During the last decade, selective plane illumination microscopy (SPIM) has proven to be one of the most suitable techniques for three-dimensional time lapse imaging. By confining the excitation light to a sheet, SPIM combines axial sectioning capability with minimal light exposure and fast, camera-based image acquisition [1]. However, the typical arrangement of two objective lenses perpendicular to each other provides a number of challenges in terms of instrument design and sample geometry, especially if the use of high numerical aperture (NA) lenses is desired. A popular approach is to dip into the sample container from the top, both lenses at a 45 degree angle with respect to the sample plane [2,3]. Instead, our new design is based on a regular inverted microscope where the sample is illuminated from the side via an accessory. A custom designed chamber is used to allow side illumination. This way, all microscope ports remain available for other purposes and there is unrestricted access from the top. Without the need of dipping into the sample container, smaller sample volumes (< 1 ml) can be realized and the use of high NA lenses is facilitated. Also, isolation of optics and sample allows imaging of sealed sample containers when demanded, e.g., for samples treated with potent toxins. Further, in this design, the orientation of the imaging plane is parallel to the surface of the sample container which is desirable for flat samples where it maximizes the field of view.Work supported in part by NIH grants P50 GM076516 and P41 GM103540.[1] Huisken, J. et al., Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy. Science 305, 1007–1009 (2004).[2] Hedde, P.N. et al., Rapid Measurement of Molecular Transport and Interaction inside Living Cells Using Single Plane Illumination. Sci. Rep. 4, 7048 (2014).[3] Hedde, P.N. et al., 3D fluorescence anisotropy imaging using selective plane illumination microscopy. Opt. Express 23, 22308–22317 (2015).