Single-cell Organelle Extraction with Cellular Indexing.

Bulk methods to fractionate organelles lack the resolution to capture single-cell heterogeneity. While microfluidic approaches attempt to fractionate organelles at the cellular level, they fail to map each organelle back to its cell of origin-crucial for multiomics applications. To address this, we developed VacTrap, a high-throughput microfluidic device for isolating and spatially indexing single nuclei from mammalian cells.

Enhancing the Efficiency of Conventional Surface Immunoassays Within Standard Labware Using Microscale Flows.

In this chapter, we present the design and fabrication of a device and implementation of a protocol to realize increased efficiency of immunoassays within microtiter plates. The device, WellProbe, is a 3D-structured probe that can be used to deliver precise flows at the bottom of standard well plates to establish concentric areas of shear stress intensities using hydrodynamically confined flows. The protocols involve both operation and data analysis.

Space in cancer biology: its role and implications.

Tumor cells present complex behaviors in their interactions with other cells. This intricate behavior is driving the need to develop new tools to understand these ecosystems. The surge of spatial technologies allows evaluation of the complexity of relationships between cells present in a tumor, giving insights about tumor heterogeneity and the tumor microenvironment while providing clinically relevant metrics for tumor classification.

Quantifying Antibody Binding Kinetics on Fixed Cells and Tissues Fluorescence Lifetime Imaging.

We present a method for monitoring spatially localized antigen-antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates.

Quantification of tumor heterogeneity: from data acquisition to metric generation.

Tumors are unique and complex ecosystems, in which heterogeneous cell subpopulations with variable molecular profiles, aggressiveness, and proliferation potential coexist and interact. Understanding how heterogeneity influences tumor progression has important clinical implications for improving diagnosis, prognosis, and treatment response prediction. Several recent innovations in data acquisition methods and computational metrics have enabled the quantification of spatiotemporal heterogeneity across different scales of tumor organization.

Spatial protein heterogeneity analysis in frozen tissues to evaluate tumor heterogeneity.

A new workflow for protein-based tumor heterogeneity probing in tissues is here presented. Tumor heterogeneity is believed to be key for therapy failure and differences in prognosis in cancer patients. Comprehending tumor heterogeneity, especially at the protein level, is critical for tracking tumor evolution, and showing the presence of different phenotypical variants and their location with respect to tissue architecture.

Advection-Enhanced Kinetics in Microtiter Plates for Improved Surface Assay Quantitation and Multiplexing Capabilities.

Surface assays such as ELISA are pervasive in clinics and research and predominantly standardized in microtiter plates (MTP). MTPs provide many advantages but are often detrimental to surface assay efficiency due to inherent mass transport limitations. Microscale flows can overcome these and largely improve assay kinetics.

Open Space Diffusive Filter for Simultaneous Species Retrieval and Separation.

We present a novel method for the local retrieval of surface bound species and their rapid in-line separation using an open space microfluidic device. Separation can be performed in less than 30 s using the difference in diffusivities within parallel microfluidic flows. As a proof-of-principle, we report the rapid and efficient filtration of polystyrene beads from small molecules and surface bound red blood cells from dimethyl sulfoxide for antigen typing.

Shake It or Shrink It: Mass Transport and Kinetics in Surface Bioassays Using Agitation and Microfluidics.

Surface assays, such as ELISA and immunofluorescence, are nothing short of ubiquitous in biotechnology and medical diagnostics today. The development and optimization of these assays generally focuses on three aspects: immobilization chemistry, ligand-receptor interaction, and concentrations of ligands, buffers, and sample. A fourth aspect, the transport of the analyte to the surface, is more rarely delved into during assay design and analysis.

Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity.

Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes.

Nip the bubble in the bud: a guide to avoid gas nucleation in microfluidics.

Gas bubbles are almost a routine occurrence encountered by researchers working in the field of microfluidics. The spontaneous and unexpected nature of gas bubbles represents a major challenge for experimentalists and a stumbling block for the translation of microfluidic concepts to commercial products. This is a startling example of successful scientific results in the field overshadowing the practical hurdles of day-to-day usage.

Quantitative microimmunohistochemistry for the grading of immunostains on tumour tissues.

Immunohistochemistry is the gold-standard method for cancer-biomarker identification and patient stratification. Yet, owing to signal saturation, its use as a quantitative assay is limited as it cannot distinguish tumours with similar biomarker-expression levels. Here, we introduce a quantitative microimmunochemistry assay that enables the acquisition of dynamic information, via a metric of the evolution of the immunohistochemistry signal during tissue staining, for the quantification of relative antigen density on tissue surfaces.

High-Quality Immunohistochemical Stains Through Computational Assay Parameter Optimization.

Accurate profiling of tumors using immunohistochemistry (IHC) is essential in cancer diagnosis. The inferences drawn from IHC-stained images depend to a great extent on the quality of immunostaining, which is in turn affected strongly by assay parameters. To optimize assay parameters, the available tissue sample is often limited.

Tissue lithography: Microscale dewaxing to enable retrospective studies on formalin-fixed paraffin-embedded (FFPE) tissue sections.

We present a new concept, termed tissue lithography (TL), and its implementation which enables retrospective studies on formalin-fixed paraffin-embedded tissue sections. Tissue lithography uses a microfluidic probe to remove microscale areas of the paraffin layer on formalin-fixed paraffin-embedded biopsy samples. Current practices in sample utilization for research and diagnostics require complete deparaffinization of the sample prior to molecular testing.