The formal expressions presented by Abbe for lateral and axial resolution in the optical microscope are: In the lateral ( x, y) plane, the focal spot features progressively dwindling external concentric rings and is referred to as an Airy disk, whereas in the axial dimension, the elliptical pattern is known as the point-spread function ( PSF). The diffraction limit in optical microscopy is governed by the fact that when imaging a point source of light, the instrument produces a blurred and diffracted finite-sized focal spot in the image plane having dimensions that govern the minimum distance at which two points can be distinguished. Limited spatial resolution, however, precludes the ability to resolve important structures including synaptic vesicles, ribosomes, or molecular interactions, which all feature size ranges that lie beneath the limits of detection in fluorescence microscopy. Various imaging modes in fluorescence are often used to dynamically track proteins and signal peptides, as well as for monitoring other interactions in living cells.
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.Ĭurrently, modern and well-established fluorescence microscopy techniques can readily resolve a variety of features in isolated cells and tissues, such as the nucleus, mitochondria, Golgi complex, cytoskeleton, and endoplasmic reticulum. Between these two extremes in resolving power lies optical microscopy. 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 1). 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. 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.
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