Journal: bioRxiv

Article Title: Cerebellar Granule Cells Develop Non-neuronal 3D Genome Architecture over the Lifespan

doi: 10.1101/2023.02.25.530020

Figure Lengend Snippet: (A) Schematic of overall study design. To understand the genomic underpinning for cerebellar development and aging, we created a 3D genome atlas (filled dots and squares) of 13,161 cells from the cerebellum and cerebral cortex (and additionally, hippocampus in mouse) across the human (0.1–86 years) and mouse (0–2 years) lifespan, using our diploid chromosome conformation capture (Dip-C) , population-scale Dip-C (Pop-C), and virus-enriched Dip-C (vDip-C) methods (lower right). We additionally created a multi-ome (simultaneous transcriptome and chromatin accessibility) atlas (circles) of 63,768 cells from the developing cerebellum (human: 0.1–2.3 years and 1 adult; mouse: postnatal day (P) 14) (upper right; Methods). Data were compared to mouse cerebral cortex and hippocampus 3D genome data from previous work . (B) Integrative transcriptome analysis of the 7 human multi-ome samples revealed transcriptionally immature granule cells (dashed outlines) in the newborn cerebellum. We visualized the transcriptome portion of our human multi-ome atlas with t-distributed stochastic neighbor embedding (t-SNE) after cross-sample integration (left: each dot represents a single cell). Granule cells existed in both mature form, which we termed transcriptional (T) stage 5 (T5; darkest purple), and a variety of immature forms, which we termed T1–T4 (dashed outlines; lighter shades of purple). Transcriptionally immature granule cells were abundant (14–34%) in the first postnatal year, but vanished (<1%) in the 2.3- and 37.6-year-old donors (right). (C) Representative gene expression profiles of cell type–specific marker genes (see for granule cells).

Article Snippet: Wide application of the new 3D genome profiling technologies (Pop-C and vDip-C), along with their corresponding analysis pipelines, to many brain regions and tissues of the human body may contribute to solving longstanding challenges such as dissecting the genetic basis of inter-individual 3D genome variability, characterizing ultra-rare cell types, and revealing the full extent of the diversity and dynamics of 3D genome organization.

Techniques: Virus, Gene Expression, Marker

Journal: bioRxiv

Article Title: Cerebellar Granule Cells Develop Non-neuronal 3D Genome Architecture over the Lifespan

doi: 10.1101/2023.02.25.530020

Figure Lengend Snippet: (A) Traditional 3C/Hi-C methods such as Dip-C measure one sample at a time (left); in contrast, the new population-scale Dip-C (Pop-C) method simultaneously profiles many samples at once, by pooling samples and computationally demultiplexing single cells based on linear DNA sequences (genotypes), thereby eliminating batch effects and substantially reducing labor and cost (right). This approach was found to be highly reproducible; note consistency between the same sample (Donor 4) assayed both individually (left) and by Pop-C (right), visualized with t-SNE of the single-cell 3D genome (based on single-cell chromatin A/B compartment values; scA/B) ; each dot represents a single cell). (B) Rare cell types, such as adult Purkinje cells (<0.5% in mouse), were often missed by conventional Dip-C (left); in contrast, virus-enriched Dip-C (vDip-C) enabled efficient isolation of adult Purkinje cells with minimally invasive, fixation-robust fluorescent labeling in wild-type mouse tissue (right). (C – D) With Pop-C and vDip-C, we created a high-resolution, cross-species 3D genome atlas for the developing and aging cerebellum (with cerebral cortex as counterpoint), and resolved the first 3D genome structures of single cerebellar cells (bottom (D)). In both species (human (C); mouse (D)), all samples were visualized together with t-SNE of scA/B; 3D genome structure types were identified with hierarchical clustering of scA/B (top). Cerebellar granule cells (shades of purple) exhibited by far the most dramatic structural transformation—born with an immature structure type, which we termed structural (S) stage S1 (lightest purple), that closely resembled forebrain neurons (shades of brown), and evolving progressively into new structure types, which we termed stages S2–S5 (darker shades of purple), that drastically differed from all other neurons as the cerebellum developed and aged. Abundances of the various S stages peaked around ages 0.2, 1, 10, 30, and 80 yr in human, and around P3, P14, P21, P56 (~2 months), and P365 (~12 months) in mouse (bottom, right).

Article Snippet: Wide application of the new 3D genome profiling technologies (Pop-C and vDip-C), along with their corresponding analysis pipelines, to many brain regions and tissues of the human body may contribute to solving longstanding challenges such as dissecting the genetic basis of inter-individual 3D genome variability, characterizing ultra-rare cell types, and revealing the full extent of the diversity and dynamics of 3D genome organization.

Techniques: Hi-C, Virus, Isolation, Labeling, Transformation Assay

Journal: bioRxiv

Article Title: Cerebellar Granule Cells Develop Non-neuronal 3D Genome Architecture over the Lifespan

doi: 10.1101/2023.02.25.530020

Figure Lengend Snippet: (A) The most prominent architectural changes in granule cells (first 5 columns) were emergence of ultra-long-range (10–100 Mb) intra-chromosomal contacts (dashed boxes) thought to be exclusive to non-neuronal cells ( , ) such as microglia (second to last column). Distribution of genomic distances of chromatin contacts (in base pairs (bp); on logarithmic scale) quantified by histogram for each 3D genome structure type (top) in human (middle) and mouse (bottom). (B) Emergent ultra-long-range contacts in granule cells formed prominent checkerboard patterns on contact maps—suggesting strong phase separation between the newly formed chromatin A/B compartments; this effect was generally stronger in the gene-poor, heterochromatic compartment B. For each species, an aggregated contact map of each structural (S) stage (S1–S5) of granule cell maturation was shown for an example chromosome (lower left triangles) and for an example zoomed-in (50-Mb) genomic region (upper right triangles). Contact maps (matrices of contact frequencies) were visualized with Juicebox , both as absolute values (first and third rows) and as relative changes compared to stage S1 (second and fourth rows). Zoomed-in regions are homologous between human and mouse; mouse coordinates were inverted for synteny. Dashed boxes highlight prominent changes during granule cell maturation. Bin size: 250 kb. (C) Granule cells formed specific inter-chromosomal contacts during development and aging; note increasing interactions between certain chromosomes—most prominently within a multi-chromosome hub of Chr 1/9/11/14/15/16/17/21/22, and between chromosome pairs such as Chr 2/9, Chr 4/14, Chr 8/11, Chr 13/20 in human. In each species, aggregated contact maps are shown for 2 example chromosome pairs. Dashed boxes highlight prominent changes during granule cell maturation. Bin sizes: 6 Mb (human genome-wide); 5 Mb (mouse genome-wide); 500 kb (zoom-in).

Article Snippet: Wide application of the new 3D genome profiling technologies (Pop-C and vDip-C), along with their corresponding analysis pipelines, to many brain regions and tissues of the human body may contribute to solving longstanding challenges such as dissecting the genetic basis of inter-individual 3D genome variability, characterizing ultra-rare cell types, and revealing the full extent of the diversity and dynamics of 3D genome organization.

Techniques: Genome Wide

Journal: bioRxiv

Article Title: Cerebellar Granule Cells Develop Non-neuronal 3D Genome Architecture over the Lifespan

doi: 10.1101/2023.02.25.530020

Figure Lengend Snippet: (A) Relationship between transcriptional and architectural changes in granule cells. We calculated the mean scA/B of each 1-Mb genomic region at each structural (S) stage (S1–S5; columns) of granule cell maturation, and identified the top 20% dynamic regions (rows) based on between-stage variance. Dynamic regions (rows) are shown in a heatmap (left) ordered by hierarchical clustering of scA/B correlation, and clustered into 2 temporal modes: continuous up- or down-regulation across the lifespan. Each mode was additionally visualized with aggregated scA/B of all its regions on the 3D genome t-SNE plot (right). (B) Continuously-progressing scA/B changes correlated with expression of mature granule cell–specific genes—suggesting continued 3D genome rewiring well after initial transcriptional up-regulation. Mean scA/B of each 1-Mb genomic region harboring conserved, granule cell–specific marker genes (Supplementary Table 5 of ) at each S stage reveals that the majority of such regions continually increased scA/B across the lifespan (left). Shown: aggregated scA/B of all such regions on the 3D genome t-SNE plot (right). (C) The granule cell–specific marker gene GABRA6 (arrows) steadily increased scA/B by losing contacts with two nearby gene-poor regions over the lifespan (dashed boxes; note the gene-poor regions formed strong contacts with each other over time) in both species. Contact maps were visualized with Juicebox , both as absolute values (first and third rows) and as relative changes compared to stage S1 (second and fourth rows). Zoom-in regions homologous between human and mouse; mouse coordinates inverted for synteny. Bin size: 250 kb. (D) Functional perturbation by disrupting chromatin remodelers. Bulk Dip-C on whole adult cerebellum (chiefly granule cells) of mice with clinically-relevant heterozygous deletion of autism-implicated genes Arid1b (middle) or Chd8 (bottom) had little effect on 3D genome (top; stage S4 chosen to match ages). Contact maps visualized both as absolute values (left and middle) and as relative changes (right). Dashed boxes highlighted prominent changes during granule cell maturation. Bin size: 250 kb.

Article Snippet: Wide application of the new 3D genome profiling technologies (Pop-C and vDip-C), along with their corresponding analysis pipelines, to many brain regions and tissues of the human body may contribute to solving longstanding challenges such as dissecting the genetic basis of inter-individual 3D genome variability, characterizing ultra-rare cell types, and revealing the full extent of the diversity and dynamics of 3D genome organization.

Techniques: Expressing, Marker, Functional Assay

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: Measurement images of the master mold and imprinted single lens obtained with a micro 3D profiler. Scale bar: 20 μm.

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: 3D profiler measurement data for master mold.

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: 3D profiler measurement data for 1st mold replica (PDMS).

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: 3D profiler measurement data for 2nd single-mold replica (stamp).

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: 3D profiler measurement data for 3rd mold replica (WSM).

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: 3D profiler measurement data for 4th WSM replica (PUA on PET film).

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: 3D profiler measurement data for 4th WSM replica (PDMS).

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: 3D profiler measurement data for 5th replica of the final product (402).

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: 3D profiler measurement data for 5th replica of the final product (OM 625).

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: 3D profiler measurement data for 5th replica of the final product (OrmoComp).

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: Micromachines

Article Title: Shrinkage-Considered Mold Design for Improvement of Micro/Nano-Structured Optical Element Performance

doi: 10.3390/mi11100941

Figure Lengend Snippet: 3D profiler measurement data for 5th replica of the final product (OrmoClear FX).

Article Snippet: Sagging depths of the micro lens were measured by a 3D profiler (Keyence VK-250K), and the curvatures of single micro lens were observed by a white interferometer (NV-3200 Nano).

Techniques:

Journal: bioRxiv

Article Title: Under-Oil Autonomously Regulated Oxygen Microenvironments: A Goldilocks Principle-Based Approach For Microscale Cell Culture

doi: 10.1101/2020.12.16.423117

Figure Lengend Snippet: The kinetics of oxygen diffusion in multiliquid-phase microsystems and 3D diffusion profiles from COMSOL Multiphysics. a) and b) Oxygen level (O 2 , %) versus time plots and 3D diffusion profiles for the influence of media depth. c), d), e), and f) Oxygen level (O 2 , %) versus time plots and 3D diffusion profiles for the influence of oil type [fluorinated oil (FC-40) versus silicone oil (SO)], depth, and viscosity [5 cSt (SO5) and 500 cSt (SO500) of silicone oil]. g) and h) Oxygen level (O 2 , %) versus time plots and 3D diffusion profiles from PDMS (elastomer)-based microdevices. i), j), and k) Oxygen level (O 2 , %) versus time plots and 3D diffusion profiles from PDMS (elastomer)-based microdevices with oil (i.e. fluorinated oil) or double-oil (i.e. silicone oil + fluorinated oil) overlay.

Article Snippet: The 3D diffusion profiles were generated in COMSOL with a color-coded O 2 , % bar denoting 0% and 21% O 2 in culture media.

Techniques: Diffusion-based Assay, Viscosity