Nonlinear Optical Microscopy applied to study the histological features of human and murine colon

ADUR J; BIANCHI M; VIALE S; IZAGUIRRE MF; ALMEIDA D; CASCO VH; CARVALHO HF; CESAR CL

Simposio; XI Brazilian Symposium on Extracellular Matrix and VI International Symposium on Extracellular Matrix; 2011

Hematoxylin and eosin (H&E) staining is one of the most popular methods for pathologists to perform medical diagnosis. The samples analysis is achieved by white light microscopy, where thin sections are needed. Multiphotonic fluorescence microscopy (MPM) uses pulsed long-wavelength light from a femtosecond pulsed laser to excite fluorophores within the specimen being observed. With the exception of two photon excitation fluorescence (TPEF), which is firmly established as a valuable approach for biomedical imaging; second and third harmonic generations (SHG and THG respectively) have recently become the most widely used contrast approach in nonlinear optical (NLO) microscopy. Connective tissue is visualized because of the high sensitivity of SHG to supramolecular structure, while THG microscopy provides information from interfaces. Since this methodology could represent several advantages over classical staining, it is very important that the data obtained allow an adequate histological interpretation. In this work, we assess the capacity of NLO microscopy to distinguish morphological features in mouse normal colon and human colon cancer. The combination of TPEF, SHG and THG provides rich information about the structural architecture and molecular distribution within the sample, comparable with that obtained from classical histological H&E staining. SHG offers detailed information from collagen fiber (which usually is altered in several connective tissue disorders). THG provides valuable information from nuclei shape, size and number (very important when the aim is to detect dysplasia, for example in cancer studies). The present study suggest that the combined application of these techniques can give us richer information than the obtained by classic histopathological techniques. Additionally SHG and THG can be carried out with minimal photobleaching and phototoxicity to cells and images of adequate quality can be obtained. Also, we have demonstrated that multicontrast nonlinear microscopy has the capacity to differentiate between cancerous and healthy tissue, especially with qualitative information about collagen and nuclei provided by the THG and SHG microscopy.