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 CH (mainly in proteins) and CH 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.
A novel, Yb-fiber laser based, handheld 2PEF/SHG microscope imaging system is introduced. It is suitable for imaging of murine skin at an average power level as low as 5 mW at 200 kHz sampling rate. Amplified and compressed laser pulses having a spectral bandwidth of 8 to 12 nm at around 1030 nm excite the biological samples at a ~1.89 MHz repetition rate, which explains how the high quality two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG) images are obtained at the average power level of a laser pointer. The scanning, imaging and detection head, which comprises a conventional microscope objective for beam focusing, has a physical length of ~180 mm owing to the custom designed imaging telescope system between the laser scanner mirrors and the entrance aperture of the microscope objective. Operation of the all-fiber, all-normal dispersion Yb-fiber ring laser oscillator is electronically controlled by a two-channel polarization controller for Q-switching free mode-locked operation. The whole nonlinear microscope imaging system has the main advantages of the low price of the fs laser applied, fiber optics flexibility, a relatively small, light-weight scanning and detection head, and a very low risk of thermal or photochemical damage of the skin samples.
A 20 MHz repetition rate, sub ps Ti:sapphire (Ti:S) laser system is proposed for real time, high chemical contrast dual vibration resonance frequency (DVRF) CARS imaging of the skin suitable for in vivo histology.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.