Mesoscale standing wave imaging

Standing wave (SW) microscopy is a method that uses an interference pattern to excite fluorescence from labelled cellular structures and produces high‐resolution images of three‐dimensional objects in a two‐dimensional dataset. SW microscopy is performed with high‐magnification, high‐numerical aperture objective lenses, and while this results in high‐resolution images, the field of view is very small. Here we report upscaling of this interference imaging method from the microscale to the mesoscale using the Mesolens, which has the unusual combination of a low‐magnification and high‐numerical aperture. With this method, we produce SW images within a field of view of 4.4 mm × 3.0 mm that can readily accommodate over 16,000 cells in a single dataset. We demonstrate the method using both single‐wavelength excitation and the multi‐wavelength SW method TartanSW. We show application of the method for imaging of fixed and living cells specimens, with the first application of SW imaging to study cells under flow conditions. LAY DESCRIPTION With a standard microscope, it is possible to interfere two or more beams of light and produce a contour map of cell structures using a technique known as standing wave microscopy. This method is usually applied with high power objective lenses, and only a tiny area can be studied, usually showing structural detail from one or two cells at most. The Mesolens is a unique objective lens that has been designed and manufactured to image large populations of cells in a single image. Two‐dimensional images of cells can be obtained with the Mesolens using regular illumination methods, but the cell topography is lost. Here we have combined standing wave illumination with the Mesolens to perform topographical imaging of large numbers of cells in a single image. We first proved the method with a non‐biological specimen comprised of a glass lens, and then we applied the technique to imaging of fixed and living cells: in the case of red blood cells, we showed that we can study the topography of more than 16,000 cells in a single image. With the much larger area of study afforded by the Mesolens, we also used standing wave illumination for the first time monitor red blood cell topography when the cells are subject to flow environment. This new application of standing wave imaging at unusually large spatial scales may prove useful for studying behaviour in the circulatory system.