High-throughput lensfree imaging and characterization of a heterogeneous cell solution on a chip

A high-throughput on-chip imaging platform that can rapidly monitor and characterize various cell types within a heterogeneous solution over a depth-of-field of approximately 4 mm and a field-of-view of approximately 10 cm(2) is introduced. This powerful system can rapidly image/monitor multiple layers of cells, within a volume of approximately 4 mL all in parallel without the need for any lenses, microscope-objectives or any mechanical scanning. In this high-throughput lensless imaging scheme, the classical diffraction pattern (i.e., the shadow) of each micro-particle within the entire sample volume is detected in less than a second using an opto-electronic sensor chip. The acquired shadow image is then digitally processed using a custom developed "decision algorithm" to enable both the identification of the particle location in 3D and the characterization of each micro-particle type within the sample volume. Through experimental results, we show that different cell types (e.g., red blood cells, fibroblasts, etc.) or other micro-particles all exhibit uniquely different shadow patterns and therefore can be rapidly identified without any ambiguity using the developed decision algorithm, enabling high-throughput characterization of a heterogeneous solution. This lensfree on chip cell imaging platform shows a significant promise especially for medical diagnostic applications relevant to global health problems, where compact and cost-effective diagnostic tools are urgently needed in resource limited settings.     

Whole-body imaging of tumor cells by azaelectrocyclization: Visualization of metastasis dependence on glycan structure

Noninvasive imaging of cancer metastasis through the efficient cell labeling constitutes a major technological breakthrough for cancer research and patient monitoring post-surgery. In the current work, we expanded our cell surface labeling technique on the whole-body fluorescence imaging of tumor metastasis in BALB/c nude mice. Four kinds of human cancer cells (two cancer cell lines, MKN45 and HCT116, and their transfected versions expressing surface glycan-related genes, MKN45-GnT-V and HCT116-GMDS) were labeled by azaelectrocyclization with Hilyte Fluor 750 for 10 min and without affecting cell viability. Fluorescence-labeled cancer cells were injected into the abdominal cavities of BALB/c mice and whole-body scans were performed with an eXplore Optix device. In accordance with previous findings, the fluorescence imaging clearly showed that tumor metastasis was dependent upon the cell surface glycans: A larger polylactosamine structure or the loss of fucosylation on the cancer cell surfaces, respectively, enhanced the metastatic potential of the tumor cells. Our noninvasive technique provides the landmark opportunity for sensitively monitoring the dynamics of the cancer cells depending on their surface structures and/or the host environments, thus impacts on the cancer prognosis and the therapeutic applications.     

High-throughput RNAi screening by time-lapse imaging of live human cells

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.     

A continuous wave technique for the measurement of the elastic properties of cortical bone

A continuous wave technique is described for measuring the nine independent orthotropic elastic coefficients from a single cubic specimen. The side dimensions of this cubic specimen are on the order of 5 mm. Because of the small size of the specimen, the spatial resolution of material inhomogeneity using this technique is quite good. Although it is possible to apply this technique to any elastic material such as woods or metals, the elastic properties of human and canine cortical femora are presented here. The orthotropic elastic coefficients and the variation of these coefficients are presented as a function of anatomical position.     

Real-time fluorescence detection of multiple microscale cell culture analog devices in situ.

We investigated multiple microscale cell culture analog (microCCA) assays in situ with a high-throughput imaging system that provides quantitative, nondestructive, and real-time data on cell viability. Since samples do not move between measurements, captured images allow accurate time-course measurements of cell population response and tracking the fate of each cell type on a quantitative basis. The optical system was evaluated by measuring the short-term response to ethanol exposure and long-term growth of drug-resistant tumor cell lines with simultaneous samples. The optical system based on epi-fluorescent excitation consists of an LED and a CCD as well as discrete optical components for imaging a large number of cells simultaneously. HepG2/C3A and MESSA cell lines were cultured in two microCCA systems for continuous cell status monitoring in cell death experiments with ethanol and long-term cell growth. The experiment that tested ethanol uptake showed that ethanol immediately caused cell death. The system was applied to extracting dynamic constants in the uptake process. In the long-term cell growth experiment, growth of MESSA cells was followed by a stationary phase and eventual cell death attributed to nutrient and oxygen depletion and a change in the pH because of the accumulation of wastes by cell metabolism. HepG2/C3A cells were subject to contact inhibition and cell number did not change significantly over time. Issues related to long-term assays are also discussed. The quantitative results have been consistent with qualitative images and confirm the applicability of the portable optical system, and potential application to high-throughput analysis of cell-based assays to measure long-term dynamics.     

Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability

In this report, we describe a highly reproducible femtosecond near-infrared (NIR) laser-based nanoprocessing technique that can be used both for non-invasive intra-tissue nanodissection of plant cell walls as well as selective destruction of a single plastid or part thereof without compromising the viability of the cells. The ultra-precise intra-tissue nanoprocessing is achieved by the generation of high light intensity (10(12)W cm(-2)) by diffraction-limited focusing of the radiation of an NIR (lambda = 740 and 800 nm) femtosecond titanium-sapphire laser to a sub-femtolitre volume and subsequent highly localized instantaneous plasma formation. Following nanosurgery, electron microscopical analysis of the corresponding cellular target areas revealed clean non-staggering lesions across the cell wall with a cut width measuring less than 400 nm. To our knowledge, this is the smallest cut made non-invasively within a plant tissue. Further evidence, including two-photon imaging of chlorophyll fluorescence, revealed that a single target chloroplast or part thereof can be completely knocked out using intense ultra-fast NIR pulses without any visible deleterious effect on the adjacent plastids. The vitality of the cells after nanoprocessing has been ascertained by exclusion of propidium iodide from the cells as well as by the presence of cytoplasmic streaming. The potential applications of this technical advance include developmental biology applications, particularly studies addressing spatio-temporal control of ontogenetic events and cell-cell interactions, and gravitational biology applications.     

High-throughput single-cell manipulation in brain tissue

The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution.     

High-throughput and multiplexed regeneration buffer scouting for affinity-based interactions

Affinity-based analyses on biosensors depend partly on regeneration between measurements. Regeneration is performed with a buffer that efficiently breaks all interactions between ligand and analyte while maintaining the active binding site of the ligand. We demonstrated a regeneration buffer scouting using the combination of a continuous flow microspotter with a surface plasmon resonance imaging platform to simultaneously test 48 different regeneration buffers on a single biosensor. Optimal regeneration conditions are found within hours and consume little amounts of buffers, analyte, and ligand. This workflow can be applied to any ligand that is coupled through amine, thiol, or streptavidin immobilization.     

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite effective, this classical approach has certain limitations for miniaturization of the imaging platform to make it compatible with the advanced state of the art in microfluidics. In this report, we introduce experimental details of a lensless on-chip imaging concept termed LUCAS (Lensless Ultra-wide field-of-view Cell monitoring Array platform based on Shadow imaging) that does not require any microscope objectives or other bulky optical components to image a heterogeneous cell solution over an ultra-wide field of view that can span as large as ~18 cm². Moreover, unlike conventional microscopes, LUCAS can image a heterogeneous cell solution of interest over a depth-of-field of ~5 mm without the need for refocusing which corresponds to up to ~9 mL sample volume. This imaging platform records the shadows (i.e., lensless digital holograms) of each cell of interest within its field of view, and automated digital processing of these cell shadows can determine the type, the count and the relative positions of cells within the solution. Because it does not require any bulky optical components or mechanical scanning stages it offers a significantly miniaturized platform that at the same time reduces the cost, which is quite important for especially point of care diagnostic tools. Furthermore, the imaging throughput of this platform is orders of magnitude better than conventional optical microscopes, which could be exceedingly valuable for high-throughput cell-biology experiments.Download video file.^{(92M, mp4)}     

Cell monitoring with optical coherence tomography

Background aimsWe evaluated a commercially available instrument, OCTiCell (chromologic.com/octicell), for monitoring cell growth in suspended agitated bioreactors based on optical coherence tomography. OCTiCell is an in-line, completely non-invasive instrument that can operate on any suspended-cell bioreactor with a window or transparent wall. In traditional optical coherence tomography, the imaging beam is rastered over the sample to form a three-dimensional image. OCTiCell, instead uses a fixed imaging beam and takes advantage of the motion of the media to move the cells across the interrogating optical beam.ResultsWe found strong correlations between the non-invasive, non-contact, reagent-free OCTiCell measurements of cell concentration and viability and those obtained from the automated cell counter, and the XTT viability assay, which is a colorimetric assay for quantifying metabolic activity.ConclusionsThis novel cell monitoring method can adapt to different bioreactor form factors and could reduce the labor cost and contamination risks associated with cell growth monitoring.     

Drop-on-demand single cell isolation and total RNA analysis

Technologies that rapidly isolate viable single cells from heterogeneous solutions have significantly contributed to the field of medical genomics. Challenges remain both to enable efficient extraction, isolation and patterning of single cells from heterogeneous solutions as well as to keep them alive during the process due to a limited degree of control over single cell manipulation. Here, we present a microdroplet based method to isolate and pattern single cells from heterogeneous cell suspensions (10% target cell mixture), preserve viability of the extracted cells (97.0±0.8%), and obtain genomic information from isolated cells compared to the non-patterned controls. The cell encapsulation process is both experimentally and theoretically analyzed. Using the isolated cells, we identified 11 stem cell markers among 1000 genes and compare to the controls. This automated platform enabling high-throughput cell manipulation for subsequent genomic analysis employs fewer handling steps compared to existing methods.     

Automatic alignment and reconstruction of images for soft X-ray tomography

Soft X-ray tomography (SXT) is a powerful imaging technique that generates quantitative, 3D images of the structural organization of whole cells in a near-native state. SXT is also a high-throughput imaging technique. At the National Center for X-ray Tomography (NCXT), specimen preparation and image collection for tomographic reconstruction of a whole cell require only minutes. Aligning and reconstructing the data, however, take significantly longer. Here we describe a new component of the high throughput computational pipeline used for processing data at the NCXT. We have developed a new method for automatic alignment of projection images that does not require fiducial markers or manual interaction with the software. This method has been optimized for SXT data sets, which routinely involve full rotation of the specimen. This software gives users of the NCXT SXT instrument a new capability - virtually real-time initial 3D results during an imaging experiment, which can later be further refined. The new code, Automatic Reconstruction 3D (AREC3D), is also fast, reliable, and robust. The fundamental architecture of the code is also adaptable to high performance GPU processing, which enables significant improvements in speed and fidelity.     

Integrated optical molecular imaging system for four-dimensional real-time detection in living single cells

A novel, multifunctional optical imaging system was developed by integrating four-dimensional (4D) real-time confocal microscopy (RT-CM), multicolor total internal reflection microscopy (TIRFM), and Nomarski differential interference contrast (DIC) microscopy based on an epifluorescence microscope platform. A microcell incubator was combined with the imaging system for extended, real-time monitoring of living cells. The 4D images were generated by a combination of 3D images and multiple spatial or time images of a specimen, obtained at 10 nm intervals. Optical sectioning was accomplished with a z-motor, which obtained 4D information with sequential layered sections. The integrated imaging system showed excellent detection sensitivity at the single-molecule level and 3D-spatial resolution (20 nm x-y and 10 nm z-axis) without moving the cell sample. This could be a tool for obtaining crucial information needed to develop approaches for characterizing and understanding the dynamics of biomolecules and nanoparticles in individual living cells and molecular interactions at the single-molecule level.     

Fluorescence-based cell viability screening assays using water-soluble oxygen probes

A simple luminescence-based assay for screening the viability of mammalian cells is described, based on the monitoring of cell respiration by means of a phosphorescent water-soluble oxygen probe that responds to changes in the concentration of dissolved oxygen by changing its emission intensity and lifetime. The probe was added at low concentrations (0.3 microM to 0.5 nM) to each sample containing a culture of cells in the wells of a standard 96-well plate. Analysis of oxygen consumption was initiated by applying a layer of mineral oil on top of each sample followed by monitoring of the phosphorescent signal on a prompt or time-resolved fluorescence plate reader. Rates of oxygen uptake could be determined on the basis of kinetic changes of the phosphorescence (initial slopes) and correlated with cell numbers (10(5) to 10(7) cells/mL for FL5.12 lymphoblastic cell line), cell viability, or drug/effector action using appropriate control samples. The assay is cell noninvasive, more simple, robust, and cost-effective than existing microplate-based cell viability assays; is compatible with existing instrumentation; and allows for high-throughput analysis of cell viability.     

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based β-lactam probes

Development of protein labeling techniques with small molecules is enthralling because this method brings promises for triumph over the limitations of fluorescent proteins in live cell imaging. This technology deals with the functionalization of proteins with small molecules and is anticipated to facilitate the expansion of various protein assay methods. A new straightforward aggregation and elimination-based technique for a protein labeling system has been developed with a versatile emissive range of fluorophores. These fluorophores have been applied to show their efficiency for protein labeling by exploiting the same basic principle. A genetically modified version of class A type β-lactamase has been used as the tag protein (BL-tag). The strength of the aggregation interaction between a fluorophore and a quencher plays a governing role in the elimination step of the quencher from the probes, which ultimately controls the swiftness of the protein labeling strategy. Modulation in the elimination process can be accomplished by the variation in the nature of the fluorophore. This diversity facilitates the study of the competitive binding order among the synthesized probes toward the BL-tag labeling method. An aggregation and elimination-based BL-tag technique has been explored to develop an order of color labeling from the equimolar mixture of the labeling probe in solutions. The qualitative and quantitative determination of ordering within the probes toward labeling studies has been executed through SDS-PAGE and time-dependent fluorescence intensity enhancement measurements, respectively. The desirable multiple-wavelength fluorescence labeling probes for the BL-tag technology have been developed and demonstrate broad applicability of this labeling technology to live cell imaging with coumarin and fluorescein derivatives by using confocal microscopy.     

Developmental imaging: Insights into the avian embryo

The study of embryonic events using different animal model systems is crucial for gaining insights into human development and birth defects. Biological imaging plays a major role in this effort by providing a spatiotemporal framework to link complex cell movements with molecular data. However, depending on the age of the embryo and the location of a morphogenetic event, visualization often requires the design of novel culture and imaging techniques. One of the primary model systems for biological imaging is the avian embryo, due to its accessibility to manipulation, relatively two-dimensional morphogenesis early on, and viability when grown in culture. Significant work in avian embryo culture and cell labeling, together with advances in imaging technology, now make it possible to monitor many developmental events within the period from egg laying to hatching. Here, we present the latest in avian developmental imaging, focusing on cell labeling, embryo culture, and imaging technologies.     

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.     

Novel approach for the prediction of cell densities and viability in standardized translucent cell culture biochips with near infrared spectroscopy

Near infrared spectroscopy is a rapid and nondestructive method for compositional analysis of biological material. The technology is widely used within bioreactors and possesses potential as a standardized method for quality control in miniaturized microfluidic cell culture systems. Here, we established a method for quantification of cell density and viability of adherent HepaRG cells cultured in a translucent, miniaturized cell culture biochip. The newly developed statistical models for interpretation of near infrared spectroscopy from biochips are the basis for a novel method of fast, continuous, and contact‐free analysis of cell viability and real‐time monitoring of cell growth. The technique thus paves the way for a robust and reliable high‐throughput analysis of biochip‐embedded cell cultures.     

The in vivo reproductive potential of density separated cells

Murine ascites cells (L1210, L5178Y, Ehrlich ascites) were labelled with ¹³¹I-iododeoxyuridine and subjected to buoyant density centrifugation on a continuous, linear Ficoll gradient. Cell losses sustained during density centrifugation were evaluated by recording the amount of ¹³¹I recovered in the final cell fractions. The viability and proliferative capacity of the density separated cells were tested by monitoring the rate of ¹³¹I excretion following inoculation of the recovered cells into new, non-radioactive hosts.Density separation in Ficoll appeared to cause few, if any, adverse effects. Cell recovery under properly regulated experimental conditions was virtually complete (97% or higher). The reproductive potential of density-separated cells was identical to that of control cells. However, considerable cell mortality could be induced by permitting cellular aggregation in medium free of antiagglutinin or by exposure of excessive quantities of cells to a density gradient.Viability indices obtained with trypan blue proved unsuitable for predicting long-term survival. In some experiments the trypan blue data provided a 90–100% viability reading when in fact the entire cell population had been inactivated by irradiation or heat incubation. Since the trypan blue test also did not reveal the full extent of mortality among aggregated cells or cells recovered from overloaded gradients, it was concluded that the dye exclusion test, in spite of its utility for monitoring immediate cell death and membrane destruction, was of limited value for evaluating the reproductive potential of mammalian cells.     

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.