Daniel R. Gossett

Label-free cell separation and sorting in microfluidic systems

AbstractCell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. FigureA wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties

Hydrodynamic stretching of single cells for large population mechanical phenotyping

Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.

High-throughput single-microparticle imaging flow analyzer

Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system’s utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million.

Particle Focusing Mechanisms in Curving Confined Flows

Particles in finite-inertia confined channel flows are known to segregate and focus to equilibrium positions whose number corresponds with the fold of symmetry of the channels cross section. The addition of curvature into channels presumably modifies these equilibrium inertial focusing positions, because of the secondary flow induced in curved channels. Here, we identify the critical interaction of the secondary flow field with inertial lift forces to create complex sets of particle focusing positions that vary with the channel Reynolds number (Re(C)) and the inertial force ratio, which is a new dimensionless parameter that is based on the ratio of inertial lift to drag forces from the secondary flow. We use these results to identify microfluidic channel geometries to focus particles at rates an order of magnitude higher than previously shown (channel Reynolds number, Re(C) = 270) and develop design criteria for the focusing of potentially arbitrary-sized particles. In addition, our results indicate that channel curvature can lead to microfluidic designs with reduced fluidic resistance, useful for lower power inertial focusing or separation. These results will enable design of practical particle/cell separation, filtration, and focusing systems for critical applications in biomedicine and environmental cleanup.

Quantitative Diagnosis of Malignant Pleural Effusions by Single-Cell Mechanophenotyping

Single-cell biophysical properties were used for diagnosing malignant pleural effusions from patients. Cytometry Device Helps (De)form a Diagnosis Is it benign, or malignant? That is the main concern of cytopathologists as they screen cells in pleural effusions, taken from the lungs of patients suspected of having infections or cancer. This process is subjective and time-intensive and requires an expert’s eye. So, to quickly “prescreen” samples for malignancy (and follow-up), Tse et al. describe deformability cytometry (DC)—an approach that relies on microfluidic forces to diagnose pleural effusion samples as malignant, or not. The authors’ device accelerates effusion samples through two opposing microfluidic channels. At the channels’ four-way intersection, the cells are rapidly decelerated as they encounter the opposing flow, and then exit out the side channels. This leads to cell deformation, changing them from sphere-like shapes to pancakes. High-speed video of this intersection allowed Tse et al. to quantify cellular squishing: the more deformable the cell, the more malignant it is. The authors took 119 pleural effusion samples from patients with known clinical outcomes—negative for malignant cells (benign), acute inflammation, chronic/mixed inflammation, atypical cells, and malignant pleural effusions (MPEs)—to develop a diagnostic scoring system on a scale of 1 to 10, with 1 being benign. DC showed the best predictive abilities in two high-confidence regimes: 1 to 6 and 9 to 10. Scores of 7 and 8 were more difficult to diagnose, so these may be the types of samples where a cytopathologist’s initial input would be necessary. Importantly, the authors looked at samples from patients that were cytology-negative with concurrent malignancy, such as a tumor, but 6 months later were diagnosed with disseminated disease. Five of 10 patients with high-grade cancers that were cytology-negative at sample collection scored high using DC. This suggests that the DC tool could be used to screen early for MPE. Using deformability as a marker of disease will require additional validation in pleural effusion samples from patients with many different types of cancer. Nevertheless, owing to the ease of use and objective readout, with further clinical testing, DC should be useful as a quick screening tool to form an early diagnosis of MPEs. Biophysical characteristics of cells are attractive as potential diagnostic markers for cancer. Transformation of cell state or phenotype and the accompanying epigenetic, nuclear, and cytoplasmic modifications lead to measureable changes in cellular architecture. We recently introduced a technique called deformability cytometry (DC) that enables rapid mechanophenotyping of single cells in suspension at rates of 1000 cells/s—a throughput that is comparable to traditional flow cytometry. We applied this technique to diagnose malignant pleural effusions, in which disseminated tumor cells can be difficult to accurately identify by traditional cytology. An algorithmic diagnostic scoring system was developed on the basis of quantitative features of two-dimensional distributions of single-cell mechanophenotypes from 119 samples. The DC scoring system classified 63% of the samples into two high-confidence regimes with 100% positive predictive value or 100% negative predictive value, and achieved an area under the curve of 0.86. This performance is suitable for a prescreening role to focus cytopathologist analysis time on a smaller fraction of difficult samples. Diagnosis of samples that present a challenge to cytology was also improved. Samples labeled as “atypical cells,” which require additional time and follow-up, were classified in high-confidence regimes in 8 of 15 cases. Further, 10 of 17 cytology-negative samples corresponding to patients with concurrent cancer were correctly classified as malignant or negative, in agreement with 6-month outcomes. This study lays the groundwork for broader validation of label-free quantitative biophysical markers for clinical diagnoses of cancer and inflammation, which could help to reduce laboratory workload and improve clinical decision-making.

Three Dimensional, Sheathless, and High‐Throughput Microparticle Inertial Focusing Through Geometry‐Induced Secondary Flows

A novel inertial focusing platform creates a single-stream microparticle train in a single-focal plane without sheath fluids and external forces, all in a high-throughput manner. The proposed design consists of a low-aspect-ratio straight channel interspersed with a series of constrictions in height arranged orthogonally, making use of inertial focusing and geometry-induced secondary flows. Focusing efficiency as high as 99.77% is demonstrated with throughput as high as 36 000 particles s(-1) for a variety of different sized particles and cells.

Inertial Manipulation and Transfer of Microparticles Across Laminar Fluid Streams

A general strategy for controlling particle movement across streams would enable new capabilities in single-cell analysis, solid-phase reaction control, and biophysics research. Transferring cells across streams is difficult to achieve in a well-controlled manner, since it requires precise control of fluid flow along with external force fields or precisely manufactured mechanical structures. Herein a strategy is introduced for particle transfer based on passive inertial lift forces and shifts in the distribution of these forces for channels with shifting aspect ratios. Uniquely, use of the dominant wall-effect lift parallel to the particle rotation direction is explored and utilized to achieve controllable cross-stream motion. In this way, particles are positioned to migrate across laminar streams and enter a new solution without significant disturbance of the interface at rates exceeding 1000 particles per second and sub-millisecond transfer times. The capabilities of rapid inertial solution exchange (RInSE) for preparation of hematological samples and other cellular assays are demonstrated. Lastly, improvements to inline flow cytometry after RInSE of excess fluorescent dye and focusing for downstream analysis are characterized. The described approach is simply applied to manipulating cells and particles and quickly exposing them to or removing them from a reacting solution, with broader applications in control and analysis of low affinity interactions on cells or particles.

Hybrid Dispersion Laser Scanner

Laser scanning technology is one of the most integral parts of todays scientific research, manufacturing, defense, and biomedicine. In many applications, high-speed scanning capability is essential for scanning a large area in a short time and multi-dimensional sensing of moving objects and dynamical processes with fine temporal resolution. Unfortunately, conventional laser scanners are often too slow, resulting in limited precision and utility. Here we present a new type of laser scanner that offers ∼1,000 times higher scan rates than conventional state-of-the-art scanners. This method employs spatial dispersion of temporally stretched broadband optical pulses onto the target, enabling inertia-free laser scans at unprecedented scan rates of nearly 100 MHz at 800 nm. To show our scanners broad utility, we use it to demonstrate unique and previously difficult-to-achieve capabilities in imaging, surface vibrometry, and flow cytometry at a record 2D raster scan rate of more than 100 kHz with 27,000 resolvable points.

Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy

A confocal fluorescence microscopy scheme that maps the image to the radiofrequency spectrum by beating together two optical fields offers enhanced read-out speeds at kilohertz frame rates. It provides a new way for observing dynamic phenomena in cells.

Rapid inertial solution exchange for enrichment and flow cytometric detection of microvesicles

Exosomes, nanosized membrane-bound vesicles released by cells, play roles in cell signaling, immunology, virology, and oncology. Their study, however, has been hampered by difficulty in isolation and quantification due to their size and the complexity of biological samples. Conventional approaches to improved isolation require specialized equipment and extensive sample preparation time. Therefore, isolation and detection methods of exosomes will benefit biological and clinical studies. Here, we report a microfluidic platform for inline exosome isolation and fluorescent detection using inertial manipulation of antibody-coated exosome capture beads from biological fluids.