FASPRI: a new method for increased spatial resolution in surface plasmon imaging of unlabelled living cells

FASPRI：一种提高未标记活细胞表面等离子体成像空间分辨率的新方法

• 批准号: BB/T011602/1
• 负责人: Gail McConnell
• 金额: $ 17.56万
• 依托单位: University of Strathclyde
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT011602%2F1
• 关键词: FASPRI; new; method; increased; spatial

Surface plasmon resonance (SPR) can be used to monitor molecular events such as the binding of an antibody to its target. It is a highly sensitive optical method. In this method, a parallel beam of light is directed into a glass prism, coated on the hypotenuse face with a metal (often gold), so that it reflects off the metal film and emerges from the prism. At a certain angle of incidence, the light excites collective oscillations of free electrons knows as surface plasmons and the gold ceases to reflect light. This is seen as a sharp dip in the plot of reflected light intensity versus angle of incidence. The position and the depth of the dip change dramatically when an antibody binds to the antigen-coated gold, and the degree of binding can thus be measured accurately, even when the bound antibody layer is only one molecule thick. Attempts to form high-resolution microscope images with SPR have failed because the beam of light reflected is parallel, and such beams cannot form detailed images. We propose here to transform SPR into a high-resolution microscope method for imaging events involving small numbers of molecules in living cells without the need to label them with specific dyes. The reflected beam in the standard SPR method is essentially reflected by a metal mirror. It is a basic physical principle that in such a situation the incidence and reflected light interfere to produce a so-called standing wave, which has zero intensity at the mirror surface. It has been established since the 19th century that no light can be detected at the mirror surface. It may be useful to think of the standing waves that can be produced by hand in a skipping rope tethered to a wall at one end. At the wall no motion can be detected in the rope. We propose to place a thin fluorescent layer made of fluorescent organic dye or nanoscale crystals between the glass and the gold film. This will ordinarily not fluoresce because it is in the aforementioned zone of zero intensity. However, in any region where SPR occurs, the reflected beam will be substantially reduced in intensity (i.e. the mirror will cease to reflect) and instead of a standing wave, there will be an ordinary propagating wave passing into the gold layer, creating resonance. Fluorescence will then be excited. Since the fluorescence will radiate in all directions it will be ideal for imaging: we will use a microscope specifically optimised for high-resolution and sensitive imaging of light radiating from fluorescent particles. We call this approach FASPRI, which stands for Full-Aperture Surface Plasmon Resonance Imaging, since filling the full aperture of the lens is our key improvement over other attempts.First, we will sandwich a fluorescent layer between a gold film and a microscope coverslip. Next, we will check that the fluorescence appears at the plasmon resonance angle, which we will measure by reflectance. We will then adapt our existing fluorescence microscope and then perform FASPRI imaging of living algal and mammalian cells.

表面等离子体共振(SPR)可用于监测分子事件，如抗体与其靶标的结合。这是一种高度灵敏的光学方法。在这种方法中，一束平行的光束被引导到一个玻璃棱镜上，在斜边表面涂上一层金属(通常是金)，这样它就会从金属薄膜上反射出来，从棱镜中出来。在一定的入射角度下，光激发自由电子的集体振荡，即表面等离子激元，金停止反射光。这被视为反射光强度随入射角的曲线急剧下降。当抗体与包被抗原的金结合时，浸渍的位置和深度发生显著变化，因此即使结合的抗体层只有一个分子厚，也可以准确地测量结合的程度。用SPR形成高分辨率显微镜图像的尝试失败了，因为反射的光束是平行的，这样的光束无法形成详细的图像。我们建议将SPR转变为一种高分辨率显微镜方法，用于成像涉及活细胞中少量分子的事件，而不需要用特定的染料标记它们。在标准的SPR方法中，反射光束本质上是由金属反射镜反射的。这是一个基本的物理原理，在这种情况下，入射和反射光干涉产生所谓的驻波，该驻波在镜面上的强度为零。自19世纪以来，人们就已经确定，在镜面上检测不到光。想想在一端系在墙上的跳绳上可以用手产生的驻波可能是有用的。在墙边，绳子上没有任何动静。我们建议在玻璃和金膜之间放置一层由荧光有机染料或纳米晶体制成的薄荧光层。这通常不会发出荧光，因为它在前面提到的零强度区域。然而，在发生表面等离子体共振的任何区域，反射光束的强度将大幅降低(即反射镜将停止反射)，并且将有一个普通的传播波进入金层，产生共振，而不是驻波。然后荧光就会被激发。由于荧光将向各个方向辐射，这将是成像的理想选择：我们将使用专门为荧光粒子辐射的光进行高分辨率和高灵敏度成像而优化的显微镜。我们称这种方法为FASPRI，代表全口径表面等离子体共振成像，因为填充透镜的全孔径是我们相对于其他尝试的关键改进。首先，我们将在金膜和显微镜盖片之间夹一层荧光层。接下来，我们将检查荧光是否出现在等离子激元共振角，我们将通过反射率来测量该角度。然后，我们将改装我们现有的荧光显微镜，然后对活的藻类和哺乳动物细胞进行FASPRI成像。

项目成果

Application of Light-Sheet Mesoscopy to Image Host-Pathogen Interactions in Intact Organs.

• DOI: 10.3389/fcimb.2022.903957
• 发表时间: 2022
• 期刊: Frontiers in cellular and infection microbiology
• 影响因子: 5.7

Multimodal optical mesoscopy reveals the quantity and spatial distribution of gram-positive biofilms in ex vivo tonsils

多模态光学介观镜揭示离体扁桃体革兰氏阳性生物膜的数量和空间分布

• DOI: 10.1101/2023.07.03.547470
• 发表时间: 2023
• 作者: Clapperton M

Enhanced fluorescence from semiconductor quantum dot-labelled cells excited at 280 nm

半导体量子点标记细胞在 280 nm 激发时发出增强的荧光

• DOI: 10.1101/2021.11.08.467709
• 发表时间: 2021
• 作者: McFarlane M

Time-lapse mesoscopy of Candida albicans and Staphylococcus aureus dual-species biofilms reveals a structural role for the hyphae of C. albicans in biofilm formation

• DOI: 10.1101/2023.08.31.555792
• 发表时间: 2023-09
• 期刊: Microbiology
• 影响因子: 1.5
• 作者: K. Baxter;Fiona A. Sargison;J. R. Fitzgerald;Gail McConnell;P. Hoskisson

Enhanced fluorescence from semiconductor quantum dot-labelled cells excited at 280 nm.

半导体量子点标记细胞在 280 nm 处激发的增强荧光。

• DOI: 10.1088/2050-6120/ac5878
• 发表时间: 2022
• 期刊: Methods and applications in fluorescence
• 影响因子: 3.2
• 作者: McFarlane M