Abstract
Stimulated emission depletion (STED) microscopy is able to image fluorescence labeled samples with nanometer scale resolution. STED microscopy is typically a point-scanning method, limited by the high intensity requirement of the depletion beam. With the development of high peak power lasers, two dimensional parallel STED microscopy has been developed. Here, we develop the theoretical basis for extending STED microscopy to three dimensional imaging in parallel. This method uses structured illumination (SI) to generates a three dimensional depletion pattern. Compared to the two dimensional parallel STED microscopy, the 3D SI-STED microscopy generates intensity modulation along the light propagation direction without requiring higher laser power. This method not only achieves axial super-resolution of STED microscopy but also greatly reduces photobleaching and photodamage for 3D volumetric imaging.
Highlights
Super-resolution microscopy is able to image fluorescence labeled samples with nanometer scale resolution
We develop the theoretical basis for extending Stimulated emission depletion (STED) microscopy to three dimensional imaging in parallel
structured illumination (SI)-Stimulated emission depletion (STED) microscopy potentially can be used for large volume super-resolution imaging of biological samples
Summary
Super-resolution microscopy is able to image fluorescence labeled samples with nanometer scale resolution. STED microscopy is a point-scanning method [1], because of the limited power of lasers for depletion. The point-scanning method limits the imaging speed of STED microscopy. 2D STED microscopy overlaps two incoherent fringe patterns in the orthogonal directions to generate a grid in the focal plane. The donut-shape spot matrix is generated by structured illumination in the parallel 2D STED microscopy [9]. This grid allows 2D fast imaging of thin samples, this approach has no depth sectioning ability for thick samples. The 2D grid pattern propagating outside of the focal plane causes photobleaching of samples
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