3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.

The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research

Fabrication of a Flexible Amperometric Glucose Sensor Using Additive Processes

This study details the use of printing and other additive processes to fabricate a novel amperometric glucose sensor. The sensor was fabricated using a Au coated 12.7 μm thick polyimide substrate as a starting material, where micro-contact printing, electrochemical plating, chloridization, electrohydrodynamic jet (e-jet) printing, and spin coating were used to pattern, deposit, chloridize, print, and coat functional materials, respectively. We have found that e-jet printing was effective for the deposition and patterning of glucose oxidase inks with lateral feature sizes between ∼5 to 1000 μm in width, and that the glucose oxidase was still active after printing. The thickness of the permselective layer was optimized to obtain a linear response for glucose concentrations up to 32 mM and no response to acetaminophen, a common interfering compound, was observed. The use of such thin polyimide substrates allow wrapping of the sensors around catheters with high radius of curvature ∼250 μm, where additive and microfabrication methods may allow significant cost reductions

Chemotherapy-induced senescent cancer cells engulf other cells to enhance their survival.

In chemotherapy-treated breast cancer, wild-type p53 preferentially induces senescence over apoptosis, resulting in a persisting cell population constituting residual disease that drives relapse and poor patient survival via the senescence-associated secretory phenotype. Understanding the properties of tumor cells that allow survival after chemotherapy treatment is paramount. Using time-lapse and confocal microscopy to observe interactions of cells in treated tumors, we show here that chemotherapy-induced senescent cells frequently engulf both neighboring senescent or nonsenescent tumor cells at a remarkable frequency. Engulfed cells are processed through the lysosome and broken down, and cells that have engulfed others obtain a survival advantage. Gene expression analysis showed a marked up-regulation of conserved macrophage-like program of engulfment in chemotherapy-induced senescent cell lines and tumors. Our data suggest compelling explanations for how senescent cells persist in dormancy, how they manage the metabolically expensive process of cytokine production that drives relapse in those tumors that respond the worst, and a function for their expanded lysosomal compartment

Solution-based deposition of functional thin films : development of a glucose biosensor and thermal processing of CZTS nanoparticle films

The development of solution-based methods for deposition of different thin film material is presented as an alternative to high cost vacuum-based methods. For certain materials, vacuum techniques are unsuitable for processing. Additionally, vacuum-based processes present high capital costs associated with equipment, and slow processing times. Atmospheric pressure solution based techniques are attractive and lend themselves to high-throughput processing using methods such as graphic patterning or roll-to-roll processing. This research details the development of solution-based outer layers for a continuous glucose biosensor. The biosensor is based on a biological ink deposited by electrohydrodynamic printing (EHDP), encapsulated by a biocompatible permselective membrane which enhances the sensor function and working lifetime. EHDP is a noncontact patterning method where ink is deposited by the application of high electric fields to a conductive microcapillary, resulting in the jetting of ionized ink droplets with precise placement. This research also examines the processing window of radiant arc plasma pulsed thermal processing (PTP) on thin films of copper zinc tin sulfide (CZTS) nanoparticles. CZTS is an attractive solar absorbing material for low cost thin film solar cells. CZTS has a near-optimal band gap of 1.5 eV and a high absorption coefficient. In this study, CZTS nanoparticles are synthesized using a continuous flow reactor, and deposited by spin-coating on substrates for PTP. The effects of PTP on film morphology and crystalline phase are investigated using scanning electon microscopy (SEM), X-ray diffraction (XRD), and Raman spectroscopy

DuXiaosongChemBioEnvEngFabricationFlexible.pdf

This study details the use of printing and other additive processes to fabricate a novel amperometric glucose sensor. The sensor was fabricated using a Au coated 12.7 μm thick polyimide substrate as a starting material, where micro-contact printing, electrochemical plating, chloridization, electrohydrodynamic jet (e-jet) printing, and spin coating were used to pattern, deposit, chloridize, print, and coat functional materials, respectively. We have found that e-jet printing was effective for the deposition and patterning of glucose oxidase inks with lateral feature sizes between ∼5 to 1000 μm in width, and that the glucose oxidase was still active after printing. The thickness of the permselective layer was optimized to obtain a linear response for glucose concentrations up to 32 mM and no response to acetaminophen, a common interfering compound, was observed. The use of such thin polyimide substrates allow wrapping of the sensors around catheters with high radius of curvature ∼250 μm, where additive and microfabrication methods may allow significant cost reductions

DuXiaosongChemBioEnvEngFabricationFlexible.pdf

This study details the use of printing and other additive processes to fabricate a novel amperometric glucose sensor. The sensor was fabricated using a Au coated 12.7 μm thick polyimide substrate as a starting material, where micro-contact printing, electrochemical plating, chloridization, electrohydrodynamic jet (e-jet) printing, and spin coating were used to pattern, deposit, chloridize, print, and coat functional materials, respectively. We have found that e-jet printing was effective for the deposition and patterning of glucose oxidase inks with lateral feature sizes between ∼5 to 1000 μm in width, and that the glucose oxidase was still active after printing. The thickness of the permselective layer was optimized to obtain a linear response for glucose concentrations up to 32 mM and no response to acetaminophen, a common interfering compound, was observed. The use of such thin polyimide substrates allow wrapping of the sensors around catheters with high radius of curvature ∼250 μm, where additive and microfabrication methods may allow significant cost reductions