Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy.

Jacky C K Chan,Bahram Jalali,Najva Akbari,Eric D Diebold,Brandon W Buckley,Sien Mao

doi:10.1364/boe.5.004428

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.

Highlights

Fluorescence lifetime spectroscopy is an optical technique which provides additional chemical information to the fluorescence intensity given its sensitivity to inter-molecular chemical environments and intra-molecular biochemical interactions
Dye cells, consisting of a 0.12mm-thick adhesive silicone spacer sandwiched between a cover slip and a microscope slide, are filled with 10-μl volumes of fresh sample for each measurement
The measurement error of the values reported here is similar to, but larger than that of literature values. This discrepancy can mainly be attributed to the use of a side-on PMT, which has a higher transit time spread than the head-on micro-channel plate (MCP) detectors used in [13,26,31]

Summary

Introduction

Fluorescence lifetime spectroscopy is an optical technique which provides additional chemical information to the fluorescence intensity given its sensitivity to inter-molecular chemical environments and intra-molecular biochemical interactions. In the frequency domain approach, an intensity modulated excitation beam illuminates a fluorescent sample and generates a phase-delayed fluorescence emission signal at the same modulation frequency. Phase measurements at a single RF frequency are theoretically sufficient to resolve the lifetime, systematic errors [20] or the presence of extra lifetime components [21] often limit their accuracy. Since these systematic errors become prohibitively large when measuring samples involving multi-exponential decays or mixed lifetime samples, multiple RF frequencies are typically necessary in lifetime imaging techniques to accurately resolve the lifetime at each pixel in the image. While multifrequency measurements can be made by sweeping the excitation frequency, this approach is too slow to image dynamic samples over a large field of view [21]

Results

Discussion

Conclusion