# Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application
## 4 Scanning Localized Surface Plasmon Microscopy (SLSPM)  
Surface plasmon polaritons (SPPs) is the collected oscillation of electrons at metal-dielectric interface. As SPPs is evanescent wave along metal-dielectric interface, the excitation of SPPs requires the wavevector matching condition satisfied by prism, waveguide, and gratings. Due to their high sensitivity to refractive index change near interface, in 1984, SPPs was firstly used as gas detector and bio-sensor based on Kretschmann configuration [92; 93], then SPPs became an essential tool for biological and chemical sensing [94; 95; 96; 97]. Due to non-intrusive, label-free and high sensitivity surface plasmon microscopy (SPM) is a suitable technology for near-field imaging to the cell-substrate interface [98]. However, the resolution of SPM is limited by propagation length of SPPs, e.g., being 7 \(\upmu\)m for gold film illuminated by 633 nm wavelength. In order to improve the resolution, [PERSON] et al. firstly presented SLSPM, in which the SPPs is excited by a focused laser beam as sensing probe to measure refractive index and film thickness [99; 100]. Polarization of incident light is an important factor to increase resolution of SLSPM. Axially symmetric radial-polarized light optimizes the probe with full width at half maximum of 210 nm that increases the resolution [101]. [PERSON] et al. used the SLSPM to image microspheres with diameter being 1.5 \(\upmu\)m [100]. Nowadays, SLSPM have improved lateral resolution of SPP imaging up to \(\sim\)200 nm by using high numerical aperture (_NA_) objective [96; 101]. Figure 6: (**a**) Representative examples of FFT analysis for benign (grade 0), grade I, grade II and grade III breast tissues [86] (reprinted with permission from [86], Copyright 2020 Wiley-VCH). (**b**) PIPO (polarization-in, polarization-out) SHGM of collagen in (A1–A8) Periducal, (B1–B8) Lobular, (C1–C8), Parenchymal in pancreas tissues, and (D1–D8) Tumor with (A1, B1, C1, D1) Bright-field images, (A2, B2, C2, D2) SHG intensity images, (A3, B3, C3, D3) Color-coded maps of the fitted R values, (A4, B4, C4, D4) Occurrence histograms of the R values, (A5, B5, C5, D5) Color-coded maps of the fitted C values, (A6, B6, C6, D6) Occurrence histograms, (A7, B7, C7, D7) Degree of linear polarization values, and (A8, B8, C8, D8) Occurrence histograms [87] (reprinted with permission from [87], Copyright 2019 Frontiers Media SA). The setup of SLSPM is shown in Figure 7a [102], the collimated incident light illuminates objective after transforming through lenses, mirrors and radial polarizer, which ensures that incident light is TM-polarization in each incident plane of focal beam. The incident light is focused into spot with diffraction limit dimension to excite SPPs. The maxima of incident angle is determined by \(\mathbf{NA}\) of objective as \(NA=~{}n~{}sin\theta_{M}\) (\(\theta_{M}\) is maximum angle of incident light). The intensity distribution on back focal plane (BFP) is recorded by a CCD camera, and a high-resolution image of specimen is acquired by using two-dimensional scanning. Figure 7b shows how the SPP evanescent field penetrates the cytosol on gold film. A typical BFP image is shown in Figure 7c, in which the dark ring indicates the SPPs excited by the incident light and ring radius represents propagation constant of SPPs. Along the radial direction, the reflective intensity profile dependent to incident angle is illustrated in Figure 7d. The refractive index and thickness of specimen can be obtained by fitting the resonant angle using a theoretical multilayer model [103; 104]. To gain better imaging quality and greater application, researchers have made many attempts to improve the performance and expand its application range. SLSPM was demonstrated to detect oligonucleotide-functionalized nanoarray by [PERSON], et al. [105]. In order to improve the image contrast, [PERSON] et al. introduced a \(Z\)-direction movement mechanism to the two-dimensional scanning mechanism.