ISM-assisted tomographic STED microscopy.

Stimulated emission depletion (STED) microscopy theoretically provides unlimited resolution. However, in practice the achievable resolution in biological samples is essentially limited by photobleaching. One method which overcomes this problem is tomographic STED (tomoSTED) microscopy. In tomoSTED microscopy, one-dimensional depletion patterns facing in different directions are successively applied in order to acquire a highly-resolved image in two dimensions. In this context, the number of addressed directions depends on the desired angular homogeneity of the point spread function or the optical transfer function and thus on the resolution increase as compared to diffraction-limited imaging. At a reasonable angular homogeneity the light dose and thus bleaching can be reduced, as compared to conventional STED microscopy. Here, we propose and demonstrate for the first time, to our knowledge, that the number of required depletion pattern orientations can be reduced by combining tomoSTED microscopy with the concept of image scanning microscopy (ISM). With our realization of an ISM-tomoSTED microscope, we show that approximately a factor of 2 lower number of orientations are required to achieve the same resolution and image quality as in tomoSTED microscopy.