Molecular probes for fluorescence lifetime imaging

Abstract

Visualization of biological processes and pathologic conditions at the cellular and tissue levels largely relies on the use of fluorescence intensity signals from fluorophores or their bioconjugates. To overcome the concentration dependency of intensity measurements, evaluate subtle molecular interactions, and determine biochemical status of intracellular or extracellular microenvironments, fluorescence lifetime (FLT) imaging has emerged as a reliable imaging method complementary to intensity measurements. Driven by a wide variety of dyes exhibiting stable or environment-responsive FLTs, information multiplexing can be readily accomplished without the need for ratiometric spectral imaging. With knowledge of the fluorescent states of the molecules, it is entirely possible to predict the functional status of biomolecules or microevironment of cells. Whereas the use of FLT spectroscopy and microscopy in biological studies is now well-established, in vivo imaging of biological processes based on FLT imaging techniques is still evolving. This review summarizes recent advances in the application of the FLT of molecular probes for imaging cells and small animal models of human diseases. It also highlights some challenges that continue to limit the full realization of the potential of using FLT molecular probes to address diverse biological problems and outlines areas of potential high impact in the future.

Figure 1

Representative fluorophore systems commonly used in lifetime imaging and associated photoluminescence lifetimes. These fluorophores can be used in their native forms and/or after conjugation to other entities.

Figure 2

(A) Structures of the NIR dyes cypate (ex/em: 792/810 nm) and 3,3′-diethylthiatricarbocyanine iodide (DTTCI; ex/em: 771/800 nm) used in this study. (B) Intensity images (top row), FLIM images (middle row), and FLT distributions (bottom row) of cells treated with cypate alone. (C) Intensity images (top row), FLIM images (middle row), and FLT distributions (bottom row) of cells treated with DTTCI alone. (D) Intensity images (top row), FLIM images (middle row), and FLT distributions (bottom row) of cells treated with either cypate or DTTCI. (E) Intensity images (top row), FLIM images (middle row), and FLT distributions (bottom row) of cells treated with both cypate and DTTCI. Reprinted with permission from ref . Copyright 2012 Royal Microscopical Society.

Figure 3

(A) Structure of LS482. (B) Spectroscopic FLT vs pH; pKa = 5.39. (C, D) FLT tomography imaging of three phantoms (Ac, acidic; N, neutral; and B, basic) implanted into a mouse. A vertical slice from the tomography reconstructed yield (C) and lifetime (D) is overlaid on a white-light image of the mouse. (Reprinted from Biophysical Journal, vol. 100, no. 8, Berezin et al., Near-infrared fluorescence lifetime pH-sensitive probes, pp. 2063-72, Copyright (2011), with permission from Elsevier.)

Figure 4

FLT images of (A) CdSe–ZnS and (B) CdSe–ZnS:dopamine QDs incubated with NIH 3T3 fibroblast cells. The color scale represents the lifetimes in ns; white scale bars are 10 μm. Note the inhomogeneous distribution of lifetimes in cells incubated with QD:dopamine conjugates compared to the structured lifetime distribution in cells incubated with QDs alone. Information on arrows is available in the original article. (Reproduced from the article by Carlini et al., with permission of The Royal Society of Chemistry. DOI: http://dx.doi.org/10.1039/C3CC36326K.)

Figure 5

(A) Structure of MQAE and its Cl^- bound form. Dissected salivary glands of a cockroach, labeled with MQAE and placed in buffers with 174 mM NaCl (B) and 2 mM NaCl (C). (Courtesy of Carsten Hille, Physical Chemistry group, University of Potsdam.)

Figure 6

Figure 6. (A) Chemical structure of fluorescent polymeric thermosensitive probe. (B) FLT images of the fluorescent polymeric thermometer in live cells. (C) Histograms of FLT derived from the cells. (D) FLT images of the control copolymer in live cells. (E) Histograms of FLT derived from the control cells. 〈τf〉 represents an average lifetime of the histogram. Scale bar represents 10 μm. Reprinted with permission from Okabe et al. Copyright 2012 Macmillan Publishers Ltd.

Figure 7

(A) Structure of the viscosity-sensitive probes PY3304, PY3174, and PY3184. (B) Left: Fluorescence decays acquired from artificial membranes stained with PY3304 showing longer lifetimes in ordered membranes (red) than in disordered membranes (black). (Right) Plots of residuals from fitting fluorescence decays. (C) Fluorescence decays and plots of residuals from artificial membranes stained with PY3174 (D) Fluorescence decays and plots of residuals from artificial membranes stained with PY3184. (E) FLT image of live HeLa cells stained with PY3304 (F) FLT image of live HeLa cells stained with PY3174 (G) FLT image of live HeLa cells stained with PY3184. FLT images show an increased order at the plasma membrane. Scale bar = 10 μm. Reprinted from ref .