Label-Free Myelin Imaging By Stimulated Raman Scattering Microscopy And Stimulated Raman Scattering Imaging By Continuous Wave Laser Excitation

Based on nonlinear optics process of coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), the coherent Raman scattering (CRS) microscopic imaging technique recruits chemical vibration to provide imaging contrast and avoids the internal ultra-structural deformations caused by exogenous labeling agents. CRS microscopy advancing in label-free and noninvasive imaging manner, has found important applications in biology and medicine. Over two decades development, CRS microscopy has been widely used in biological and medical fields. The characteristics of molecular vibration imaging enable CRS microscopy to map the molecular composition in normal and disease tissues.Myelin sheath is one of the most important components in nervous system, which is responsible for transducing neural signals over long distances, ensuring rapid propagation of action potentials in a saltatory manner. The development and maturation of myelin sheath is the fundament of the operation of brain. The research of myelin sheath is important for us to understand the mechanism of brain. In myelin related diseases, the damaged myelin sheath impairs conduction of neural signal, cause deficiencies in sensation, movement, cognition, and other functions depending on which nerves are involved. Thus, demyelination has been extensively studied to understand the basic cellular and molecular mechanisms, which devise a potential to promote remyelination. Currently, the imaging tools for myelin sheath research have relied heavily on electron microscopy (EM), traditional histomorphology, laser scanning fluorescence microscopy, and magnetic resonance imaging (MRI). EM has been used to reveal the myelin sheath’s ultrastructure; histomorphology provides a platform for the diagnosis of myelin related disease; fluorescence microscopy is a useful tool to identify the location and the function of myelin assembly proteins. However, observing dynamic behaviors of the myelin sheath and node of Ranvier using these imaging techniques has been a challenge as these techniques can mainly be used on fixed samples. Moreover, the incorporation of exogenous fluorescence proteins with intrinsic proteins might interfere with the normal physiological functions and properties of myelin. MRI is an excellent tool for diagnosing myelin-related diseases; however, its resolution is quite limited and is certainly not sufficient for ultra-structural investigation of the myelin sheath. Stimulated Raman scattering microscopic imaging avoids the internal ultra-structural deformations caused by exogenous labeling agents, as it recruits chemical bonds to provide imaging contrast. And the nonlinear process enables SRS microscopy an inherent3D section ability. The unique characteristics of transparent body before metamorphosis make Xenopus laevis tadpole excellent for in vivo observation of dynamic cellular behaviors, and avoid any surgical exposure. By in vivo SRS imaging, we monitor the process of myelin sheath formation in a single Schwann cell, maturation of a node of Ranvier, and degradation of a damaged nerve VII over time. The application of SRS microscopy for myelin imaging provides a new powerful platform to evaluate demyelination and remyelination.Current CARS or SRS microscopy uses two synchronized picosecond or femtosecond ultrafast pulsed lasers as excitation sources. However, the big hurdle against broad use of this label-free imaging modality is the high cost and complexity of the ultrafast laser sources. In order to lower the cost of CRS microscopy, we report a highly cost-effective SRS microscope using continuous-wave (cw) lasers as excitation sources. The cw-SRS spectroscopy was demonstrated before the cw-SRS microscopy, two cw diode lasers are employed to provide pump and Stokes beam for excitation, in which the pump laser has a tunable wavelength range from765nm to781nm, while Stokes laser has a fixed central wavelength at982nm. A function generator was used to modulate Stokes laser at5.4MHz. The pump and Stokes beams were collinearly overlapped, and were focused into sample by a10mm lenses. The transmitted pump beam was then detected by a photodiode, and the stimulated Raman loss (SRL) signal were extracted by a lock in amplifier, and sent to a DAQ card. The wavelength of the pump beam was scanned from766nm to772nm (0.6nm per step), which correspond to Raman shift from2750to2900cm-1. The SRL signal from multi-wavelength were normalized by excitation power and plotted as SRS spectra. We recorded the cw-SRS spectra of olive oil, methanol, and cyclohexane, which are identical with the spontaneous Raman spectrum. However, by single modulation of Stokes laser, there is a big offset in the cw-SRS spectrum, in which the offset is5times larger than SRS signal. The offset is originated from the modulation of Stokes laser, which leaked into the power supply of the pump laser at the same frequency as the detected SRS signal. To remove the parasitic modulation of the pump beam and eliminate the offset, we apply dual modulation scheme in which the pump beam was modulated at0.8MHz and the Stokes beam was modulated at4.6MHz. The SRS signal was detected at the sum frequency of5.4MHz. As the SRS signal occur at5.4MHz, which avoids the modulation frequency, the offset was eliminated completely. The cw-SRS microscopy was performed based on dual modulation scheme. The on-and off-resonant SRS imaging of olive oil are obtained at2765and2850cm-1, respectively. And the cw-SRS imaging of lipids in fatty liver is demonstrated by excitation of C-H stretch vibration at the Raman shift of2850cm-1. Compared with pulsed laser excitation, the cw-SRS signal is about103times weaker, which is because the peak power of pulsed laser is103times higher. As the cw laser has a higher photo damage threshold than pulsed laser, it is promising to bring the cw-SRS signal back by using higher excitation power. The demonstration of cw-SRS imaging of fatty liver sample by cw diode lasers shows a highly cost-effective cw-SRS microscope, which is a promising modality for label-free spectroscopic imaging of medical and biological samples.