# Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application
## 3 Second Harmonic Generation Microscopy (SHGM)  
SHG process is a special case of the sum frequency process, also called \"frequency doubling\". As shown in Figure 4a [66], by using two incident lights with same frequency, the output is twice the frequency of the incident lights. The incident light is called fundamental frequency and the output with frequency doubling is called second harmonic. In 1961, SHG was first confirmed with a quartz sample by [PERSON] et al. [67]. In 1974, [PERSON] and [PERSON] firstly integrated SHG and microscopy by imaging SHG signals from polycrystalline ZnSe [68]. A typical setup of SHGM is shown in Figure 4b [69]. The excitation wavelength was 780 nm. A galvo scanner was used to direct the beam in a raster-scan pattern. The beam was reflected by a dichroic mirror and focused onto the sample using a high _NA_ water-immersion objective 1. The emitted backward SHG signal was collected by the same objective, whereas the forward signal was collected by a low _NA_ objective 2. In both geometries the same two filters were used: one filter was used to block  
Figure 3: (**a**) SRS images of regions in the same white matter region before and after tissue-clearing [52] (reprinted with permission from [52], Copyright 2019 PNAS). (**b**) In Vivo SRSM images of human glioblastoma multiforme xenografts [58] (reprinted with permission from [58], Copyright 2013 AAAS). (**c**) Individual SRS images of a 1-mm-thick fresh mouse brain section at (**i**) 1658 cm\({}^{-1}\), (**ii**) 1670 cm\({}^{-1}\), and (**iii**) 1680 cm\({}^{-1}\) and (**iv**) the composite three-color image showing the distribution of lipid (green), normal protein (blue), and amyloid plaque (magenta) [59] (reprinted with permission from [59], Copyright 2019 AAAS). (**d**) SRSM of untreated cell walls [61] (reprinted with permission from [61], Copyright 2012 AAAS). the fundamental laser, and the other was a band-pass filter to transmit the SHG signal at 390 nm. The photomultiplier tubes in both geometries were used to record the forward and backward SHG images. In 1986, the first biological SHG imaging experiments were done by [PERSON] and [PERSON] to study the orientation of collagen fibers in rat tail tendon [70]. In 1993, [PERSON] examined the second-harmonic response of biological membrane in electric fields via using chiral styryl dye [71]. He also demonstrated his work on live cell imaging [72]. In 2010, [PERSON] extended SHG to whole animal in vivo imaging [66]. The review by [PERSON] showed how SHGM achieved multimodal microscopic images of biological tissues, namely backward, forward and polarization SHG imaging [73]. However, the spatial resolution of SHGM is vastly restricted due to the near infrared excitation wavelength. Many methods have been carried out in order to improve the spatial resolution. Radially-polarized laser illumination is an effective and easy way for dramatically enhancing the resolution of SHGM [74; 75; 76]. [PERSON] et al. demonstrated that spatial resolution was improved more than 21% by using radially-polarized beam focused by elliptical mirror compared with lens [77]. The work of [PERSON] et al. showed that the subtractive imaging method could been extended to SHGM that increased the resolution to 210 nm and obtained a better contrast simultaneously [78]. [PERSON] et al. combined SHGM with structured illumination based on point-scanning, which improves the resolution by factors of ~1.4 in the lateral and 1.56 in the axial directions, respectively [74]. [PERSON] proposed pixel reassignment, in which the point detector of the traditional confocal microscopy is replaced by a multi-element array detector [79]. Combining with deblurring operation, this method showed a spatial resolution enhancement of about 1.87 compared with conventional SHGM [80]. Nowadays, SHGM has been extensively used in visualization to structures and functions of cells and tissues.