Multidimensional custom-made non-linear microscope: from ex-vivo to in-vivo imaging

Year: 2008

Authors: Cicchi R., Sacconi L., Jasaitis A., O’Connor R.P., Massi D., Sestini S., De Giorgi V., Lotti T., Pavone FS.

Autors Affiliation: LENS and Department of Physics, University of Florence, Via Nello Carrara 1, 50019 Sesto Fiorentino, Italy; Department of Human Pathology and Oncology, University of Florence, Viale G.B. Morgagni 85, 50134 Florence, Italy; Department of Dermatology, University of Florence, Via della Pergola 58, 50121 Florence, Italy

Abstract: We have built a custom-made multidimensional non-linear microscope equipped with a combination of several non-linear laser imaging techniques involving fluorescence lifetime, multispectral two-photon and second-harmonic generation imaging. The optical system was mounted on a vertical honeycomb breadboard in an upright configuration, using two galvo-mirrors relayed by two spherical mirrors as scanners. A double detection system working in non-descanning mode has allowed both photon counting and a proportional regime. This experimental setup offering high spatial (micrometric) and temporal (sub-nanosecond) resolution has been used to image both ex-vivo and in-vivo biological samples, including cells, tissues, and living animals. Multidimensional imaging was used to spectroscopically characterize human skin lesions, as malignant melanoma and naevi. Moreover, two-color detection of two photon excited fluorescence was applied to in-vivo imaging of living mice intact neocortex, as well as to induce neuronal microlesions by femtosecond laser burning. The presented applications demonstrate the capability of the instrument to be used in a wide range of biological and biomedical studies.


Volume: 92 (3)      Pages from: 359  to: 365

KeyWords: Fluorescence; Harmonic generation; Lasers; Light emission; Luminescence; Mirrors; Optical systems; Photons; Pulsed laser applications; Sensors, Biological samples; Detection systems; Ex-vivo; Experimental set ups; Femto-second laser; Fluorescence life-time; Human skin; In-vivo; In-vivo imaging; Living animals; Malignant melanomas; Multi-spectral; Non-linear; Non-linear laser imaging; Optical-; Photon counting; Second-harmonic generation; Spherical mirrors; Two photons; Two-color; Two-photon excited fluorescence, Imaging techniques
DOI: 10.1007/s00340-008-3130-3

Citations: 13
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