TartanSW: a new method for spectrally-resolved standing wave cell microscopy and mesoscopy

细胞运动等重要的生物过程依赖于细胞表面形状的动态变化。除了在运动性和细菌病原体的摄取方面，细胞膜在神经细胞之间形成突触和免疫系统细胞处理抗原的过程中也积极地改变形状。神经元生长锥显示了在几秒钟的时间尺度上发生的突起，在许多活动细胞中看到了更快的运动。不幸的是，传统的显微镜方法无法准确回答一个基本问题：细胞膜的形状是什么，对于附着的细胞来说，它比底物高出多高？50年来一直使用反射干涉对比法，但这种方法实际上报告的是细胞内靠近膜的质量分布，而不是膜的位置。我们最近报道了一种驻波荧光成像方法，以超分辨率深度绘制细胞膜表面，使用一种在拥有标准资源和基础设施的生物医学实验室中几乎免费实施的方法。在我们的驻波工作中，我们将染有荧光的红细胞放在一面简单的镜子上，而不是放在显微镜载玻片上，并使用驻波(SW)来创建次衍射限制的照明平面。我们观测到的轴向分辨率约为90 nm，这与所描述的其他超分辨率技术相当，但由于我们生成多平面图像，因此我们可以很容易地在此分辨率下获得样品的3D信息。这一建议的实质是在这个驻波作品中增加了一种新的方法，我们称之为TartanSW(因为有色条纹图案与纺织品图案的相似性)。没有标出高度的等高线没有什么价值，但我们发现，通过使用多波长窄带探测，我们可以根据驻波波动点的颜色识别它们的顺序，从而区分丘陵和山谷。我们建议首先开发一种简单的成像显微镜系统，能够以每秒100幅图像的速度同时记录多个波长，以提供关于细胞结构的超分辨率3D信息。我们将首先用染料单分子层和模型标本来表征显微镜，然后将TartanSW成像扩展到准备了标记细胞膜的荧光标记的单个红细胞。基于我们的初步工作，我们希望能够检测到红细胞膜结构的非常微小但高速的变化。我们还将应用该方法来研究神经元的高动态骨架结构，并跟踪细胞边缘随时间的生长。我们还建议使用介晶镜进行TartanSW成像，这是一种新的巨型物镜，能够以亚细胞分辨率成像大组织标本，目前是我们实验室独有的。通过将TartanSW应用于中层透镜，将有可能以比目前中层透镜所能处理的更高的3D分辨率来成像数百个细胞。我们将应用TartanSW介镜来研究前面描述的相同的红细胞和神经元样本，在以高分辨率同时对数百个细胞进行成像时，我们预计将更容易检测到可能指示疾病发病的罕见事件或异常细胞，就像我们已经研究的受疟疾感染的红细胞一样。我们将帮助和鼓励其他实验室采用超分辨率TartanSW显微镜，这种显微镜可以在任何已配备荧光显微镜的实验室以低成本实施，尽管Mesolens目前是Strathclyde独有的，但现有的Mesolab设施将支持广泛获得拟议的技术。

Important biological processes such as cell movement depend on dynamic changes in the shape of the cell surface. As well as in motility and the ingestion of bacterial pathogens, the cell membrane changes shape actively in the formation of synapses between nerve cells and the handling of antigens by cells of the immune system. The neuronal growth cone shows protrusions occurring over a time scale of seconds and much faster movements are seen in many motile cells.Unfortunately, conventional microscope methods fail to provide exact answers to one of the basic questions: 'what is the shape of the cell membrane and how high is it above the substrate in the case of attached cells?'. For 50 years reflection interference contrast has been used but this method actually reports the distribution of mass within the cell near to the membrane rather than the position of the membrane.We have recently reported a standing wave method of fluorescence imaging to map the surface of the cell membrane with super-resolution in depth, using a method that is almost cost-free to implement in a biomedical sciences laboratory with standard resource and infrastructure. In our standing-wave work, we placed fluorescently-stained red blood cells atop a simple mirror instead of a microscope slide and using a standing wave (SW) to create sub-diffraction limited planes of illumination. We observed an axial resolution of around 90 nm, which is comparable to other the super-resolution techniques described, but because we generate multi-planar images, we can readily obtain 3D information on the specimen at this resolution. The essence of this proposal is to add to this standing-wave work a new method which we call TartanSW (because of the similarity of the coloured fringe patterns to textile patterns). A contour map without heights marked on the lines is of little value, but we have discovered that by using multiple wavelength narrowband detection we can recognize the order of the standing wave antinodes by their colours and so tell the difference between hills and valleys. We propose to first develop a simple imaging microscope system, capable of recording multiple wavelengths simultaneously at speeds of up to 100 images per second, to provide super-resolved 3D information on cell structure. We will first characterise the microscope with dye monolayers and model specimens, and then extend the TartanSW imaging to individual red cells prepared with a fluorescent label that stains the cell membrane. Based on our preliminary work we expect to be able to detect very tiny but high-speed changes in the structure of the red cell membrane. We will also apply the method to study the highly dynamic skeletal structure of neurones and follow the growth of the cell edge over time.We also propose to perform TartanSW imaging with the Mesolens, a new giant objective lens that is capable of imaging large tissue specimens with sub-cellular resolution and which is at present unique to our laboratory. By applying TartanSW with the Mesolens, it will be possible to image hundreds of cells at even higher 3D resolution than the Mesolens can manage at present. We will apply TartanSW mesoscopy to study the same red cell and neurone specimens described previously, and in imaging hundreds of cells with high resolution simultaneously we expect it will be easier to detect rare events or abnormal cells that may indicate onset of disease, as in the malaria infected red cells which we have already studied.We will aid and encourage other laboratories to take up super-resolution TartanSW microscopy, which could be implemented at low cost in any lab already equipped with a fluorescence microscope, and although the Mesolens is presently unique to Strathclyde, the existing Mesolab facility will support wide access to the proposed technology.