Developing Stimulated Raman Spectroscopic Techniques For Imaging Below the Optical Diffraction Limit

Abstract

Stimulated Raman spectroscopy (SRS) is a technique that amplifies the normally weak Raman scattering process using an additional laser beam, resulting in increased signal amplitudes. For this reason, it has been developed as a biological imaging platform with the potential to be used as an alternative to fluorescence microscopy due to its chemical specificity. This eliminates the need for fluorescent tags, which can photobleach or disrupt the structure or dynamics of the system of interest. However, due to the optical diffraction limit SRS cannot compete with the spatial resolution that super-resolution fluorescence techniques are capable of. An SRS-based technique capable of breaking the diffraction limit would therefore allow for nanoscale research to occur on systems for which super-resolution fluorescence is not an option. To that end, we developed a method to improve spatial resolution in SRS using a toroidal beam to deplete SRS signal. As a result, signal is only generated in a reduced area at center of the beam. Initial experiments demonstrated up to 97% depletion of the signal and explored the properties of the depletion process. Additionally, we improved spatial resolution by approximately a factor of two using the toroidal beam to deplete signal while scanning the laser beams over the edge of a diamond plate. While the proof-of-concept experiments were successful, they were performed with a laser with high peak power and a relatively low repetition rate of 1 kHz. These high powers were not compatible with soft matter samples, causing significant photodamage. We therefore adapted super-resolution SRS on laser with a 2.04 MHz repetition rate to average faster and increase the peak power flexibility. Experiments on the 2.04 MHz laser corroborated many proof-of-concept results, including resolution improvement by about a factor of two. However, depletion iv was not achieved with the same efficiency and further improvements in resolution were not forthcoming. This is likely due to the inconsistent phase of the laser’s fundamental pulse profile, highlighting the importance of consistent and reproducible pulses when driving sensitive nonlinear optical processes. Additionally, we demonstrate the use of a new Raman tag using carboranes. By scanning a thin film of a carborane-terminated poly(N-isopropylacrylamide) (pNIPAAm), we show that their high density of B-H bonds and their unique vibrational frequency in the cell silent region make carboranes useful Raman imaging tags that expand multiplexing options. Carboranes’ role as reversible addition-fragmentation chain transfer (RAFT) polymerization agents make them especially good endogenous probes for polymers produced in this manner. Finally, we discuss planned experiments to further improve signal-to-noise ratio (SNR) and explore the mechanism of signal depletion. We also discuss applications of Raman imaging in lipid dynamics, using both diffraction-limited and sub-diffraction techniques. We propose possible methods to compare results from Raman and fluorescence microscopy to determine the impact of fluorescent tags on dynamics. In the research described herein, we develop and explore new Raman imaging methods and highlight the potential power of super-resolution SRS as a versatile chemical imaging tool.