# Clinical Applications of In Vivo and Ex Vivo Confocal Microscopy
## 3 Ex Vivo Confocal Laser Scanning Microscopy  
Ex vivo CLSM applied to skin analysis has been available on the market since 2013, so it is a relatively new and not fully explored imaging technique, allowing a real-time microscopic examination of freshly excised unfixed tissue, exploring structures and cells at a near histologic resolution. The analyzed specimen is optically sliced, scanned by a point source of light (laser) and, through a pinhole rejecting out of focus light, a thin section of tissue is obtained and displayed on a monitor [7; 82]. To date, the only commercially available device is VivaScope 2500 (MAVIG GMBH, Munich, Germany), a new version of VivaScope 2000. Ex vivo CLSM works in two different modalities: the reflectance mode (830 nm laser wavelength), mainly employed for in vivo settings and the fluorescence mode (488 or 658 nm laser wavelength), usually employed for ex vivo examination [7; 83]. Using reflectance mode, no staining is required, because the contrast is related to the different refractive index of cellular structures (i.e., melanin, keratin), similarly to in vivo RCM [83; 84]. In 2001, [PERSON] et al. reported the first pictures obtained from BCC excised during [PERSON] micrographic surgery, highlighting the necessity to improve the quality of images through a staining technique to enhance the contrast [8]. Fluorochromes in ex vivo CLSM act like dyes in histopathology, enabling the identification of cell and tissue structures, depending on the wavelength of the exciting light as well as the features of the target tissue [85]. Among these agents, acridine orange is the most largely used, followed by methylene or toluidine or Nile blue, fluorescein or Patent blue V [82]. Acridine orange has been introduced as a nuclear contrast agent, able to bind nuclear DNA, offering the best contrast into epithelial tissues without affecting subsequent frozen sections and formalin-fixed histopathology quality. [PERSON] et al. [86] compared three different concentrations of acridine orange (0.1, 0.3 and 0.6 mM), reaching the optimum at 0.6 mM for 20 s, to enhance the contrast, minimizing artifacts in the dermis. The specimen can be observed entirely \"en face\" to assess the horizontal extension, or transversally, as for conventional histology; the examination of large lesions is possible because the instrument can show wide mosaics reaching up to 20 \(\times\) 20 mm [87]. The tissue sample is embedded between two glass slides fixed together with silicon glue, enabling both sides examination. Aqueous gel or silicone can be applied in order to aid the contact of the external part of the sample and the glass slide. Furthermore, obtaining a flattened sample, by means of a \"tissue press\", is important to avoid potential artifacts resulting from an irregular contact between the surface of the specimen and the glass slide that may be related to irregular cutting. When put on the microscope stage, each specimen is scanned along the x-axis and the y-axis using a diode laser illumination. A two-dimensional, high-resolution sequence of horizontal images of 750 \(\times\) 750 \(\upmu\)m of the different tissue layers up to a thickness of 200 \(\upmu\)m is acquired and stitched together into a mosaic. The lateral and axial spatial resolutions are 1 \(\upmu\)m and 3 \(\upmu\)m, respectively. Since the microscope is confocal, only the in-focus plane can be visualized and it has to be adjusted moving the objective lens [82]. The displayed image depends on the conversion of the reflectant/fluorescent signal from nuclei of cells by a software. In reflectance mode, gray-scale images are visualized, showing dark hypo-reflective structures and bright hyper-reflective structures, overlapping to in vivo RCM appearance. On the other hand, with fluorescence contrast, non-fluorescent structures appear dark, as compared to the white fluorescent ones.