Multiple-Pulse Pumping with Time-Gated Detection for Enhanced Fluorescence Imaging in Cells and Tissue

Abstract

Fluorescence-based sensing and imaging experiments are constrained by the background signal generated on a sample. A main contribution to the background, besides direct scattering of excitation and Raman scattering of the solvent, comes from sample autofluorescence and additives used for sample preparation. Such unwanted signals from endogenous chromophores and fixatives typically are broad and spectrally overlap with the probe signal; thus becoming a major limitation for sensitive detection and quantitative imaging. Since the fluorescence lifetimes of the majority of naturally occurring chromophores are relatively short, long-lived fluorophores allow for background discrimination by time-gated detection. Unfortunately, the brightness of long-lived, red-emitting fluorescent probes is inherently very low, consequently limiting many applications. Recently we reported a simple new approach with bursts of closely spaced laser excitation pulses for excitation (multi-pulse excitation) that allows for many-fold increase in the intensity of a long-lived probe over the background signal. This technology can be easily implemented for biomedical diagnostics and imaging to significantly enhance the signal of long-lived probes over the background. In this report, we are discussing an example of the Ruthenium-based dye tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (Ru) (Sigma–Aldrich) (~2% quantum yield and ~350 ns fluorescence lifetime) that when used with the multi-pulse approach and time-gated detection allows for high quality imaging that can easily be enhanced two orders of magnitude as compared to the normal approach (imaging with typical fluorescence microscopy).