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A
NanoSIMS application in biology
This study aimed at improving understanding of some mechanisms of
deafness. A group of mice was fed food enriched with 15N Leucine
for two weeks while a control group was fed normal food. After two weeks,
the mice were sacrificed and tissues of parts of the inner ear (the cochlea)
were analyzed with the NanoSIMS 50. Images of 14N and 15N
ions were recorded and the isotopic ratio calculated from different areas
of the tissues. The terrestrial nitrogen isotopic ratio obtained from the
control group and bone sections of test group, indicated that the
instrument and measurement worked well. Cochlea tissues from different
areas in the test group showed statistically significant 15N
enrichment, thereby proving the recent incorporation of the 15N
and hence the regeneration activity of these organelles. The next step
will be to submit the mice to a sound shock, making them deaf and to study
which areas have been damaged and do not regenerate any more.
12C14N-,
log scale 12C15N-
, log scale
Mouse fed for
14 days with
15N leucine. Cochlea: Reisner membrane with one
cell and four red blood cells. Cs+, 16 keV;
beam diam.: 100 nm; field: 12 x 12 µm; 20 ms/ pixel. Acq. time: 22
min. for up to 5 simultaneous ion images.
15N/14N ratios were measured for four locations on the
Reisner membrane (Rm), two locations on the nucleus (N), two locations
on the cytoplasm (C), and for each of the four red blood cells (RBC).
|
Area
|
Image locus
|
N pixels
|
14N counts
|
15N counts
|
15N/ 14N
( % )
|
|
1
|
Rm
|
1208
|
1768130
|
7831
|
0.443
|
|
2
|
Rm
|
1461
|
3191510
|
14879
|
0.466
|
|
3
|
Rm
|
676
|
1550440
|
7078
|
0.457
|
|
4
|
Rm
|
357
|
806287
|
3675
|
0.456
|
|
5
|
N
|
2200
|
5039080
|
21468
|
0.426
|
|
6
|
N
|
1141
|
1919240
|
8032
|
0.418
|
|
7
|
C
|
662
|
648097
|
2680
|
0.414
|
|
8
|
C
|
271
|
281904
|
1217
|
0.432
|
|
9
|
RBC
|
1808
|
4243480
|
16476
|
0.388
|
|
10
|
RBC
|
1966
|
6139860
|
30160
|
0.491
|
|
11
|
RBC
|
2221
|
9420760
|
36787
|
0.390
|
|
12
|
RBC
|
2344
|
11159300
|
43767
|
0.392
|
Terrestrial ratio 15N/14N=0.3676.
Note for example, red blood cell #10. It shows strong 15N enrichment
which indicates it is younger than the three other RBC, and high regeneration
activity.
To be able to make use
of the isotopic information of the SIMS technique, the requirements are:
-
High Mass Resolution (major interference of
12C15N
with 13C14N at mass 27)
-
High transmission at HMR together with reactive
primary ions (Cs+): the precision of 14N/ 15N
ratio is mainly limited by the statistic (so the count rate) on 12C15N
ion.
-
Small spot size to have access to intracellular
structures.
Only the NanoSIMS 50 can
allow this kind of study.
Courtesy Pr. Lechene,
Harvard Medical School, Boston, USA
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