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

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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.