Stain-free Histopathology of Basal Cell Carcinoma by Dual Vibration Resonance Frequency CARS Microscopy

Basal cell carcinoma (BCC) is the most common malignancy in Caucasians. Nonlinear microscopy has been previously utilized for the imaging of BCC, but the captured images do not correlate with H&E staining. Recently, Freudiger et al. introduced a novel method to visualize tissue morphology analogous to H&E staining, using coherent anti-Stokes Raman scattering (CARS) technique. In our present work, we introduce a novel algorithm to post-process images obtained from dual vibration resonance frequency (DVRF) CARS measurements to acquire high-quality pseudo H&E images of BCC samples. We adapted our CARS setup to utilize the distinct vibrational properties of CH₃ (mainly in proteins) and CH₂ bonds (primarily in lipids). In a narrowband setup, the central wavelength of the pump laser is set to 791 nm and 796 nm to obtain optimal excitation. Due to the partial overlap of the excitation spectra and the 5-10 nm FWHM spectral bandwidth of our lasers, we set the wavelengths to 790 nm (proteins) and 800 nm (lipids). Nonresonant background from water molecules also reduces the chemical selectivity which can be significantly improved if we subtract the DVRF images from each other. As a result, we acquired two images: one for "lipids" and one for" proteins" when we properly set a multiplication factor to minimize the non-specific background. By merging these images, we obtained high contrast H&E "stained" images of BBC's. Nonlinear microscope systems upgraded for real time DVRF CARS measurements, providing pseudo H&E images can be suitable for in vivo assessment of BCC in the future.