High-resolution multiphoton optical tomography of tissues: an in vitro and in vivo study

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Abstract
Multiphoton optical tomography based on NIR (near-infrared) femtosecond laser pulses provides non-invasive optical sectioning of skin with high spatial intracellular resolution and high tissue penetration. The imaging system DermaInspect was used to perform this technology in clinical studies in vivo on patients with suspicious melanoma. Pigmented cell clusters based on non-linear luminescence were clearly distinguished from non-pigmented cells in the epidermis using the autofluorescence of endogenous fluorophores like NAD(P)H, flavins, keratin, elastin, collagen and melanin. Some of the investigated tissues showed differences in the structure of the epidermal layers and the presence of dendritic cells compared to normal skin. Multiphoton laser microscopy was used to visualize extracellular matrix (ECM) structures of native and tissueengineered heart valves. The quality of the resulting 3-D images allowed an exact differentiation between collagenous and elastic fibers. The analysis of heart valve tissues of patients with cardiomyopathy revealed a dramatic loss of its capability to generate SH (second harmonic), indicating a structural deformation of the collagenous fibers, which was virtually impossible to obtain by routine histological or immunohistological staining. These results indicate that NIR femtosecond laser scanning systems can be employed as novel non-invasive optical technology for 3-D resolved ECM component imaging and in vitro and in vivo tissue diagnosis.

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