Confocal microscopes have been the workhorses of 3-D biological imaging, but they are slow, offer limited depth penetration and collect only ballistic photons. With their inefficient use of excitation photons they expose biological samples to an often intolerably high light burden. The speed limitation and photo-bleaching risk can be somewhat relaxed in a spinning-disk geometry, due to shorter pixel dwell times and rapid re-scans during image capture. Alternatively, light-sheet microscopes rapidly image large volumes of transparent or chemically cleared samples. Finally, with infrared excitation and efficient scattered-light collection, 2-photon microscopy allows deep-tissue imaging, but it remains slow. Here, we describe a new optical scheme that borrows the best from three different worlds: the speed and direct-view from a spinning-disk confocal, deep tissue-penetration and intrinsic optical sectioning from 2-photon excitation, and a large field of view and a low invasiveness of a selective-plane illumination microscope – all with a single objective lens. We validate the performance of our 2-photon spinning-disk microscope in various applications that have in common to simultaneously require a large depth penetration, high speed and larger fields of view. Beyond biological fluorescence, we demonstrate an application in material science, imaging coherent non-linear scattering from a 3-D nano-porous network.

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that—in many aspects—recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that—unlike two- or three-lens light-sheet microscopes—it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petráň spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.