Trends in Single-Molecule Total Internal Reflection Fluorescence Imaging and Their Biological Applications with Lab-on-a-Chip Technology

Abstract

Single-molecule imaging technologies, especially those based on fluorescence, have been developed to probe both the equilibrium and dynamic properties of biomolecules at the single-molecular and quantitative levels. In this review, we provide an overview of the state-of-the-art advancements in single-molecule fluorescence imaging techniques. We systematically explore the advanced implementations of in vitro single-molecule imaging techniques using total internal reflection fluorescence (TIRF) microscopy, which is widely accessible. This includes discussions on sample preparation, passivation techniques, data collection and analysis, and biological applications. Furthermore, we delve into the compatibility of microfluidic technology for single-molecule fluorescence imaging, highlighting its potential benefits and challenges. Finally, we summarize the current challenges and prospects of fluorescence-based single-molecule imaging techniques, paving the way for further advancements in this rapidly evolving field.

Single‐Particle and Single‐Molecule Characterization of Immobilized Enzymes: A Multiscale Path toward Optimizing Heterogeneous Biocatalysts

“…[20] By definition, the spatial and temporal resolution of the enzyme process is higher in the SM (single enzyme) studies than in the SP (all enzymes bound to a single particle or bead) ones. For example, SM-based on total internal reflection fluorescence microscopy (TIRFM) [21] can reach a spatial resolution of 10-100 nm with temporal resolution in the range of hundreds of ms. The resolution can be increased to 1-2 nm using single-molecule Föster resonance energy transfer (SM-FRET).…”

Methodological Pitfalls of Investigating Lipid Rafts in the Brain: What Are We Still Missing?

“…Among the techniques utilizing fluorophores for the detection of targeted molecules, only super-resolution microscopy allows for the direct exploration of lipid rafts due to lowering the resolution up to 10 nm [109][110][111]. The actual achieved resolution and the ability to localize individual fluorophores depend on various factors, including the quality of the fluorophores and the specific experimental conditions [112,113]. Among all available microscopic methods, immunoelectron microscopy offers superior resolution for the direct observation of lipid raft compositions.…”

Lipid-Based Nanotechnology: Liposome

“…LSCFM, known for its high resolution, optical sectioning, and 3D imaging capabilities [ 79 , 80 ], serves to characterize liposomes and observe their structure, behavior, and interactions with high precision [ 81 , 82 ]. In contrast, TIRFM is well-known for its accessibility and high sensitivity in probing biomolecules properties [ 83 ]. When characterizing liposomes, TIRFM presents unique advantages by allowing observations of membrane-related phenomena and interactions at the liposome interface [ 84 , 85 ].…”