Study cellular reponses at the microscale by creating heterogenity in cultured cells using a microfluidic probe

We introduce a new approach to study cellular responses in different cell subpopulations while not disrupting the microenvironments. We believe this might become a useful tool to investigate resistance-related cellular responses in cancer cells. Please click Additional Files below to see the full abstract

Computational Immunohistochemistry: Recipes for Standardization of Immunostaining

Cancer diagnosis and personalized cancer treatment are heavily based on the visual assessment of immunohistochemically-stained tissue specimens. The precision of this assessment depends critically on the quality of immunostaining, which is governed by a number of parameters used in the staining process. Tuning of the staining-process parameters is mostly based on pathologists' qualitative assessment, which incurs inter- and intra-observer variability. The lack of standardization in staining across pathology labs leads to poor reproducibility and consequently to uncertainty in diagnosis and treatment selection. In this paper, we propose a methodology to address this issue through a quantitative evaluation of the staining quality by using visual computing and machine learning techniques on immunohistochemically-stained tissue images. This enables a statistical analysis of the sensitivity of the staining quality to the process parameters and thereby provides an optimal operating range for obtaining high-quality immunostains. We evaluate the proposed methodology on HER2-stained breast cancer tissues and demonstrate its use to define guidelines to optimize and standardize immunostaining

Reconfigurable microfluidics: real-time shaping of virtual channels through hydrodynamic forces

To break the current paradigm in microfluidics that directly links device design to functionality, we introduce microfluidic "virtual channels" that can be dynamically shaped in real-time. A virtual channel refers to a flow path within a microfluidic flow cell, guiding an injected reagent along a user-defined trajectory solely by hydrodynamic forces. Virtual channels dynamically reproduce key microfluidic functionality: directed transport of minute volumes of liquid, splitting, merging and mixing of flows. Virtual channels can be formed directly on standard biological substrates, which we demonstrate by sequential immunodetection at arrays of individual reaction sites on a glass slide and by alternating between local and global processing of surface-adherent cell-block sections. This approach is simple, versatile and generic enough to form the basis of a new class of microfluidic techniques

Fluidic Bypass Structures for Improving the Robustness of Liquid Scanning Probes

Objective: We aim to improve operational robustness of liquid scanning probes. Two main failure modes to be addressed are an obstruction of the flow path of the processing liquid and a deviation from the desired gap distance between probe and sample. Methods: We introduce a multi-functional design element, a microfluidic bypass channel, which can be operated in dc and in ac mode, each preventing one of the two main failure modes. Results: In dc mode, the bypass channel is filled with liquid and exhibits resistive behavior, enabling the probe to passively react to an obstruction. In the case of an obstruction of the flow path, the processing liquid is passively diverted through the bypass to prevent its leakage and to limit the buildup of high pressure levels. In ac mode, the bypass is filled with gas and has capacitive characteristics, allowing the gap distance between the probe and the sample to be monitored by observing a phase shift in the motion of two gas-liquid interfaces. For a modulation of the input pressure at 4 Hz, significant changes of the phase shift were observed up to a gap distance of 25 mu m. Conclusion: The presented passive design element counters both failure modes in a simple and highly compatible manner. Significance: Liquid scanning probes enabling targeted interfacing with biological surfaces are compatible with a wide range of workflows and bioanalytical applications. An improved operational robustness would facilitate rapid and widespread adoption of liquid scanning probes in research as well as in diagnostics

Hydrodynamic thermal confinement: creating thermo-chemical microenvironments on surfaces

We present a new, general concept termed Hydrodynamic Thermal Confinement (HTC), and its implementation for the creation of microscale dynamic thermo-chemical microenvironments on biological surfaces. HTC is based on a scanning probe and operates under physiological conditions. The temperature can be regulated between 30° and 80 °C with ±0.2 °C precision and temperature ramps of 5 °C s−1 over a footprint of ∼50 μm × 80 μm in a volume of ∼50 × 80 × 15 μm3 (∼50 pl).ISSN:1359-7345ISSN:1364-548

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. This imposes strong limitations in terms of the number of tests as well as the time when they can be performed on a single sample. Microscale dewaxing lifts these constraints by permitting deprotection of a fraction of a tissue for testing while keeping the remaining of the sample intact for future analysis. After testing, the sample can be sent back to storage instead of being discarded, as is done in standard workflows. We achieve this microscale dewaxing by hydrodynamically confining nanoliter volumes of xylene on top of the sample with a probe head. We demonstrate micrometer-scale, chromogenic and fluorescence-based immunohistochemistry against multiple biomarkers (p53, CD45, HER2 and β-actin) on tonsil and breast tissue sections and microarrays. We achieve stain patterns as small as 100 μm × 50 μm as well as multiplexed immunostaining within a single tissue microarray core with a 20-fold time reduction for local dewaxing as compared to standard protocols. We also demonstrate a 10-fold reduction in the rehydration time, leading to lower processing times between different stains. We further show the potential of TL for retrospective studies by sequentially dewaxing and staining four individual cores within the same tissue microarray over four consecutive days. By combining tissue lithography with the concept of micro-immunohistochemistry, we implement each step of the IHC protocol-dewaxing, rehydration and staining-with the same microfluidic probe head. Tissue lithography brings a new level of versatility and flexibility in sample processing and budgeting in biobanks, which may alleviate current sample limitations for retrospective studies in biomarker discovery and drug screening