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. Multiphoton microscopy using short‐wave infrared (SWIR) radiation, such as two‐photon fluorescence, offers a nondestructive and high‐resolution method to image through tissue, but the broad spectral peaks limit its multiplexing capabilities. Hyper‐Raman scattering offers spectrally narrow features and the use of reporter‐functionalized noble metal nanoparticles selective targeting in biological media. For the first time, we present SWIR surface‐enhanced resonance hyper‐Raman scattering (SERHRS) spectra as a two‐photon imaging method in tissue. SWIR SERHRS combines two‐photon imaging advantages with narrow vibrational peak widths, presenting future applications of multitargeted bioimaging.