The power of single molecule microscopy: from nanoparticle investigations to microbiome analysis.

摘要

Optical microscopy has been a tool of choice ever since van Leeuwenhoek used Hooke's microscope to observe biological specimens. Chief among its advantages is the fact that imaging is noninvasive. In combination with the straightforward sample preparation and general convenience, optical microscopes remain essential to many aspects of modern-day research. The advent of fluorescence microscopy further increased the importance and applicability of optical microscopy. Observing fluorescence emission instead of measuring light transmission dramatically increases the sensitivity and selectivity of the technique, and fluorescence microscopy has become a widely used and fundamental tool in the study of complex biological and biochemical problems. The sensitivity argument has accumulated in the development of single molecule microscopy. As a result, a large variety of techniques for labeling carefully targeted molecules with fluorescent dyes has been developed. Unfortunately, the price that is paid for the convenience and capability of fluorescence microscopy is that of a relatively poor spatial resolution when compared to electron or X-ray microscopy. The smallest distance between two objects that can be resolved is known as the resolution of the microscope, and in optical microscopy this fundamental limitation is roughly equal to half the wavelength of the used light, which typically corresponds to about 250 nm for blue-green excitation light (500 nm). In modern high-end research microscopes this resolution limit is not due to technical or design issues, but solely determined by this fundamental law of nature.