Single-cell correlations of mRNA and protein content in a human monocytic cell line after LPS stimulation.

The heterogeneity of mRNA and protein expression at the single-cell level can reveal fundamental information about cellular response to external stimuli, including the sensitivity, timing, and regulatory interactions of genes. Here we describe a fully automated system to digitally count the intron, mRNA, and protein content of up to five genes of interest simultaneously in single-cells. Full system automation of 3D microscope scans and custom image analysis routines allows hundreds of individual cells to be automatically segmented and the mRNA-protein content to be digitally counted. Single-molecule intron and mRNA content is measured by single-molecule fluorescence in-situ hybridization (smFISH), while protein content is quantified though the use of antibody probes. To mimic immune response to bacterial infection, human monocytic leukemia cells (THP-1) were stimulated with lipopolysaccharide (LPS), and the expression of two inflammatory genes, IL1ß (interleukin 1ß) and TNF-α (tumor necrosis factor α), were simultaneously quantified by monitoring the intron, mRNA, and protein levels over time. The simultaneous labeling of cellular content allowed for a series of correlations at the single-cell level to be explored, both in the progressive maturation of a single gene (intron-mRNA-protein) and comparative analysis between the two immune response genes. In the absence of LPS stimulation, mRNA expression of IL1ß and TNF-α were uncorrelated. Following LPS stimulation, mRNA expression of the two genes became more correlated, consistent with a model in which IL1ß and TNF-α upregulation occurs in parallel through independent mechanistic pathways. This smFISH methodology can be applied to different complex biological systems to provide valuable insight into highly dynamic gene mechanisms that determine cell plasticity and heterogeneity of cellular response.

Resonance control of acoustic focusing systems through an environmental reference table and impedance spectroscopy.

Acoustic standing waves can precisely focus flowing particles or cells into tightly positioned streams for interrogation or downstream separations. The efficiency of an acoustic standing wave device is dependent upon operating at a resonance frequency. Small changes in a system's temperature and sample salinity can shift the device's resonance condition, leading to poor focusing. Practical implementation of an acoustic standing wave system requires an automated resonance control system to adjust the standing wave frequency in response to environmental changes. Here we have developed a rigorous approach for quantifying the optimal acoustic focusing frequency at any given environmental condition. We have demonstrated our approach across a wide range of temperature and salinity conditions to provide a robust characterization of how the optimal acoustic focusing resonance frequency shifts across these conditions. To generalize these results, two microfluidic bulk acoustic standing wave systems (a steel capillary and an etched silicon wafer) were examined. Models of these temperature and salinity effects suggest that it is the speed of sound within the liquid sample that dominates the resonance frequency shift. Using these results, a simple reference table can be generated to predict the optimal resonance condition as a function of temperature and salinity. Additionally, we show that there is a local impedance minimum associated with the optimal system resonance. The integration of the environmental results for coarse frequency tuning followed by a local impedance characterization for fine frequency adjustments, yields a highly accurate method of resonance control. Such an approach works across a wide range of environmental conditions, is easy to automate, and could have a significant impact across a wide range of microfluidic acoustic standing wave systems.

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry.

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 µL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis.