Limits...
Multi-color electron microscopy by element-guided identification of cells, organelles and molecules

View Article: PubMed Central - PubMed

ABSTRACT

Cellular complexity is unraveled at nanometer resolution using electron microscopy (EM), but interpretation of macromolecular functionality is hampered by the difficulty in interpreting grey-scale images and the unidentified molecular content. We perform large-scale EM on mammalian tissue complemented with energy-dispersive X-ray analysis (EDX) to allow EM-data analysis based on elemental composition. Endogenous elements, labels (gold and cadmium-based nanoparticles) as well as stains are analyzed at ultrastructural resolution. This provides a wide palette of colors to paint the traditional grey-scale EM images for composition-based interpretation. Our proof-of-principle application of EM-EDX reveals that endocrine and exocrine vesicles exist in single cells in Islets of Langerhans. This highlights how elemental mapping reveals unbiased biomedical relevant information. Broad application of EM-EDX will further allow experimental analysis on large-scale tissue using endogenous elements, multiple stains, and multiple markers and thus brings nanometer-scale ‘color-EM’ as a promising tool to unravel molecular (de)regulation in biomedicine.

No MeSH data available.


Related in: MedlinePlus

EDX analysis reveals single cells that contain exocrine zymogen granules with endocrine glucagon and insulin.(a) Overview as in Fig. 1a. (b) Area indicated in (a) by the cyan dot with parts of two cells with different granules based on grey levels and morphology. (c–e) Elemental maps as indicated. (f) Overlay of N (red), P (green) and S (blue) allows identification of cells and granules. (g) Zoom of the center part of f, note the small purple and yellow granules, as well as the big red granule, all present in the same cell. (h) Same region, with P and S combined with the backscatter image, which was reverted to red. Note the membranes around the vesicles. (i) Interpretation of the EDX data on the large scale STEM image showing the presence of three distinct granules in an endocrine cell. Bars: 50 μm (a), 2 μm (b–f) and 0.2 μm (g–i). See for more elements Fig.S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384080&req=5

f3: EDX analysis reveals single cells that contain exocrine zymogen granules with endocrine glucagon and insulin.(a) Overview as in Fig. 1a. (b) Area indicated in (a) by the cyan dot with parts of two cells with different granules based on grey levels and morphology. (c–e) Elemental maps as indicated. (f) Overlay of N (red), P (green) and S (blue) allows identification of cells and granules. (g) Zoom of the center part of f, note the small purple and yellow granules, as well as the big red granule, all present in the same cell. (h) Same region, with P and S combined with the backscatter image, which was reverted to red. Note the membranes around the vesicles. (i) Interpretation of the EDX data on the large scale STEM image showing the presence of three distinct granules in an endocrine cell. Bars: 50 μm (a), 2 μm (b–f) and 0.2 μm (g–i). See for more elements Fig.S3.

Mentions: To explore EDX for biomedical microscopy, we used embedded material from our previous studies15, namely a diabetic prone rat. 90% of these rats spontaneously develop diabetes, with the blood sugar level as an indication for the diabetic state. This experimental animal had not developed diabetes, and showed no signs of insulitis15 (Figs 1a, 2a, 3a and dataset). Unexpectedly, typical distinct vesicles within one cell are present at multiple locations. These multi-vesicle containing cells are at the border of the endocrine Islet of Langerhans and the surrounding exocrine tissue, the latter readily identifiable by the abundant endoplasmic reticulum and large zymogen vesicles (Fig. 3b, top). The adjacent cell has all characteristics of an endocrine cell (Fig. 3b, bottom). Note the presence of the small granules with a halo (typical for insulin) and other small granules showing an overall high electron density (typical for glucagon), but also large vesicles that resemble the zymogen granules in the exocrine tissue. Using the elemental characteristics to discriminate granules (Fig. 1), we analyzed the distribution of N, P and S (Fig. 3c–e; full elemental results in Fig. S3). Three distinct granules with characteristics of zymogen (N; red), glucagon (N in red and P in green, yellow appearance of coincidence) and insulin (N in red and S in blue, purple appearance) are present (Fig. 3f), which is more prominent at higher zoom (Fig. 3g). Color-coding of the backscatter image in green, a signal mainly caused by Os, allows to superpose the signal created by Os enriched in membranes (Fig. 3h). Interpretation of the multi-hormone and zymogen containing area is modelled on the STEM image (Fig. 3i). Thus, only with EM three distinct granules can be conclusively identified within the same cell and EDX shows the different composition based on different elemental ratios, thus without prior knowledge or anticipated labeling. Based on these new observations, we decided to substantiate our notion probing protein content, and subsequent optimization of double-immunolabeling confirmed our findings (Fig. 4).


Multi-color electron microscopy by element-guided identification of cells, organelles and molecules
EDX analysis reveals single cells that contain exocrine zymogen granules with endocrine glucagon and insulin.(a) Overview as in Fig. 1a. (b) Area indicated in (a) by the cyan dot with parts of two cells with different granules based on grey levels and morphology. (c–e) Elemental maps as indicated. (f) Overlay of N (red), P (green) and S (blue) allows identification of cells and granules. (g) Zoom of the center part of f, note the small purple and yellow granules, as well as the big red granule, all present in the same cell. (h) Same region, with P and S combined with the backscatter image, which was reverted to red. Note the membranes around the vesicles. (i) Interpretation of the EDX data on the large scale STEM image showing the presence of three distinct granules in an endocrine cell. Bars: 50 μm (a), 2 μm (b–f) and 0.2 μm (g–i). See for more elements Fig.S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384080&req=5

f3: EDX analysis reveals single cells that contain exocrine zymogen granules with endocrine glucagon and insulin.(a) Overview as in Fig. 1a. (b) Area indicated in (a) by the cyan dot with parts of two cells with different granules based on grey levels and morphology. (c–e) Elemental maps as indicated. (f) Overlay of N (red), P (green) and S (blue) allows identification of cells and granules. (g) Zoom of the center part of f, note the small purple and yellow granules, as well as the big red granule, all present in the same cell. (h) Same region, with P and S combined with the backscatter image, which was reverted to red. Note the membranes around the vesicles. (i) Interpretation of the EDX data on the large scale STEM image showing the presence of three distinct granules in an endocrine cell. Bars: 50 μm (a), 2 μm (b–f) and 0.2 μm (g–i). See for more elements Fig.S3.
Mentions: To explore EDX for biomedical microscopy, we used embedded material from our previous studies15, namely a diabetic prone rat. 90% of these rats spontaneously develop diabetes, with the blood sugar level as an indication for the diabetic state. This experimental animal had not developed diabetes, and showed no signs of insulitis15 (Figs 1a, 2a, 3a and dataset). Unexpectedly, typical distinct vesicles within one cell are present at multiple locations. These multi-vesicle containing cells are at the border of the endocrine Islet of Langerhans and the surrounding exocrine tissue, the latter readily identifiable by the abundant endoplasmic reticulum and large zymogen vesicles (Fig. 3b, top). The adjacent cell has all characteristics of an endocrine cell (Fig. 3b, bottom). Note the presence of the small granules with a halo (typical for insulin) and other small granules showing an overall high electron density (typical for glucagon), but also large vesicles that resemble the zymogen granules in the exocrine tissue. Using the elemental characteristics to discriminate granules (Fig. 1), we analyzed the distribution of N, P and S (Fig. 3c–e; full elemental results in Fig. S3). Three distinct granules with characteristics of zymogen (N; red), glucagon (N in red and P in green, yellow appearance of coincidence) and insulin (N in red and S in blue, purple appearance) are present (Fig. 3f), which is more prominent at higher zoom (Fig. 3g). Color-coding of the backscatter image in green, a signal mainly caused by Os, allows to superpose the signal created by Os enriched in membranes (Fig. 3h). Interpretation of the multi-hormone and zymogen containing area is modelled on the STEM image (Fig. 3i). Thus, only with EM three distinct granules can be conclusively identified within the same cell and EDX shows the different composition based on different elemental ratios, thus without prior knowledge or anticipated labeling. Based on these new observations, we decided to substantiate our notion probing protein content, and subsequent optimization of double-immunolabeling confirmed our findings (Fig. 4).

View Article: PubMed Central - PubMed

ABSTRACT

Cellular complexity is unraveled at nanometer resolution using electron microscopy (EM), but interpretation of macromolecular functionality is hampered by the difficulty in interpreting grey-scale images and the unidentified molecular content. We perform large-scale EM on mammalian tissue complemented with energy-dispersive X-ray analysis (EDX) to allow EM-data analysis based on elemental composition. Endogenous elements, labels (gold and cadmium-based nanoparticles) as well as stains are analyzed at ultrastructural resolution. This provides a wide palette of colors to paint the traditional grey-scale EM images for composition-based interpretation. Our proof-of-principle application of EM-EDX reveals that endocrine and exocrine vesicles exist in single cells in Islets of Langerhans. This highlights how elemental mapping reveals unbiased biomedical relevant information. Broad application of EM-EDX will further allow experimental analysis on large-scale tissue using endogenous elements, multiple stains, and multiple markers and thus brings nanometer-scale ‘color-EM’ as a promising tool to unravel molecular (de)regulation in biomedicine.

No MeSH data available.


Related in: MedlinePlus