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

表面等离子体共振（SPR）可用于监测分子事件，例如抗体与其靶标的结合。这是一种高灵敏度的光学方法。在这种方法中，一束平行的光被引导到一个玻璃棱镜中，在棱镜表面涂上金属（通常是金），这样它就被金属膜反射并从棱镜中出来。在一定的入射角下，光激发自由电子的集体振荡，称为表面等离子体激元，并且金停止反射光。这在反射光强度与入射角的关系图中被视为急剧下降。当抗体与抗原包被的金结合时，凹陷的位置和深度急剧变化，因此即使结合的抗体层只有一个分子厚，也可以精确地测量结合的程度。利用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，并且这样的光束不能形成详细的图像。我们在这里建议将SPR转化为一种高分辨率的显微镜方法，用于成像活细胞中涉及少量分子的事件，而不需要用特定的染料标记它们。标准SPR方法中的反射光束基本上由金属镜反射。一个基本的物理原理是，在这种情况下，入射光和反射光干涉产生所谓的驻波，其在镜面处的强度为零。自世纪以来已经确定在镜面处不能检测到光。我们不妨想象一下，用手在一端系在墙上的跳绳上可以产生驻波。在墙上，绳子没有运动。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级晶体制成的薄荧光层。这通常不会发荧光，因为它在上述的零强度区。然而，在SPR发生的任何区域中，反射光束的强度将大幅降低（即，镜子将停止反射），并且将存在普通传播波而不是驻波，从而进入金层，产生共振。然后将激发荧光。由于荧光将向各个方向辐射，因此非常适合成像：我们将使用专门针对荧光颗粒辐射光的高分辨率和灵敏成像进行优化的显微镜。我们称这种方法为FASPRI，它代表全孔径表面等离子体共振成像，因为填充透镜的全孔径是我们对其他尝试的关键改进。接下来，我们将检查荧光是否出现在等离子体共振角，我们将通过反射率测量。然后，我们将调整现有的荧光显微镜，然后对活的藻类和哺乳动物细胞进行FASPRI成像。

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.