High-spectral-resolution Stimulated Raman Spectroscopic Imaging And Its Applications

Stimulated Raman scattering(SRS) microscopy is a newly-developed label-free imaging technique that generates images based on the vibrational signatures of molecules. As different compounds have particular vibrational frequencies, SRS microscopy could offer intrinsic chemical selectivity and chemical specificity without labeling. Besides, the nonlinear nature of SRS process automatically grants it the capability of three-dimensional sectioning. Thanks to the above-mentioned merits, SRS microscopy has become a particularly important tool in the filed of life science in the past few years. As the frontier as well as the hotspot of researches in the past two to three years, stimulated Raman spectroscopic imaging, based on either hyperspectral SRS or multiplex SRS, offers spectral information at each pixel, and as such could separate molecules with overlapped Raman bands, providing better chemical specificity and much more abundant chemical information. Yet, due to the limited development time, current research on stimulated Raman spectroscopic imaging still needs to be further improved in many aspects. In this dissertation, we study the instrumentation of high-spectral-resolution stimulated Raman spectroscopic imaging comprehensively by deploying different approaches including spectral focusing and another method that integrates nonlinear spectral compression and pulse shaping together. Meanwhile, we also explore the applications of stimulated Raman spectroscopic imaging in the detection of cellular membrane potential and chemical mapping of lignin.Theoretically, we first deduced the generation of SRS signal by using the nonlinear coupled-wave equations, and investigated the origin of imaging contrast of SRS microscopy. The spectral profile difference between SRS and CARS under various nonresonant backgrounds was further calculated and compared. For those Raman modes with different depolarization ratios, we also studied the effects of laser polarization on the SRS signal, and revealed the mechanism of polarization-modulated SRS imaging. Besides, we detailedly discussed the noise, signal-to-noise ratio(SNR), and backgrounds in SRS imaging, and pointed out that ideally all the SRS microscopes should reach shot-noise-limited detection.Next, we analyzed the principle of the spectral focusing method used for hyperspectral coherent Raman scattering microscopy. With glass rods as chirping medium, a pulse chirping based hyperspectral SRS microscope was built, and detailed protocols for the Raman shift calibration and i ntensity calibration in the spectral focusing system were further drafted. In terms of applications, for the first time, we discussed the possibility of deploying stimulated Raman scattering signals to probe the membrane potential without labeling. Using erythrocyte ghost as a model, we demonstrated vibrational spectroscopic imaging of single natural membrane, and validated the sensitivity of hyperspectral SRS microscopy. To prove the concept of probing transmembrane potential via SRS microscopy, we changed the potential across the erythrocyte ghost membrane from +10 to-10 m V by manipulating the intravesicular and extravesicular ionic compositions. We found that the SRS spectral profiles of erythrocyte ghosts showed a distinct dependence on the membrane potentials, which clearly shows the feasibility of using hyperspectral SRS microscopy to sense membrane potential in a label-free manner.On the other hand, we also noted that the spectral focusing method has a shortcoming of confined spectral resolution which may not be suited for interrogating the molecular vibrations in the crowded fingerprint region. Conversely, the pulse shaper based spectral narrowing approach provides high spectral resolution but is not efficient in the utilization of limited laser power. In other words, one of the remaining challenges for present techniques is the trade-off between excitation power and spectral resolution which are both essential for SRS imaging of weak Raman bands in the congested fingerprint region. To overcome this b ottleneck, we introduced nonlinear spectral compression into stimulated Raman spectroscopic imaging. Experimentally, we developed a flexible and compact spectral compressor that could narrow down the line-width of broadband femtosecond pulses into several wavenumbers while retaining more than half of the laser power simultaneously. By implementing the lab-built spectral compressor as high-power Stokes source, we successively constructed two hyperspectral SRS microscopes with spectral resolution better than 10 cm-1in the fingerprint and silent regions. Moreover, in the Raman-silent region, we further improved the spectral scanning device, and designed a novel intrapulse spectral scanner which employs a galvanometer to perform rapid wavelength tuning. Furthermore, considering the fact that currently hyperspectral SRS microscopy has been widely used in the studies of animal cells, tissues, and model organisms while its use in the plant field still remains unexploited and underestimated, we also carried out the research of analyzing and mapping the lignin chemical composition using vibrational fingerprints. With wild-type Arabidopsis and cad-c cad-d mutant, we investigated and validated the capacities of hyperspectral SRS microscopy for quantitatively differentia ting different chemical contents in lignin and real-time monitoring of the dynamic change of lignin composition. Next, we further examined bristle grass and corn stover, and performed in situ SRS imaging of vascular bundle. By multivariate curve resolution(MCR) analysis of the hyperspectral images, for the first time, we unveiled a spatially distinct distribution of aldehyde and alcohol groups in the plant cell wall.In addition, we also demonstrated a new multiplex SRS imaging scheme which utilizes oppositely chirped pulses as excitation sources to broaden the detection spectral range. With positive and negative chirps introduced by glass rods and grating pair respectively, an oppositely chirped pulses based multiplex SRS imaging system was built, and further applied to image the DMSO sample. Through testing the system with DMSO, the feasibility of the proposed method was also verified.Taken together, the results presented in this dissertation would significantly promote the development of instrumentation and applications of stimulated Raman spectroscopic imaging. In the meantime, we expect that our work would contribute to the further evolution of SRS microscopy as well.