Recently, light microscopy-based techniques have been extended to live mammalian models leading to the development of a new imaging approach called intravital microscopy (IVM). Although IVM has been introduced at the beginning of the last century, its major advancements have occurred in the last twenty years with the development of non-linear microscopy that has enabled performing deep tissue imaging. IVM has been utilized to address many biological questions in basic research and is now a fundamental tool that provide information on tissues such as morphology, cellular architecture, and metabolic status. IVM has become an indispensable tool in numerous areas. This study presents and describes the practical aspects of IVM necessary to visualize epithelial cells of live mouse mammary gland with multiphoton techniques. © 2013 OSA-SPIE.8797 The Optical Society,The Society of Photo-Optical Instrumentation Engineers (SPIE)Amornphimoltham, P., Masedunskas, A., Weigert, R., Intravital microscopy as a tool to study drug delivery in preclinical studies (2011) Adv. Drug. Deliv. Rev., 63, pp. 119-128Andresen, V., Alexander, S., Heupel, W.M., Hirschberg, M., Hoffman, R.M., Friedl, P., Infrared multiphoton microscopy: Subcellular-resolved deep tissue imaging (2009) Curr. Opin. Biotechnol., 20 (1), pp. 54-62Kedrin, D., Gligorijevic, B., Wyckoff, J., Verkhusha, V.V., Condeelis, J., Intravital imaging of metastatic behavior through a mammary imaging window (2008) Nat. Methods., 5 (12), pp. 1019-1021Provenzano, P.P., Eliceiri, K.W., Keely, P.J., Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment (2009) Clin. Exp. Metastasis, 26 (4), pp. 357-370Zipfel, W.R., Williams, R.M., Webb, W.W., Nonlinear magic: Multiphoton microscopy in the biosciences (2003) Nat. Biotechnol., 21 (11), pp. 1369-1377Campagnola, P.J., Loew, L.M., Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms (2003) Nat. Biotechnol., 21 (11), pp. 1356-1360Levitt, J.A., Matthews, D.R., Ameer-Beg, S.M., Suhling, K., Fluorescence lifetime and polarization-resolved imaging in cell biology (2009) Curr. Opin. Biotechnol., 20 (1), pp. 28-36Muller, M., Zumbusch, A., Coherent anti-Stokes Raman scattering microscopy (2007) Chemphyschem, 8 (2), pp. 2156-2170Vakoc, B.J., Lanning, R.M., Tyrrell, J.A., Padera, T.P., Bartlett, L.A., Stylianopoulos, T., Munn, L.L., Bouma, B.E., Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging (2009) Nat. Med, 15 (10), pp. 1219-1223Drew, P.J., Shih, A.Y., Driscoll, J.D., Knutsen, P.M., Blinder, P., Chronic optical access through a polished and reinforced thinned skull (2010) Nat. Methods, 7 (12), pp. 981-984Flusberg, B.A., Nimmerjahn, A., Cocker, E.D., Mukamel, E.A., Barretto, R.P., High-speed, miniaturized fluorescence microscopy in freely moving mice (2008) Nat. Methods., 5 (11), pp. 935-938Looney, M.R., Thornton, E.E., Sen, D., Lamm, W.J., Glenny, R.W., Stabilized imaging of immune surveillance in the mouse lung (2011) Nat. Methods., 8 (1), pp. 91-96Weigert, R., Sramkova, M., Parente, L., Amornphimoltham, P., Masedunskas, A., Intravital microscopy: A novel tool to study cell biology in living animals (2010) Histochem. Cell. Biol., 133 (5), pp. 481-491Cohen, M., Georgiou, M., Stevenson, N.L., Miodownik, M., Baum, B., Dynamic filopodia transmit intermittent Delta-Notch signaling to drive pattern refinement during lateral inhibition (2010) Dev. Cell., 19 (3), pp. 78-89Serrels, A., Timpson, P., Canel, M., Schwarz, J.P., Carragher, N.O., Realtime study of E-cadherin and membrane dynamics in living animals: Implications for disease modeling and drug development (2009) Cancer. Res., 69 (7), pp. 2714-2719Adur Pelegati, J.V.B., De Thomaz, A.A., Baratti, M.O., Andrade, L.A., Second harmonic generation microscopy as a powerful diagnostic imaging modality for human ovarian cancer (2012) J. Biophotonics, , doi: 10.1002/jbio.201200108Adur, J., Pelegati, V.B., De Thomaz, A.A., Baratti, M.O., Almeida, D.B., Optical biomarkers of serous and mucinous human ovarian tumor assessed with nonlinear optics microscopies (2012) PLoS One, 7, pp. e47007. , doi: 10.1371/journal.pone.0047007