Significance: In breast-preserving tumor surgery, the inspection of the excised tissue boundaries for tumor residue is too slow to provide feedback during the surgery. The discovery of positive margins requires a new surgery which is difficult and associated with low success. If the re-excision could be done immediately this is believed to improve the success rate considerably.

Aim: Our aim is for a fast microscopic analysis that can be done directly on the excised tissue in or near the operating theatre.

Approach: We demonstrate the combination of three nonlinear imaging techniques at selected wavelengths to delineate tumor boundaries. We use hyperspectral coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excited fluorescence (TPF) on excised patient tissue.

Results: We show the discriminatory power of each of the signals and demonstrate a sensitivity of 0.87 and a specificity of 0.95 using four CARS wavelengths in combination with SHG and TPF. We verify that the information is independent of sample treatment.

Conclusions: Nonlinear multispectral imaging can be used to accurately determine tumor boundaries. This demonstration using microscopy in the epi-direction directly on thick tissue slices brings this technology one step closer to clinical implementation.