human 3d cerebral organoid medium  (Hopstem Biotechnology)

Journal: bioRxiv

Article Title: 3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

doi: 10.1101/672584

Figure Lengend Snippet: a , The system works in epi-fluorescence mode. A large volume within the 3D imaging range is excited simultaneously by a highly-inclined illumination and collected by the DAOSLIMIT. The inhomogeneous distribution of the refractive index in multicellular specimens produces strong spatially nonuniform aberrations at the back-pupil plane, which can be segmented for correction with adaptive optics. For illustration, light from different sub-apertures is labelled with different colours. A microlens array is inserted at the image plane for parallel acquisition of multiplexed phase-space measurements, whose resolution is further enhanced by the scanning process with a two-dimensional galvo scanning system. During reconstruction, we first realign the pixels from the raw data into the high-resolution multiplexed phase-space. Then, a mutual iterative tomography algorithm is employed to obtain the high-resolution volume with pixel-wise wavefront corrections. b , The multiplexed phase-space measurements can be synthesized for 3D reconstruction with the digital beam propagation. However, the sample-induced aberration will result in a distorted focus. We can digitally shift the sub-aperture point spread function (PSF), akin to applying a correction wavefront estimated during the volume reconstruction, to create a perfect focus. Both the spatial overlap induced by scanning and the frequency aliasing induced by the small aperture of each microlens facilitate the incoherent synthetic aperture during the 3D reconstruction, up to the diffraction limit of the whole objective’s NA.

Article Snippet: Human 3D cerebral organoids were generated and maintained in human 3D cerebral organoids medium (Cat # HopCell-3DM-001, Hopstem Bioengineering) by Hopstem Bioengineering according to the manufacturer's protocols.

Techniques: Fluorescence, Imaging, Tomography, Synthesized

Journal: bioRxiv

Article Title: 3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

doi: 10.1101/672584

Figure Lengend Snippet: a , Orthogonal MIPs from 16-μm-thick slabs of the focal stack captured by WFM, including 90 axial slices at 200-nm steps. The sample used here is a HeLa cell labelled with actin (green) and nuclei (blue). The Fourier transforms of the MIPs are shown on the right to indicate the degrees of information recovery in low-SNR conditions. The yellow dashed circle corresponds to the spatial frequency of the Abbe diffraction limit for comparison. b , Results reconstructed by applying 3D RL deconvolution to the focal stack in a, with enhanced SNR and contrast. c , Results obtained by traditional LFM with 3D deconvolution, showing much lower spatial resolution and reconstruction artefacts close to the native object plane. d , Results reconstructed by DAOSLIMIT with a 67% overlap ratio (corresponding to 3 × 3 lateral shifts), indicating higher SNR and resolution especially in the x-z and y-z planes (notably only 9 images are used for 3D reconstruction, 10 times less than WFM). Scale bar: 20 μm.

Article Snippet: Human 3D cerebral organoids were generated and maintained in human 3D cerebral organoids medium (Cat # HopCell-3DM-001, Hopstem Bioengineering) by Hopstem Bioengineering according to the manufacturer's protocols.

Techniques:

Journal: bioRxiv

Article Title: 3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

doi: 10.1101/672584

Figure Lengend Snippet: a , Colour-coded MIP of the 3D volume reconstructed by sLFM without DAO. Different colours encode different depths. Scale bar: 10 μm. b , Colour-coded MIP of the 3D volume reconstructed by DAOSLIMIT. Scale bar: 10 μm. c , Different corrected wavefronts applied to different areas across the field of view in a. d , Orthogonal MIPs from 2-μm-thick slabs of the selected area in a, with a cross-section profile illustrating the resolution loss from aberration. The white arrow points to a structure blurred by the aberration. Scale bar: 2 μm. e , Orthogonal MIPs from 2-μm-thick slabs of the selected area in b, with a cross-section profile illustrating the diffraction-limited performance with DAO. The average correction wavefront of the selected region is shown in the inset at the unit of wavelength. The white arrow points to the ring structure resolved after DAO. Scale bar: 2 μm. f - g , Colour-coded MIPs of the selected areas in b at different time stamps marked at the bottom row. The arrow in f indicates a fast-moving mitochondrion in 3D, whereas the arrow in g indicates a mitochondrial fusion process happening in 3D. Scale bar: 1 μm. h , The distributions of the average speed and tracking length with the same tracking algorithm applied to 3D videos without DAO, 2D videos with DAO and 3D videos with DAO. More accurate speed can be estimated in 3D, whereas the SNR and resolution enhancements by DAO facilitate longer tracking duration and more mitochondria under tracking.

Article Snippet: Human 3D cerebral organoids were generated and maintained in human 3D cerebral organoids medium (Cat # HopCell-3DM-001, Hopstem Bioengineering) by Hopstem Bioengineering according to the manufacturer's protocols.

Techniques:

Journal: bioRxiv

Article Title: 3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

doi: 10.1101/672584

Figure Lengend Snippet: a , Orthogonal MIPs of the 3D membrane structures reconstructed by WFM with 3D deconvolution (left panel) and DAOSLIMIT (right panel). b , 100 Hz 3D imaging of vesicle dynamics and potential vesicle trafficking in one zebrafish epithelial cell in the gastrulation stage (Supplementary Video 2). The colour-coded MIP of the volume at 0 s is shown in the left panel with different colours corresponding to different depths. The 3D tracking trajectories of every vesicle are shown in the right panel. c , The colour-coded MIP of the in vivo membrane dynamics reconstructed without DAO and time-loop algorithms are shown on the left, whereas that reconstructed with DAO, and time-loop algorithms is shown on the right, indicating improvement in both SNR and resolution (Supplementary Video 3). d , The effectiveness of the time-weighted algorithm on the compensation of the sacrificed temporal resolution resulting from lateral scanning (Supplementary Video 3). Orthogonal MIPs across 5-μm-thick slabs of the selected area in c are displayed with different time stamps marked at the bottom row. The results without the time-weighted algorithm are shown on the first row, and results with the time-weighted algorithm are shown on the second row, which eliminates the motion blur of a moving vesicle (white arrow) with the same spatial resolution. ( e-g ) Orthogonal MIPs of the selected areas in c with different time stamps marked at the bottom row (Supplementary Video 4). Dynamic filopodia retraction processes can be observed in e and g. A cell division process showed gradual membrane enrichment dynamics at the boundary of daughter cells in f. h – i , The colour-coded MIP of the 3D membrane dynamics in vivo at 10 Hz. Different zoom-ins with orthogonal MIPs in h show the 3D movements of a migrasome and a clear cell migration in 3D at high spatiotemporal resolution (Supplementary Video 6). High-speed fluctuations of the filopodia membrane can be observed in i, including filopodia retraction process and migrasome movements (Supplementary Video 7). Scale bar: 10 μm.

Article Snippet: Human 3D cerebral organoids were generated and maintained in human 3D cerebral organoids medium (Cat # HopCell-3DM-001, Hopstem Bioengineering) by Hopstem Bioengineering according to the manufacturer's protocols.

Techniques: Imaging, In Vivo, Migration

Journal: bioRxiv

Article Title: 3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

doi: 10.1101/672584

Figure Lengend Snippet: a , Colour-coded MIP of the GCamp6s-labelled human 3D cerebral organoids obtained by DAOSLIMIT. Different colours represent different depths. b , Temporal-coded MIP of the selected area in a for the start time (the time point when the signal reaches 10% of the maximum intensity). Different colours correspond to the start points of the spontaneous calcium response, and the intensity represents the MIP of the temporal trace standard deviation for each voxel. Spontaneous 3D calcium propagation evoked from the intersection of two neurons can be clearly observed. c , Temporal-coded MIP of the selected area in a, illustrating the rise time (the time required to increase from 20% to 80% of the maximum intensity). Different colours correspond to the rise time of the spontaneous calcium response, and the intensity represents the MIP of the temporal trace standard deviation for each voxel. d , Orthogonal MIPs of the selected areas in a with different time stamps marked at the bottom row (Supplementary Video 9). The video was captured at 30 Hz. e , The temporal traces (ΔF/F 0 ) of the ROIs labelled in a. f , Colour-coded MIP of the Drosophila larval Cho neurons with the jGCaMP7s indicator. Different colours represent different depths. g, Temporal-coded MIP for start time. h , Temporal-coded MIP for rise time. i , The temporal traces (ΔF/F 0 ) of the ROIs labelled in f. The red arrow indicates the time point when we applied the 500 Hz sound stimulus. j, Orthogonal MIPs of the selected areas in f with different time stamps marked at the bottom row (Supplementary Video 10). The video was captured at 100 Hz. Scale bar: 10 μm.

Article Snippet: Human 3D cerebral organoids were generated and maintained in human 3D cerebral organoids medium (Cat # HopCell-3DM-001, Hopstem Bioengineering) by Hopstem Bioengineering according to the manufacturer's protocols.

Techniques: Standard Deviation