Imaging of mouse experimental melanoma in vivo and ex vivo by combination of confocal and nonlinear microscopy

KEY WORDS second harmonic generation; two-photon microscopy; mouse melanoma; hyper-thermia treatmentABSTRACT We investigated possibilities of the combination of the one- and two-photon excita-tion microscopy for examination of the experimental melanoma tissue in vivo, in mice under gen-eral anesthesia, and ex vivo on freshly harvested specimens. Our aim was to obtain sufficiently in-formative images of unstained tumor tissues and their modifications after hyperthermia treat-ment. The mouse experimental melanoma structure was studied and compared with normal tissuefrom the same animal by using confocal and nonlinear microscopy techniques based on (i) one-pho-ton excitation (1PE) fluorescence, (ii) 1PE reflectance, (iii) second harmonic generation imaging,and (iv) two-photon excitation autofluorescence. We checked different spectral conditions and othersettings of image acquisition, as well as combinations of the above imaging modalities, to fullyexploit the potential of these techniques in the evaluation of treated and untreated cancer tissuemorphology. Our approach enabled to reveal the collagen fiber network in relation with the othertissues, and to identify invasive tumor cells. It also proved to be useful for the examination of inter-relationships between functional and morphological aspects based on optical properties of the tis-sues, especially in studies of changes between the tumor and control tissue, as well as changesinduced by physical treatments, e.g., delivery of microwave hyperthermia treatment. These differ-ences were also evaluated quantitatively, when we found out that the maximum Euler–Poincare´characteristic reflects well the melanoma morphological structure. The results showed that theproposed investigative approach could be suitable also for a direct evaluation of tissue modifica-tions induced by clinical interventions. Microsc. Res. Tech. 72:411–423, 2009.