# Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application
## 2 Laser Scanning Coherent Raman Scattering Microscopy (CRSM)
### Laser Scanning Stimulated Raman Scattering Microscopy (SRSM)  
Although CARSM has good performance on high sensitivity and high spatial resolution imaging to biological and chemical specimen, it is not suitable for quantitative chemical imaging, and has to restrain the strong non-resonant background. As SRS signals depend linearly on concentration of signaling molecule inside sample with background-free contrast, SRSM is a good candidate for quantitatively analyzing the molecule map information inside specimen. The excitation process of SRS is displayed in Figure 2a [43]. Similar with CARS, when the frequency difference between pump and Stokes beams is equal to Raman active vibrational frequency of molecules, amplification of the Raman signal is achieved by virtue of stimulated excitation. Consequently, the intensity of Stokes beam \(I_{s}\), experiences a gain \(\Delta I_{s}\) (stimulated Raman gain, SRG), and the intensity of pump beam \(I_{p}\), experiences a loss \(\Delta I_{p}\) (stimulated Raman loss, SRL), as shown in Figure 2b. Therefore, unlike CARS, SRL and SRG do not exhibit a nonresonant background. For SRSM, the Stokes beam is modulated at a high frequency, and the amplitude modulation of the pump beam can be detected by using SRL (Figure 2c). As shown in Figure 2d, the Stokes beam of SRSM is modulated by an electro-optic modulator. A photodiode is used to detect the transmitted and reflected light after filtering. SRL is measured by a lock-in amplifier to provide image pixels, and a fast scanning is performed for 3D imaging. Linear dependence of SRL signal intensity on concentrations of target molecule is shown in Figure 2e. Figure 2: (**a**) Energy diagram for Stimulated Raman Scattering (SRS). (**b**) Input and output spectra of SRS. SRS leads to an intensity increase with a gain \(\Delta I_{s}\) (stimulated Raman gain (SRG)) in the Stokes beam and an intensity decrease with a loss \(\Delta I_{p}\) (stimulated Raman loss (SRL)) in the pump beam. (**c**) Detection scheme of SRL. (**d**) Stimulated Raman Scattering Microscopy (SRSM) with both forward and epi detection. (**e**) Linear dependence of SRL on concentrations of retinol in ethanol at 1595 cm\({}^{-1}\)[43] (reprinted with permission from [43], Copyright 2008 AAAS). SRS was first discovered by [PERSON] and [PERSON] in 1962 [44; 45]. Then SRSM was introduced by [PERSON] et al. for imaging polystyrene beads in 2007 [46]. In 2008, [PERSON]'s group reported a high speed, high sensitivity, and three-dimensional multiphoton vibrational imaging technique based on SRS, which showed powerful label-free chemical contrast capability in bio-imaging [43]. The sensing range of typical SRSM is \(\upmu\)M-mM of molecules concentration. Through using a microsecond resonant delay line, the detection sensitivity approaches 21.2 mM at 83 \(\upmu\)s acquisition time [47]. Meanwhile, the super-resolution SRS imaging has been proposed and implemented by combining concepts from stimulated emission depletion (STED) microscopy and femtosecond stimulated Raman spectroscopy (FSRS) [48]. The far-field spatial resolution of SRSM has achieved \(\sim\)130 nm with excitation wavelength of 800\(\sim\)1064 nm [49]. A virtual sinusoidal modulation method was also proposed for retrieving super-resolution SRS images, which demonstrated a spatial resolution of 255 nm [50]. As volumetric imaging provides global understanding of 3D complex systems, [PERSON] et al. demonstrated the Bessel-beam-based simulated Raman projection microscopy and tomography for label-free volumetric imaging with spatial resolution being 0.83 \(\upmu\)m [51]. Typically, the imaging depth of SRSM is 100 \(\upmu\)m for high scattering biological samples and 300-500 \(\upmu\)m for less scattering biological samples. [PERSON] et al. developed a volumetric chemical imaging method that couples Raman-tailored tissue-clearing with SRSM, which achieves greater than 10 times depth increase compared with the standard SRS (Figure 3a) [52].