Abstract
Selective plane illumination microscopy (SPIM) represents a preferred method in dynamic tissue imaging, because it combines high spatiotemporal resolution with low phototoxicity. The OpenSPIM system was developed to provide an accessible and flexible microscope set-up for non-specialist users. Here, we report Structured SPIM (SSPIM), which offers an open-source, user-friendly and compact toolbox for beam shaping to be applied within the OpenSPIM platform. SSPIM is able to generate digital patterns for a wide range of illumination beams including static and spherical Gaussian beams, Bessel beams and Airy beams by controlling the pattern of a Spatial Light Modulator (SLM). In addition, SSPIM can produce patterns for structured illumination including incoherent and coherent array beams and tiling for all types of the supported beams. We describe the workflow of the toolbox and demonstrate its application by comparing experimental data with simulation results for a wide range of illumination beams. Finally, the capability of SSPIM is investigated by 3D imaging of Drosophila embryos using scanned Gaussian, Bessel and array beams. SSPIM provides an accessible toolbox to generate and optimize the desired beam patterns and helps adapting the OpenSPIM system towards a wider range of biological samples.
Highlights
Research on the structure and function of living matter often relies upon imaging of biological samples within the μm to nm range using conventional optical microscopy
After validating the various outputs of Structured SPIM (SSPIM) informed spatial light modulator (SLM) generated patterns for beam shaping, we examined the capability of SSPIM to apply the desired beam patterns when imaging a biological sample
We introduced the SSPIM toolbox which was built for beam shaping within the OpenSPIM environment[18]
Summary
Research on the structure and function of living matter often relies upon imaging of biological samples within the μm to nm range using conventional optical microscopy. A well-known and robust method for high-resolution 3D imaging is selective plane illumination microscopy (SPIM). SPIM is characterized by minimal photo bleaching and low photo toxicity, and its most attractive application represents the imaging of relatively large and living biological specimens at a high spatial and temporal resolution (Table 1). In order to suppress the shadows, a pivoting method is applied in which the static light sheet is scanned in different angles within each plane using a scanning mirror[3]. This problem can be overcome by using different illumination beams, such as a circular Gaussian beam, which is scanned over the www.nature.com/scientificreports/. The imaging properties of the SPIM can be further improved by more sophisticated, non-diffracting beams, such as the Airy beam or the Bessel beam
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