Superresolution microscopy

Abstract

There are many types of optical microscopy systems that produce superresolution. This discussion centers on optical microscopy techniques that have the potential to extract features from objects that are one the scale of 100 nm or less, which is much smaller than what can be achieved with a classical optical microscope. In order to achieve resolution on the order of 100 nm with visible-light photons, more information must be obtained from the system than a single image using classical illumination can produce. In some cases, the extra information is in the form of a series of images with a customized illumination pattern. In other systems, the object displays response characteristics that effectively reduce the size of the scanning laser spot used to illuminate it. In yet other systems, light is forced through a nanosized aperture and then scanned over the object. None of the systems described in this chapter actually change the characteristics or physics of the optical systems used to collect photons. Instead, classical optical systems are cleverly combined with advanced illumination techniques and postprocessing that produce superresolution images. In the introduction, basic concepts regarding classical resolution are reviewed, and terms are defined that are important with respect to understanding how superresolution microscopy works. Subsequent sections describe superresolution techniques, including scanning aperture techniques, 4-Pi microscopy, enhancement/depletion techniques, photoactivated localization, lattice light-sheet microscopy, and structured illumination. A short comparison of techniques for live-cell imaging is also provided. Although examples of the techniques are given, this chapter is not intended to be a state-of-the-art review inclusive of all variations. Instead, the intent is to provide a basic understanding of the primary classes of superresolution microscopy techniques. It is notable that work in this area has generated several recent Nobel prizes, because of the importance to science of being able to resolve structures and physiology at the nanoscale [26.1].