Ultra-sensitive spectroscopies and micromanipulation techniques for the study of biological processes at single cell and single molecule level

In the biophysics and medicine field, researchers act as detectives working hard to unravel the mysteries surrounding cells. One type of spectroscopy in particular, Raman spectroscopy, has proven especially useful in providing detailed chemical composition of biological samples, constituting a sort of its chemical fingerprint. However the limited spatial resolution and sensitivity available is not sufficient to be able to resolve sub-cellular species. Furthermore, studies are normally performed on multiple cells grown or fixed on a surface which leaves a question about the influence of surface adhesion and cell to cell heterogeneities and interactions upon the biochemical state of the cells studied. Quantitative imaging and analysis of single normal and diseased cells is a challenging analytical goal. The principal objective of this project is to develop further a biophotonics technique that combine novel Raman spectroscopy methods with Optical Tweezers and apply this technique in obtaining time- and space-resolved information on biochemical and biophysical reactions inside single healthy and diseased living cells.In order to detect the distribution of assigned chemicals within a single selected cell, without physically touching it or needing to absorb it to a surface, it will be developed a new wide field spectral Raman imaging based on the use of Spatial Light Modulator (SLM) combined with an Optical Cell Rotator (OCR). This system will allow the acquisition of several spectra simultaneously from a large uniform area of interest of the optically trapped cell and incredibly will reduce the required acquisition time. So far, single living cells and subcellular compartments can be molecularly examined rapidly, non-invasively, without the use of external markers or fluorescent dyes and free from the interferences with the other cells.Although Raman spectroscopy is a useful technique to identify and quantify the biochemical composition of a cell, it can be severely limited in its applicability by sensitivity limits. Recently, numerous Raman based techniques have been developed in order to enhance the sensitivity of the basic technique. Among these, Surface-Enhanced Raman Scattering (SERS) is a process whereby the Raman scattering is enhanced, up to 14 orders of magnitude, when a Raman-active molecule is close to a metallic nanoparticle. Taking advantages from the major characteristics of both these new ultra-sensitive spectroscopic techniques (wide-field Raman imaging and SERS), it will be possible . to determine where assignment biochemical transformations occur on malignant cell membrane with spatial resolution of a few nanometers and sensitivity up to single molecules. All that has relevant implication for diagnostic and therapy of many diseases.Other important information can be obtained correlating such malignant biochemical transformations with the mechanical structure of normal and diseased cells. Recently, it has been demonstrated that mechanical stress can modify the protein expression in cancer cells membrane and consequently their susceptibility to the immune system detection. In particular, the project will focalize on the spectroscopic study of stress induced specific proteins in the various cellular compartments. This will require the development of micromanipulation apparatus (Optical Stretcher) that will be integrated with the ultra-sensitive spectroscopic techniques. Micromanipulation techniques will allow selecting a single cell and applying controlled mechanical stress (stretching) on it and ultra-sensitive spectroscopic techniques will allow the direct measurement of changes at molecular level (protein expression, cytoskeleton re-organization, etc.) that are directly related with mechanical stress.

The presented research project aims to develop new experimental techniques to characterize the biological complex mechanism that happens in cancer cells. This three years duration of the fellowship will enable me to tackle such complex problem and will give me the possibility to provide useful information for development of Raman-based devices for non-invasive evaluation and diagnosis of the cancer. The importance and the interdisciplinary of such subject render it interesting for the international community. The relevant implication of the explained research for diagnostic and therapy of many diseases could be of great impact on human health and international development. At this purpose, the collaboration with the Prof. Herrington's group of Bute Medical School is crucial for essentially two reasons: 1. He represents the first link with who will benefit from this research, i.e. the patient; 2. He will help to create new links between the research and the application. Equally important is the collaboration with Prof. Sasso's group; in fact, he will ensure the internationalization of the project, and its good engagement and communication. The experimental development and applications of such ultra-sensitive spectroscopies and micromanipulation techniques will be additionally disseminated through peer-reviewed international journals, conferences and freely downloadable numerical packages based. To achieve maximum scientific dissemination, I plan to publish new and timely developments in fast response journals like Optics Express, Biophysics Journal and Physics Review Letters. International conferences offer the best way to ensure widest possible dissemination of work while offering collaborative and exploitation opportunities. Potential industrial applications will be considered for patents through the St Andrews University Research and Enterprise Services.