Abstract
Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.
Highlights
The smaller a structure, such as a cell organelle, a filament or a molecule, the faster it can move because of less friction
In this study we have shown that thousands of super-resolved, high contrast images of dynamic, unlabelled cells can be acquired at high frame rates without loss in image quality
In our recent study[16], we have shown by experiments, computer simulations and mathematical theory that total internal reflection rotating coherent scattering (TIR-ROCS) microscopy generates its superior resolution and contrast by interference effects
Summary
The smaller a structure, such as a cell organelle, a filament or a molecule, the faster it can move because of less friction. Super-resolving microscopy techniques have increased in speed and image contrast[1,2,3,4,5] or have forged ahead to image structures of a sub-100 nm length scale[6,7,8,9,10], they all have the problem that the image acquisition process needs much time or many photons or both. In a similar approach with a rotating laser, but based on a different contrast formation and without post-processing[15], we could demonstrate high-contrast super-resolution imaging of a 2D layer of small, fixed latex beads by rotating www.nature.com/scientificreports/. In this article we show that despite multiple scattering of the laser illumination light it is possible to record super-resolved images of living, highly dynamic cells based on multiple interferences. The images of adherent J774 mouse macrophages and of swimming helical bacteria offer an exceptionally high contrast, while thousands of images are recorded at 63 and 100 Hz without any loss in image quality and without any post-processing
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