Emerging applications of stimulated Raman scattering microscopy in materials science

Abstract

The conventional spontaneous Raman microscopy is a powerful tool that images chemical bonds in materials, but it suffers from the intrinsically weak Raman cross-sections and requires long acquisition time. As an emerging imaging tool, stimulated Raman scattering (SRS) microscopy significantly amplifies the Raman scattering signal. It has higher chemical sensitivity, faster imaging speed, and fine spatial resolution. Meanwhile, it is label-free and capable of 3D imaging. With its fast speed and superb chemical sensitivity, it can visualize the dynamic process in materials science, such as ion transport. SRS microscopy was originally invented for biomedical studies so it is not well known by the materials science community yet. In this review paper, we introduce the principle and instrumentation of SRS microscopy, applications in materials science, and discuss future potentials and challenges. We believe that SRS microscopy will become a valuable instrument for materials science. Stimulated Raman scattering (SRS) is a nonlinear Raman scattering process that can amplify Raman scattering by up to 108 times under modern microscopy configuration. SRS microscopy has emerged as a powerful chemical imaging technique due to its high chemical, spatial, and temporal resolution. While SRS microscopy was originally designed for biomedical applications, it has drawn increasingly more attention from the materials science community in recent years. The high-speed and high-chemical sensitivity of SRS are attractive for both high-throughput material characterizations and capturing transient dynamics in chemical transport and reactions. It has been explored in various topics, such as 2D materials, energy storage and conversion, and polymerizations with great success. In this review, we discuss principles, instrumentation, and current applications of SRS microscopy in materials science, followed by our perspectives on future exciting topics to be studied by SRS microscopy. Stimulated Raman scattering (SRS) is a nonlinear Raman scattering process that can amplify Raman scattering by up to 108 times under modern microscopy configuration. SRS microscopy has emerged as a powerful chemical imaging technique due to its high chemical, spatial, and temporal resolution. While SRS microscopy was originally designed for biomedical applications, it has drawn increasingly more attention from the materials science community in recent years. The high-speed and high-chemical sensitivity of SRS are attractive for both high-throughput material characterizations and capturing transient dynamics in chemical transport and reactions. It has been explored in various topics, such as 2D materials, energy storage and conversion, and polymerizations with great success. In this review, we discuss principles, instrumentation, and current applications of SRS microscopy in materials science, followed by our perspectives on future exciting topics to be studied by SRS microscopy. 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Vibrational spectroscopies, such as spontaneous Raman scattering spectroscopy, have long served as key measures for material characterizations.46Larkin P. Infrared and Raman Spectroscopy: Principles and Spectral Interpretation. Elsevier, 2011Google Scholar, 47Butler H.J. Ashton L. Bird B. Cinque G. Curtis K. Dorney J. Esmonde-White K. Fullwood N.J. Gardner B. Martin-Hirsch P.L. et al.Using Raman spectroscopy to characterize biological materials.Nat. Protoc. 2016; 11: 664-687Crossref PubMed Scopus (556) Google Scholar, 48Schmid T. Opilik L. Blum C. Zenobi R. Nanoscale chemical imaging using tip-enhanced Raman spectroscopy: a critical review.Angew. Chem. Int. Ed. 2013; 52: 5940-5954Crossref PubMed Scopus (217) Google Scholar Conventional spontaneous Raman spectroscopy uses one single laser (ωp) shining onto the sample (Figure 2A). At the molecular level, all Raman scattering events involve the inelastic interactions between photons and molecules, which result in a photon energy change of either a loss (Stokes) or gain (anti-Stokes). By collecting the scattered photons, the vibrational signatures and concentrations of chemical bonds are acquired. This information is different from other characterization methods, such as crystallography (coordinates of specific atoms), UV-vis spectroscopy (energy differences between molecular orbitals), or mass spectroscopy (mass of entire molecules or fragments). This feature endows the vibrational Raman spectroscopy with high chemical specificity and provides rich bonding and structural information. Raman is less sensitive to polar bonds, hence it is more tolerant to a water background compared with infrared (IR) microscopy.49Cheng J.X. Xie X.S. 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In typical practice, spontaneous Raman spectroscopy requires a few seconds to obtain the spectrum at a single spot, so Raman mapping can take minutes to hours. To address the sensitivity issue, resonant Raman scattering techniques were also exploited to enhance the weak Raman signal by coupling electronic and vibrational motions of chromophores.50Asher S.A. UV resonance Raman studies of molecular-structure and dynamics—applications in physical and biophysical chemistry.Annu. Rev. Phys. Chem. 1988; 39: 537-588Crossref PubMed Scopus (183) Google Scholar,51Schellenberg P. Johnson E. Esposito A.P. Reid P.J. Parson W.W. Resonance Raman scattering by the green fluorescent protein and an analogue of its chromophore.J. Phys. Chem. B. 2001; 105: 5316-5322Crossref Scopus (74) Google Scholar SRS differs in many ways from the abovementioned Raman scattering phenomena.28Freudiger C.W. Min W. Saar B.G. Lu S. Holtom G.R. He C.W. Tsai J.C. Kang J.X. Xie X.S. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy.Science. 2008; 322: 1857-1861Crossref PubMed Scopus (1539) Google Scholar It utilizes one extra laser beam (ωs, Stokes beam) that is spatially and temporally synchronized with the pump beam (ωp) to stimulate the vibrational transition rate and greatly promote the scattering cross-section (σRaman) by roughly 108 (Figure 2A). When the ground-state molecule is excited to the virtual state by a pump photon, the Stokes beam dramatically accelerates its relaxation process to the vibrational excited state as the energy difference between pump and Stokes photons matches the vibrational energy gap. During this transition event, a pump photon is consumed while a new Stokes photon is emitted. This results in a net loss of pump intensity and a gain of Stokes intensity. By modulating the pump or Stokes beam, the gain or loss can be detected by a high-frequency (∼MHz) lock-in amplifier. The signal size of SRS can be interpreted from the stimulated Raman loss, typically proportional to Ns × σRaman × Ip × Is, where Ns is the number of target bonds, Ip and Is are the intensity of pump and Stokes beams, respectively. As a consequence of the strong quantum amplification, the chemical sensitivity of SRS is much higher than that of spontaneous Raman. The spatial resolution of SRS microscopy, on the other hand, is similar to conventional Raman microscopy, since they are both diffraction limited. It is worth noting that the standard SRS setup (Figure 2) with a picosecond laser pulse train only records the information of one wavenumber (ωp – ωs) at a time. It sacrifices multiple-channel information to acquire a much high temporal and chemical resolution. Therefore, SRS microscopy has great advantages when high temporal resolution and chemical sensitivity are required, such as studying the ion diffusion process. Spontaneous Raman microscopy should be chosen when spectrometric information on each spot is needed, such as distinguishing multiple species simultaneously. Compared with other nonlinear Raman scattering processes, SRS has many unique advantages. Firstly, SRS is background free, as it directly probes the energy exchange between the laser field and chemical bonds. Secondly, compared with other coherent Raman scattering microscopies, such as coherent anti-Stokes Raman scattering, the SRS signal is free from the non-resonant background and is usually proportional to the concentration of chemical bonds if the peak position does not shift with varied concentration. This greatly simplifies data interpretation and facilitates quantification.28Freudiger C.W. Min W. Saar B.G. Lu S. Holtom G.R. He C.W. Tsai J.C. Kang J.X. Xie X.S. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy.Science. 2008; 322: 1857-1861Crossref PubMed Scopus (1539) Google Scholar Thirdly, unlike SERS where the sample must be adsorbed by metal surfaces, SRS does not require any substrates. The enhancement factors from the SERS effect vary substantially, making it less applicable for imaging. In comparison, SRS can faithfully interrogate the spectral details while showing great linear concentration dependence as a far-field effect.22Cheng Q. Wei L. Liu Z. Ni N. Sang Z. Zhu B. Xu W. Chen M. Miao Y. Chen L.-Q. Operando and three-dimensional visualization of anion depletion and lithium growth by stimulated Raman scattering microscopy.Nat. Commun. 2018; 9: 2942Crossref PubMed Scopus (83) Google Scholar,32Hu F. Shi L. Min W. Biological imaging of chemical bonds by stimulated Raman scattering microscopy.Nat. Methods. 2019; 16: 830-842Crossref PubMed Scopus (97) Google Scholar These features make SRS suitable for materials studies, and allow us to track the dynamic variation of chemical concentration in a rapidly changing environment. As the SRS stimulation requires an extra light source with a distinct wavelength, an SRS microscope is typically equipped with two laser sources: the initial incident beam for excitation (pump) and the stimulating beam (Stokes). The frequency difference (ωp – ωs) between the two beams matches exactly with the energy of specific vibrational modes of interest (Figure 2B). For full-spectrum measurement, it is obvious that the energy difference between the two lasers needs to be tuned. The typical SRS setup uses an optical parametric oscillator (OPO) to achieve wavelength sweeping. Moreover, the two beams are spatially and temporally synchronized before being guided into the microscope to achieve optimal synergy. Temporally, both beams are pulsed lasers to maximize the peak power, due to the nonlinear nature of stimulated scattering. Spatially, a confocal laser scanning microscope is needed to tightly focus the laser beams on a sample. Notably, although the scattering cross-sections are greatly enhanced via stimulated scattering, the relative intensity changes (pump loss and Stokes gain) are still small compared with the intensities of incident beams (e.g., ∼10−4 of the incident beams). To extract the relatively weak scattering signal, either the pump or Stokes beam is modulated by an electro-optic modulator, so that the pump loss or Stokes gain signal can be created at a radio frequency (at which the technical noise of the laser is vanishing) and subsequently detected by a lock-in amplifier. This signal will be digitized into pixel intensity, and collectively an image will be obtained by raster scanning of the laser beams across the sample. Because detection sensitivity, imaging speed, and spatial resolution are three key considerations in evaluating microscopy, next we discuss them to provide a basic evaluation of this emerging microscopy. Firstly, as the initial motivation to use stimulated emission, the sensitivity of SRS is multiple orders of magnitude better than the spontaneous Raman. Besides, thanks to the high-frequency modulation, SRS detection is immune to noise, such as laser fluctuation and fluorescence background, at low frequency. Typically, the detection limits of SRS microscopy are around μM to mM.52Wei L. Hu F. Shen Y. Chen Z. Yu Y. Lin C.C. Wang M.C. Min W. Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering.Nat. Methods. 2014; 11: 410-412Crossref PubMed Scopus (283) Google Scholar The exact sensitivity closely depends on the Raman spectrum, such as the cross-section of individual chemical bonds (σRaman) and the number of bonds (Ns) presented. Because Raman scattering originates from the polarizability change during oscillation, non-polar bonds generally show stronger Raman cross-sections than polar bonds. Hence, the backbone bonds, such as C=C, C≡C, C≡N, and C=O, become hot spots for imaging.53Zhao Z. Shen Y. Hu F. Min W. Applications of vibrational tags in biological imaging by Raman microscopy.Analyst. 2017; 142: 4018-4029Crossref PubMed Google Scholar,