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

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.

We have previously demonstrated how a red blood cell membrane can be imaged with a confocal microscope, and its contour mapped with 90 nm axial resolution, simply by placing the specimen on a first-surface mirror. This simple apparatus gives super-resolution along the instrument axis at negligible cost to a lab already equipped with a fluorescence microscope. We also discovered that because of polychromatism the emission was not simply a sine-squared function of distance from the reflector. This showed that multi-spectral detection and analysis could allow unequivocal identification of the order of the internode and thus of the absolute distance from the reflector. We have also demonstrated in unpublished work that the SW method can be performed on a standard wide-field epi-fluorescence microscope at imaging speeds of 100 frames/second.

We propose here to extend this proof-of-concept research by developing new technologies that will further improve the position sensing and temporal resolution of our new SW method, which we call TartanSW. We will first build a multi-channel microscope to support individual live cell 3D imaging with an axial resolution below the diffraction limit at speeds of up to 100 Hz. We will prove the utility of TartanSW imaging by studying red cells prepared with lipophilic membrane dyes to study fast 3D changes in cell membrane, and we will also perform TartanSW imaging of fluorescently-tagged neuronal growth cones at the same high imaging speed in response to chemical gradients.

Using the same cell specimens will also evaluate the TartanSW method with the Mesolens, a giant 4x/0.47 N.A. objective lens which supports imaging of large tissue volumes with sub-cellular resolution. This will extend the TartanSW method from imaging a single cell to being able to image several hundreds of cells simultaneously, which will improve cell statistics and aid the detection of rare events or abnormalities that may indicate onset of disease.

Our first publication in this field has already aroused a lot of interest, notably from parasitologists and neurobiologists, and we now plan to maximise the non-academic impact.

For impact to arise from research, a combination of novel technology, obvious utility and ready accessibility is necessary. Standing wave imaging is not new, but we have shown, better than ever before, its utility in our red cell membrane work (Scientific Reports, 4 7359 (2014)). Our intention is to make this novel technology more widely accessible to researchers and non-academic beneficiaries. We will achieve this in two ways. The first is by using commercially available hardware for TartanSW microscopic imaging, so that all biomedical scientists can test this resource at the development stage. The second is to use the existing Mesolab facility mechanisms to give the UK research community access to TartanSW mesoscopic imaging. As with confocal microscopy, which progressed from a physics lab curiosity to a universal working tool in only two years, the impact of TartanSW at both the microscopic and mesoscopic scale is likely to be significant, once the applications have been demonstrated.

Initial impacts are likely to be in terms of economic benefits to UK industry, once the phase of academic development is complete. The UK laser research and development industry is buoyant, and the applicants have excellent links to the key manufacturers of ultra-short pulsed lasers. Through organisation of the European Molecular Biology Organisation Practical Course in Advanced Optical Microscopy, the applicants have good relationships with manufacturers of microscopes and photo-detectors both in the UK and overseas. Despite the fact that the initial work on super-resolution optical microscopy was done overseas, this work is will help to steer the focus of low-cost 'practical' super-resolution microscopy towards the UK.

Public sector beneficiaries will, in the longer-term, include healthcare professionals in haematology and infectious diseases. Locally, the new South Glasgow Hospitals campus houses a new biomedical imaging facility and we are already in discussion with the NHS regarding new technologies which may offer improvements over existing imaging methods.

The general public will gain increased awareness and understanding of the science through the engaging demonstrations proposed in the 'Pathways to Impact' statement, and ultimately gaining health benefits through discoveries in fundamental biomedical science.

In general, the UK science community has been a spectator of super-resolution developments, which have occurred in Germany or the USA because of better funding and better links between engineers, physicists and biomedical scientists. However, one UK scientist's reaction to our first publication in this field was to ask 'Does this mean we could have had super-resolution years ago?'. We believe that the answer is yes and in this proposal we are providing an opportunity for industry in the UK to get into this field, using a method which is possible even in a period of funding austerity.