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Why can FLIM break the limitations of traditional microscopes? Explore the mystery of fluorescence lifetime!
Fluorescence lifetime imaging microscopy (FLIM) has attracted widespread attention in the scientific community in recent years because it reveals a new imaging method that breaks through various limitations of traditional microscopes. FLIM not only relies on the intensity of fluorescent molecules in a sample, but also delves into their fluorescence lifetime (FLT), a property that makes it invaluable in many applications.
The fluorescence lifetime depends on the microenvironment of the fluorescent molecule, such as pH, viscosity and chemical concentration, which allows FLIM to provide second-to-none information and avoid erroneous measurements caused by changes in light source brightness or background light intensity.
FLIM focuses on the time it takes for fluorescent molecules to return to the ground state. Each fluorescent molecule has its own specific extinction rate during this process. This means that even if materials emit light at the same wavelength but fade at different rates, FLIM can still clearly distinguish them. As an imaging technology, FLIM can be used in a variety of situations such as confocal microscopy, two-photon excitation microscopy, and multi-photon computed tomography, and can effectively reduce photon scattering problems caused by sample thickness.
FLIM’s advantages
By measuring the lifetime of fluorescent molecules, FLIM technology provides in-depth information about biomolecules and their environment. Depending on different environmental parameters, changes in fluorescence lifetime can be used as an important indicator. For example, when the microenvironment of the probe changes, including pH value, viscosity of the solution, and even intermolecular interactions, FLIM can respond and reflect it immediately.
Because FLIM does not depend on light intensity, it eliminates the effects of light source fluctuations, background brightness changes, and photobleaching on experimental data, making it more reliable in practical applications.
How to perform FLIM measurements
FLIM implementation methods mainly include time-correlated single photon counting (TCSPC), pulse excitation and phase modulation and other technologies. Through these methods, FLIM can achieve high-resolution and fast imaging effects. These techniques allow researchers to observe the behavior of fluorescent molecules in minute detail in living cells or other rapidly changing environments.
Time-correlated single photon counting (TCSPC)
TCSPC is an important technology in FLIM, used to record the decay curve of fluorescent molecules. Using commercial TCSPC equipment, researchers can record fluorescence decay curves with a time resolution of up to 405 femtoseconds, which provides a basis for analyzing the dynamic behavior of molecules.
Pulse excitation method
Pulse excitation is a technique often used when measuring FLIM. It involves the use of reflected excitation light and the ability to accurately capture fluorescence signals, a method that further improves FLIM's temporal resolution.
Phase modulation technology
Phase modulation is a method to obtain fluorescence lifetime by modulating a laser light source at high frequency to detect phase differences. Its ability to image quickly makes it particularly suitable for live cell research.
Data analysis and result interpretation
The core of FLIM data analysis is to extract a pure decay curve and estimate its lifetime, which is usually achieved by fitting a single or multi-exponential function. The most commonly used technique is least-squares iterative reconvolution, a process that is continuously optimized in a step-by-step process that provides accurate lifetime estimates.
Although FLIM is challenging, its potential range of applications and the high-quality data it can provide make it irreplaceable in the scientific research community.
Explore future applications
FLIM has a wide range of applications, including biomedicine, materials science and environmental science. With the advancement of technology, FLIM is expected to play a more important role in more complex biological systems in the future, helping scientists to gain insight into small changes, thereby promoting the development of new medical diagnostic technologies.
In this technological revolution, will FLIM become the benchmark for future microscopy technology?
