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Heraghty, D.

Publications and source records attributed to Heraghty, D..

2 recordsLinked to original sources

Patch deconvolution for Fourier light-field microscopy

Imaging flow cytometry using Fourier light-field microscopy enables high-throughput three-dimensional cellular imaging, capable of capturing thousands of events per second. However, volumetric reconstruction speed remains orders of magnitude slower than the acquisition speed. The current state of art uses Richardson-Lucy algorithm, restricted to just 5-10 reconstructed per second with GPU acceleration. This limitation hinders real-time applications such as cell sorting and thus has bottlenecked the widespread adoption of 3D imaging flow cytometry. We introduce patch deconvolution, an optimisation compatible with the Richardson-Lucy framework that significantly accelerates convergence, achieving over 100-200 reconstructions per second on standard GPUs, a 20-40 fold improvement over Richardson-Lucy. Validated on both simulated and experimental datasets, patch deconvolution achieves reconstruction quality comparable to Richardson-Lucy in both static and flow data. This supports rapid cell sorting based on spatial features and enables advanced applications, such as detecting rare spatial events in large cell populations, which would otherwise be indistinguishable in traditional flow cytometry.

biophysics↗

Imaging the interactions of chimeric antigen receptor-expressing T-cells with colorectal cancer cells

Chimeric antigen receptor (CAR) T-cell therapy has shown unprecedented success in haematological cancers but faces challenges in solid tumours. Although carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is differentially expressed in many solid tumours, anti-CEACAM5 CAR T-cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting primary CAR T-cells with colorectal cancer (CRC) cells using fluorescence microscopy. We found that CRC cells glycocalyx is much deeper than that of the CAR T cell causing delayed activation. Oscillating calcium fluxes, indicative of non-sustained CAR T cell activation and reduced cytotoxicity, were observed when CAR T cells interacted with CRC cells, which increased with increasing cell-seeding time. Imaging revealed that this effect correlated with a progressive loss of accessible CEACAM5 antigen on the CRC cell surface, possibly due to their sequestration in the intercellular junction, rendering CAR T cell engagement less effective. Local proteolytic treatment with trypsin to disrupt the CRC cell monolayer, using a micropipette, increased CEACAM5 availability, decreased glycocalyx thickness, and restored sustained CAR T cell calcium fluxes, increasing the killing of CRC cells. Similar enhanced interaction was observed after treatment of CRC cell monolayer with hyaluronidase, approved for use in humans. We observed limited availability of CEACAM5 on human colorectal cancer tissues, whereas treatment with trypsin or hyaluronidase increased accessibility. Our results reveal why CAR T cells targeting CEACAM5 are ineffective and suggest possible routes to improved therapy for CRC.

immunology↗