameters of the GNPs using the relationship between the particle size and the scattering light peak wavelength; this conversion was carried out using Matlab program based on an RGB-To-Wavelength (RTW) process. This approach is more convenient, less time-consuming, and enables observation under arbitrary conditions compared to the scanning electron microscopy (SEM) technique. The differences between the diameters of the GNPs that were calculated using this method and those that were measured using SEM were less than 5 nm. The RTW method has also been applied in the monitoring of the refractive index of the media surrounding the GNPs, and their dynamic acting within cells in real-time.     

Fluorescent Polymer Nanoparticles for Cell Barcoding In Vitro and In Vivo

Fluorescent polymer nanoparticles for long‐term labeling and tracking of living cells with any desired color code are developed. They are built from biodegradable poly(lactic‐co‐glycolic acid) polymer loaded with cyanine dyes (DiO, DiI, and DiD) with the help of bulky fluorinated counterions, which minimize aggregation‐caused quenching. At the single particle level, these particles are ≈20‐fold brighter than quantum dots of similar color. Due to their identical 40 nm size and surface properties, these nanoparticles are endocytosed equally well by living cells. Mixing nanoparticles of three colors in different proportions generates a homogeneous RGB (red, green, and blue) barcode in cells, which is transmitted through many cell generations. Cell barcoding is validated on 7 cell lines (HeLa, KB, embryonic kidney (293T), Chinese hamster ovary, rat basophilic leucemia, U97, and D2A1), 13 color codes, and it enables simultaneous tracking of co‐cultured barcoded cell populations for >2 weeks. It is also applied to studying competition among drug‐treated cell populations. This technology enabled six‐color imaging in vivo for (1) tracking xenografted cancer cells and (2) monitoring morphogenesis after microinjection in zebrafish embryos. In addition to a robust method of multicolor cell labeling and tracking, this work suggests that multiple functions can be co‐localized inside cells by combining structurally close nanoparticles carrying different functions.     

Enzymatic activation of a peptide functionalised gold nanoparticle system for prodrug delivery

Gold nanoparticles (GNPs) with a monolayer of peptides were synthesized as a potential tumour activated cancer drug delivery system. The prodrug system was achieved by the attachment of two varying lengths of peptides to GNPs: An 18 amino acid peptide sequence encompassing a shorter fluorescent labelled (coumarin) six amino acid peptide sequence. The longer peptide chain included the sequence D-AFK that is selectively cleavable by the over-expression of proteases in the vicinity of cancer cells. The protease-mediated exposure of the coumarin was demonstrated by the incubation of peptide capped GNPs with adenocarcinomic human alveolar basal epithelial A549 cells and madin-darby bovine kidney epithelial cells. Confocal laser scanning microscopy studies revealed enhanced fluorescence emission intensities in the cancer cell line as compared to the intensity exhibited by the healthy cell line. This work suggests that GNPs functionalised with a cytotoxic agent or fluorophore encapsulated by longer peptide strands may find useful applications for development of GNPs with therapeutic or diagnostic studies.     

Detection of single photoluminescent diamond nanoparticles in cells and study of the internalization pathway

Diamond nanoparticles are promising photoluminescent probes for tracking intracellular processes, due to embedded, perfectly photostable color centers. In this work, the spontaneous internalization of such nanoparticles (diameter 25 nm) in HeLa cancer cells is investigated by confocal microscopy and time-resolved techniques. Nanoparticles are observed inside the cell cytoplasm at the single-particle and single-color-center level, assessed by time-correlation intensity measurements. Improvement of the nanoparticle signal-to-noise ratio inside the cell is achieved using a pulsed-excitation laser and time-resolved detection taking advantage of the long radiative lifetime of the color-center excited state as compared to cell autofluorescence. The internalization pathways are also investigated, with endosomal marking and colocalization analyses. The low colocalization ratio observed proves that nanodiamonds are not trapped in endosomes, a promising result in prospect of drug delivery by these nanoparticles. Low cytotoxicity of these nanoparticles in this cell line is also shown.     

Liposome delivery of quantum dots to the cytosol of live cells

An increasing number of studies have demonstrated the multiple advantages of using nanocrystals, such as Quantum dots, for biological imaging. Quantum dots functionalized with biomolecules on their surfaces were shown to be able to bind to specific extracellular targets via specific recognition and to be internalized inside the cells, thereby allowing the imaging of intracellular pathways. However, the use of Quantum dots for live tracking of intracellular molecules is relatively limited because of the difficulties encountered during the induction of Quantum dots across living cell membranes. In this study we show that cationic liposomes can deliver low concentrations of non-targeted Quantum dots into the cytosol of living cells via a lipid-mediated fusion with the cell membrane. The intracellular Quantum dots exhibit aggregation that appears dependent upon their concentration, but does not visibly affect cell viability. Our results point towards the use of cationic liposomes as an effective delivery system for targeted Quantum dots within the cell cytosol, which would facilitate live cell imaging of the labeled molecules.     

Enhanced single-particle brightness and photostability of semiconductor polymer dots by enzymatic oxygen scavenging system

Semiconductor polymer dots (Pdots) are emerging as an excellent fluorescent probe in biology and medicine. However, the photostability of Pdots can't meet the requirements of long term single-particle imaging and tracking applications. Here we describe the enhanced single-particle brightness and photostability of Pdots by using an efficient enzymatic oxygen scavenging system (OSS). Pdots with particle diameters of 21 nm and 43 nm (PFBT21 and PFBT43) were prepared by a nanoprecipitation method. Single-particle imaging and photobleaching were performed to investigate the effect of OSS on the per-particle brightness and photostability of Pdots. Our results indicate that the single-particle brightness of the PFBT21 Pdots in OSS was enhanced nearly two times as compare to the PFBT21 Pdots in water. The photobleaching percentages of PFBT21 and PFBT43 in OSS were determined to be 29% and 33%, respectively. These values are decreased by 2–3 times as compared to those of the same Pdots in water, indicating the significantly improved photostability of Pdots by OSS. This study provides a promising approach for enhancing photostability of Pdots in long term single-particle tracking.     

Tracking mesenchymal stem cells with iron oxide nanoparticle loaded poly(lactide-co-glycolide) microparticles

Monitoring the location, distribution and long-term engraftment of administered cells is critical for demonstrating the success of a cell therapy. Among available imaging-based cell tracking tools, magnetic resonance imaging (MRI) is advantageous due to its noninvasiveness, deep penetration, and high spatial resolution. While tracking cells in preclinical models via internalized MRI contrast agents (iron oxide nanoparticles, IO-NPs) is a widely used method, IO-NPs suffer from low iron content per particle, low uptake in nonphagocytotic cell types (e.g., mesenchymal stem cells, MSCs), weak negative contrast, and decreased MRI signal due to cell proliferation and cellular exocytosis. Herein, we demonstrate that internalization of IO-NP (10 nm) loaded biodegradable poly(lactide-co-glycolide) microparticles (IO/PLGA-MPs, 0.4-3 μm) in MSCs enhances MR parameters such as the r(2) relaxivity (5-fold), residence time inside the cells (3-fold) and R(2) signal (2-fold) compared to IO-NPs alone. Intriguingly, in vitro and in vivo experiments demonstrate that internalization of IO/PLGA-MPs in MSCs does not compromise inherent cell properties such as viability, proliferation, migration and their ability to home to sites of inflammation.     

Cancer‐Cell Targeting and Photoacoustic Therapy Using Carbon Nanotubes as “Bomb” Agents

A unique approach using the large photoacoustic effect of single‐walled carbon nanotubes (SWNTs) for targeting and selective destruction of cancer cells is demonstrated. SWNTs exhibit a large photoacoustic effect in suspension under the irradiation of a 1064‐nm Q‐switched millisecond pulsed laser and trigger a firecracker‐like explosion at the nanoscale. By using such an explosion, a photoacoustic agent is developed by functionalizing the SWNTs with folate acid (FA) that can selectively bind to cancer cells overexpressing folate receptor on the surface of the cell membrane and kill them through SWNT explosion inside the cells under the excitation of millisecond pulsed laser. The uptake pathway of folate‐conjugated SWNTs into cancer cells is investigated via fluorescence imaging and it is found that the FA‐SWNTs can enter into cancer cells selectively with a high targeting capability of 17–28. Under the treatment of 1064‐nm millisecond pulsed laser, 85% of cancer cells with SWNT uptake die within 20 s, while 90% of the normal cells remain alive due to the lack of SWNTs inside cells. Temperature changes during laser treatment are monitored and no temperature increases of more than ± 3 °C are observed. With this approach, the laser power used for cancer killing is reduced 150–1500 times and the therapy efficiency is improved. The death mechanism of cancer cells caused by the photoacoustic explosion of SWNTs is also studied and discussed in detail. These discoveries provide a new way to use the photoacoustic properties of SWNTs for therapeutic applications.     

Automated tracking of liquid velocities in a refractive index matched porous medium

Abstract

Particle tracking velocimetry is applied to flow inside a porous column at Reynolds number Re = 28. The column is composed of refractive‐index‐matched solid and liquid materials, allowing seeding particles to be tracked in a laser‐illuminated axial slice. To complement earlier results acquired for 7 mm spheres, we conduct new experiments with larger 12 mm spheres. By improving the image acquisition and analysis, we are able to process the new experiments using fully automated algorithms instead of manual tracking. As a result, greater vector yields, more accurate velocity data, and a more complete spatial coverage are achieved.     

Confinement of Dyes inside Boron Nitride Nanotubes: Photostable and Shifted Fluorescence down to the Near Infrared

Fluorescence is ubiquitous in life science and used in many fields of research ranging from ecology to medicine. Among the most common fluorogenic compounds, dyes are being exploited in bioimaging for their outstanding optical properties from UV down to the near IR (NIR). However, dye molecules are often toxic to living organisms and photodegradable, which limits the time window for in vivo experiments. Here, it is demonstrated that organic dye molecules are passivated and photostable when they are encapsulated inside a boron nitride nanotube (dyes@BNNT). The results show that the BNNTs drive an aggregation of the encapsulated dyes, which induces a redshifted fluorescence from visible to NIR-II. The fluorescence remains strong and stable, exempt of bleaching and blinking, over a time scale longer than that of free dyes by more than 10⁴. This passivation also reduces the toxicity of the dyes and induces exceptional chemical robustness, even in harsh conditions. These properties are highlighted in bioimaging where the dyes@BNNT nanohybrids are used as fluorescent nanoprobes for in vivo monitoring of Daphnia Pulex microorganisms and for diffusion tracking on human hepatoblastoma cells with two-photon imaging.     

一种数字荧光显微系统及其在单粒子追踪实验中的应用

为实现对活细胞表面受体的追踪,我们在制冷ＣＣＤ的基础上研制了一套高性能数字荧光显微系统。利用这套系统和经过我们改善的单粒子追踪（ＳＰＴ）技术,根据受体荧光图象呈二维高斯分布的特性,使每幅图象中受体位置的匹配精度达到了２５ｎｍ。经改善的ＳＰＴ技术不但可以精确测量受体的光强,而且还可以提供高时间分辨力。本系统和方法为单个活细胞显微图象的检测和分析奠定了较好基础。     

Coherent Imaging Through a Random Screen

Abstract

Recent work on imaging coherently illuminated objects through a time-varying random screen has demonstrated the possibility of recovering the Fourier modulus of the object spectrum. We suggest an approach which, although limited to restricted conditions, enables both the modulus and the phase of the object to be recovered from the time-averaged image spectrum.     

A PIID-DTBT based semi-conducting polymer dots with broad and strong optical absorption in the visible-light region: Highly effective contrast agents for multiscale and multi-spectral photoacoustic imaging

As a hybrid imaging technique, photoacoustic imaging (PAI) can provide multiscale morphological information of tissues, and the use of multi-spectral PAI (MSPAI) can recover the spatial distribution of chromophores of interest, such as hemoglobin within tissues. Herein, we developed a contrast agent that can very effectively combine multiscale PAI with MSPAI for a more comprehensive characterization of complex biological tissues. Specifically, we developed novel PIID-DTBT based semi-conducting polymer dots (Pdots) that show broad and strong optical absorption in the visible-light region (500–700 nm). The performances of gold nanoparticles (GNPs) and gold nanorods (GNRs), which have been verified as excellent photoacoustic contrast agents, were compared with that of the Pdots based on the multiscale PAI system. Both ex vivo and in vivo experiments demonstrated that the Pdots have better photoacoustic conversion efficiency at 532 nm than GNPs and showed similar photoacoustic performance with GNRs at 700 nm at the same mass concentration. Photostability and toxicity tests demonstrated that the Pdots are photostable and biocompatible. More importantly, an in vivo MSPAI experiment indicated that the Pdots have better photoacoustic performance than the blood and therefore the signals can be accurately extracted from the background of vascular-rich tissues. Our work demonstrates the great potential of Pdots as highly effective contrast agents for the precise localization of lesions relative to the blood vessels based on multiscale PAI and MSPAI.

Open image in new window

     

Sizing subcellular organelles and nanoparticles confined within aqueous droplets

This article describes two complementary techniques, single-particle tracking and correlation spectroscopy, for accurately sizing nanoparticles confined within picoliter volume aqueous droplets. Single-particle tracking works well with bright particles that can be continuously illuminated and imaged, and we demonstrated this approach for sizing single fluorescent beads. Fluorescence correlation spectroscopy detects small intensity bursts from particles or molecules diffusing through the confocal probe volume, which works well with dim and rapidly diffusing particles or molecules; we demonstrated FCS for sizing synaptic vesicles confined in aqueous droplets. In combination with recent advances in droplet manipulations and analysis, we anticipate this capability to size single nanoparticles and molecules in free solution will complement existing tools for probing cellular systems, subcellular organelles, and nanoparticles.     

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.