# Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application
## 4 Scanning Localized Surface Plasmon Microscopy (SLSPM)
They found that if the sample is slightly shifted from the focus by about 1 \(\upmu\)m, the image contrast (refractive index sensitivity) will be improved and the refractive index can be retrieved from the interference fringes [106; 107]. In 2012, [PERSON] et al. proposed a confocal system integrated scanning SPM, which provides a simpler and more stable alternative [108]. [PERSON] et al. reported a high-resolution metal-clad waveguide scanning microscopy with a diffraction-limited resolution, which can be operated in both TM and TE waveguide modes with radially and azimuthally polarized beams, respectively, and allows both refractive index and topography of dielectric objects to be evaluated at high resolution and sensitivity [106]. [PERSON] reported a high resolution imaging with lateral resolution of ~170 nm and thickness resolution against the deposited lipid bilayer being ~0.33 nm [109]. [PERSON] et al. proposed a workflow to make SLSPM as a complement for EM studies (Figure 8a, which allows for the quantification of the impact of chemical fixation on the cell-substrate interface [102]. As SPPs is evanescent wave, the SLSPM is particularly attractive for studying cell adhesion and dynamics without staining the samples [110; 111; 112]. Time-lapse long-term imaging of living adherent cells was reported via combining SLSPM and fiber interferometer [113]. Adherence and motility of living C2C12 myoblast cells are monitored for  
Figure 7: **(a)** The entire back focal plane (BFP) is illuminated with radially polarized light resulting in an illumination of the sample with a broad range of angles. **(b)** Illuminating the sample under surface plasmon polaritons (SPP) excitation angles induces the SPP evanescent field penetrating the cytosol. **(c)** The BFP imaging at each scanning point allows for the extraction of **(d)** reflectance curve [102] (reprinted with permission from [102], Copyright 2020 Wiley-VCH). 50 h, revealing the dynamics of these cells during their migration (Figure 8b) [114]. Further understanding at the bio-interface has growing importance in fundamental neuroscience and biomedical research. With SLSPM, cell-substrate interface was real-time monitored and its cleft gap distance was measured in-situ [115]. [PERSON] et al. showed a temporal and spatial mapping to changes of both refractive index and the cell-substrate distance which strongly correlates with the action potentials. They also reconstructed the three-dimensional profiles of the basal cell membrane and its dynamics, which reached an actual measurement accuracy of 2.3 nm (Figure 8c) [103]. By using SLSPM, they also analyzed the refractive index of cell organelles quantitatively in a noninvasive and label-free manner with a lateral resolution of diffraction limit [104]. Figure 8: (a) a, d, Correlation of scanning localized surface plasmon microscopy (SLSPM) and electron microscope (EM) for two individual cells. b, e, The cell substrate distances are evaluated along the cross-section with surface plasmon resonance microscopy (SPRM). c, f, Focused ion beam milling combined with scanning electron microscopy (FIB-SEM) for the same cells, [102] (reprinted with permission from [102], Copyright 2020 Wiley-VCH). (b) (i)–(vi) SLSPM images of living C2C12 cells at different times. The arrows have the following meaning: filopodia (F), lamellipodia (L), nucleus (N) and fronts (B) [114] (reprinted with permission from [114], © The Optical Society). (c) Nanometer-resolved mapping of cell-substrate distances of contracting Cardiomycetes. Cell image under live-imaging mode and scanned area (white rectangular box) (left), cell–substrate distance and refractive index change over time (middle), morphology distribution under SLSPM [103] (reprinted with permission from [103], Copyright 2018 ACS Publications).