## 3 EM Techniques for CH Diagnosis
### Microscopy
#### 3.1.3 Practical Application of Microscopy to CH
Given the significance of the artwork, samples were obtained in a limited number (the red numbered dots in Figure 2) to examine the layered structure and to reveal non-original retouching [13]. \begin{table}
\begin{tabular}{p{142.3 pt} p{142.3 pt}} \hline \hline \multirow{4}{*}{Super-Resolution Fluorescence Microscopy} & **Techniques:** Stimulated Emission Depletion (STED), Photo-Activated Localization Microscopy (PALM), Stochastic Optical Reconstruction Microscopy (STORM) & **Techniques:** Approximately 20–50 nm \\  & **Capabilities:** Breaks the diffraction limit of light microscopy, allowing visualization of individual proteins and molecular complexes. \\  & **Limitations:** Requires specialized equipment, fluorescent labeling, and complex data analysis. \\ \hline \multirow{4}{*}{Atomic Force Microscopy (AFM)} & **Resolution:** 1 nm laterally, 0.1 nm vertically \\  & **Capabilities:** Provides topographical maps of surfaces at nanometer resolution, applicable to live or fixed samples. \\ \cline{1-1}  & **Limitations:** Limited to surface features and small scan areas. \\ \hline \hline \end{tabular}
\end{table} 
Table 6: _Cont._ \begin{table}
\begin{tabular}{p{142.3 pt} p{142.3 pt}} \hline \hline \multirow{2}{*}{Light Microscopy (EM)} & Light microscopy is generally the most accessible and cost-effective option for many laboratories. \\ \hline \multirow{2}{*}{Electron Microscopy (EM)} & Electron microscopy (TEM and SEM) provides high resolution but is costly, complex, and requires specialized training, limiting routine use to specialized research centers. \\ \hline \multirow{4}{*}{Super-Resolution Fluorescence Microscopy (AFM)} & Super-resolution methods like STED (Stimulated Emission Depletion Microscopy), PALM (Photo-Activated Localization Microscopy), and STORM (Stochastic Optical Reconstruction Microscopy) offer high resolution but are costly, complex, and require specialized equipment and expertise, limiting use to advanced research labs. \\ \hline \multirow{2}{*}{Atomic Force Microscopy (AFM)} & AFM provides high-resolution surface imaging of live samples but is costly, requires training, and has limited scan area, making it less versatile for broad biological use. \\ \cline{1-1}  & \\ \hline \hline \end{tabular}
\end{table} 
Table 7: Comparative overview of cost-effectiveness and accessibility of various microscopy techniques. Nine out of ten samples were embedded in a bioplastic resin for cross-section examination under a stereomicroscope with visible and ultraviolet light. The microscopic techniques applied to this masterpiece include:  
* Visible and fluorescent light microscopy (VLM and FLM);
* SEM with EDS (as in [PERSON] et al. [69]);
* Stereomicroscopy (which allows higher levels of magnification and provides a stereoscopic view of the specimen, aiding the examination of photographic features [98]). Table 8 reports the results of pigment analyses obtained solely through microscopy techniques, while Figure 3 provides an impressive overview of the different layers of this painting, from glue size to paint layer. Optical and electron microscopy are also used to quantify the side effects of irradiation [103] in parchment [103; 104; 105] and paper [106]. The effects of laser cleaning on pigments are also investigated through microscopy [100]. Figure 4 is a high-resolution (HR) SEM image that shows the difference between cellulose fibers of ancient paper, which are long, and those of modern paper, which are shorter and mixed with granular material. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Color** & **Pigment** & **Technique** \\ \hline Red & Possible red lake with alum-derived substrate & FLM, SEM-EDS \\ Brown & Umber & SEM-EDS \\ Black & Bone black & SEM-EDS \\ Transparent & Quartz & SEM-EDS \\ \hline \hline \end{tabular}
\end{table} 
Table 8: Pigment analysis results by the mean of only microscopy technique applied in the study of [PERSON]’s Salvator Mundi. Data have been taken from [13] (p. 4, Table 3).