Novel label-free detection technologies for 3D microscopy of biological specimens

There is a need for rapid microscopic imaging techniques in biomedicine. In conventional laser scanning microscopy (LSM) an excitation laser is scanned over the sample and a pixel by pixel response is detected. This is not ideal for monitoring dynamic processes. 3D imaging is also slow since such pixel by pixel scanning needs to be carried out at different stack depths. Light sheet microscopy uses planar illumination to visualise the full field of view of a sample from an orthogonal direction without raster scanning of the laser on the sample.

Light sheet microscopy is increasingly gaining popularity in biological research with all major microscope manufacturers (Leica, Zeiss, Nikon) offering light sheet microscopes. However, all current light sheet microscopes (as well as other laser scanning microscopes) are designed for detecting fluorescence. This is because typically molecules or cell structures are labelled with fluorescent endogenous proteins or exogenously added dyes/stains. Both these processes are invasive, can affect the native biochemistry and/or are only applicable to fixed (dead) specimens. Furthermore, fluorescence detection reports only those molecules which are labelled and therefore such studies do not give holistic information.

In this project the aim is to develop novel label-free detection methodologies based on scattering for light sheet microscopy. Raman and Rayleigh scattering are techniques which provide label-free information. Raman scattering, is a well-established analytical technique in chemical sciences that provides vibrational 'fingerprint' molecular information by probing the polarizability of chemical bonds. While Rayleigh scattering is also generated by interacting with molecular polarizability, the signal is emitted at the same wavelength as the incident excitation, and therefore the chemical information gets encoded only in its change of the polarisation. Thus Rayleigh polarisation anisostropy (ratio of parallel and orthogonally polarised emitted signals and also of different types of polarization as for instance circular versus linear) becomes a novel label-free measure, especially suited for imaging nearly transparent biological specimens such as cells. Size based scattering possible from some biological structures can be overcome by relying on the broadening characteristics of Rayleigh scattered light. Raman and Rayleigh light sheet imaging will thus be developed for imaging 3D biological specimens. Overall this project aims to deliver truly novel microscopic modalities implemented on a light sheet microscope. The provision of the 'highly informative' Raman and the 'simple' Rayleigh techniques on a light sheet system will be a world leading development with huge applications in biology and medicine.