Abstract
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.
Highlights
The development of pharmacological treatments for neuropsychiatric and neurodegenerative diseases has been hampered by the poor availability of preclinical models that adequately capture the complexity of human disorders (Gonzalez et al, 2017).Human inducible pluripotent stem cells offer a promising platform for disease modeling and drug screening
A comparably new technique is the directed differentiation and reprogramming of patient fibroblasts into neurons, astrocytes, microglia and oligodendro-cytes. Their combinational culture permits the growth of embryonic bodies (EBs) and brain organoids, 3-D cultures that—in many aspects—recapitulate the development of the human brain (Fatehullah et al, 2016; Giandomenico and Lancaster, 2017)
When Human inducible pluripotent stem cells (hIPSCs) had reached about 80% confluence, they were detached with versene (ThermoFisher)
Summary
Human inducible pluripotent stem cells (hIPSCs) offer a promising platform for disease modeling and drug screening. A comparably new technique is the directed differentiation and reprogramming of patient fibroblasts into neurons, astrocytes, microglia and oligodendro-cytes. Their combinational culture permits the growth of embryonic bodies (EBs) and brain organoids, 3-D cultures that—in many aspects—recapitulate the development of the human brain (Fatehullah et al, 2016; Giandomenico and Lancaster, 2017). HIPSCs, EBs, and brain organoids enable observations and experiments that were previously inconceivable, neither on human subjects nor in animal models (Lancaster et al, 2013; Kelava and Lancaster, 2016). Fully vascularized brain organoids have spurred hopes of growing even larger 3-D cell assemblies (Mansour et al, 2018), bringing the hitherto theoretical ‘‘brain in a vat’’1 within reach of the imaginable
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