Method of Simultaneous Frequency-Sweeping Lifetime Measurements on Multiple Excitation Wavelengths

摘要

A fast fluorescence lifetime microscopic system images FRET between multiple labels in live cells and deep tissue, using a quantitative analysis method to reconstruct the molecular machinery behind the multiplexed FRET phenomenon. The system measures fluorescence lifetime, intensity and anisotropy as images of excitation-emission matrices (EEM) in real time and high speed within a single image scan, performs high-resolution deep-penetrating 3D FRET imaging in live samples, and fully analyzes all possible photon pathways of multiplexed FRET. The system provides a way for systematic and dynamic imaging of biochemical networks in cells, tissue and live animals, which will help to understand mechanisms of genetic disorders, cancers, and more.

主权项

1. An apparatus for measuring time-resolved excitation-emission of a sample, comprising:
an interferometer that includes an optical delay line; a multi-wavelength radiation source providing an input laser beam of radiation of multiple wavelengths to the interferometer, said interferometer providing an excitation radiation beam of said multiple wavelengths for scanning the sample and exciting fluorophores in the sample, said fluorophores emitting an emission radiation beam in response to the excitation radiation beam; wherein said optical delay line comprises a rotating polygonal mirrored surface and optics that cause the input beam to impinge onto the polygonal mirrored surface at least four times, said polygonal mirrored surface reflecting said input beam to provide said excitation radiation beam, and wherein frequency-sweeping laser modulations from 0 to at least 10 MHz through interference in the excitation radiation beam are generated at an interference modulation frequency that is linear to the path-length scanning speed of the rotating polygonal mirrored surface; a mechanism scanning the excitation radiation beam across the sample; a plurality of modulation detectors detecting separately different wavelength components of said excitation radiation beam to provide excitation modulation signals; a plurality of emission detectors detecting separately different wavelength components of said emission radiation beam to acquire an image of the sample to provide emission signals; and an instrument analyzing the excitation modulation signals and the emission signals to determine with at least nanosecond resolution a time-resolved signal or signals of one or more excitation-emission photon pathways in the sample.