超靈敏明場顯微鏡可實現無標記單一蛋白質偵測

Abstract

能夠解析奈米尺度生物物件的無標記光學顯微技術，對於在不受螢光標記干擾的情況下研究分子層級的生物過程至關重要。近年來，干涉散射顯微術（interferometric scattering microscopy, iSCAT）的發展，使得研究人員得以利用奈米粒子本身的折射率對比，直接偵測尺寸極小的物件。為了達到高靈敏度，傳統的 iSCAT 系統多採用共路徑反射式幾何架構，使奈米粒子所產生的微弱散射場與基板反射所形成的參考場產生干涉。反射式 iSCAT 已展現極高的偵測能力，甚至可達到單一蛋白質的無標記偵測；然而，在細胞或複雜生物樣本中的應用，常受到強烈背景反射（特別是來自細胞膜）的限制，導致胞內奈米結構的訊號被掩蓋。 在本論文中，我們採用相干明場顯微術（Coherent Brightfield Microscopy, COBRI）——一種以穿透式架構實作的 iSCAT 形式，並進一步將其靈敏度提升至可進行無標記單一蛋白質偵測的層級。透過調控雷射照明的空間相干性，並結合主動回授控制以提升光學系統的機械穩定性，我們大幅抑制了相干雜訊，成功獲得來自奈米尺度散射體的穩定干涉訊號。此外，藉由對後焦平面（back focal plane）進行瞳函數工程設計，進一步增強干涉對比度，最佳化對弱散射奈米物件的偵測能力。配合進階影像處理與分析方法，我們得以解析對應於單一蛋白質在基板界面結合的動態事件。 透過此方法，本研究達成的靈敏度下限相當於散射截面低至 10⁻⁶ nm²。此一靈敏度使我們能夠偵測多種單一蛋白質，包括 免疫球蛋白 G（IgG）、去鐵蛋白（apoferritin）、甲狀腺球蛋白（thyroglobulin）以及 免疫球蛋白 M（IgM），其分子量範圍涵蓋 150 至 915 kDa。更重要的是，量測到的干涉對比度與分子量呈現線性關係，使得在穿透式幾何架構下可進行定量分子質量分析，其準確度約為 2%。本研究所展示的超高靈敏度穿透式 iSCAT 顯微技術，拓展了無標記干涉成像在生物相關環境中的應用範疇，並為在複雜系統中進行單分子交互作用與動態行為的定量研究開啟了新的可能性。

Label-free optical microscopy techniques capable of resolving nanoscale biological objects are essential for studying molecular processes without perturbation from fluorescent labels. Recent advances in interferometric scattering (iSCAT) microscopy have enabled direct detection of nanoscopic objects through their intrinsic refractive-index contrast. To achieve high detection sensitivity, conventional iSCAT implementations typically rely on a common-path reflection geometry, in which the weak scattering signal from a nanoparticle interferes with a reference field reflected from the substrate. While reflection-mode iSCAT has demonstrated exceptional sensitivity—including label-free detection of single proteins—its application to cellular and complex biological samples is hindered by strong background reflections, particularly from cell membranes, which obscure signals from intracellular structures. In this thesis, we employ coherent brightfield (COBRI) microscopy, an iSCAT modality implemented in a transmission configuration, and extend its sensitivity to the regime of label-free single-protein detection. By tailoring the spatial coherence of the laser illumination and enhancing the mechanical stability of the optical system through active feedback control, coherent noise is substantially suppressed and stable interferometric signals from nanoscale scatterers are achieved. Furthermore, interference contrast is enhanced via back-focal-plane pupil function engineering, optimizing the detection of weakly scattering nano-objects. Advanced image processing and analysis methods are applied to resolve dynamic events corresponding to individual protein binding at the substrate interface. Using this approach, we achieve a sensitivity limit corresponding to scattering cross-sections as low as 10⁻⁶ nm². This sensitivity enables the detection of individual proteins including immunoglobulin G (IgG), apoferritin, thyroglobulin, and immunoglobulin M (IgM), spanning molecular masses from 150 to 915 kDa. Importantly, the measured interferometric contrast scales linearly with molecular mass, allowing quantitative mass analysis in a transmission geometry with an accuracy of approximately 2%. The demonstrated ultrasensitive transmission-mode iSCAT microscopy expands the applicability of label-free interferometric imaging to biologically relevant environments and opens new opportunities for quantitative studies of single-molecule interactions and dynamics in complex systems.