An application of spatial deconvolution to a capillary‐based high‐pressure chamber for fluorescence microscopy imaging

Abstract

Capillary-based high-pressure chambers for which the wall serves as both the optical window and mechanical support have been reported for fluorescence microscopy imaging. Although capillary chambers are straightforward and economical to construct, the curved capillary wall introduces image aberrations. The significance of these aberrations in imaging sub-cellular-dimension objects has yet to be assessed. Using a capillary chamber that is routinely pressurized to between 20 and 30 MPa, a pressure range suitable for studying a wide variety of cellular processes, we demonstrate sub-cellular-dimension spatial resolution in the imaging of fluorescent micro-spheres. Objectives with a range of numerical apertures (0.5-1.3) and working distances (0.1-7.4 mm) are considered. We show that spatial (or point-spread function, PSF) deconvolution improves image contrast in capillary-based images by comparing deconvolution results with those obtained from slide-mounted controls. Furthermore, similar deconvolution results between a measured PSF and a calculated, flat-geometry PSF indicate that the capillary wall is optically flat on cellular length scales. Results here facilitate the application of contemporary techniques in fluorescence microscopy to high-pressure imaging fields.