Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D spatial and data analysis workflow 3D spatial analysis of 3D-IF stained and optically cleared samples with UltraMicroscope Blaze™ light sheet microscope (Step 16). Post processing (stitching) in case data was acquired with tile-scanning (Step 18). Surfaces generation of autofluorescence and target region with Imaris software (Oxford Instruments) and target plane definition with “Oblique Slicer” tool (Step 20 and 20).

Article Snippet: Figure 7 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”).

Techniques: Staining, Microscopy, Software

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: Overview of schematic process steps to define and analyze region of interest within 3D spatial analysis data Visualization of process Steps 20-29 (A). Definition of target plane to optimize intersection with region of interest using “Oblique Slicer” tool of Imaris software (Step 20). Adding additional ”Oblique Slicer” for either natural or artificial reference plane (Step 21 and B). Series of simulated optical sections passing through previously defined target plane, to generate a cutting path as a quality control during physical sectioning and exemplary snapshots (Step 25 and C–H). Angle determination (Step 26) and preparation of respective agarose block for angle correction (Step 29). Scale bars: (C–H) 500 μm.

Article Snippet: Figure 7 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”).

Techniques: Software, Control, Blocking Assay

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: Illustration of MACSima™ Imaging Cyclic Staining (MICS) principle MICS technology was applied (Step 46). (0) Image acquisition of 3D-IF staining in autofluorescence channel, followed by Photobleaching. (2–4) Multi-cyclic imaging: Rounds of 2D-IF staining with FITC, PE and APC coupled antibody fluorochrome-conjugate, image acquisition of respective FITC, PE and APC-channels and signal erasure by photobleaching.

Article Snippet: Figure 7 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”).

Techniques: Imaging, Staining

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: Figure 7 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”).

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Human OvCa) (A) Imaris 3D surface rendering of autofluorescence (cyan) and CD326 positive cells (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) with target plane in yellow. (C) Light sheet guided target plane selection representing CD326 positive cell (purple), CD45 positive cells (red), and CD3 positive cells (green). (D) DAPI overview image of selected tissue slice for 2D MACSima™ imaging. (E) Magnified merged six color multiparameter MICS image with CD45 (green), CD326 (cyan), FOLR1 (purple), Collagen III (red), Collagen IV (red), and CD31 (yellow). (F–L) Single staining MICS images (white) of DAPI (F), CD45 (G), CD326 (H), FOLR1 (I), Collagen III (J), Collagen IV (K), and CD31 (L) (gray) (see “Antibodies”). Scale bars: (A–F) 1 mm; (E) 250 μm; (F–L) 500 μm.

Article Snippet: Figure 7 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”).

Techniques: Imaging, Comparison, Selection, Staining

Journal: bioRxiv

Article Title: The structured hairpin region of the bacterial ESCRT-III protein IM30 orchestrates stress-induced condensate formation

doi: 10.64898/2026.02.28.708693

Figure Lengend Snippet: (A) Super-resolution fluorescence images showing Synechocystis cells expressing mVenus-tagged IM30 wt and IM30*. Chlorophyll signals representing TMs are colored in red (1 st column) and mVenus signals colored green (2 nd column). Scale bar = 2 μm. (B) Zoom-in image of a Synechocystis cell expressing mVenus-tagged IM30*. An IM30 punctum as well as protein attached to the cytoplasmic membrane are visible. Scale bar = 1 μm. (C) 3D-rendering of TM (red) and mVenus (green) fluorescence signals show that IM30 proteins form spherical assemblies. Scale bar = 1 μm.

Article Snippet: For 3D rendering, raw data was collected at 0.110 μm slice intervals and processed using the SIM 2 leap model, and 3D rendering was performed in Imaris (version 10.1.1).

Techniques: Fluorescence, Expressing, Membrane

Journal: Npj Biomedical Innovations

Article Title: Engineering scalable vascularized kidney organoids for in vivo glomerular filtration with human endothelial integration

doi: 10.1038/s44385-025-00063-5

Figure Lengend Snippet: A 3D image reconstruction of glomerular surfaces in day 21 STR organoids. PODXL + 3D glomerular surface was generated via 3D surface rendering of Z-stack images by IMARIS Labkit segmentation and machine learning analysis. n = 15 organoids from 5 independent batches (3 organoids in each batch). Scale bars represent 50 μm. B Glomerular and stromal surfaces of STR organoids in time-course experiments. 3D surface rendering by IMARIS displays PODXL+ podocyte/glomerular and PDGFRβ + stromal surfaces in a time-course manner. The lower graph displays the ratio of PDGFRβ + surface volume to its nuclear counterpart. C The vascular surface of STR organoids in time-course experiments. 3D surface rendering by IMARIS displays CD31+ vascular surfaces in a time-course manner. Scale bars in ( B , C ) represent 200 and 100 μm, respectively. D Representative electron microscopy images of proximal tubular regions of STR organoids on days 35 and 70. The asterisks show multiple Mitochondria with their evident cristae structures, while the arrowheads point out Endoplasmic reticulum networks enveloping and interacting with adjacent Mitochondria in the metabolically active tubular epithelial cells. The black dashed line encircles the Golgi apparatus. The scale bars of the left low-magnification images represent 10 μm, while the scale bars of the right high-magnification images display 800 μm, respectively. Asterisks in the bar graphs ( A – C ) indicate p values derived from one-way ANOVA with Tukey’s multiple comparisons tests. Means ± SEM. (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001); ns, not significant.

Article Snippet: 3D surface rendering by IMARIS displays CD31+ vascular surfaces in a time-course manner.

Techniques: Generated, Electron Microscopy, Metabolic Labelling, Derivative Assay

Journal: Npj Biomedical Innovations

Article Title: Engineering scalable vascularized kidney organoids for in vivo glomerular filtration with human endothelial integration

doi: 10.1038/s44385-025-00063-5

Figure Lengend Snippet: A Vessel sprouts in polarized podocyte clusters with CD146+ capillary network supported by MEIS1/2/3+ stromal cells in day 21 STR organoids (left set of two images) compared to the podocyte clusters of day 21 control static organoids (right set of four images). White arrows in the STR images indicate the vascularized glomeruli surrounded by polarized podocytes. Scale bars of STR images represent 100 μm and 10 μm (zoomed STR image), respectively. Rectangular white dashed lines point out the zoomed images of non-vascularized podocyte clusters of the static organoid. Scale bars of static organoid images represent 100 μm. B 3D surface reconstruction of CD146+ capillary network and surrounding PODXL+ podocyte clusters as vascularized glomerular components. Z-stack images were acquired at a step size of 2 μm. The scale bar represents 30 μm. C Transmission electron microscopy images show multiple vascular lumens (left image) and accompanying podocyte foot processes around the vascular lumen (right image) within organoid glomerular structures on day 35. The asterisk indicates the nucleus of the podocyte surrounding the vascular lumen, while the arrowheads mark podocyte foot processes. Black scale bars at the corners: 2 μm. D Percentages of vascularized podocyte clusters containing CD146 endothelial surface in STR versus static organoids via 3D surface reconstruction by machine learning. n = 3 static and n = 9 STR organoids from 3 independent batches differentiated from BJFF.6 or HUES62 cells. E ACTA2+ mesangial-like cells surrounded by polarized PODXL+ podocytes in Bowman’s space of day 35 STR and static organoids. STR organoids were generated by the transfer of the same-batch static organoids into STR wells on day 14 of differentiation. The graph represents the percentage of organoids containing ACTA2+ cells ( n = 5 static and n = 6 STR organoids from 2 independent batches differentiated from H9 or HUES62 cells). F Upregulation of certain GO terms associated with extracellular matrix organization, cell adhesion, and migration in day 35 STR organoids compared to static organoids. “Regulation of cell migration”, “Positive regulation of cell adhesion”, “Regulation of cell adhesion”, and “Extracellular matrix organization” terms constitute GO biological processes while “Extracellular matrix”, “Collagen-containing extracellular matrix”, “Actin-based cell projection”, and “Basement membrane”, “Collagen network”, and “Basement membrane collagen trimer” terms can be classified as GO cellular components, and “Assembly of collagen fibrils and other multimeric structures” is a part of Reactome pathways. G Inhibition of glomerular vascularization of STR organoids by the application of α2β1 integrin inhibitor, BTT3033 (stock concentration of 10 mM), with a final concentration of 1 μM for 7 or 18 days, in contrast to the control vehicle-treated organoids. The white arrows in the confocal microscopy images indicate the vascularized glomeruli surrounded by polarized podocytes. The scale bars represent 100 μm. H 3D surface reconstruction of CD146+ capillary network and surrounding PODXL+ podocyte clusters as vascularized glomerular surfaces in vehicle- and BTT3033-treated organoids. Z-stack images were acquired at a step size of 2 μm. The scale bar represents 50 μm. Y-axes of graphs represent two parameters: the percentage of vascularized podocyte clusters and the volume percent ratio of CD146+ vascular surface in these podocyte clusters to the whole PODXL+ podocyte surface, respectively ( n = 7). Asterisks in the bar graphs ( D , E , H ) indicate p values derived from two-tailed unpaired t-tests. Means ± SEM. (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: 3D surface rendering by IMARIS displays CD31+ vascular surfaces in a time-course manner.

Techniques: Control, Transmission Assay, Electron Microscopy, Generated, Migration, Membrane, Inhibition, Concentration Assay, Confocal Microscopy, Derivative Assay, Two Tailed Test

Journal: Npj Biomedical Innovations

Article Title: Engineering scalable vascularized kidney organoids for in vivo glomerular filtration with human endothelial integration

doi: 10.1038/s44385-025-00063-5

Figure Lengend Snippet: A Workflow for the implantation of nephron sheets generated from STR organoids into dorsal skinfold chambers (DSFCs) of NSG mice. Implantation of STR organoids into mice was performed six times from three independent organoid batches. B Phase 1 left photo shows the self-assembled nephron sheet formed by STR organoids. Instead of implantation, this sheet was fixed with 4% PFA on day 28 to confirm the self-assembly process. The right image represents the stained nephron sheet with the designated markers (LTL, CD31, CDH1, CD146, PODXL, MEIS1/2/3) following the fixation process. The scale bars represent 1 mm. C Phase 2 photos include the NSG mouse with a dorsal skin fold chamber (DSFC) on its back (left photo) and the implanted nephron sheet generated from day 24 STR organoids inside the DSFC (right photo). D Phase 3 multiphoton intravital microscopy (MP-IVM) of the implanted sheet reveals the nephron units (designated by a circular white dashed line) with the dynamic flow of both high-molecular-weight (HMW, 500 kDa-Cy5) and low-molecular-weight (LMW, 3 kDa-FITC) dextrans in glomerular vascular-like structures (designated by 3D-reconstructed gray surface), accompanied by the filtration of LMW dextran into the surrounding space. The 3D-reconstructed cellular surface was generated via 3D surface rendering of Hoechst in Z-stack images by IMARIS. The scale bar displays 20 μm. E Phase 4 left photo shows the gross vascularization of the implanted nephron sheet with murine vessels on the 6th day of implantation, following its extraction and fixation. 3D reconstructed image (right) demonstrates the ongoing CD146+ human vascular network inside the same phase 4 nephron sheet (PODXL, CD146, LTL, MEIS123). Z-stack images were acquired at a step size of 100 μm. The scale bars represent 1 mm. F The confocal microscopy image showing the anastomosis of MECA-32-CD146+ human (white arrows, single-positive red) and MECA-32+ murine vascular endothelial networks. The scale bar displays 150 μm.

Article Snippet: 3D surface rendering by IMARIS displays CD31+ vascular surfaces in a time-course manner.

Techniques: Generated, Staining, Intravital Microscopy, High Molecular Weight, Molecular Weight, Filtration, Extraction, Confocal Microscopy

Journal: Npj Biomedical Innovations

Article Title: Engineering scalable vascularized kidney organoids for in vivo glomerular filtration with human endothelial integration

doi: 10.1038/s44385-025-00063-5

Figure Lengend Snippet: A Confocal microscopy images show the persistence of the CD146+ human endothelial network that is anastomosed with the MECA-32+ murine endothelial network. Yellow arrowheads indicate the human capillaries infiltrating the nephron structures. Scale bars: 150 μm. B 3D reconstructed surfaces showing human vessel penetration into nephron structures. Z-stack images were acquired at a step size of 5 μm. The scale bars: 50 μm. C 3D rendering of explanted nephron sheets indicates the persistence of the CD146+ human glomerular endothelial network supported by MEIS1/2/3+ stromal cells inside the podocyte clusters (markers: PODXL, CD146, LTL, MEIS1/2/3). The upper and lower white arrowheads pointed out the human glomerular capillary network and accompanying afferent arteriole, respectively. The Z-stack images of these nephron structures demonstrate the presence of LMW dextran in Bowman’s space and its uptake by proximal tubular epithelial cells and stromal cells, whereas HMW dextran (two white arrows) is detected only inside the vessels. Z-stack images were acquired with a step size of 2 μm, as Z1 represents the top stack of 3D structures. Scale bars: 20 μm. D Fixed tissue images showing a distinct distribution of LMW and HMW dextran, suggestive of filtration and flow of LMW dextran from vascular lumens to Bowman’s space and tubular lumens, in contrast to HMW dextran. Z-stack analysis revealed the uptake of LMW dextran by LTL+ proximal tubules and PODXL+ podocytes, while HMW dextran was strictly limited to CD146+ vascular lumen and absent from the tubules. Scale bars: 20 μm.

Article Snippet: 3D surface rendering by IMARIS displays CD31+ vascular surfaces in a time-course manner.

Techniques: Confocal Microscopy, Filtration