Figure 8

Distinguishing between an artifact and the subnucleolar heterogeneity of ¹⁵N-uridine incorporation. (a,b) Parallel quantitative mass images of (a) ¹²C¹⁴N^- and (b) ¹²C¹⁵N^- images of a fibroblast cultured in the presence of ¹⁵N-uridine. Ncl, nucleoli; NM, nuclear membrane. Field: 40 × 40 μm (image has been cropped); acquisition time 20 min. (c-e) High-resolution parallel mass images at ¹²C^-, ¹²C¹⁴N^- and ¹²C¹⁵N^- of the large nucleolus seen in (a,b). Field: 8 × 8 μm; acquisition time 30 min. (c) ¹²C^- image, arising from both tissue and embedding medium; the dark spot (red arrow) was caused by accidental exposure to a stationary high-intensity primary Cs⁺ ion beam. (d) ¹²C¹⁴N^- image. (e) ¹²C¹⁵N^- image, showing subnucleolar areas of low local ¹⁵N incorporation (white arrows). (f) Ratio of the (d) ¹²C¹⁴N^- and (e) ¹²C¹⁵N^- images; here, the 'dark spot' (red circle) is barely visible because the value of the ¹²C¹⁵N^-/¹²C¹⁵N^- ratio is close to that of the surrounding area. (g) HSI image of the ¹²C¹⁵N^-/¹²C¹⁴N^- ratio (the numerator has been multiplied by 10,000). The 'dark spot' isotope ratio is close to that of the surrounding area. Subnucleolar regions of low incorporation of ¹⁵N-uridine stand out in both the (f) ratio and the (g) HSI images. (h) Calibration with ¹⁵N-uridine. The graph shows the intranucleolar accumulation of ¹⁵N-uridine (measured as ¹²C¹⁵N^-/¹²C¹⁴N^- (experimental – control)/control) as a function of the concentration of ¹⁵N-uridine in the culture medium.