Tracing engineered nanomaterials in biological tissues using coherent anti-Stokes Raman scattering (CARS) microscopy – A critical review

Nanomaterials (NMs) are used in an extremely diverse range of products and are increasingly entering the environment, driving a need to better understand their potential health effects in both humans and wildlife. A major challenge in nanoparticle (eco)toxicology is the ability to localise NMs post exposure, to enable more targeted biological effects analyses. A range of imaging techniques have been applied to do so, but they are limited, requiring either extensive processing of the material, staining or use of high intensity illumination that can lead to photo damage and/or have limited tissue penetration. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free imaging technique, providing contrast based on the intrinsic molecular vibrations of a specimen, circumventing the need for chemical perturbation by exogenous labels. CARS uses near infra-red excitation wavelengths which allow microscopy at depths of several hundred microns in intact tissues and minimises photo-damage to live and delicate samples. Here we provide an overview of the CARS process and present a series of illustrative examples demonstrating its application for detecting NMs within biological tissues, ranging from isolated cells to whole organisms and including materials spanning metals to polymers. We highlight the advantages of this technique which include chemically selective live imaging and substantial depth penetration, but we also discuss its limitations when applied to nanotoxicology, which most notably include the lack of resolution for studies on single nanoparticles.

纳米材料（Nanomaterials, NMs）已被广泛应用于品类繁多的各类产品中，且正持续大量进入自然环境，这使得学界亟需更系统地厘清其对人类与野生生物的潜在健康风险。纳米粒子（生态）毒理学（nanoparticle (eco)toxicology）领域的核心挑战之一，在于如何在暴露后精准定位纳米材料，从而开展更具针对性的生物学效应分析。目前已有多种成像技术被用于实现该目标，但均存在显著局限：要么需要对材料进行复杂预处理、染色标记，要么需采用高强度照明光源——后者不仅可能引发光损伤，且组织穿透能力较为有限。相干反斯托克斯拉曼散射（Coherent anti-Stokes Raman scattering, CARS）显微镜技术作为一种无标记成像手段，可基于样本的固有分子振动产生成像对比度，无需借助外源性标记物进行化学扰动。该技术采用近红外激发波长，能够在完整组织中实现数百微米深度的显微成像，同时最大限度降低对鲜活脆弱样本的光损伤。本文综述了CARS技术的原理流程，并展示一系列示例性案例，阐明其在生物组织内检测纳米材料的应用场景：检测对象涵盖从分离细胞到完整生物体的各类样本，材料类型则覆盖金属至聚合物的诸多品类。本文重点阐述了该技术的优势，包括可实现化学选择性活体细胞成像以及优异的组织穿透深度；同时也讨论了其在纳米毒理学应用中存在的局限，其中最突出的问题是无法实现单纳米粒子水平的分辨率观测。