Seminar by Dr. Melvin 'Probing biological questions with optical approaches'

Speaker:
Dr. Tracy Melvin, Optoelectronics Research Centre, University of Southampton, UK

Seminar Title:
Probing biological questions with optical approaches

Abstract:
Employing light to manipulate and evaluate biologically relevant processes at the molecular, cellular and tissue level is at the forefront of interdisciplinary research.  Firstly I will discuss the use of optical tweezers, which have been employed to study cell mechanics and the interaction of the cell membrane with its surrounding environment. My group has been investigating the effect of the main cellular components on the whole cell mechanics and orientation in optical tweezers.   We have recently demonstrated that the response to an optical field is cell-specific and depends on the main cellular constituents, namely the cytoplasm and nucleus. We have been able to gain a fundamental insight on the optical properties of cells and can provide evidence that undifferentiated human embryonic stem cells can be distinguished from differentiated cells using this non contact, label free method.
Coherent anti-Stokes Raman scattering (CARS) microscopy can be applied in living cells and provides vibrational contrast at a speed that is orders of magnitude higher than conventional Raman microscopy.  The majority of CARS studies on living systems so far, have focused on the investigation of lipids; an example includes lipid vesicles inside HeLa cells.  Lipids are relatively easy molecules to probe by CARS due to the high densities within structures in vivo and the strong signal at 2845 cm^-1 assigned to the C-H vibrational stretch.  Other regions of the spectrum can give information about other important molecules; for example, imaging at 1090 cm^-1 of phosphate groups of chromosomes within cells undergoing mitosis.  Since the Raman spectral region from ~1000 cm^-1 to ~1800 cm^-1 contains bands that can be considered as ‘fingerprints’ for the structure of proteins, given a level of knowledge/reference data it is possible to gain an understanding of the structure of protein molecules directly from the Raman spectrum of the ‘pure’ protein. Examples include Raman marker bands for α-helix (1650 and 1340 cm^-1), β-sheet (1690, 1740 and 1240 cm^-1) and disordered protein structures (1670 and 1220 cm^-1).   Recently we have investigated the use of CARS microscopy for the investigation of protein structure in vivo.   We decided to focus on the protein aggregates typical of neurodegenerative diseases, we first interrogated polyglutamine (polyQ) aggregates, that are implicated in Huntington’s disease.  This genetic disease is caused by a CAG/polyQ expansion from a non-pathological length (Q20-Q40) to a disease-associated length (>Q40).  Above this Q40 threshold polyQ-containing peptides/proteins misfold, oligomerise and form amyloid-like fibrils inside cells, a process that has been modelled extensively in the test-tube, cell and animal models. The structural changes associated with the polyQ aggregation process and its complex interplay with other proteins that recognise abnormal proteins (molecular chaperones) has not been elucidated in living systems to date.  In our first studies we have applied CARS microscopy approaches for the evaluation of the structure of these polyQ aggregates in vivo.