Baugher Ryan N, Nehmetallah George, Raub Christopher B
Department of Biomedical Engineering, The Catholic University of America, Washington, District of Columbia.
Department of Electrical Engineering and Computer Science, The Catholic University of America, Washington, District of Columbia.
J Histochem Cytochem. 2025 May-Jun;73(5-6):223-236. doi: 10.1369/00221554251348502. Epub 2025 Jun 24.
Immunocytochemistry (IHC) and immunofluorescence (IF) offer crucial diagnostic insights in clinical settings. Most IF assays are performed using fluorophore-conjugated antibodies, but these fluorophores can be subject to issues, such as photobleaching and autofluorescence, that result in lower signal-to-noise ratios (SNR). Gold nanoparticles provide greater signal stability and scatter light well, making them easily separable from the background of biological tissues and improving SNR. In this study, we sought to determine the labeling efficiency, signal quality, and image artifacts of 2.2, 10, and 40 nm diameter gold nanoparticle probes conjugated to antibodies in IF applications on fixed, permeabilized cells in comparison to traditional fluorophores. Overall, micrographs of nanoparticle labels had higher SNR due to lower background signal, and punctate appearances as compared with the continuously distributed signal of the immunofluorescent label. Signal-to-noise ratios varied with nanoparticle diameter, and signal fidelity was worse for keratin versus epithelial growth factor receptor (EGFR). Labeling of EGFR was successful using both extracellular and intracellular epitopes, while poor labeling of keratin 19 with 10 nm diameter nanoparticles was improved by pretreatment with heat and sonication, suggesting hindrance of nanoparticle labels within the fixed, permeabilized cell.
免疫细胞化学（IHC）和免疫荧光（IF）在临床环境中提供了至关重要的诊断见解。大多数IF检测是使用荧光团偶联抗体进行的，但这些荧光团可能会出现诸如光漂白和自发荧光等问题，从而导致较低的信噪比（SNR）。金纳米颗粒具有更高的信号稳定性且散射光良好，使其易于与生物组织背景分离并提高信噪比。在本研究中，我们试图确定与传统荧光团相比，直径为2.2、10和40 nm的金纳米颗粒探针在固定、通透细胞的IF应用中与抗体偶联后的标记效率、信号质量和图像伪影。总体而言，由于背景信号较低，纳米颗粒标记的显微照片具有更高的信噪比，并且与免疫荧光标记的连续分布信号相比呈现点状外观。信噪比随纳米颗粒直径而变化，角蛋白的信号保真度比表皮生长因子受体（EGFR）更差。使用细胞外和细胞内表位均成功标记了EGFR，而用10 nm直径纳米颗粒对角蛋白19的标记不佳通过加热和超声预处理得到改善，这表明纳米颗粒标记在固定、通透细胞内存在阻碍。
