Quantitative imaging of lipid droplets in single cells.

The combination of next generation sequencing (NGS) and automated liquid handling platforms has led to a revolution in single-cell genomic studies. However, many molecules that are critical to understanding the functional roles of cells in a complex tissue or organs, are not directly encoded in the genome, and therefore cannot be profiled with NGS. Lipids, for example, play a critical role in many metabolic processes but cannot be detected by sequencing. Recent developments in quantitative imaging, particularly coherent Raman scattering (CRS) techniques, have produced a suite of tools for studying lipid content in single cells. This article reviews CRS imaging and computational image processing techniques for non-destructive profiling of dynamic changes in lipid composition and spatial distribution at the single-cell level. As quantitative CRS imaging progresses synergistically with microfluidic and microscopic platforms for single-cell genomic analysis, we anticipate that these techniques will bring researchers closer towards combined lipidomic and genomic analysis.

Ultralarge Modulation of Fluorescence by Neuromodulators in Carbon Nanotubes Functionalized with Self-Assembled Oligonucleotide Rings.

Noncovalent interactions between single-stranded DNA (ssDNA) oligonucleotides and single wall carbon nanotubes (SWNTs) have provided a unique class of tunable chemistries for a variety of applications. However, mechanistic insight into both the photophysical and intermolecular phenomena underlying their utility is lacking, which results in obligate heuristic approaches for producing ssDNA-SWNT based technologies. In this work, we present an ultrasensitive "turn-on" nanosensor for neuromodulators dopamine and norepinephrine with strong relative change in fluorescence intensity (Δ F/ F0) of up to 3500%, a signal appropriate for in vivo neuroimaging, and uncover the photophysical principles and intermolecular interactions that govern the molecular recognition and fluorescence modulation of this nanosensor synthesized from the spontaneous self-assembly of (GT)6 ssDNA rings on SWNTs. The fluorescence modulation of the ssDNA-SWNT conjugate is shown to exhibit remarkable sensitivity to the ssDNA sequence chemistry, length, and surface density, providing a set of parameters with which to tune nanosensor dynamic range, analyte selectivity and strength of fluorescence turn-on. We employ classical and quantum mechanical molecular dynamics simulations to rationalize our experimental findings. Calculations show that (GT)6 ssDNA form ordered rings around (9,4) SWNTs, inducing periodic surface potentials that modulate exciton recombination lifetimes. Further evidence is presented to elucidate how dopamine analyte binding modulates SWNT fluorescence. We discuss the implications of our findings for SWNT-based molecular imaging applications.

Microfluidic single-cell whole-transcriptome sequencing.

Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.

Label-Free Digital Quantification of Lipid Droplets in Single Cells by Stimulated Raman Microscopy on a Microfluidic Platform.

Quantitative characterization of a single-cell phenotype remains challenging. We combined a scalable microfluidic array of parallel cell culture chambers and stimulated Raman scattering (SRS) microscopy to quantitatively characterize the response of lipid droplet (LD) formation to free-fatty-acid stimuli with single-LD resolution at the single-cell level. By enabling the systematic live-cell imaging with SRS microscopy in a microfluidic device, we were able to quantify the morphology of over a thousand live cells in 10 different chemical environments and with 8 replicates for each culture condition, in a single experiment, and without relying on fluorescent labeling. We developed an image processing pipeline for cell segmentation and LD morphology quantification using dual-channel SRS images. This allows us to construct distributions of the morphological parameters of LDs in the cellular population and expose the vast phenotypic heterogeneity among genetically similar cells. Specifically, this approach provides an analytical tool for quantitatively investigating LD morphology in live cells in situ. With this high-throughput, high-resolution, and label-free method, we found that LD growth dynamics showed considerable cell to cell variation. Lipid accumulation in nonadipocyte cells is mainly reflected in the increase of LD number, as opposed to an increase in their size or lipid concentration. Our method allows statistical single-cell quantification of the LD distribution for further investigation of lipid metabolism and dynamic behavior, and also extends the possibility to couple with other "omics" technologies in the future.

Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging.

Quantitative single-cell analysis enables the characterization of cellular systems with a level of detail that cannot be achieved with ensemble measurement. In this Feature we explore quantitative cellular imaging applications with nonlinear microscopy techniques. We first offer an introductory tutorial on nonlinear optical processes and then survey a range of techniques that have proven to be useful for quantitative live cell imaging without fluorescent labels.

High-throughput single-molecule optofluidic analysis.

We describe a high-throughput, automated single-molecule measurement system, equipped with microfluidics. The microfluidic mixing device has integrated valves and pumps to accurately accomplish titration of biomolecules with picoliter resolution. We demonstrate that the approach enabled rapid sampling of biomolecule conformational landscape and of enzymatic activity, in the form of transcription by Escherichia coli RNA polymerase, as a function of the chemical environment.

Mechanical phenotyping reveals unique biomechanical responses in retinoic acid-resistant acute promyelocytic leukemia.

All-trans retinoic acid (ATRA) is an essential therapy in the treatment of acute promyelocytic leukemia (APL), but nearly 20% of patients with APL are resistant to ATRA. As there are no biomarkers for ATRA resistance that yet exist, we investigated whether cell mechanics could be associated with this pathological phenotype. Using mechano-node-pore sensing, a single-cell mechanical phenotyping platform, and patient-derived APL cell lines, we discovered that ATRA-resistant APL cells are less mechanically pliable. By investigating how different subcellular components of APL cells contribute to whole-cell mechanical phenotype, we determined that nuclear mechanics strongly influence an APL cell's mechanical response. Moreover, decondensing chromatin with trichostatin A is especially effective in softening ATRA-resistant APL cells. RNA-seq allowed us to compare the transcriptomic differences between ATRA-resistant and ATRA-responsive APL cells and highlighted gene expression changes that could be associated with mechanical changes. Overall, we have demonstrated the potential of "physical" biomarkers in identifying APL resistance.

Ostwald ripening of clusters during protein crystallization.

Contrary to classical nucleation theory, protein crystals can nucleate via a two-step process in which the molecular arrangement of the ordered solid phase is preceded by nucleation of a dense amorphous phase. We study the growth of these precrystalline clusters in lysozyme using a combination of dynamic light scattering, optical microscopy, and microfluidics. Clusters display Ostwald ripening growth kinetics but deviate from this trend after nucleation of the crystal phase. This behavior arises from the metastable relationship between clusters and the ordered solid and is explained numerically using a population balance model.

Controller for microfluidic large-scale integration.

Microfluidic devices with integrated valves provide precise, programmable fluid handling platforms for high-throughput biological or chemical assays. However, setting up the infrastructure to control such platforms often requires specific engineering expertise or expensive commercial solutions. To address these obstacles, we present a Kit for Arduino-based Transistor Array Actuation (KATARA), an open-source and low-cost Arduino-based controller that can drive 70 solenoid valves to pneumatically actuate integrated microfluidic valves. We include a python package with a GUI to control the KATARA from a personal computer. No programming experience is required.

Single-Cell Transcriptional Analysis.

Despite being a relatively recent technological development, single-cell transcriptional analysis through high-throughput sequencing has already been used in hundreds of fruitful studies to make exciting new biological discoveries that would otherwise be challenging or even impossible. Consequently, this has fueled a virtuous cycle of even greater interest in the field and compelled development of further improved technical methodologies and approaches. Thanks to the combined efforts of the research community, including the fields of biochemistry and molecular biology, technology and instrumentation, data science, computational biology, and bioinformatics, the single-cell RNA-sequencing field is advancing at a pace that is both astounding and unprecedented. In this review, we provide a broad introduction to this revolutionary technology by presenting the state-of-the-art in sample preparation methodologies, technology platforms, and computational analysis methods, while highlighting the key considerations for designing, executing, and interpreting a study using single-cell RNA sequencing.

Microfluidics for biological measurements with single-molecule resolution.

Single-molecule approaches in biology have been critical in studies ranging from the examination of physical properties of biological macromolecules to the extraction of genetic information from DNA. The variation intrinsic to many biological processes necessitates measurements with single-molecule resolution in order to accurately recapitulate population distributions. Microfluidic technology has proven to be useful in the facilitation and even enhancement of single-molecule studies because of the precise liquid handling, small volume manipulation, and high throughput capabilities of microfluidic devices. In this review we survey the microfluidic "toolbox" available to the single-molecule specialist and summarize some recent biological applications of single-molecule detection on chip.

Chip in a lab: Microfluidics for next generation life science research.

Microfluidic circuits are characterized by fluidic channels and chambers with a linear dimension on the order of tens to hundreds of micrometers. Components of this size enable lab-on-a-chip technology that has much promise, for example, in the development of point-of-care diagnostics. Micro-scale fluidic circuits also yield practical, physical, and technological advantages for studying biological systems, enhancing the ability of researchers to make more precise quantitative measurements. Microfluidic technology has thus become a powerful tool in the life science research laboratory over the past decade. Here we focus on chip-in-a-lab applications of microfluidics and survey some examples of how small fluidic components have provided researchers with new tools for life science research.

Simultaneous measurement of amyloid fibril formation by dynamic light scattering and fluorescence reveals complex aggregation kinetics.

An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, ß-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports ß-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.

Optical imaging of non-fluorescent nanodiamonds in live cells using transient absorption microscopy.

We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.