# Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application
## 2 Laser Scanning Coherent Raman Scattering Microscopy (CRSM)
### Laser Scanning Coherent Anti-Stokes Raman Scattering Microscopy (CARSM)  
In 1965, [PERSON] and [PERSON] discovered the CARS phenomenon. They found a blue-shift of the Raman scattering spectrum when the frequency difference of two incident lasers match the benzene Raman scattering [10]. Then [PERSON] and coworkers demonstrated the first laser scanning CARSM in 1982 [11]. As shown in Figure 1a [12], the CARS is a coherent nonlinear optical process. Two laser beams (frequency \(f_{p}\)) with one for pump and probe and another for Stokes laser (frequency \(f_{s}\)), are impinged to target molecules within the sample. When the frequency difference between pump and Stokes laser satisfies the Raman active vibrational frequency of molecules \(f_{vib}=f_{p}-f_{s}\), the CARS occurs with frequency being \(f_{CARS}=2f_{p}-f_{s}\). Specifically, the molecule absorbs a pump photon to a lower virtual state and emits a Stokes photon for returning to a higher energy ground state. Then the molecule immediately absorbs a probe photon to a higher virtual state, emits a photon with frequency of \(f_{p}+f_{vib}\) and returns to the ground state, as shown in Figure 1a. The optical schematic of laser scanning CARSM is shown in Figure 1b [13]. Two incident lasers are collimated and tightly focused on sample to excite the CARS signal. A time-delay is used to control the temporal overlap of two excitation beams for effective excitation of CARS. The signal of backward propagating is collected by a high _NA_ objective, which is also used to focus incident light. The signal of forward propagating is collected by another objective on transmission light path. The CARS signal is detected by PMT (Photomultiplier tubes) detector after filtering the incident lasers. The CARSM began a rapid development via improving the detection speed, broadband, and sensitivity. [PERSON] et al. developed a high-speed CARS vibrational imaging method, which is two orders of magnitude faster than traditional CARSM by combining high repetition rate laser scanning and analog signal detection scheme [14]. Then a CARSM  
Figure 1: **(a)** Principle of coherent anti-Stokes Raman scattering (CARS) shown by Jablonski diagram (energy level diagram) [12] (reprinted with permission from [12], Copyright 2014 SPIE). **(b)** Diagram of a collinear laser-scanning CARS microscopy [13] (reprinted with permission from [13], Copyright 2018 SPIE). with imaging speed of video-rate was developed for vibrational imaging of tissues in vivo by [PERSON]'s group via detecting strong backscattering of forward CARS signal with video-rate scanning microscopy [15]. Today, higher speed imaging has been achieved by employing more techniques, such as Fourier-transform [16] and deep-learning noise reduction [17]. The spectral bandwidth of CARSM has also shown great increase by multiplex [18; 19] and broadband CARSM techniques [20; 21]. [PERSON] and coworkers reported a two-pulse multiplex CARS microspectroscopy using a picosecond pump beam and a femtosecond Stokes beam to demonstrate the polarization CARS imaging [18]. [PERSON] et al. demonstrated the three-color CARS with continuum pulse of different frequency components as pump and Stokes and a narrowband pulse as probe [22]. As sensitivity of CARSM is restrained by the strong non-resonant background noise, polarization CARSM [23], phase and polarization coherent control CARSM [24], and femtosecond adaptive spectroscopic technique via CARS were introduced for suppressing background noise [25]. [PERSON]'s group developed a broadband CARS imaging platform by using intrapulse three-color excitation and exploiting the strong non-resonant background to amplify the inherently weak fingerprint signal, which provides an advantageous combination of speed, sensitivity and spectral bandwidth [26]. Recently, the study on improving the resolution of CARSM has attracted lots of attentions. By using laser beam shaping and higher-order CARS signal, resolution of CARSM has been remarkably enhanced.