The second indicator presented in this article is a
ratio on two major components of the tissue, the use
of a ratio eliminates all the previous bias enumerated
before and will bring more reliable results. Finding a
change in Tryptophan-Collagen ratio seems realistic
in tumorous tissues, knowing that tryptophan is
linked to vascular region and the tumorous tissue
present increased vascularity. Necrosis tissues in
glioblastoma are poor in collagen such as control
tissues and the change in tryptophan can be
highlighted through this ratio, however in other
tumorous tissues such as metastasis, a collagen
matrix spreads to organize cells migration inducing
both an increase of tryptophan and collagen,
therefore this ratio will not change as significantly as
in glioblastoma, giving false negative. It will be
interesting to look at other ratio and increase the
number of tumor types in the cohort.
The data showed that in this excitation range we
were able to fit the NADH component, this molecule
plays an essential role in metabolism, as a coenzyme
in redox reactions. And appears in the literature as a
major indicator in endogenous fluorescence. Its
behavior in tissues under visible and two photon
excitation has been well documented (Huang et al.,
2002; Skala et al., 2007). Articles looked at its cross
section over the excitation range or the redox ratio.
Knowing the role of this component we followed it
in deep UV, with a study over the excitation
wavelength from 310 to 340nm. The maximum in
excitation for the NADH in solution is at 345nm, so
we should get a curve increasing with the
wavelength. In our result we noticed a decrease at
340nm in the glioblastoma tissue. It could be either a
new indicator for cancerous tissue or just an
experimental artifact, due to the fact that this
measurement has been done on a very small number
of sample. Increasing the statistic of this analysis
could give significance to this result and highlight an
important phenomenon in tumorous metabolism.
Wide field images of endogenous fluorescence
allow us to correlate an area on each sample to the
H&E staining, the gold standard in histology to
validate the tumorous nature of a sample. This
correlation was possible thanks to the help of
anatomopathologists from Sainte-Anne hospital.
Correlation allowed us to demonstrate that high cells
density area in H&E images correspond to darker
area in wide-field images. Green channel in images
represent Tryptophan filter, these areas could
correspond to a loss of tryptophan or an increase if
the other channels, especially the collagen one.
Correlation with two-photon imaging could be an
interesting way to find more information.
All this promising resulfts encourage to increase
the cohort in order to have a better statistic on the
results.
ACKNOWLEDGEMENTS
This Work as a part of the MEVO and IMOP project
was supported by “Plan Cancer” program founded
by INSERM (France), by CNRS with “Défi
instrumental” grant, and the Institut National de
Physique Nucléaire et de Physique des Particules
(IN2P3).
We would like to thank Synchrotron SOLEIL for
beamtime under project #20160206. Thanks also to
PIMPA Platform partly funded by the French
program “Investissement d’Avenir” run by the
“Agence Nationale pour la Recherche” (grant
“Infrastructure d’avenir en Biologie Santé – ANR –
11-INBS-0006”).
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