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.