UC Berkeley

The fundamental properties of the stimluated Raman scattering (SRS) interaction make it attractive for application in biological microscopy. The signal is linear in both the concentration of scatterers, as well as the power of both excitation sources used. In turn, this allows for quantitative concentration measurements to be made within a sample. Furthermore, the SRS spectrum, an exact match to the spontaneous Raman spectrum, is chemically specific. I.e., individual molecules can be specifically and uniquely identified by their spectra. As a result, in principle, SRS microscopy allows for quantitative label-free measurements to be made of biological molecules.

In practice, however, outside of a few applications, SRS microscopy is neither widely adopted nor applied in biology. It is often thought that the specificity of fluorescence cannot be achieved in a label-free manner with SRS due to the complexity of the biological systems under investigation. Additionally, as the standard implementation of SRS microscopy requires laser scanning for the formation of an image, fundamental limits on acquisition speed are thought to make the application of SRS microscopy to the study of dynamic processes challenging, if not infeasible.

The following dissertation presents new experimental and computational methods to broaden the applications of SRS imaging within the life sciences. An overview of the fabrication of the standard SRS imaging system is provided, with attention to the implications of certain design choices. This helps establish the limits within which the SRS microscopy technique currently operates, as well as make the technique more accessible to a general audience. Best practices for performing measurements and analyzing the results are presented. The application of SRS microscopy to the imaging of small molecule neurotransmitters, in a specific manner, is demonstrated, highlighting the capability of SRS microscopy to achieve of the specificity of fluorescence imaging. Finally, extensions to the standard imaging system are presented which lay the groundwork for future applications with faster acquisition speeds. In aggregate, the work presented in this dissertation serves to extend the range of problems to which SRS microscopy can be applied in biology.