Super-resolution imaging in live cells

Abstract

Over the last twenty years super-resolution fluorescence microscopy has gone from proof-of-concept experiments to commercial systems being available in many labs, improving the resolution achievable by up to a factor of 10 or more. There are three major approaches to super-resolution, stimulated emission depletion microscopy, structured illumination microscopy, and localisation microscopy, which have all produced stunning images of cellular structures. A major current challenge is optimising performance of each technique so that the same sort of data can be routinely taken in live cells. There are several major challenges, particularly phototoxicity and the speed with which images of whole cells, or groups of cells, can be acquired. In this review we discuss the various approaches which can be successfully used in live cells, the tradeoffs in resolution, speed, and ease of implementation which one must make for each approach, and the quality of results that one might expect from each technique.

Keywords: Localisation microscopy; Stimulated emission depletion microscopy; Structured illumination microscopy; Super-resolution microscopy.

Fig. 1

Fluorescence super-resolution methods. (a) Structured illumination microscopy. When a sample (left) is imaged, the resultant image is convolved with the point spread function of the microscope (right). This image has information at a range of frequencies, up to the transmission frequency of the microscope (middle). (b) When a grating is projected onto the sample, then in frequency space the sample information is reproduced at the grating frequency (blue). Due to this shift in frequency, frequencies not visible in the widefield image are shifted into the visible range (red). The resulting image (green) is the sum of the original image (yellow), and the positive (blue/red) and negative (dotted) frequency shifted copies. (c) By taking multiple images at different angles, the shifted higher frequency information can be extracted, giving a super-resolution image. (d) Stimulated emission depletion microscopy. In confocal microscopy, a diffraction limited point of light is scanned across the sample (left) giving a diffraction limited image (right). (e) If a doughnut shaped depletion beam is used, the effective beam size is smaller (left) and so the image from naturally emitted light is sharper (right). (f) Localisation microscopy. Sparse sets of fluorophores are excited (left) and imaged (right). The centers of the diffraction limited spots are localised (red). (g) By repeating this process many times (left) a super-resolution image of the sample can be built up from these localised centres (right). (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this paper.)