Vibrational two-photon microscopy for tissue imaging: Short-wave infrared surface-enhanced resonance hyper-Raman scattering

Multiphoton microscopy using short-wave infrared (SWIR) radiation offers nondestructive and high-resolution imaging through tissue. Two-photon fluorescence (TPF), for example, is commonly employed to increase the penetration depth and spatial resolution of SWIR imaging, but the broad spectral peaks limit its multiplexing capabilities. Hyper-Raman scattering, the vibrational analog of TPF, yields spectral features on the order of 20 cm^-1 and reporter-functionalized noble metal nanoparticles (NPs) provide a platform for both hyper-Raman signal enhancement and selective targeting in biological media. Herein we report the first tissue imaging study employing surface-enhanced resonance hyper-Raman scattering (SERHRS), the two-photon analog of surface-enhanced resonance Raman scattering. Specifically, we employ multicore gold-silica NPs (Au@SiO₂ NPs) functionalized with a near infrared-resonant cyanine dye, 3,3'-diethylthiatricarbocyanine iodide as a SERHRS reporter. SWIR SERHRS spectra are efficiently acquired from mouse spleen tissue. SWIR SERHRS combines two-photon imaging advantages with narrow vibrational peak widths, presenting future applications of multitargeted bioimaging.