Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

Abstract

Background: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points.

Results: Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes.

Conclusion: We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.

Fig. 1

Setup and resolution of Airy-beam light sheet microscopy (ALSM). a ALSM system is equipped with 2 water dipping objectives—10 × 0.3 NA illumination and 20X 0.5 NA detection objective—positioned at a 45° angle. Biological specimens are placed on the X-Y-Z stage. Movement of the stage is controlled by a Z-axis Stepper motor (ZSM) which moves the sample along the optical axis of the detection objective. The system is equipped with 405 nm, 488 nm, 561 nm and 640 nm wavelength lasers. b Illumination objective focuses the Airy-beam (green) on to sample and the detection objective captures the emission (yellow) at 90 degrees. The geometry of the objectives gives the system a (hypothetical) working distance of 3.5 mm. c An example of the Airy-beam produced by focusing the 488 nm laser though water. d Representative image of a 500 nm Tetraspeck fluorescent beads imaged at 405, 488, 561 and 640 wavelengths; image has not undergone deconvolution. Characteristic Airy lobes can be seen in the axial plane. e Deconvolved image of the fluorescent bead in C). Axial Airy lobes have been removed following deconvolution. f Image of a single 500 nm Tetraspeck fluorescent bead; the PSF of a single 500 nm bead is 0.625 μm diameter at full width at half maximum (FWHM) when imaged at 488 nm. Scale bars = 500 nm unless stated otherwise

Fig. 2

Confocal imaging of wholemount day 7 human cortical spheroids (hCSs) is limited at depth. a Representative confocal images of intact (wholemount) day 7 hCS showing strong immunostaining for the neuroepithelial/NPC markers Nestin and Sox2. b Representative confocal image of wholemount day 7 hCS demonstrates hallmarks of the emergence of neural rosettes, as determined by N-cadherin (N-cad) staining of apical membrane and surrounding Pax6-positive cells. c Maximally projected and orthogonal views of Z stacks of intact Day 7 hCSs acquired by confocal imaging. Confocal imaging displays a drop-off in fluorescence intensity correlated with acquisition depth (Z planes). (i) DAPI, a non-antibody stain for DNA shows incomplete detection throughout the tissue-like structure. Similarly, (ii) Nestin-positive and (iii) Sox2-positive immunostaining displays even greater drop off in depth-detection, shown in (iii) as a fluorescence-intensity heatmap. d Graphic depicting changes in fluorescent intensity (A.U.) with increasing imaging depth (μm) of Ci–iii. Scale bars = 50 μm

Fig. 3

Confocal imaging of cryo-sectioned day 17 hCSs reveals incomplete acquisition of neural rosette 3D morphology. a–d Representative confocal images of sectioned (20 µm) day 17 hCSs; a ZO-1 staining of apical membrane is surrounded by apico-basal polarised Nestin filaments; b Pax6-positive cells self-organise around N-cad positive foci; c Immature DCX-positive neuronal processes are found outside of neural rosette structures; and d Map2-positive neuronal processes are also found to surround Pax6-positive NPCs organised into rosettes. e Confocal imaging of 60 μm sectioned day 17 hCSs express signs of cortical rosette formation. Newly formed PKCλ-positive membranes are seen at the edge of the tissue as indicated by yellow arrows. f Within thicker sections (60 μm), internalised tubular structures positive for N-cad are observed, suggesting that the inner lumen of neural rosettes may form into tube-like structures. g Phalloidin 488-positive actin filaments (RDYPROBE) show a concentrated organisation around ventricle-like structures. Magenta arrows in e–g images highlight the ovoid formation of cortical rosettes with a pinched end, whereas blue arrows highlight formation of a tubular-like structure. Scale bars = 100 µm (a–d); 50 μm (e–g)

Fig. 4

Visualising internal structures of hCSs using Airy-beam light sheet fluorescent microscopy. a Clearvolume 3D representation of multiple internalised rosette lumen of differing sizes shown by RDYPROBE staining (F-actin) with dimensional scale. b Magnified region (a, dotted line) showing characteristic ovoid or tubular shaped lumens expected of a ventricle-like structure. c Composite (i) and individual channel max-projected images of (ii) RDYPROBE (F-actin) staining and (iii) Nestin-positive filaments around apical membranes, showing the presence of multiple large rosettes, Scale bars = 100 μm. d 3D render of magnified region from ci (dotted line) displaying of a singular rosette lumen co-stained for radial Nestin filaments and RDYPROBE (F-actin) with dimensional scale. e Orthogonal views of stained tissues permit examination of a cortical rosette in all axes. Scale bar = 50 μm

Fig. 5

ALSM imaging of cleared hCSs. a Representative 3D volume image (600 × 600 × 600 µm) of cleared day 30 hCS immunostained with ZO-1 (green) and Sox2 (magenta) and imaged by ALSM. b Schematic image of ALSM optical sections of cleared hCS shown in c. c ALSM optical Sects. (600 × 600 × 50 µm) at 450, 500 and 550 µm depth, of cleared hCS shown in a. Cyan arrows indicate ZO-1-positive rosette lumen with surrounding radially organised Sox2-positive NPCs. Green and red bars indicate scale in X and Y planes (600 µm)—blue scale bar indicates scale in Z plane (600 µm in a and 50 µm in c)

Fig. 6

Visualisation and quantification of neural rosette lumen morphology in intact cleared hCSs. a Representative 3D volume images of ZO-1-positive apical lumen membranes of cleared day 30 hCS. b Rosette lumens were defined as objects in Volocity (PerkinElmer) by thresholding volumetric regions based on ZO-1 channel. Objects with volumes of less than 150 µm³ were discarded. c, d Frequency distribution of neural rosette lumen volume (µm³; c) and surface area (µm²; d). Measurements of these morphological parameters revealed a single neural rosette with a lumen with a volume of 192,484.21 µm³ and surface area of 146,697.61 µm². e, f Frequency distribution of neural rosette lumen length (µm; e) and shape factor f, g Digital zoom of largest neural rosette lumen. Green channel indicates ZO-1-positive apical membrane; magenta channel indicates Sox2-positive NPCs surrounding central lumen. h Threshold image of ZO-1-positive lumen demonstrating morphology of structure in 3D. i Representative 3D render of largest neural rosette lumen demonstrating the radial organisation of Sox2-positive NPCs surrounding ZO-1-positive (green) rosette lumen