Presentation number: 4A16

Investigation of cellular heterogeneity in Glioblastoma: IRimaging study
Bhawana Singh a, Vani Santosh b, Kumaravel Somasundaram c and Siva Umapathy a*a; IPC department, Indian Institute of Science, Bangalore, 560012
b; National Institute of Mental Health and Neuro Sciences, Bangalore, 560029
c; MCBL department, Indian Institute of Science, Bangalore, 560012

Introduction
Fourier transform infrared (FTIR) microspectroscopy has emerged as a potential
technique for the analysis of biological samples (1 - 3). In our study, we report the applicationof FTIR imaging to study grade IV brain cancer. Even though WHO guidelines are available
but still histopathological classification of brain cancer remains a challenging area. In
medical community, increasing need has been felt for interdisciplinary methodologies toimprove the diagnosis of difficult to determine cases in a shorter time frame.

Experiment
Normal and brain cancer tissue samples were collected from National Institute of
Mental Health and Neuro Sciences (NIMHANS) Bangalore, India. Sample collection andexperiments were approved by the human ethics committee of NIMHANS. Liquid nitrogencooled FPA detector having 64 × 64 detector elements was used to collect IR spectroscopic
images in reflection mode. Multiple IR images were recorded from a tissue section by
moving the sample stage. FTIR hyper-spectral data images were recorded in the range of
950-4000 cm-1 at 4 cm-1 spectral resolution and data were analyzed using Cytospec,
resolution pro (Varian) and Origin software programmes.

Results and Discussion
This study not only presents the characterization and classification of normal andgrade IV brain cancer tissue specimen but also shows the potential FTIR microspectroscopy
in identifying different cell types within grade IV brain cancer tissue samples. The advantageof this technique is that it is capable of bringing out the inherent chemical heterogeneity inconjunction with the morphology of the sample. At first, we characterized and classified
normal and grade IV brain cancer (glioblastoma) tissue specimen and then within grade IVbrain cancer we identified different cell types. Imaging experiments gave idea about theinherent biochemical nature of the samples which directly co-related with the morphology ofthe samples. It is well known that grade IV brain cancer show large heterogeneity in tissuemorphology. Based on this fact our aim was to take the maximum cellular heterogeneity into
account and recognize spectral markers for them. Thus, in our study, within grade IV brain
cancer, morphologically different cell types namely fibrillary, pleomorphic, small, giant andlipidized were differentiated spectroscopically.

In short, this study contributes towards improving our understanding towards possible
future use of IR imaging in the field of biomedicine for diagnostic purposes, especially it canplay an important role, in determining tumour margins or while dealing with frozen sectionsof soft tissues like brain which often lose their morphology at low temperature.

References:(1) Fernandez, Daniel C., Bhargava, Rohit, Hewitt, Stephen M., and Levin, Ira W., Nature
Biotech, 23, 469 (2005).
(2) M. Diem, M. Romeo, S. Boydston-White, M. Miljkovic and C. Matthaus, Analys,t 129,880 (2004).
(3) A. Pacifico, Luis A. Chiriboga, P. Lasch and M. Diem, Vibrational Spectroscopy, 32, 107(2003).