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Single-molecule fluorescence resonance energy transfer: How to reveal the mystery of biomolecules?
In biophysics, single-molecule fluorescence resonance energy transfer (smFRET) is rapidly becoming a key technology that can measure distances between single biomolecules with an accuracy of 1 to 10 nanometers. This technique not only provides a closer look at molecules, but also reveals many dynamic behaviors that cannot be probed in traditional collective measurements.
The power of single-molecule FRET lies in its ability to resolve the FRET signal of each molecule, thereby revealing its kinetic information.
Using FRET technology, researchers can observe the dynamic processes within and between molecules, such as the folding and unfolding of DNA, RNA and proteins. This method is of great significance for applications such as chemical sensing and biological detection, because through observation at the single-molecule level, scientists can obtain the most realistic molecular behavior data.
Measurement methods
Single-molecule FRET measurements are typically performed using fluorescence microscopy, where the molecules may be immobilized on a surface or freely diffusing. Researchers use a strong light source, such as a laser, to excite the FRET pair, generating enough fluorescent signal for single-molecule detection. To reduce noise, studies often combine full-field multiphoton microscopy with total internal reflection fluorescence (TIRF) microscopy.
Single-molecule FRET signal changes help reveal the dynamic behavior of the system in equilibrium.
In surface-immobilized experiments, biomolecules carrying fluorescent tags are immobilized on the cover glass surface and fluorescent images are continuously collected. Such experiments allow the observation of many molecules switching simultaneously for a long time until fluorescence quenching occurs. In contrast, free-diffusion smFRET can be observed as the molecules in the liquid sample diffuse naturally, making the tracking of changes more flexible.
Data Analysis
smFRET data typically involve time traces of a two-dye system, the donor and the acceptor fluorescence intensities. Researchers use optical filters or beam splitters to separate the emissions of the two dyes, and the data is then calculated to obtain the FRET efficiency. Since the data may contain signals from incomplete labeling or aggregation, various complex analysis software are usually required to ensure the accuracy of the data.
Noise control is also an important part of data processing. The noise detected can come from camera readout, shutter noise, and variations in the sample itself, so it is critical to remove invalid data and improve the signal-to-noise ratio.
State Identification and Rate Analysis
Many algorithms have been developed to analyze smFRET data, the most commonly used include thresholding and hidden Markov models (HMM). These methods make it possible to identify state transitions from time trajectories and further calculate the transfer rate constants between states. This analysis is crucial to understanding how molecules interact and transform in time and space.
Through smFRET, scientists can observe tiny changes in molecular structure and dynamics from a completely new perspective.
Single-molecule fluorescence resonance energy transfer technology continues to expand our understanding of biological systems, and its potential in biomedicine and materials science is increasingly recognized. With the continuous advancement of technology, we may be able to explore the mysteries of the molecular world more accurately. Will this change the direction of our research in the field of biological sciences?
