Nonlinear Imaging Microscopy: Methodological setup and applications for epithelial cancer diagnosis

ADUR J; CARVALHO HF; CESAR CL; CASCO VH

Nonlinear Optics. Fundamentals, Applications, and Technological Advances

Nova Science Publishers

Año: 2014; p. 97 - 136

Nowadays, two-photon excitation and other nonlinear optical approaches have been successfully used within the microscopy framework which evolved as an alternative to conventional single-photon confocal microscopy. In general, they are collectively known as nonlinear microscopy, which includes two-photon excited fluorescence (TPEF) microscopy, second and third harmonic generation (SHG, THG) microscopy, and coherent anti-Stokes Raman scattering (CARS) microscopy. TPEF approach provides high-resolution images of molecular autofluorescence signals in living tissues, SHG microscopy has been successfully used to study structural protein arrays, such as collagen, and THG signal can be used both to reveal the presence of lipid bodies and to highlight the nuclei inside the thickness of a tissue. In the first part of this chapter will be described the nonlinear contrast mechanism of each technique, and how to combine and build these setup, on a single commercial multimodal confocal system platform. It will be demonstrate that it is easy-to-operate, suitable to perform TPFE, SHG, and THG imaging, using only the standard femtosecond Ti:sapphire laser. Also, will be explained the modifications over the scan head and the detector and also the physical evidences of the higher harmonics will be submitted. Following, the usefulness of these techniques to detect early changes associated with the progression of epithelial cancer will be showed. Thereby, this multi-contrast nonlinear optics microscopy setup, emerge as a valuable tool to differentiate between cancerous, benign, and healthy tissue.