# Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application
## 5 Discussion
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Type**} & **Work** & **Work** & **Repetition** & & **Lateral** & **Asial** & **Ref.** \\  & **Principle** & **(nm)** & **Rate** & & **Resolution** & **Resolution** & **Resolution** & **Ref.** \\ \hline \multirow{8}{*}{**CRS**} & supercritical & \multirow{2}{*}{\begin{tabular}{c} coherent \\ the relative \\ phase \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} 835 (pump) \\ 1107 (Stokes) \\ \end{tabular} } & \multirow{2}{*}{76 MHz} & \multirow{2}{*}{\
\begin{tabular}{c} 3 mW (pump) \\ 10 mW (Stokes) \\ \end{tabular} } & \multirow{2}{*}{295} & \multirow{2}{*}{3.32 μm} & \multirow{2}{*}{[28]} \\  & & & & & & & \\ \cline{3-8}  & & coherent & & & & & \\  & & controlling of & 728 (Stokes) & & & & & \\  & the relative & 727.8 (pump) & - & - & 130 & - & [29] \\  & & phase & & & & & \\ \cline{3-8}  & & higher-order & 1041 (Stokes) & & \multirow{2}{*}{80 MHz} & \multirow{2}{*}{1.8 \(\times\) 10\({}^{9}\)-\(3.6\times 10^{21}\) W/cm\({}^{2}\)} & \multirow{2}{*}{196} & \multirow{2}{*}{-} & \multirow{2}{*}{[30]} \\  & & & & & & & \\  & & (pump) & & (Stokes) & & & \\ \hline \multirow{8}{*}{**SRSM**} & near- & \multirow{2}{*}{
\begin{tabular}{c} resonance \\ enhancement \\ \end{tabular} } & \multirow{2}{*}{450} & \multirow{2}{*}{80 MHz} & \multirow{2}{*}{4.2 mW (pump)} & \multirow{2}{*}{130} & \multirow{2}{*}{-} & \multirow{2}{*}{[49]} \\  & & resonance & & & & & \\  & enhancement & & & & & & \\ \cline{3-8}  & & & & & & & \\ \cline{3-8}  & & & & & & & \\ \hline \multirow{8}{*}{**SHGM**} & structured & \multirow{2}{*}{1064} & \multirow{2}{*}{40 MHz} & \multirow{2}{*}{30 mW} & \multirow{2}{*}{235} & \multirow{2}{*}{693 nm} & \multirow{2}{*}{[74]} \\  & illumination & & & & & & \\ \cline{3-8}  & & subractive & & & & & \\ \cline{3-8}  & imaging & 800 & 80 MHz & - & 210 & - & [78] \\  & pixel & & & & & & \\  & reassignment & & & & & & \\ \hline \multirow{8}{*}{**SLSPM**} & scanning & \multirow{2}{*}{632.8} & \multirow{2}{*}{-} & \multirow{2}{*}{-} & \multirow{2}{*}{186} & \multirow{2}{*}{2.3 nm} & \multirow{2}{*}{[103]} \\  & localized SPP & & & & & & \\ \cline{1-1} \cline{6-6}  & scanning & & & & & & \\ \cline{1-1} \cline{6-6}  & localized SPP & & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Comparison of three laser scanning microscopic technologies. involving only virtual state without energy deposition, SHGM brings little photodamage or photobleaching by increasing the repetition rate that keeps the peak intensity below damage threshold [122; 123]. The laser powers and repetition rates of label-free LSMs are indicated in Table 1.  
However, there are still some limitations for current label-free laser scanning microscopies. CARSM is relatively easy to implement but with non-resonance background from a four-wave mixing process inside sample. This impedes accessibility of CARSM to higher resolution and sensitivity in biological imaging. Compared to CARSM, SRSM is background-free imaging, but the extraction of SRS signal requires complex lock-in detection for modulation and demodulation. SHGM is suitable for samples without inversion symmetry, such as collagen and myosin, which limits its scope of application. SLSPM is limited to image samples within evanescent penetration depth of SPPs and have a slow imaging speed due to scanning mode and sophisticated data process. From the above discussion, no single imaging technology can present a global understanding to the complex biological structure and process. Therefore, multi-technique combination is a trend for imaging and information analysis to complicated samples [124; 125; 126]. New imaging schemes will make these label-free laser scanning microscopies to be more powerful biological imaging tool when combine with spectroscopy, laser and detection technology. Higher resolution, sensitivity and faster imaging speed are still the most important pursuits of microscopy for more detailed and accurate morphology and dynamic process.