Revolutionary microscopy: 3D views of living cells revealed!

Revolutionary microscopy: 3D views of living cells revealed!

Light sheet fluorescence microscopy (LSFM) has established itself as a groundbreaking technology in biochemical research. This method allows researchers like Zeyu Zhang to obtain a three-dimensional view of fluorescent samples, especially parts of living cells. A prominent feature of LSFM is the use of the time-correlated single photon counting (TCSPC) technique, which allows the luminescence duration of molecules in the samples to be precisely measured. Dr. Meike Hofmann, research associate at the Department of Technical Optics, highlights the cruciality of the lighting duration for understanding biological processes.

The microscope system detects photons and tracks their arrival time and location. These precise results require a large amount of photons, which similarly leads to the need to collect many data points to produce a comprehensive picture. This is what experts report from the nature.com that recent developments in fluorescence lifetime microscopy (FLIM) offer the potential to represent the complexity of biological systems with unprecedented clarity.

The mechanics of FLIM

FLIM is a fluorescence-based imaging technique that measures the lifetimes of excited fluorescent molecules instead of fluorescence intensity. The comparison between conventional fluorescence microscopy and FLIM shows the significant differences in fluorescence lifetimes, which provide important information about the molecular environment and energy transfer processes. For example, a decrease in fluorescence lifetimes may indicate Förster resonance energy transfer, which is important for research into protein interactions.

The fluorescence lifetime indicates the average time that a molecule remains in the excited state before returning to the ground state. This lifetime is inversely proportional to the sum of the decay rates of the radiative and non-radiative processes. Because the fluorescence lifetime depends on the identity and chemical environment of the dye, researchers can use this method to create precise images for each pixel.

Applications and innovations

The measurement methods within FLIM include pulsed excitation, in which the temporal decay of fluorescence is analyzed, as well as intensity-modulated excitation with phase shift. The excitation intensity is adjusted so that only one photon can be detected per pulse. The use of histograms from individual measurements allows the fluorescence lifetime to be precisely determined.

The technological advances as seen in the research of Wikipedia include the integration of state-of-the-art image sensors such as CCD cameras and avalanche photodiode fields for detection, as well as the use of ICCD cameras that use image intensifiers for sensitization. These developments make a decisive contribution to dramatically improving measurement accuracy and the associated applications in biomedicine and molecular biology.