2019 年 27 巻 p. 13-22
The quantitative aspect of fluorescence microscopy is becoming significant. It is essential to determine the exact number of molecules in order to evaluate the mechanisms underlying major life phenomena. For industrial and clinical diagnostic applications, day-to-day quality assurance and inter-laboratory comparability are necessary. By determining the absolute concentration of molecules from fluorescence images, datasets collected under different system conditions and in different laboratories can be directly compared without additional calibration procedures. Absolute quantitation requires determining the exact number of molecules, such as proteins and nucleic acids. However, it is difficult to precisely determine the molecular number (or concentration) in fluorescence microscopy images. Fluorescence correlation spectroscopy (FCS) and its related methods are promising for evaluating molecular numbers in solutions and living cells, owing to their simplicity and straightforward interpretation of the obtained data. In this review, we present an overview of the usefulness of and current limitations associated with various FCS techniques for standardizing quantitative confocal fluorescence imaging by evaluating the microscope system performance in collecting relevant image data.