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Photon Counting - Fundamentals and Applications || Detectors for Super-Resolution & Single-Molecule Fluorescence Microscopies

DOI:10.5772/intechopen.71943 出版年份:2018 更新时间:2025-09-23 15:23:52
摘要: The resolution of light microscopy was thought to be limited to 250–300 nanometers based on the work of Ernest Abbe. This Abbe diffraction limit was believed to be insurmountable until the invention of Super-resolution microscopic techniques in the late 20th century. These techniques remove this limit and have provided unprecedented detail of cellular structures and dynamics down to several nanometers. An emerging goal in this field is to quantitatively measure individual molecules. Measurement of single-molecule dynamics, such as diffusion coefficients and complex stoichiometries, can be accomplished using fluorescence fluctuation techniques to reveal nanosecond-to-microsecond temporal reactions. These powerful complimentary experimental approaches are made possible by sensitive low-light photodetectors. In this chapter, an overview of the principles of super-resolution and single-molecule microscopies are provided. The different types of photodetectors employed in these techniques are explained. In addition, the advantages and disadvantages for these detectors are discussed, as well as the development of next generation detectors. Finally, example super-resolution and single-molecule cellular studies that take advantage of these detector technologies are presented.
作者: Robert T. Youker
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To provide an overview of the principles of super-resolution and single-molecule microscopies, explain the different types of photodetectors used in these techniques, discuss their advantages and disadvantages, and present examples of cellular studies utilizing these detectors.

Advances in photodetector technologies have enabled super-resolution and single-molecule fluorescence microscopies by providing high sensitivity, temporal resolution, and low noise. EMCCD and sCMOS cameras are preferred for probe-based SRM, while APD and hybrid detectors are common for PSF-engineered SRM and FFTs. Future developments aim to combine the best attributes of point-like and array detectors, with de-noising algorithms improving SNR. No single detector is suitable for all techniques, and researchers should choose based on experimental needs.

The paper is a review and does not present original experimental data, so it lacks technical constraints or optimization areas from specific experiments. It discusses general limitations of detector technologies, such as low quantum efficiency in PMTs, noise issues in array detectors, and the need for empirical determination of SNR cross-over points.

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