Fig 2: 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 3: Spatial analysis of organelle colocalization in BM MSCs and UC MSCs. (a) Boxplots of Pearson’s correlation of marker pairs per cell between all BM MSCs (blue) and UC MSCs (orange) categorized by nuclear, cytoskeleton, and organelle markers. Box plots show the median, first and third quartile, minimum, and maximum (excluding outliers). The outliers are marked as individual points. Stars denote the statistical significance for pairwise comparison. p value was calculated using the Mann–Whitney test (ns: p > = 0.05, ****p < = 0.0001). BM MSCs and UC MSCs exhibit the greatest differences in colocalized expression among pairs that include mitochondria (TOM20, HSP60), cytoskeleton (Beta Tubulin, Phalloidin), and endoplasmic reticulum (Concanavalin A). (b) Boxplots of pixel overlap colocalization values of marker pairs per cell between all BM MSCs (blue) and UC MSCs (orange) categorized by nuclear, cytoskeleton, and organelle markers. Box plots show the median, first and third quartile, minimum, and maximum (excluding outliers). The outliers are marked as individual points. Stars denote the statistical significance for pairwise comparison. p value was calculated using the Mann–Whitney test (ns: p > = 0.05, ****p < = 0.0001). UC MSC markers express a higher fraction of colocalized pixels in ER (ATF6, Concanavalin A) and mitochondria (HSP60) than those of BM MSCs, suggesting more active crosstalk of organelles in UC MSCs. (c) Comparison of the total area of 11 markers per cell between all BM MSCs (blue) and UC MSCs (orange) using boxplots. Box plots show the median, first and third quartile, minimum, and maximum (excluding outliers). The outliers are marked as individual points. Stars denote the statistical significance for pairwise comparison. p value was calculated using the Mann–Whitney test (ns: p > = 0.05, ****p < = 0.0001). UC MSC markers generally express larger and more variable areas than those of BM MSCs, suggesting that these organelles serve a higher energetic role in UC MSCs. (d) Comparison of the total intensity of 11 markers per cell between all BM MSCs (blue) and UC MSCs (orange) using boxplots. Box plots show the median, first and third quartile, minimum, and maximum (excluding outliers). The outliers are marked as individual points. Stars denote the statistical significance for pairwise comparison. p value was calculated using the Mann–Whitney test (ns: p > = 0.05, ****p < = 0.0001. Markers in UC MSCs express a larger range of intensities than in BM MSCs and thus these UC MSC organelles are in a more active state.

Fig 4: Comparison of protein markers targeting the same organelles in BM MSCs and UC MSCs. (a) Single BM MSC example with various organelles across the columns. Each organelle is shown by a marker pair: HSP60 & TOM20 (mitochondria, left), GOLPH4 & Sortilin (Golgi, middle), ATF6 & Concanavalin A (ER, right). All scale bars 10 µm. The bottom row shows intensity scatter plots with 50,000 pixels for each marker pair colocalized within the same organelle. The x and y-axis indicate the min–max scaled intensity values of the markers. More colocalized pixels appear closer to the y = x diagonal while less colocalized pixels appear closer to either axis, belonging more to that particular marker. In BM MSCs, mitochondria antibodies are more colocalized than either Golgi or ER. (b) Single UC MSC example with various organelles across the columns. Each organelle is shown by a marker pair: HSP60 & TOM20 (mitochondria, left), GOLPH4 & Sortilin (Golgi, middle), ATF6 & Concanavalin A (ER, right). All scale bars 10 µm. The bottom row shows intensity scatter plots with 50,000 pixels for each marker pair colocalized within the same organelle. The x and y-axis indicate the min–max scaled intensity values of the markers. More colocalized pixels appear closer to the y = x diagonal while less colocalized pixels appear closer to either axis, belonging more to that particular marker. In UC MSCs, ER antibodies are more colocalized than either mitochondria or Golgi.

Fig 5: 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.