Abstract
Super-resolution microscopy techniques break the diffraction limit of conventional optical microscopy to achieve resolutions approaching tens of nanometres. The major advantage of such techniques is that they provide resolutions close to those obtainable with electron microscopy while maintaining the benefits of light microscopy such as a wide palette of high specificity molecular labels, straightforward sample preparation and live-cell compatibility. Despite this, the application of super-resolution microscopy to dynamic, living samples has thus far been limited and often requires specialised, complex hardware. Here we demonstrate how a novel analytical approach, Super-Resolution Radial Fluctuations (SRRF), is able to make live-cell super-resolution microscopy accessible to a wider range of researchers. We show its applicability to live samples expressing GFP using commercial confocal as well as laser- and LED-based widefield microscopes, with the latter achieving long-term timelapse imaging with minimal photobleaching.
Highlights
The development of fluorescence microscopy in the 20th century was a major advancement in cell biology, allowing researchers to dynamically observe the intracellular behaviour of labelled molecules and organelles in living cells
Assuming that the emitting fluorophores in each frame are separated by distances greater than the diffraction limit, computational analysis can be used to pinpoint the centres of the emitting molecules with high accuracy and as such a super-resolution ‘map’ of the imaged structure can be generated
In addition to examples present in the literature, that Super-Resolution Radial Fluctuations (SRRF) is a versatile and straightforward method for livecell super-resolution imaging
Summary
The development of fluorescence microscopy in the 20th century was a major advancement in cell biology, allowing researchers to dynamically observe the intracellular behaviour of labelled molecules and organelles in living cells. SIM is a widefield technique, which generates interference patterns between the labelled structure in the sample and the periodically patterned (rather than homogeneous) illumination field From these patterns, an image of the structure can be calculated at a resolution beyond the diffraction limit. While the above-mentioned super-resolution techniques have established themselves as an important part of cell biology research, the major advantage of super-resolution microscopy, i.e. live-cell compatible imaging, is still not widely exploited. This is due to some fundamental limitations of the techniques. Methods for adapting SMLM techniques for live-cell imaging are a active area of research
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