Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.

Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics. © 2018 International Society for Advancement of Cytometry

Biomimetic and Biophysical Approach to Profile Metastatic Cancer Cell Migration

Honors Research ScholarshipCancer metastasis is a complex process by which cells in a primary tumor acquire an aggressive phenotype, and travel to distant, secondary sites in the body. One aspect of cancer metastasis is cell migration toward the vascular system, called invasion. Multiple modalities of single cell invasion exist, including amoeboid migration and mesenchymal migration. Amoeboid migration is less well understood, and in particular, the forces involved in amoeboid migration have yet to be fully elucidated at a subcellular scale. Cellular traction force microscopy, or CTFM, is one method used to probe migration forces. However, this approach is largely limited to two dimensions, and is limited by the size of the pillars on the substrate. To address these limitations, we developed a system using microfluidics and DNA origami capable of real-time force measurement of cell migration on a subcellular scale with a 10 pN resolution. Microfluidic devices were made using soft lithography and replica molding in our laboratory. DNA origami were made using protocols developed by Michael Hudoba and Dr. Carlos Castro in the Nanoengineering and Biodesign Laboratory. The devices were imaged using TIRF microscopy to study dwell times of the sensors in the open and closed states, and the devices were analyzed with an AFM to determine that they are best suited for measuring shear forces. Further, the presence of streptavidin protein was found to have a significant effect on DOFS binding with a p-value less than 0.05. DOFS concentrations around 1 nM were found to provide the most coverage while minimizing structure aggregation. Thus, our microfluidic devices are able to be functionalized with DNA origami force sensors with a high degree of attachment. This platform is thus capable of measuring cell migration and adhesion forces, and future work should harness this system to create 3D maps of cell migration to gain insight into invasion.Institute for Materials ResearchSecond-Year Transformational Experience Program (STEP)A one-year embargo was granted for this item.Academic Major: Biomedical Engineerin

Optically-controlled platforms for transfection and single- and sub-cellular surgery

Improving the resolution of biological research to the single- or sub-cellular level is of critical importance in a wide variety of processes and disease conditions. Most obvious are those linked to aging and cancer, many of which are dependent upon stochastic processes where individual, unpredictable failures or mutations in individual cells can lead to serious downstream conditions across the whole organism. The traditional tools of biochemistry struggle to observe such processes: the vast majority are based upon ensemble approaches analysing the properties of bulk populations, which means that the detail about individual constituents is lost. What are required, then, are tools with the precision and resolution to probe and dissect cells at the single-micron scale: the scale of the individual organelles and structures that control their function. In this review, we highlight the use of highly-focused laser beams to create systems providing precise control and specificity at the single cell or even single micron level. The intense focal points generated can directly interact with cells and cell membranes, which in conjunction with related modalities such as optical trapping provide a broad platform for the development of single and sub-cellular surgery approaches. These highly tuneable tools have demonstrated delivery or removal of material from cells of interest, but can simultaneously excite fluorescent probes for imaging purposes or plasmonic structures for very local heating. We discuss both the history and recent applications of the field, highlighting the key findings and developments over the last 40 years of biophotonics researc

Applications of light-sheet microscopy in microdevices

Light-sheet fluorescence microscopy (LSFM) has been present in cell biology laboratories for quite some time, mainly as custom-made systems, with imaging applications ranging from single cells (in the micrometer scale) to small organisms (in the millimeter scale). Such microscopes distinguish themselves for having very low phototoxicity levels and high spatial and temporal resolution, properties that make them ideal for a large range of applications. These include the study of cellular dynamics, in particular cellular motion which is essential to processes such as tumor metastasis and tissue development. Experimental setups make extensive use of microdevices (bioMEMS) that provide better control over the substrate environment than traditional cell culture experiments. For example, to mimic in vivo conditions, experiment biochemical dynamics, and trap, move or count cells. Microdevices provide a higher degree of empirical complexity but, so far, most have been designed to be imaged through wide-field or confocal microscopes. Nonetheless, the properties of LSFM render it ideal for 3D characterization of active cells. When working with microdevices, confocal microscopy is more widespread than LSFM even though it suffers from higher phototoxicity and slower acquisition speeds. It is sometimes possible to illuminate with a light-sheet microdevices designed for confocal microscopes. However, these bioMEMS must be redesigned to exploit the full potential of LSFM and image more frequently on a wider scale phenomena such as motion, traction, differentiation, and diffusion of molecules. The use of microdevices for LSFM has extended beyond cell tracking studies into experiments regarding cytometry, spheroid cultures and lab-on-a-chip automation.JR acknowledges funding from EU H2020 FET Open project SENSITIVE, ID 801347 and Spanish Ministry of Economy and Competitiveness (MINECO) Grant FIS2016-77892-R. AM-B acknowledges funding from the MINECO (TEC2013-48552-C2-1-R, TEC2015-73064-EXP, TEC2016-78052-R), Beca Leonardo a Investigadores y Creadores Culturales 2017, Fundacion BBVA and EU Open project SENSITIVE. The CNIC is supported by the Ministerio de Ciencia, Innovacion y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505)

Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology.

Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology

Development of an Inverted Epifluorescence Microscope for Long-Term Monitoring of Bacteria in Multiplexed Microfluidic Devices

Developing more efficient methods for antibiotic susceptibility testing is a pressing issue in novel drug development as bacterial resistance to antibiotics becomes increasingly common. Microfluidic devices have been demonstrated to be powerful platforms that allow researchers to perform multiplexed antibiotic testing. However, the level of multiplexing within microdevices is limited, evidencing the need of creating simple, low-cost and high-resolution imaging systems that can be integrated in antibiotic development pipelines. This paper describes the design and development of an epifluorescence inverted microscope that enables long-term monitoring of bacteria inside multiplexed microfluidic devices. The goal of this work is to provide a simple microscope powerful enough to allow single-cell analysis of bacteria at a reduced cost. This facilitates increasing the number of microscopes that are simultaneously used for antibiotic testing. We prove that the designed system is able to accurately detect fluorescent beads of 100 nm, demonstrating comparable features to high-end commercial microscopes and effectively achieving the resolution required for single-cell analysis of bacteria. The proposed microscope could thus increase the efficiency in antibiotic testing while reducing cost, size, weight, and power requirements, contributing to the successful development of new antibiotic drugs.This work was partially funded by project TEC2016-78052-R from the Spanish Ministry of Economy and PID2019-109820RB-I00 from the Spanish Ministry of Science and Innovation. The research leading to these results received funding from the Innovative Medicines Innitiative 2 Joint Undertaking (JU) under grant agreement No 853989. The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA and Global Alliance for TB Drug Development non profit organisation, Bill & Melinda Gates Foundation and University of Dundee