Super-resolution microscopy is opening up new avenues in the biosciences, however all widely-used techniques require addition of fluorescent probes. Now, we have demonstrated that optical-super-resolution imaging is possible in unlabelled living cells, using the phenomenon of optical super-oscillation. Super-oscillation is an intriguing mathematical phenomenon, first described in the context of quantum mechanics. It is widely accepted that any function that is band-limited (in frequency) oscillates no faster (in time) than its fastest Fourier component. However, a band-limited super-oscillatory function may oscillate arbitrarily fast in regions of relatively low intensity [1]. In the context of optics, this means that we can create an arbitrarily small hotspot far from any lens or surface, simply using precisely engineered interference of light. Super-oscillatory hotspots, beyond the dark perimeter, are necessarily surrounded by sidebands that contain a significant fraction of the optical power – trading efficiency for resolution. To use this spot for imaging, we replace the conventional focusing lens in a confocal microscope with a super-oscillatory lens and use a confocal imaging arrangement to reject the light scattered from the sidebands. The resolution of the image formed is determined by the size of the super-oscillatory hotspot. We previously demonstrated far-field imaging with sub-wavelength resolution on non-fluorescent manufactured samples [2]. We have developed the system further and can now image unlabelled cells at super-resolution. To do this we combine our super-oscillatory microscope with an advanced form of polarisation contrast imaging. The instrument is a modification of a standard confocal microscope, with two key components: spatial light modulators (SLMs) to shape the laser beam entering the microscope, and a liquid crystal (LC) panel to control the input polarisation. We capture four different super-resolved images of the sample with different incident polarisations, from which we can calculate the anisotropy and orientation angle of each pixel (using a similar method to ref [3]). This highlights those parts of a cell with significant molecular structuring, such actin filaments, microtubules, and even protein enriched lipid bilayers such as vesicles and cell membranes. We have applied this to cells, illustrated in figure 1, showing it is able to reveal new levels of information in biological samples. Figure 1a shows a schematic of our setup, and 1b shows a metal sector star sample illustrating the polarisation contrast, where brightness shows level of anisotropy and hue shows the polarisation angle at which most light is reflected. In c and d we show images of a live, unlabelled MG63 cell. The super-oscillatory image is sharper than the equivalent confocal image of the same system, seen even more clearly in the zoomed in insets i and ii.
Advances in Bio-Imaging (Warwick, UK) (2016) Proc Physiol Soc 36, C03
Poster Communications: Super-oscillatory polarisation contrast for super-resolution imaging of unlabelled cells
E. T. Rogers2,1, S. Quraishe4,2, T. A. Newman4,2, J. E. Chad3,2, N. I. Zheludev1,5, P. J. Smith2,3
1. Optoelectronics Research Centre, Faculty of Physical Sciences and Engineering, University of Southampton, Southampton, Hampshire, United Kingdom. 2. Institute for Life Sciences, University of Southampton, Southampton, Hampshire, United Kingdom. 3. Biological Sciences, Faculty of Natural and Environmental Science, University of Southampton, Southampton, Hampshire, United Kingdom. 4. Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, Hampshire, United Kingdom. 5. Centre for Disruptive Photonic Technologies, Nanyang Technological University, Singapore, Singapore.
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