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Photon Counting - Fundamentals and Applications || Detectors for Super-Resolution & Single-Molecule Fluorescence Microscopies
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.
关键词： biophysical techniques,STORM,nanoscopy,STED,protein dynamics,palm,spectroscopy,molecular brightness,fluorescence fluctuation
更新于2025-09-23 15:23:52
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Lifetime super-resolution optical fluctuation imaging
摘要： In this paper, we propose a promising super-resolution imaging scheme in fluorescence lifetime domain (lifetime super-resolution optical fluctuation imaging, ltSOFI). ltSOFI has the potential to obtain super-resolution images by taking advantage of fluorescence lifetime blinking under wide-field lifetime detection. The proof-of-concept for ltSOFI was demonstrated through numerical simulation of high-order cumulant analysis on fluorescence lifetime blinking emitters. As a tentative experimental demonstration, we obtained super-resolution lifetime imaging from time-lapse FLIM recording of HeLa cells expressing a cAMP sensor using ltSOFI method. ltSOFI is expected to initiate a new dimension in the lifetime domain for blinking-based super-resolution microscopy.
关键词： fluorescence fluctuation,lifetime blinking,super-resolution
更新于2025-09-19 17:15:36
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A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts
摘要： A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
关键词： Issue 142,cell-cell adhesion,fluorescence correlation spectroscopy,cell-cell interactions,Protein-protein interactions,number and brightness,fluorescence fluctuation spectroscopy,Biochemistry,N&B
更新于2025-09-09 09:28:46