The MechanoBiology Institute prides itself having some of the fastest and most accurate optical tracking machinery for force sensing by deflection measurements of transparent polydimethyl­siloxane pillars. Optical tracking allows for long term in-vivo observation of dense (up to about one vector per square mm) force maps with low pN accuracy. This translates into hundreds of frames per second recording at better than 4nm localization. The convection of the medium is used to provide local cooling for the region where the pump light penetrates the biological material and the immersion medium itself must feature a low absorption of the tracking wavelengths in order to serve as a coolant.

The refractive index difference of the pillars from this medium provides both the necessary contrast mechanism as well as a noticeable distortion of the recorded optical image of those pillars. Scattering in the specimen itself, small devices brought into the specimen, or the small chamber above the specimen further harm the imaging of the pillars. We believe that – as contrast and artefact are generate by the same mechanism – that these distortions can be minimized but not entirely avoided.

We present some design steps to limit the optical distortion of the images and some image processing insights that allow for the discrimination of the pillar projection and scattering artifacts along the beam path.

As a result the useful range of these observations and the scope of where these observations are accurate are greatly expanded and allow for some simplifications of crucial cellular force sensing experiments.

Quantitative single-cell analysis enables the characterization of cellular systems with a level of detail that cannot be achieved with ensemble measurement. I am going to show some of our recent work on developing better approaches for single-cell sequencing, including whole genome amplification and whole transcriptome analysis, using droplets that generated by various microfluidic approaches.

Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single- nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. I will present a new approach, emulsion WGA (eWGA), to overcome these problems. We divide single-cell genomic DNA into a large number (> 100,000) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.

Following similar design, we also develop a robust and simple single-cell RNA-seq method, named 'easier-seq' to perform whole transcriptome analysis. This new method is capable of capturing novel transcripts without polyA tails.

Such droplet-based amplification method has been shown to exhibit great potential to facilitate sequencing of single cells, as well as other highly-quantitative measurement of limited input sample, for example, the digital PCR reactions.

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Three-dimensional (3D) refractive-index (RI) microscopy is suitable for live-cell imaging due to its label-free and fast 3D imaging capabilities. 3-D RI maps are reconstructed from sequences of two-dimensional quantitative phase images at various illumination angles obtained with off-axis interferometry. We achieved fast retrieval and unwrapping of quantitative phase images using parallel computing on graphics processing units. The results of applying discrete cosine transform to phase wrapping showed over ten times improvement in computation speed and slight reduction of spatial phase noise compared to FFT-based phase unwrapping. The improved efficiency and accuracy of phase retrieval and unwrapping processes will enable real-time extraction and quantitative visualization of phase maps and RI images of living cells. As an example, 3D RI distributions of white blood cells (WBC) will be presented. The feasibility of analyzing light scattering properties of individual WBC based on their 3D RI maps will be demonstrated.

Techniques based on continuous fractionation by hydrodynamic interaction are attractive, due to their simple structure, robustness and potential high throughput. We developed a new approach for particle separation by introducing viscosity difference of the sheath flows to form an asymmetric focusing of sample particle flow. This approach relies on the high-velocity gradient in the asymmetric focusing of the particle flow to generate a lift force, which plays a dominated role in the particle separation. The larger particles migrate away from the original streamline to the side of the higher relative velocity, while the smaller particles remain close to the streamline. Under high-viscosity (glycerol–water solution) and low-viscosity (PBS) sheath flows, a significant large stroke separation between the smaller (1.0 μm) and larger (9.9 μm) particles was achieved in a sample micro fluidic device. We demonstrate that the flow rate and the viscosity difference of the sheath flows have an impact on the interval distance of the particle separation that affects the collected purity and on the focusing distribution of the smaller particles that affects the collected concentration. This separation method proposed in our work can potentially be applied to biological and medical applications due to the wide interval distance and the narrow focusing distribution of the particle separation, by easy manufacturing in a simple device.

Infrared (IR) spectro-microscopy (FTIR) is a label-free chemical imaging technique which has been widely used as a diagnostic and research tool to obtain detailed molecular information in biological specimens. However, its application as an imaging technology to study living systems has been limited due to the strong absorbance of water in the mid-IR; even in a thin layer, water can completely overwhelm the chemical information from the sample. Microfluidic technology can provide a platform to overcome this limitation, with benefits in terms of measurement accuracy and integration of FTIR with other technics (e.g. flow cytometry).

The non-transparency to IR light of standard materials used in microfluidic, such as glass, plastics and PDMS, has so far limited the availability of practical set-up for the application of FTIR to live cells analysis. Here we present our approach for the easy and cheap fabrication of plastic microfluidic devices suitable for both optical and IR imaging. The plastic device is produced via standard soft-lithographic process; an embedded transparent view-port allows for imaging using both visible and IR photons, while the plastic body eases the connection with external pumping systems for the injection of the samples, handling of fluids and collection of waste. Chemical maps of REF52 cells seeded acquired while they were maintained inside the device show the potential application of FTIR for single-cell analysis.

Metal nanoparticles (mNPs) also have strong interactions with light to generate localized SPR (LSPR) that leads to unique optical properties suitable for bioanalytical applications in various fields. Ultra small metal nanoclusters (mNCs) (i.e. < 2 nm with several to a few hundreds of metal atoms) have strong quantum confinement in the sub-2 nm size regime, thus exhibit unique molecular-like properties, such as strong fluorescence. I will present our contribution to this field, include (1) synthesis of anisotropic mNPs [1, 2], (2) coupling complementary sensing materials for bioassay development for a wide range of analytes (including protein [3], DNA [4], drug [5], pesticides, bacteria cells [6] etc), (3) mNPs embedded paper sensors for food/environmental monitoring, and (4) drug screening using mNCs [7]. Through these work, we have introduced novel analytical concepts of inverse sensitivity (for circulating DNA [8]) and synthesis-cum-sensing strategies (for drug screening [7] and UV-exposure detection [9]).

Disordered optics is a fascinating area, yet not fully understood, as it increasingly attracts interest due to the virtues of multiple scattering of light in diffusive materials and their numerous applications to imaging through opaque media, to spectroscopy as well as more fundamental optical transport properties including Anderson localization. Among the various kinds of disordered media, Black Silicon is a randomly micro-structured surface exhibiting several outstanding optical and wetting properties, which makes it increasingly used in various applications that will be discussed. On the other hand, complex fluids and especially colloidal suspensions have the specific feature of the time-dependence of the disorder; even in the static equilibrium regime of no flow, complex fluids exhibit Brownian motion of the embedded particles leading to an additional dimension to the disorder. The heterogeneous nature of complex fluids induces specific behavior at the microscale as well as collective effects at larger scales. This holds true not only regarding rheology but also in the optical domain, where one can define an effective refraction index in a medium subject to microscale effects of light scattering. Furthermore, it is also intuitive to think of a possible time-dependence of those properties. This poses the question of the most appropriate length scale and time scale to perform measurements in such media.

In recent years, low-dimensional semiconductor structures have attracted extensive research interest. However, due to the large surface-to-volume ratio, surface states play very important roles on the optical properties of the materials. In this talk, I will introduce the manipulation of optical property of low-dimensional semiconductors through different approaches, for example, surface passivation, introduction of localized states, strain, energy transfer, etc. The combination of these methods will enable the improvement of the optical property of materials and their practical device applications.

Metallic nanocavities sustain localized surface plasmons which enable deep subwavelength light concentration and manipulation. The nanocavity in between two nanoparticles, i.e., a dimer, is a typical geometry for light concentration, with typical electric field enhancement of 10² - 10³ under resonant excitation. The smaller the gap distance is, the larger the field enhancement will be. However, direct probing of the field enhancement is prohibited since the probe (e.g. a scanning tip) cannot be inserted into the tiny gap of the nanocavity. In this talk, I will talk about how to probe the field enhancement within a subnanometer gap in a nanoparticle over mirror (NPOM) configuration using Surface enhanced Raman spectroscopy (SERS). The strong field enhancement in the gap can also be used to amplify the interaction between light and exciton. Furthermore, I will also show that the nanocavity can be used as a plasmon sensor with unprecedented sensitivity.

In the human body, the mechanical aspects of cell matrices are critical in maintaining cell differentiation and growth patterns. Both the static rigidity of the matrix and the stretching activity of the matrix are critical factors. Using PDMS pillars of submicrometer diameters, we first determined that the static rigidity of the matrix was measured by local contractions of 100 nm by sarcomere-like units of about 2 micrometers (Wolfenson et al., 2016).  These modular rigidity-sensing machines contained many cytoskeletal proteins like tropomyosin, a-actinin, actin and myosin. When they were missing, cells did not sense soft surfaces and grew inappropriately. When rigidity sensors were present, cells would die on soft surfaces but growth was rescued if the soft surfaces were stretched. 1-5% cyclic stretching over a frequency range of 0.01 to 10 Hz caused spreading and growth (optimum 0.1 Hz) (Cui et al., 2015). Of possible factors linked to fibroblast growth, MRTF-A (Myocardin-related transcription factor-A) moved to the nucleus in 2 hrs of cyclic stretching and reversed upon cessation; but, YAP (Yes-associated protein) moved much later. Knockdown of either MRTF-A or YAP blocked stretch-dependent growth. Thus, we suggest that the repeated pulling from a soft matrix can substitute for a stiff matrix in stimulating spreading and growth.  More generally, mechanical activation of cell substrates can be used to control cell growth and even differentiation.