Subcellular imaging of isotopically labeled carbon compounds in a biological sample by ion microprobe (NanoSIMS).
Here we demonstrate the technique of nanoscale secondary ion mass spectrometry, utilizing the Cameca NanoSIMS50 ion microprobe, to detect and image the metabolism of an isotopically labeled compound (NaH(13)CO(3)) in a biological sample. In particular, we have designed and verified protocols for imaging the subcellular distribution and determining the relative abundance of labeled (13)C, within the coral Galaxea fascicularis. Analyses were conducted on 1-mum thick sections of resin-embedded material, using both scanned (mapping) and static (spot analysis) Cs(+) primary ion beam of approximately 100 nm diameter. Using these samples we establish that NanoSIMS has adequate mass resolution to reliably distinguish (13)C from potential isobaric interference by (12)C(1)H and that data extracted from ion maps are comparable to those acquired by spot analyses. Independent of the method of acquisition, ratioing of (13)C to the naturally abundant (12)C is essential if meaningful data, which can be statistically compared to standard and control samples, are to be obtained. These results highlight the potential of NanoSIMS for intracellular tracking of a variety of organic and inorganic compounds labeled with stable isotopes of C, N, O, S, P, and halogens.
Clode
Peta L
PL
Centre for Microscopy and Microanalysis, The University of Western Australia, Crawley, Western Australia, Australia. pclode@cmm.uwa.edu.au
Stern
Richard A
RA
Marshall
Alan T
AT
D000818:Animals; D037421:Anthozoa / Q000382:microbiology / Q000648:ultrastructure; D002247:Carbon Isotopes; D056890:Eukaryota / Q000648:ultrastructure; D007089:Image Enhancement / Q000295:instrumentation / Q000379:methods; D008854:Microscopy, Electron; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D018629:Spectrometry, Mass, Secondary Ion / Q000295:instrumentation* / Q000379:methods*; D013559:Symbiosis
D002247:Carbon Isotopes