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Methodology designed to circumnavigate the classical Abbe diffraction barrier in optical microscopy is rapidly advancing using both ensemble and single-molecule techniques.
Over the past several decades, fluorescence microscopy has become an essential tool for examining a wide variety of biological molecules, pathways, and dynamics in living cells, tissues, and whole animals. In contrast to other techniques (such as electron microscopy), fluorescence imaging is compatible with cells that are being maintained in culture, which enables minimally invasive optical-based observation of events occurring on a large span of timescales. In terms of spatial resolution, several techniques including positron-emission tomography, magnetic resonance imaging, and optical coherence tomography can generate images of animal and human subjects at resolutions between 10 centimeters and 10 micrometers, whereas electron microscopy and scanning probe techniques feature the highest spatial resolution, often approaching the molecular and atomic levels (see Figure ). Between these two extremes in resolving power lies optical microscopy. Aside from the benefits derived from being able to image living cells, the most significant drawback to all forms of fluorescence microscopy (including widefield, laser scanning, spinning disk, multiphoton, and total internal reflection) are the limits to spatial resolution that were first elucidated and described by Ernst Abbe in the late 1800s.
The Abbe diffraction limit (or at least the recognition of this limit) stood for almost a century before inventive microscopists began to examine how their instruments could be improved to circumvent the physical barriers in order to achieve higher resolution. Due to the fact that axial resolution is far lower than lateral resolution (by at least a factor of two), much of the work conducted in the latter part of the twentieth century addressed improvements to performance in the axial dimension. Researchers discovered that laser scanning confocal instruments produced very modest increases in resolution at the cost of signal-to-noise, and that other associated technologies (including multiphoton, structured illumination, and spinning disk) could be used for optical sectioning, but without significant improvement in axial resolution. An important concept to note, and one of the most underappreciated facts associated with optical imaging in biology, is that the achieved microscope resolution often does not reach the physical limit imposed by diffraction. This is due to the fact that optical inhomogeneities in the specimen can distort the phase of the excitation beam, leading to a focal volume that is significantly larger than the diffraction-limited ideal. Furthermore, resolution can also be compromised by improper alignment of the microscope, the use of incompatible immersion oil, coverslips having a thickness outside the optimum range, and improperly adjusted correction collars.
The most significant advances in superresolution imaging have been achieved in what is termed far-field microscopy and involve either spatially or temporally modulating the transition between two molecular states of a fluorophore (such as switching between a dark and bright state) or by physically reducing the size of the point-spread function used in the excitation illumination. Among the methods that improve resolution by PSF modification, the most important techniques are referred to by the acronyms STED (stimulated emission depletion; from the Stefan Hell laboratory) and SSIM (saturated structured illumination microscopy; pioneered by Mats Gustafsson). Techniques that rely on the detection and precise localization of single molecules include PALM (photoactivated localization microscopy; introduced by Eric Betzig and Harald Hess) and STORM (stochastic optical reconstruction microscopy; first reported by Xiaowei Zhang). As will be discussed, there are many variations on these techniques, as well as advanced methods that can combine or even improve the performance of existing imaging schemes. Even more importantly, new superresolution techniques are being introduced with almost breathtaking speed (relative to traditional advances in microscopy) and it is not unreasonable to suggest that at some point in the near future, resolution of a single nanometer may well be attainable in commercial instruments.
Via Dr. Stefan Gruenwald