Fig 2: Multiplexed protein analysis of organelle markers in BM MSCs and UC MSCs. (a) Average Pearson’s correlation of the total intensity of 5 markers in BM MSCs and UC MSCs per marker per cell plotted as a heatmap. BM MSCs are shown on the left, UC MSCs are shown in the middle, and combined Pearson’s for each cell is shown on the right. The comparison of the correlation of 5 markers between BM MSCs (n = 7) and UC MSCs (n = 7) was provided as a heatmap using the average linkage method based on the correlation distance. Larger correlation values are shown in red and smaller correlation values are shown in blue. BM MSCs possess more separation between nuclear and cytosolic organelles while UC MSCs are less separated, which illustrates that UC MSC organelles are more spread across the cell. The right side shows the single-cell heatmap for all marker pairs. UC MSCs exhibit higher correlations in DAPI_ATF6, TOM20_ATF6, and Nucleolin_ATF6, suggesting more crosstalk between nuclear and cytosolic organelles. Single BM MSCs exhibit stronger correlations within nuclear or cytosol pairs e.g. Nucleolin_DAPI, GOLPH4_ATF6, TOM20_GOLPH4, implying that nucleus and cytosol organelles are more segregated in BM MSCs, which agrees with the left heatmap. (b) Average pixel overlap colocalization between 5 markers in BM MSCs and UC MSCs per marker per cell plotted as a heatmap. The comparison of pixel overlap between BM MSCs (left) and UC MSCs (middle) was provided as a heatmap using the average linkage method based on the contact frequency distance. The combined pixel overlap on a single cell level is shown on the right. Large pixel overlap values are shown in red and small pixel overlap values are shown in blue. UC MSCs possess slightly higher pixel overlap values among organelles in different compartments (nuclear vs cytoskeleton) but this difference is less pronounced than in (a). A few UC MSCs (10, 12, 13, 14) show distinct patterns from other UC MSCs in terms of weaker pixel overlap in Nucleolin_DAPI, GOLPH4_ATF6, TOM20_GOLPH4, Nucleolin_ATF6, TOM20_Nucleolin. UC MSCs exhibit higher variability in pixel overlap. (c) Heatmaps were generated to compare the morphological features and to determine any close relationships or associations between markers and between BM MSCs (red) and UC MSCs (teal). Morphology is defined as area (left), minor axis (middle), and major axis (right). Both cell types exhibit more differences in minor and major axes and fewer differences in terms of area, implying that organelles differ more in shape and less in expression area. The most notable difference is TOM20, suggesting a difference in mitochondrial energetic activity. Most differences, such as TOM20 and ATF6, are attributable to UC MSCs, suggesting more single-cell variability among UC MSCs. (d) Kolmogorov–Smirnov (K–S) hypothesis test was conducted between organelle marker pairs targeting the mitochondria (TOM20 and HSP60; left) and Golgi (GOLPH4 and Sortilin; right) to study if similar proteins express different spatial distributions within each cell. Single BM MSCs are shown in red while single UC MSCs are shown in teal. In terms of spatial mitochondrial expression, BM MSCs have more single-cell variability while UC MSCs express more uniformly. BM MSCs possess more single-cell variability than UC MSCs concerning spatial ER expression.

Fig 3: Pixel level clustering of organelle markers in BM MSCs and UC MSCs. (a) K-Means clustering of intensities of markers for a single BM MSC. 10 clusters were chosen and colored back on the original cell (large left). Each cluster represents one distinct expression profile of the protein markers in the cells. A pair of images are shown for each organelle (mitochondria, Golgi, Nucleus, ER): (1) an overlay of the two markers that target the organelle (right) and (2) the colocalization of the two markers obtained by multiplying the two marker images pixel-wise from the right to highlight areas of overlap (left). Example: ATF6 and Concanavalin A forming the ER localization. Yellow values indicate areas of higher overlap while red values indicate areas of lower overlap. The right side shows the heatmap of marker intensity with a dendrogram based on marker intensities (right, top) and clustering results (right, bottom). From the dendrogram based on marker intensities, GOLPH4 and TOM20 are directly linked, along with ATF6. This suggests moderate crosstalk between ER and mitochondria, especially in cluster 8. Nuclear organelles DAPI and Nucleolin are associated in a separate cluster. (b) K-Means clustering of intensities of markers for a single UC MSC. 10 clusters were chosen and colored back on the original cell (large left). Each cluster represents one distinct expression profile of the protein markers in the cells. A pair of images are shown for each organelle (mitochondria, Golgi, Nucleus, ER): (1) an overlay of the two markers that target the organelle (right) and (2) the colocalization of the two markers obtained by multiplying the two marker images pixel-wise from the right to highlight areas of overlap (left). Example: ATF6 and Concanavalin A forming the ER localization. Yellow values indicate areas of higher overlap while red values indicate areas of lower overlap. The right side shows the heatmap of marker intensity with a dendrogram based on marker intensities (right, top) and clustering results (right, bottom). From the dendrogram based on marker intensities, ATF6 and TOM20 exhibit moderate crosstalk (clusters 3, 7, 8) but not as intensely as the BM MSC in (a).

Fig 4: Ten-plex organelle mapping in mesenchymal stem cells using rapid multiplexed immunofluorescence (RapMIF). (a) Schematic of RapMIF for organelle analysis in MSCs. Each cycle contains 3 conjugated antibodies plus 4',6-diamidino-2-phenylindole (DAPI) followed by bleaching of the signal before the next cycle consisting of 3 new antibodies. Imaging of the 3 antibodies before and after bleaching confirms the presence of signal and then signal removal. Multiplex imaging consists of multiple cycles (n) of antibody labeling and bleaching. BM MSCs (brown) and UC MSCs (cyan) are labeled with the same multiplex antibodies that target the same set of organelles. All images are acquired on Nikon widefield and registered across cycles to produce a final set of multiplex-labeled images. Example images show Beta Tubulin (magenta, left) and TOM20 (red, right) overlaid with the nucleus in DAPI (blue). Created with BioRender.com. (b) Visualization of organelle markers in single cells from BM MSCs and UC MSCs. Each row corresponds to a distinct single cell. The top 2 rows show BM MSCs and the bottom 2 rows show UC MSCs. Multiplexed markers for the same cell are displayed across 4 columns (Column 1: ATF6 & Concanavalin A, Column 2: Beta Tubulin & Phalloidin, Column 3: GOLPH4 & Sortilin, Column 4: HSP60 & TOM20, Column 5: Nucleolin & WGA). Each image displays DAPI with a pair of organelle markers in magenta and green. DAPI is used to register the signals across cycles. Signal removal is confirmed with a widefield microscope after bleaching each cycle. All scale bars 10 µm.

Fig 5: Virtual reality-based visualization and analysis of organelle imaging data in MSCs. (a) Visualization of organelle protein images (10 markers) for one BM MSC in ConfocalVR62. For each cell, the top shows the RGB image of the combined markers, while the bottom two show a single marker image in either red or green with DAPI. Different organelle markers are illustrated across the columns. Merged images are shown in the first row. Column 1: ATF6 (middle row) & Concanavalin A (bottom row), Column 2: Beta Tubulin (middle row) & Nucleolin (bottom row), Column 3: GOLPH4 (middle row) & Sortilin (bottom row), Column 4: Phalloidin (middle row) & WGA (bottom row), Column 5: TOM20 (middle row) & HSP60 (bottom row). Handset toggle switches used to interact with the image are shown on the left and right sides. (b) Visualization of organelle protein images (10 markers) for one BM MSC in Genuage63. The histogram of pixel count in 10 cylindrical bins (shown in white) of width 12.5 pixels (108 nm/pixel, bin width 1.354 µm) was plotted in the software. The top left image is an illustration explaining how cells are binned across the length to gauge spatial variability. In each box, red and blue colors illustrate the marker according to the upper left label. Beta Tubulin, ATF6, TOM20, and GOLPH4 show discontinuous regions of signal, supported by the uneven histograms, while the other signals are more continuous with more Gaussian histograms.