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the probe from 3 to 13,8 which raises the signal-to-noise ratio
for 19F by more than four-fold. However, the use of a probe
based on the 15N/14N ratio provides a much more elegant
solution to the contamination problem.
TriazNF1 also enables the simple estimation of the copy number
of tags measured from the preparation. While the number of tags
cannot be derived from 19F, due to non-specific contaminating
signals, it can be derived from the isotope counts for 14N and 15N
(ESI,† Section 5). When sufficiently large areas are measured, to
compensate for the pixel-to-pixel variation in the ratio, a 15N/14N
ratio close to the natural abundance (0.00367) is found for the non-
transfected cells. In the presence of TriazNF1, the ratio increases
significantly (Fig. 1C). This increase can be translated into actual
copy numbers of TriazNF1 molecules by a simple equation (ESI,†
Section 5), which is based on the 14N and 15N counts alone. This can
be estimated for each voxel, if the voxels are sufficiently large to
counteract the effects of isotope counting noise. We present such an
estimation in Fig. 1D, using voxels of B156 Â 156 nm in the
imaging plane, and a depth of B20 nm. This figure panel shows
estimates, rather than exact copy numbers. The latter, however, can
be obtained by calibrating the NanoSIMS instrument with standard
isotopic samples, to turn the 14N and 15N counts into precise isotope
numbers.
Fig. 1 TriazNF1 specifically labeled proteins for visualisation in NanoSIMS.
We incorporated PRK into syntaxin 1 while expressing it in BHK cells. We
then fixed the samples and labelled them by click reaction with TriazNF1,
followed by embedding in LR White, and thin-sectioning. (A) Representative
images of a cell expressing syntaxin 1 labelled with TriazNF1. The top panels
show the fluorescence image of TriazNF1 (Star635 fluorescence), in con-
focal microscopy, and NanoSIMS images of the 14N and 15N isotopes, as well
as their ratio. An overlay of this ratio with the fluorescence image confirms
the good colocalisation of the two signals. The bottom panels show further
NanoSIMS images of the same cell: 19F and its ratio to 14N, 32S, and finally a
ratio of 13C to 12C, as an indication of cellular turnover (based on the
incorporation of L-leucine-2-13C into newly secreted proteins). The ratio
images only show pixels with more than 300 14N counts; lower values
represent regions outside of the cell, in which the ratios are not meaningful.
The last image in this row indicates the possibility of combining TriazNF1
with immunostainings, in fluorescence microscopy (the endoplasmic reti-
culum protein calnexin is shown). (B) NanoSIMS measurements for a non-
transfected cell. Corresponding images of the transfected and non-
transfected cell are identically scaled. (C) The mean 15N/14N ratios for cells
expressing different SNARE proteins are significantly higher (***, P o 0.001
compared to control in Student’s t-test) than the ratio for non-transfected
cells. The Y axis starts at the value of the natural abundance ratio (0.00367).
For each condition we analysed a number of circular cellular regions of
interest, of B0.123 mm2: 15 regions for SNAP-25, 54 for syntaxin 1, 31 for
syntaxin 13, and 137 for non-transfected cells. The error bars indicate the
standard errors. (D) Image depicting syntaxin 1 copy number inferred from
the 15N/14N ratio. Scale bar for all images, 2 mm.
TriazNF1 can now be used as a label for specific proteins,
and enables the investigator to analyse the composition of the
organelles containing the respective proteins. For example, the
cells can be treated with isotopically labelled amino acids, whose
incorporation into cellular proteins gives a direct indication of
cellular turnover, on the subcellular scale.4,14–16 L-Leucine-2-13C
was applied for 24 h, containing one 13C atom. Its incorporation
into the cell can be compared with the TriazNF1 position
(Fig. 1A). In addition, the analysis of the isotopic composition
of the cell is not limited to N or C isotopes. Many other organic
elements can be visualised (for example 32S, Fig. 1A).
Finally, the fluorophore moiety of TriazNF1 enables the
simple correlation of the isotopic images with further fluorescence
images, obtained from immunostaining or other procedures
(Fig. 1A, calnexin panel, in which an immunostaining for this
endoplasmic reticulum marker protein is shown).
SIMS imaging is increasingly used as a technique to investigate
the organisation and structure of cells and organelles.3 The main
difficulty with this and similar techniques is that, while many labels
can be used to investigate, for example, the turnover of all cellular
proteins, very few possibilities exist for the tagging of individual
biomolecules. Some proposed methods are similar to the techniques
used in fluorescence imaging and immunoelectron microscopy,
coupling the protein of interest to elements that are not present in
than approaches based on imaging elements that are not normally the cells, such as 19F, as discussed above,8 or lanthanide metals
present in the preparation, such as 19F. This is evident in Fig. 1A, coupled to antibodies.17 These probes, as seen in Fig. 1A, are
where a relatively high 19F signal is visible in the nucleus of the inherently sensitive to contamination by 19F or lanthanides in the
cell. As the transmembrane protein syntaxin 1 (shown in Fig. 1A) sample. In contrast, a probe based on a change in the 15N/14N ratio,
cannot reach the nucleus, we are forced to conclude that the 19F like TriazNF1, offers the same advantages, these being specific
signal present here is due to a contamination with unknown labelling and flexibility in both isotopic and fluorescence micro-
fluorinated molecules in the nucleus, which, however, partly scopy, while being entirely insensitive to contamination.
compromises the 19F imaging procedure (Fig. 1A). We solved this
issue partly in the past by increasing the number of 19F atoms in Ecology and Ecosystem Management, Centre of Life and Food
We would like to thank Johann Lugmeier (Department
This journal is ©The Royal Society of Chemistry 2015
Chem. Commun.
Kabatas, Selda
