## 2 Visual and Microscopical Analyses
Optical microscopy and SEM are also applied for the identification and study of these microorganisms, which possibly can contaminate the parchment objects and cause the microbial degradation of the library materials [4; 15; 17]. As an example of the application of the SEM analyses in the parchment materials, Figure 2 reveals the microbial damage to the collagen fibers and the presence of characteristic cell chains that are typical of filamentous bacteria being on the surface of parchment from the Archimedes Palimpsest [18]. The SEM image shows that an outer layer of the parchment is still intact, but it is dotted with bacterial cells (mostly spore chains filaments that are smaller than one micron, which are likely to be actinomycetes). In the lower left corner of the image, the outer layer is eroded with collagen fibers, showing cavities and holes due to the occurrence of biological activity (collagenolytic exoenzymes can be deduced). Another state-of-the-art technique is light transmission analysis (LTA), which is used to assess the damage that occurs to the native parchment collagen. The LTA equipment consists of a microscopy system that is used to register the images in polarized light, thus recording what happens during denaturation (i.e., shrinkage or loss of birefringence of the fibrous tissue). The images are then linked to the specific characteristics of the LTA curve [19], which is obtained during the experiment. The application of LTA for the analysis of collagen composite materials allows for the quantitative assessment of the state of integrity of the material by measuring its chemical stability through the hydrothermal denaturation temperature [19; 20]. During the LTA analysis, the parchment fibers are heat-jellified in an aqueous solution, while a signal that is generated by light passing through the sample is recorded. The fluctuations of the signal during the transformation process are converted into a denaturation curve as a function of temperature. The curve is defined by specific peaks, which indicate the denaturation temperature and provide the semi-quantitative information on the chemical stability of the collagen pool. The higher the  
Figure 2: Scanning electron microscopical (SEM) image. The surface of parchment from the Archimedes Palimpsest [18]. The image was obtained in high vacuum mode using an EVO50 SEM (CarlZeiss) on a gold-metalized sample without further preparation. Picture shows the surface of collagen fibers of parchment at a magnification of about 20,000 X (20 KX). Picture and legend: Dr. [PERSON]. temperature that corresponds to the position of the peak is, then the higher the chemical stability is. Recent studies have applied complementary analyses, including the LTA method, to investigate the phenomenon of purple stain biodeterioration on parchment [5; 20; 21; 22]. These analyses have helped to highlight the nature of a microbial attack, which modifies the collagen populations throughout the biodegradation process. The results of this complementary approach pointed to a salt-mediated origin of the purple stain damage [20], revealed the presence of bacteriorhodopsin, and supported the presence of _Halobacteria_ by detecting their DNA by using the high-throughput sequencing (NGS) technology (see Section 4.2). In addition, the LTA analysis revealed that the deterioration had spread to the highly structured collagen fibers over time.