Label-free Cells Research Articles

A novel electrochemical biosensor based on polyadenine modified aptamer for label-free and ultrasensitive detection of human breast cancer cells

Simple, rapid, sensitive, and specific detection of cancer cells plays a pivotal role in the diagnosis and prognosis of cancer. A sandwich electrochemical biosensor was developed based on polyadenine (polydA)-aptamer modified gold electrode (GE) and polydA-aptamer functionalized gold nanoparticles/graphene oxide (AuNPs/GO) hybrid for the label-free and selective detection of breast cancer cells (MCF-7) via a differential pulse voltammetry (DPV) technique. Due to the intrinsic affinity between multiple consecutive adenines of polydA sequences and gold, polydA modified aptamer instead of thiol terminated aptamer was immobilized on the surface of GE and AuNPs/GO. The label-free MCF-7 cells could be recognized by polydA-aptamer and self-assembled onto the surface of GE. The polydA-aptamer functionalized AuNPs/GO hybrid could further bind to MCF-7 cells to form a sandwich sensing system. Characterization of the surface modified GE was carried out by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) using Fe(CN)63-/4- as a redox probe. Under the optimized experimental conditions, a detection limit of 8 cellsmL−1 (3σ/slope) was obtained for MCF-7 cells by the present electrochemical biosensor, along with a linear range of 10–105 cellsmL−1. By virtue of excellent sensitivity, specificity and repeatability, the present electrochemical biosensor provides a potential application in point-of-care cancer diagnosis.

Prevention of electron beam transmittance for biological cell imaging using electron beam excitation-assisted optical microscope

We demonstrated the high-spatial-resolution imaging of label-free biological cells using an electron beam excitation-assisted optical (EXA) microscope without irradiation damage by the electron beam. An EXA microscope can be used to observe a specimen with a nanometric light source excited in the \(\mathrm{Si}_{\mathrm{3}}\mathrm{N}_{\mathrm{4}}\) membrane by an electron beam. The incident electron beam penetrates the \(\mathrm{Si}_{\mathrm{3}}\mathrm{N}_{\mathrm{4}}\) membrane and damages the specimen. To suppress the irradiation damage of the specimen, we prevented the transmittance of the electron beam by coating the \(\mathrm{Si}_{\mathrm{3}}\mathrm{N}_{\mathrm{4}}\) membrane with a gold thin film. To obtain an electron beam transmittance through the \(\mathrm{Si}_{\mathrm{3}}\mathrm{N}_{\mathrm{4}}\) of 0%, a gold film of 15 nm thickness was required. By adding the gold layer, a label-free cellular structure was observed with 135-nm spatial resolution.

Scattering pulse of label free fine structure cells to determine the size scale of scattering structures.

Scattering pulse is sensitive to the morphology and components of each single label-free cell. The most direct detection result, label free cell's scattering pulse is studied in this paper as a novel trait to recognize large malignant cells from small normal cells. A set of intrinsic scattering pulse calculation method is figured out, which combines both hydraulic focusing theory and small particle's scattering principle. Based on the scattering detection angle ranges of widely used flow cytometry, the scattering pulses formed by cell scattering energy in forward scattering angle 2°-5° and side scattering angle 80°-110° are discussed. Combining the analysis of cell's illuminating light energy, the peak, area, and full width at half maximum (FWHM) of label free cells' scattering pulses for fine structure cells with diameter 1-20 μm are studied to extract the interrelations of scattering pulse's features and cell's morphology. The theoretical and experimental results show that cell's diameter and FWHM of its scattering pulse agree with approximate linear distribution; the peak and area of scattering pulse do not always increase with cell's diameter becoming larger, but when cell's diameter is less than about 16 μm the monotone increasing relation of scattering pulse peak or area with cell's diameter can be obtained. This relationship between the features of scattering pulse and cell's size is potentially a useful but very simple criterion to distinguishing malignant and normal cells by their sizes and morphologies in label free cells clinical examinations.

Plasmon-Enhanced Autofluorescence Imaging of Organelles in Label-Free Cells by Deep-Ultraviolet Excitation.

We demonstrate the observation of organelles in label-free cells on an aluminum thin film using deep-ultraviolet surface plasmon resonance (DUV-SPR). In particular, the Kretschmann configuration is used for the excitation of DUV-SPR. MC3T3-E1 cells are directly cultured on the aluminum thin film, and DUV-SPR leads to autofluorescence of in the label-free MC3T3-E1. We found that nucleic acid and mitochondria in these label-free MC3T3-E1 cells quite strongly emit the autofluorescence as a result of DUV-SPR. Yeast cells are also deposited on the aluminum thin film. Tryptophan and mitochondrial nicotinamide adenine dinucleotide (NADH) in the yeast cells are subsequently excited, and their autofluorescence is spectrally analyzed in the UV region. On the basis of these results, we conclude that DUV-SPR constitutes a promising technique for the acquisition of highly sensitive autofluorescence images of various organelles in the cells.

Label-free imaging immune cells and collagen in atherosclerosis with two-photon and second harmonic generation microscopy

Atherosclerosis has been recognized as a chronic inflammation disease, in which many types of cells participate in this process, including lymphocytes, macrophages, dendritic cells (DCs), mast cells, vascular smooth muscle cells (SMCs). Developments in imaging technology provide the capability to observe cellular and tissue components and their interactions. The knowledge of the functions of immune cells and their interactions with other cell and tissue components will facilitate our discovery of biomarkers in atherosclerosis and prediction of the risk factor of rupture-prone plaques. Nonlinear optical microscopy based on two-photon excited autofluorescence and second harmonic generation (SHG) were developed to image mast cells, SMCs and collagen in plaque ex vivo using endogenous optical signals. Mast cells were imaged with two-photon tryptophan autofluorescence, SMCs were imaged with two-photon NADH autofluorescence, and collagen were imaged with SHG. This development paves the way for further study of mast cell degranulation, and the effects of mast cell derived mediators such as induced synthesis and activation of matrix metalloproteinases (MMPs) which participate in the degradation of collagen.

Enhanced Enrichment and Purification of Blood-Derived T-Cells Using a Novel Hydrogel Technology

Introduction: The rapid growth of cell-based immunotherapy research in academic and industry laboratories has highlighted a clear need for an optimal method to isolate specific immune cell populations from heterogeneous samples with high viability, purity and recovery efficiency. This is a particular concern in the area of CAR T-cell immunotherapy research, since isolated cells must be both viable and free of any magnetic bead labels for clinical use (N Engl J Med 2014; 371:1507-17). Herein, we report a novel approach for isolating desired T-cell populations from heterogeneous samples, including peripheral and umbilical cord blood, using a biologically-friendly hydrogel technology (QuickGel™) with an interior magnetic bead carrier (MagCloudz™). MagCloudz™ have been engineered with an exterior streptavidin binding surface, which readily allows for target cell isolation using a biotinylated antibody directed against the cellular marker of interest.Methods: Target cells were isolated from human samples and analyzed for marker expression and viability as follows. CD3+ T-cells were first labeled with biotinylated antibody and then bound to the MagCloudz™ streptavidin during a short incubation step. These target CD3+ T-cells were then separated from the undesired cell populations using a magnetic stand. Any cells non-specifically adhered to the MagCloudz™ were removed during a wash step and then the CD3+ T-cells were released from the MagCloudz™ using a specially designed buffer, which dissolved the QuickGel™ and effectively de-coupled the T-cells from the magnetic bead carriers. The magnetic beads were then removed using the magnetic stand. CD3/CD45 expression on isolated T-cells were determined by flow cytometry. Viability of recovered cells was determined by 7-AAD and Annexin-V staining.Results: We demonstrate successful CD3+/CD45+ T-cell enrichment from both peripheral and umbilical cord vein blood samples. In peripheral blood, flow cytometry analysis indicated that CD3+/CD45+ T-cell populations were enriched 1.8-fold from 56% in the starting population to 98.3% CD3+/CD45+ in the isolated T-cell population, with recovery >80% and viability >90%. In umbilical cord blood, CD3+/CD45+ T-cells were enriched 4.8-fold from 17.5% in the starting population to 83.7% CD3+/CD45+ in the isolated T-cell population. Viability of the recovered T-cells was 96%, making them ideal for use in further functional studies (ongoing).Conclusions: These data demonstrate that the novel MagCloudz™ with QuickGel™ approach described here has the potential to drastically improve the enrichment, purity and viability of isolated of immune cell populations from whole blood. We expect that this approach will reduce the cost and time of bio-processing for immunotherapy research and provide a means to deliver magnetic label-free cells for clinical applications such as CAR T-Cell therapy (N Engl J Med 2014; 371:1507-17). DisclosuresBrehm:The Jackson Laboratory: Consultancy.

Separation of Microvesicles from Serological Samples Using Deterministic Lateral Displacement Effect

Label-free isolation and detection of extracellular vesicles are essential in many diagnostic and therapeutic approaches. Recently, there has been an interest in methods that avoid the use of biochemical labels, intrinsic biomarkers, or electrical polarizability to identify microvesicles (also referred to as microparticles) from serological samples. Here, we report a microfluidic device to separate circulating extracellular vesicles from serological samples using the deterministic lateral displacement principle. The device continuously fractionates label-free extracellular vesicles and cells according to size and membrane flexibility by displacing them perpendicularly to the fluid flow direction in a micro-fabricated array of posts. Experimental data and computational fluid dynamic simulations are presented to create a compelling argument that microvesicles from serological samples could be separated by deterministic lateral displacement arrays. Direct separation of different-sized micro- and nanospheres were demonstrated using a multi-stage separation strategy thus offering a potential route for novel cancer diagnostic approaches where microvesicles can be targeted and intercepted during cell-to-cell communication.

Abstract 4938: In vivo visualization using two-photon laser scanning microscopy of anti-c-MET antibody in peritoneal metastatic gastric cancer

Abstract Background: c-MET (hepatocyte growth factor receptor) gene is overexpressed in approximately 20% of gastric cancer. Onartuzumab, a MET-selective humanized one-armed monoclonal antibody, has been studied in metastatic Met-positive non-small cell lung cancer and expected to be introduced to MET-positive gastric cancer. Two-photon laser scanning microscopy (TPLSM) has revolutionized in vivo real-time imaging because of the benefits of higher resolution, increased tissue penetration, and reduced photodamage. We established a method of intravital imaging for intra-abdominal organs using TPLSM with an organ stabilizing system (Japanese Patent No.5268282). Aim: To visualize the binding of fluorescence labeled-antibody to its receptor of cancer cells in peritoneal metastatic xenografts of living mice using a TPLSM. Method: Human gastric cancer cell lines (NUGC4) were inoculated into the peritoneal cavity of green fluorescent protein (GFP) expressing nude mice to make macroscopic peritoneal metastases. In vitro study, the binding of Zenon Alexa Fluor 594 labeled anti-c-MET antibody to viable NUGC4 cells was imaged using fluorescence microscopy. In vivo study, the binding of fluorescence labeled-antibody to viable NUGC4 cells of peritoneal metastatic xenografts was imaged using a TPLSM. Result: Zenon Alexa Fluor 594 labeled anti-c-MET antibody (red) was bound to the surface of viable NUGC4 cells, and clearly visualized under a fluorescence microscopy. Peritoneal metastases with label-free NUGC4 cells (black) and murine stromal cells (green) were imaged under TPLSM in vivo real-time. Fluorescence labeled-antibody could be visualized within tumor vessels of peritoneal metastases after its intravenous administration. Thereafter, label-free NUGC4 cells were identified by the binding of fluorescence labeled- anti-c-MET antibody to c-MET of NUGC4 cells. Conclusion: We could visualize fluorescence labeled-antibody in peritoneal metastases of gastric cancer in living mice using a TPLSM. The binding of fluorescence labeled- anti-c-MET antibody to c-MET of viable NUGC4 cells was clearly imaged at high magnification and high resolution in vivo real-time. It is valuable to develop a method of in vivo optical pathology for therapeutic monoclonal antibodies using MPM on metastatic tumor xenografts. Citation Format: Shozo Ide, Koji Tanaka, Tadanobu Shimura, Masato Okigami, Satoru Kondo, Takahito Kitajima, Hiroki Imaoka, Yoshinaga Okugawa, Susumu Saigusa, Yuji Toiyama, Hiromi Yasuda, Masaki Ohi, Yasuhiro Inoue, Toshimitsu Araki, Keiichi Uchida, Yasuhiko Mohri, Masato Kusunoki. In vivo visualization using two-photon laser scanning microscopy of anti-c-MET antibody in peritoneal metastatic gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4938. doi:10.1158/1538-7445.AM2014-4938

Light scattering properties in spatial planes for label free cells with different internal structures

Label free cells properties of 2D scattering intensity spectra and 3D scattering images in different spatial fields and planes are studied according to three dimensional finite-difference time-domain simulation results. The correlation of intensity spectra and scattering images in different spatial regions are discussed with different cells nucleus size, nucleus off-center situations, small organelles existence and distribution. Compared the scattering information, the most sensitive spatial regions to cells structures changing are figured out which can be selected to improve label free cells distinguishing accuracy. The research results would be very important to fully discover cells spatial scattering features and provide more reliable sorting criterions for label free cells detection technologies.

Super-resolution differential interference contrast microscopy by structured illumination

We propose a structured illumination differential interference contrast (SI-DIC) microscopy, breaking the diffraction resolution limit of differential interference contrast (DIC) microscopy. SI-DIC extends the bandwidth of coherent transfer function of the DIC imaging system, thus the resolution is improved. With 0.8 numerical aperture condenser and objective, the reconstructed SI-DIC image of 53 nm polystyrene beads reveals lateral resolution of approximately 190 nm, doubling that of the conventional DIC image. We also demonstrate biological observations of label-free cells with improved spatial resolution. The SI-DIC microscopy can provide sub-diffraction resolution and high contrast images with marker-free specimens, and has the potential for achieving sub-diffraction resolution quantitative phase imaging.

Novel Label-Free Cells Structure Detection Method Study Based on Mie Scattering Principle

Cell health situation relates to its inter structures closely. Cell scattering measurement can be a non-invasive measurement method to obtain cells structure information. But normal scattering detection by original scattering spectrum can not identify cells inner structure changing, such as nucleus radii difference. Traditional scattering spectrum analysis method for identifying cells is to plot the forward scattering (FS) light intensity against side scattering (SS) light intensity. Overlapping phenomenon always occurs which leads to serious error or even mistakes in cells identification results. The Novel even scattering angle superposition algorithm and even incident angle superposition algorithm are put forward herein. In this way, the same kind of cells with different inner structures can be effectively distinguished. The rapid, convenient and label-free cells assorting and detecting can be therefore well accomplished, and these novel methods could be a kind of important diagnostic tool in cancer or other malignant cells diagnosis.

Cell analysis using a multiple internal reflection photonic lab-on-a-chip

Here we present a protocol for analyzing cell cultures using a photonic lab-on-a-chip (PhLoC). By using a broadband light source and a spectrometer, the spectrum of a given cell culture with an arbitrary population is acquired. The PhLoC can work in three different regimes: light scattering (using label-free cells), light scattering plus absorption (using stained cells) and, by subtraction of the two former regimes, absorption (without the scattering band). The acquisition time of the PhLoC is ∼30 ms. Hence, it can be used for rapid cell counting, dead/live ratio estimation or multiparametric measurements through the use of different dyes. The PhLoC, including microlenses, micromirrors and microfluidics, is simply fabricated in a single-mask process (by soft lithographic methods) using low-cost materials. Because of its low cost it can easily be implemented for point-of-care applications. From raw substrates to final results, this protocol can be completed in 29 h.

Controlled viable release of selectively captured label-free cells in microchannels.

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.

Flow focussing of particles and cells based on their intrinsic properties using a simple diamagnetic repulsion setup

The continuous flow focussing and manipulation of particles and cells are important factors in microfluidic applications for performing accurate and reproducible procedures downstream. Many particle focussing methods require complex setups or channel designs that can limit the process and its applications. Here, we present diamagnetic repulsion as a simple means of focussing objects in continuous flow, based only on their intrinsic properties without the requirement of any label. Diamagnetic polystyrene particles were suspended in a paramagnetic medium and pumped through a capillary between a pair of permanent magnets, whereupon the particles were repelled by each magnet into the central axis of the capillary, thus achieving focussing. By investigating this effect, we found that the focussing was greatly enhanced with (i) increased magnetic susceptibility of the medium, (ii) reduced flow rate of the suspension, (iii) increased particle size, and (iv) increased residence time in the magnetic field. Furthermore, we applied diamagnetic repulsion to the flow focussing of living, label-free HaCaT cells.

Fiber-laser-based photoacoustic microscopy and melanoma cell detection

For broad applications in biomedical research involving functional dynamics and clinical studies, a photoacoustic microscopy system should be compact, stable, and fast. In this work, we use a fiber laser as the photoacoustic irradiation source to meet these goals. The laser system measures 45×56×13 cm3. The stability of the laser is attributed to the intrinsic optical fiber-based light amplification and output coupling. Its 50-kHz pulse repetition rate enables fast scanning or extensive signal averaging. At the laser wavelength of 1064 nm, the photoacoustic microscope still has enough sensitivity to image small blood vessels while providing high optical absorption contrast between melanin and hemoglobin. Label-free melanoma cells in flowing bovine blood are imaged in vitro, yielding measurements of both cell size and flow speed.

Imaging label-free intracellular structures by localisation microscopy

Localisation microscopy methods allow to realize a light optical resolution far beyond the Abbe–Rayleigh limit of about 200 nm laterally and 600 nm axially. So far, this progress was achieved using labelling with appropriate fluorochromes and fluorescent proteins. Here, we describe for the first time that optical resolution of cellular structures in the λ/10 range (∼50 nm) can be achieved even in label-free cells. This was obtained using Spectral Precision Distance/Position Determination Microscopy (SPDM), a method based on the general principles of localisation microscopy. Besides a substantial resolution improvement of autofluorescent structures, SPDM revealed cellular objects which are not detectable under conventional fluorescence imaging conditions.