Supplementary Figure 9: Comparison of four-view and orthogonal dual-view imaging (x-y sections).

Enlarged views of x-y image sections for the 24 regions marked by white boxes in Supplementary Fig. 8. The images show a side-by-side comparison of multi-view deconvolved image data obtained with orthogonal four-view imaging (IsoView approach) and orthogonal dual-view imaging (diSPIM approach) for a live stage 17 Drosophila embryo expressing mRFP1 in all cell nuclei. All image data were acquired with the IsoView microscope, using either two (diSPIM) or all four (IsoView) of the microscope’s imaging arms. Imaging and multi-view deconvolution settings were otherwise identical. The line profiles next to the image panels represent normalized intensity profiles along the image y-axis at the respective x-locations indicated by the black arrows. Line profiles are shown both for dual-view imaging (magenta) and four-view imaging (blue). Numbers provided next to the line profiles indicate full-width-at-half-maximum (FWHM) measurements for the left-most intensity peak at the respective x-location. The optical axes of the two objectives employed in dual-view imaging were aligned with the dorsoventral and lateral embryonic axes. The 24 regions analyzed in this comparison cover a wide range of illumination and detection path lengths across the embryo, including regions with diSPIM illumination and detection paths up to 60 µm (ROIs 1, 3, 5 and 7), regions with dorsoventral paths >60 µm and lateral paths <60 µm (ROIs 2, 4, 6 and 8), regions with dorsoventral paths <60 µm and lateral paths >60 µm (ROIs 13-15, 17, 18, 20 and 21-24) and regions with dorsoventral and lateral paths >60 µm (ROIs 9, 10-12, 16 and 19). Dual-view imaging provides excellent spatial resolution up to a maximum depth of approximately 60 µm, yielding high-resolution coverage of approximately one quarter of the specimen. Four-view imaging typically achieved identical spatial resolution in these regions, although a quarter of our data points attest to higher resolution in the IsoView data. These differences are likely rooted in the fact that the respective regions are captured in sufficiently high quality by three out of the four views provided by IsoView, thus adding a third source of high-frequency image content. For illumination and detection path lengths exceeding 60 µm, resolution and signal strength in dual-view imaging degrade substnatially and cellular resolution is typically compromised. In contrast, high-resolution coverage of almost the entire embryo is achieved with four-view imaging, consistent with the intuition that opposing views in IsoView should effectively double optical access along both illumination and detection axes. Notably, four-view imaging still resolves neighboring cell nuclei as distinct objects in some the deepest regions of the embryo with optical path lengths close to 100 µm (ROIs 13 and 16). Scale bars, 5 µm.