The development of methodologies to identify single molecules and/or to detect/monitor molecular behavior at the single-molecule level is one of the important research topics in chemistry and biology. In this review, we summarized the state-of-the-art of single molecule measurement methods and its latest applications using nanodevices integrated with molecular-size functional nanostructures, nanopores, nanogaps, and nanofluidic channels, which detect differences in chemical species, presence or absence of translational modifications, changes in steric structure, and changes in interactions between molecules. Besides these fundamental analytical achievements of molecular identification abilities, the latest applications include the single-molecule electrical sequencing, disease diagnosis, viral testing, single-molecule drug screening, and environmental monitoring. Finally, we added some discussion on the current status of single-molecule measurement as a method and technology to solve the problems to expand the future application needs of single-molecule measurement.

Nucleic acids are important biomarkers for disease detection, monitoring, and treatment. Advances in technologies for nucleic acid analysis have enabled discovery and clinical implementation of nucleic acid biomarkers. However, challenges remain with technologies for nucleic acid analysis, thereby limiting the use of nucleic acid biomarkers in certain contexts. Here, we review single-molecule technologies for nucleic acid analysis that can be used to overcome these challenges. We first discuss the various types of nucleic acid biomarkers important for clinical applications and conventional technologies for nucleic acid analysis. We then discuss technologies for single-molecule in vitro and in situ analysis of nucleic acid biomarkers. Finally, we discuss other ultra-sensitive techniques for nucleic acid biomarker detection.

Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called \'green revolution\', has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called \'super-resolution\' fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques.

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