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How does fluorescence lifetime imaging reveal hidden information within a sample?
Fluorescence lifetime imaging (FLIM) has become an indispensable tool in modern biomedical and material science research. The technique captures images by measuring the transition of fluorescent molecules from an excited state to their ground state, a process characterized by a unique lifetime. This technology can detect hidden information inside the sample, thus opening up new perspectives for understanding the microscopic environment.
Fluorescence lifetime (FLT) measurements not only avoid errors caused by changes in light intensity, but also provide a deep understanding of the internal environment of the sample.
The principle of fluorescence lifetime imaging technology is based on the different decay rates of fluorescent molecules. When fluorescent molecules are excited by photons, they return to the ground state through a variety of decay pathways with a certain probability, the most important of which is the spontaneous emission of photons. Fluorescence intensity changes are usually affected by factors such as light source brightness changes and background light intensity, but in FLIM, images are generated based on fluorescence lifetime rather than intensity.
This gives FLIM a significant advantage in imaging thick samples because it minimizes the effects of photon scattering. For example, in biological samples, fluorescence lifetime can be used to indicate important biological parameters such as pH, viscosity, and chemical concentration.
Fluorescence lifetime and measurement technology
The length of the fluorescence lifetime is closely related to the surrounding environment of the excited molecule, so it can be used to detect changes in material properties or biological functions. In addition, using continuous pulse excitation techniques such as time-correlated single photon counting (TCSPC), the fluorescence decay curves can be accurately recorded and lifetime parameters containing useful information can be extracted.
The application of TCSPC technology is not limited to biological sciences, but also includes many fields such as material science. Through precise time measurement, it can achieve the observation and analysis of microstructures.
In recent years, the development of this technology has increasingly improved its applicability and flexibility in various fields. At the same time, through methods such as phase modulation and pulse excitation, fluorescence lifetime imaging technology can be improved to a higher resolution and application potential. In particular, these advanced versions are able to provide fast and accurate real-time imaging in living cell research.
Analysis and future prospects
In FLIM data analysis, the sweet spot is to efficiently extract the pure decay curve and estimate the lifetime. This is usually done by methods such as least squares iterative convolution, where the theoretically calculated decay curve is compared one-by-one with the actual data to obtain the best fit. However, with technological advances, emerging technologies such as non-fitting methods such as rapid lifetime determination (RLD) provide more efficient solutions for data processing.
With the easy integration from instruments to algorithms in recent years, the application of FLIM has continued to expand, becoming a hot topic and important tool for collaborative research in multiple fields.
Looking into the future, fluorescence lifetime imaging technology will be further optimized and applied to a wider range of research fields. This includes not only instant diagnosis and analysis in the biomedical field, but also precise observation of nanostructures in materials science. With the help of high-performance detectors and optimized data analysis algorithms, FLIM will open up more possible exploration paths for scientists. Let us think about how this technology will affect our understanding of the nature of life and materials in the future?
