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The amazing uses of FLIM microscopy: How to obtain ultra-high resolution images in real-time detection?
As science and technology advance at a rapid pace, microscope technology is also constantly innovating. Among them, fluorescence lifetime imaging microscopy (FLIM) has become an important tool in life science and material science research due to its unique imaging principle and practical applications. Unlike traditional microscopy, which uses fluorescence intensity as the basis for imaging, FLIM generates images based on the speed at which fluorescent molecules return from an excited state to a ground state, rather than on their intensity.
By measuring fluorescence lifetime, FLIM not only eliminates measurement errors caused by variations in light source brightness, background light intensity, or photobleaching, but also minimizes the effects of photon scattering in thick sample layers.
"The change in fluorescence lifetime depends on the local microenvironment of the fluorescent molecule and can therefore serve as an indicator of pH, viscosity, and chemical concentration, among other things."
Basic principles of fluorescence lifetime
When excited by photons, the photon molecules return to the ground state with different probabilities through electromagnetic radiation and non-radiative decay pathways. Only the pathway involving spontaneous emission of photons leads to the formation of fluorescence. According to Schott's law, the mathematical expression of the decay of fluorescence intensity over time can be simplified to an exponential function. This shows that the fluorescence lifetime is a physical quantity that is independent of the initial intensity.
FLIM measurement and implementation technology
In FLIM, the instrument uses a pulsed excitation source and then records the decay curve through the machine's response function. When a group of fluorescent molecules is excited by an ultrashort light pulse, the resulting time-resolved fluorescence will inevitably exhibit exponential decay. The most common implementation is based on time-correlated single photon counting (TCSPC), a technique that improves measurement accuracy and minimizes the impact of background noise.
"Using commercial TCSPC equipment, the time resolution of the fluorescence decay curve can reach 405 femtoseconds."
Application Scope
FLIM technology offers unique advantages in the study of living cells and their microenvironment. This technology can distinguish different molecular movements at the nanoscale, and can be used to study biomolecular interactions, changes in intracellular enzyme activity, etc.
In recent years, FLIM technology has been further applied to medical diagnosis, especially in brain tumor surgery, where it can be combined with endoscopes to implement real-time diagnosis. By measuring the fluorescence lifetime of tumor tissue, doctors can more accurately assess the boundaries and properties of the tumor.
Data Analysis and Future Outlook
In FLIM image analysis, extracting pure decay curves and estimating fluorescence lifetime are key tasks. In this regard, a variety of algorithms have been proposed, whether it is the least squares method or the rapid life determination method, they are constantly improving the accuracy and practicality of calculation.
"The development of these methods has transformed FLIM from a laboratory theory into a more practical and convenient method, allowing this technology to enter the clinic."
With the advancement of technology, the image acquisition speed of FLIM will continue to increase and the scope of application will become wider. Whether in basic scientific research or in clinical diagnosis, the potential of this technology cannot be ignored. However, as its application areas expand, we need to think about how to better combine FLIM technology with other techniques to promote the development and innovation of biomedicine?
