Fig 1.
Schematic overview of the microscopy workflows presented in this study, integrating multiple imaging modalities.
Table 1.
Materials used in the experiments.
Table 2.
Equipment used in the experiments.
Fig 2.
Growth of SV-HUC-1 and T24 spheroids over 7 days.
Phase-contrast images were taken on days 1, 2, 3, and 7 to monitor spheroid development before further processing. Scale bars, 200 µm.
Table 3.
Summary of microscopy techniques combined with spheroid preparation for analysing and characterizing spheroids.
Fig 3.
Superior morphological preservation in paraffin sections compared to cryosections.
Representative haematoxylin–eosin (HE)-stained sections of SV-HUC-1 and T24 spheroids prepared by cryosectioning (A-F) and paraffin embedding (G-L). A necrotic core (indicated by asterisks, A-B, D-E, G-H, J-K) is visible in most cross sections, except those obtained from peripheral regions. Scale bars: 500 µm (A, D, G, J), 100 µm (B, E, H, K), 50 µm (C, F, I, L).
Fig 4.
Double immunofluorescence labelling in paraffin sections of SV-HUC-1 and T24 spheroids reveals distinct adhesion molecule expression patterns between normal and cancerous urothelial cells in 2D.
Representative images of paraffin sections show E-cadherin (green) in the plasma membrane in cells of SV-HUC-1 spheroids (A-a3) and N-cadherin (red) in the plasma membrane of T24 spheroids (B-b3). Some SV-HUC-1 cells are also positive for N-cadherin (arrows, a2 and a3). Yellow insets (in A and B) are magnified (a1-a3 and b1-b3) and display individual and merged channels of E-cadherin, N-cadherin, and DAPI-stained nuclei. Scale bars: 100 µm (A, B), 20 µm (a1-a3, b1-b3).
Fig 5.
Whole-mount immunofluorescence reveals protein expression throughout entire spheroids, preserving their 3D structure.
(A1-A8) Shown are single confocal images at different z-depths showing N-cadherin immunolabelling (red) and nuclei labelling with Hoechst stain (blue). Numbers in the upper left corner indicate the depth (in μm) of the optical sections within the Z-stack. (B) 3D reconstruction of a whole spheroid made from serial Z-stack images. The Z-stack comprised 274 optical sections, each taken with a 0.896 μm step. Scale bar, 200 μm.
Fig 6.
Comparison of double immunofluorescence labelling on cryosections, paraffin sections, and whole-mount SV-HUC-1 spheroids, highlighting 3D protein distribution and spatial context revealed by whole-mount imaging beyond traditional sectioning methods.
Cryosections (A-C) and paraffin sections (D-F) demonstrated E-cadherin-positive cell membranes and Ki-67-positive cell nuclei in individual cryosections (A-C) and paraffin sections (D-F), while whole-mount immunofluorescence (G1-G9) revealed the spatial distribution of E-cadherin and Ki-67 within the complete spheroid volume. (G1-G9) Numbers in the upper left corner represent the focus position of the optical section. (H) 3D reconstruction of the whole spheroid made from serial Z-stack images. The Z-stack comprised 168 optical sections, each with a 2 μm thickness. Scale bars, 100 µm (A, D), 40 µm (B, E), 10 µm (C, F), 100 µm (G1-G9).
Fig 7.
SEM reveals distinct surface ultrastructure of SV-HUC-1 and T24 spheroids.
SV-HUC-1 (A) and T24 cells (C) form spheroids with a spherical morphology. (B) SV-HUC-1 cells are tightly attached to each other (arrows), and microvilli are seen on their surface. (D) T24 cells are loosely attached, displaying wider intercellular spaces (arrowheads) and have fewer microvilli. Scale bars: 100 µm (A, C), 10 µm (B, D).
Fig 8.
TEM reveals distinct ultrastructure of SV-HUC-1 and T24 spheroids.
Outermost cells in SV-HUC-1 spheroids display cuboidal morphology (A), whereas T24 cells are more elongated (B). The presence of cell junctions in the outermost cell layer of SV-HUC-1 (C) and T24 spheroid (D) (boxed regions) indicates a tight cellular network of the outermost cell layer. The central necrotic zone (asterisks) is filled with necrotic cells in SV-HUC-1 and T24 spheroids (E, F), as clearly demonstrated on semithin sections (G, H), prepared before ultrathin sectioning. Scale bars: 100 µm (G-H), 10 µm (A-B), 5 µm (E-F), 1 µm (C-D).