Daaboul George G, Freedman David S, Scherr Steven M, Carter Erik, Rosca Alexandru, Bernstein David, Mire Chad E, Agans Krystle N, Hoenen Thomas, Geisbert Thomas W, Ünlü M Selim, Connor John H
nanoView Diagnostics Inc., Boston, MA, United States of America.
Department of Mechanical Engineering, Boston University, Boston, MA, United States of America.
PLoS One. 2017 Jun 26;12(6):e0179728. doi: 10.1371/journal.pone.0179728. eCollection 2017.
Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (40 nm), while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (360 nm). Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.
光学显微镜是检测和分析寄生虫、真菌及原核生物的有力工具，但用于检测单个病毒颗粒却颇具挑战。未标记的病毒颗粒太小，无法用标准可见光显微镜观察到。病毒颗粒的表征通常使用诸如电子显微镜或原子力显微镜等高分辨率方法。这些方法需要将病毒粒子从其正常环境中纯化出来，需要相当专业的知识，而且每个视野只能计数少量颗粒。在此，我们利用一种名为单粒子干涉反射成像传感器（SP-IRIS）的可见光成像方法，该方法可对数千个单个病毒粒子进行自动计数和大小测量。病毒粒子直接从复杂溶液中捕获到硅芯片上，然后使用反射干涉成像方式进行检测。我们表明，使用不同的成像波长可使多种病毒颗粒可视化。使用紫光/紫外光照明，SP-IRIS技术能够检测单个黄病毒粒子（约40纳米），而绿光照明能够识别并区分水疱性口炎病毒和痘苗病毒（约360纳米）。引人注目的是，该技术能够清晰识别丝状传染性埃博拉病毒粒子和病毒样颗粒。区分和量化未标记病毒粒子的能力扩展了传统光学显微镜的用途，并且可以体现在一种简单的台式方法中，从而实现从生物流体的快速检测到用于疫苗开发和生产的病毒样颗粒分析等广泛应用。
