Identification and Imaging of 15N Labeled Cells with ToF-SIMS

Abstract

Stable isotope labeling may provide a novel method for tracking stem cells once they have been injected into a human or animal host. Here we present a simple pilot study to determine the potential for using ToF-SIMS to detect and localize ¹⁵N labeled cells in tissue biopsies for use in cell therapy studies. For this pilot study, 3T3 fibroblasts were grown in normal media and in two different media containing ¹⁵N labeled amino acids. Samples containing a mixture of ¹⁵N labeled and unlabeled cells were prepared, fixed and dried for analysis and were then imaged using a bunched Bi₃⁺ primary ion source. The cells containing ¹⁵N labeled amino acids could be readily distinguished using nitrogen containing peaks which have been previously associated with the labeled amino acids. Contrast was sufficient to allow easy identification of labeled cells in both sparsely and densely plated cultures. Multivariate analysis showed that the image contrast could be improved by including peaks originating from characteristic fragments of the labeled amino acids as well as lower mass NH₄⁺ and CH₄N⁺ peaks. Additional work is being pursued to determine and improve the longevity of the label.

Figure 1

200 × 200 μm ion images of a region from Set 1C. The image from the CH₄¹⁵N⁺ ion peak is shown at the top right. Top left shows the image for the sum of ¹⁴N peaks shown in Table 1 and bottom left shows the image for the sum of the corresponding ¹⁵N peaks. The image at the bottom right was calculated using MAF of the 973 peak image stack.

Figure 2

200 × 200 μm ion images of a region from Set 2. The image from the CH₄¹⁵N⁺ ion peak is shown at the top right. Bottom left shows the image for the sum of the 3 identified ¹⁵N peaks and top left shows the image for the sum of the corresponding ¹⁴N peaks. The image at the bottom right was calculated using MAF of the 973 peak image stack.