We have developed a multimodal optical platform for label-free imaging on the basis of nonlinear optical microscopy (NLO) for applications in biology and biomedical science. Three application areas have been chosen as examples: regenerative medicine, drug delivery monitoring and cancer diagnosis. Obtained data showed the potential of NLO microscopy for the following: (1) investigating the stem cell differentiation states prior their use in regenerative medicine and tissue repair, (2) tracking drug molecules in living cells and (3) detecting cancer by imaging biopsies ex vivo. The results of this study illustrate the potential to translate NLO microscopy from ex vivo to in vivo applications. Copyright (c) 2013 John Wiley & Sons, Ltd.