Use of MIMS to study nitrogen-fixing bacteria. (a-c) Secondary ion images from the molecular ions (a) 12C14N-, (b) 12C15N-, and (c) the HSI 12C15N-/12C14N- ratio of a sample containing both Teredinibacter turnerae (Tt; rod-like cells) and Enterococcus faecalis (Ef; bunches of rounded cells) cultured in a 15N atmosphere for 120 h. Field: 46 × 46 μm (512 × 512 pixels); acquisition time 3 min. The magenta color of the T. turnerae cells is an indication of their incorporation and fixation of 15N (see Figure 3 for explanation). (d) The effect of scaling of the HSI 12C15N-/12C14N- ratio image (the numerator has been multiplied by 100) from T. turnerae cells exposed to a 15N atmosphere for 32 h. Assigning the hue spectrum to the whole range of ratio values allows easy identification of bacteria most highly enriched in 15N (the turquoise cells in the top left panel). Compressing the hue scale (shown gradually from top left to lower right) causes images of some of the cells to saturate at the magenta level and allows us to easily recognize a succession of cells also enriched in 15N, although at a lower level. The isotope values start with 0–7 (top left; a value of 7 is 19-fold higher than the natural ratio) and go to 0–0.5 (bottom right; a value of 0.5 is 1.43 times the natural ratio). The field of view is 13 × 13 μm (256 × 256 pixels); acquisition time 20 min. (e,f) HSI image of the 12C15N-/12C14N- ratio (the numerator has been multiplied by 100) of a T. turnerae cell exposed to a 15N atmosphere for 96 h. Field: (e) 8 × 8 μm; (f) 6 × 6 μm. Acquisition time: (e) 10 min; (f) 40 min. (g,h) HSI image of (g) the 12C15N-/12C14N- ratio (the numerator has been multiplied by 100) and (h) the 13C-/12C- ratio of T. turnerae in shipworm gill bacteriocytes incubated in the presence of a 15N atmosphere for 4 h. Field: 10 μm × 10 μm (256 × 256 pixels); acquisition time 60 min. (i,j) HSI image (i) of the 12C15N-/12C14N- ratio (the numerator has been multiplied by 100) and (j) at 12C15N- of T. turnerae exposed for 96 h in a 15N atmosphere. Arrows indicate the flagella of the bacteria. Field: 60 × 60 μm (256 × 256 pixels); acquisition time 20 min. (k) Line scan across the flagellum observed in (i,j) showing 12C15N- secondary-ion counts as a function of pixel address across the flagellum. One pixel is equivalent to 234 nm. Inset: arrow points to the flagellum; the red box indicates the area of the bacterium that was used to evaluate the mean 12C15N- counts.