Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy

Abstract

Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat's-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures.

Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats

“…Spatially varying modulation of light intensity with mechanical rotation (17,18) or scanning (19) enables the mapping of spatial position to modulation frequency for scan-less full-field FLIM. More recently, fluorescence imaging by radio frequency (RF)-tagged emission (FIRE) (20)(21)(22) is demonstrated on the basis of a combination of frequency multiplexing and spectral mapping in the RF region without the need for mechanical motion. In this method, a one-dimensional (1D) array of focal spots with different RF modulation frequencies is generated by digitally synthesized optical beating with an acousto-optic deflector and is then radiated onto a sample.…”

“…One interesting approach expandable to scan-less full-field FLIM is fluorescence imaging by radiofrequency-tagged emission (FIRE) [17][18][19] , which is based on a combination of frequency multiplexing and spectral mapping in the radiofrequency (RF) region. In this method, a 1D array of focal spots with different RF modulation frequencies is generated by digitally synthesized optical beating with an acousto-optic deflector, and is then radiated onto a sample.…”

Extremely High-Throughput Parallel Microfluidic Vortex-Actuated Cell Sorting

“…However, arranged as a 4 × 4 array on chip, the pitch must be ≤1 mm to achieve sufficient sensitivity with reasonable optical components (lenses and filters ≤2-inch diameter). While several parallel flow cytometers [ 21 , 22 , 23 , 24 , 25 , 26 , 27 ] and one parallel sorter [ 20 ] have been published, no previous microfluidic cell sorters met this specification because of their size (including actuators and side-channels), low sort rates, or other complexities.…”

Cell sorting actuated by a microfluidic inertial vortex

“…However, arranged as a 4 × 4 array on chip, the pitch must be ≤1 mm to achieve sufficient sensitivity with reasonable optical components (lenses and filters ≤2 inch diameter). While several parallel flow cytometers [23][24][25][26][27][28][29] and one parallel sorter 30 have been published, until now no microfluidic cell sorters meet this specification, because of their size (including actuators and side-channels), low sort rates, or other complexities.…”