Time-Resolved Emission Imaging Microscopy Using Phosphorescent Metal Complexes: Taking FLIM and PLIM to New Lengths

Abstract

Luminescent metal complexes are increasingly being investigated as emissive probes and sensors for cell imaging using what is traditionally termed fluorescence microscopy. The nature of the emission in the case of second-and third-row metal complexes is phosphorescence rather than fluorescence, as it emanates from triplet rather than singlet excited states, but the usual terminology overlooks the distinction between the quantum mechanical origins of the processes. In steady-state imaging, such metal complexes may be alternatives to widely used fluorescent organic molecules, used in exactly the same way but offering advantages such as ease of synthesis and colour tuning. However, there is a striking difference compared to fluorescent organic molecules, namely the much longer lifetime of phosphorescence compared to fluorescence. Phosphorescence lifetimes of metal complexes are typically around a microsecond compared to the nanosecond values found for fluorescence of organic molecules. In this contribution, we will discuss how these long lifetimes can be put to practical use. Applications such as time-gated imaging allow discrimination from background fluorescence in cells and tissues, while increased sensitivity to quenchers provides a means of designing more responsive probes, for example, for oxygen. We also describe how the technique of fluorescence lifetime imaging microscopy (FLIM) -which provides images based on lifetimes at different points in the image -can be extended from the usual nanosecond range to microseconds. Key developments in instrumentation as well as the properties of complexes suitable for the purpose are discussed, including the use of two-photon excitation methods. A number of different research groups have made pioneering contributions to the instrumental set-ups, but the E. Baggaley and J.A. Weinstein Department of Chemistry, University of Sheffield, Sheffield S3 7HF, UK e-mail: liz.baggaley@sheffield.ac.uk; julia.weinstein@sheffield.ac.uk J.A.G. Williams (*) Department of Chemistry, Durham University, Durham DH1 3LE, UK e-mail: j.a.g.williams@durham.ac.uk terminology and acronyms have not developed in a systematic way. We review the distinction between time-gating (to eliminate background emission) and true timeresolved imaging (whereby decay kinetics at each point in an image are monitored). For instance, terms such as PLIM (phosphorescence lifetime imaging microscopy) and TRLM (time-resolved luminescence microscopy) refer essentially to the same technique, whilst TREM (time-resolved emission imaging microscopy) embraces these long timescale methods as well as the more well-established technique of FLIM.Keywords Cell imaging Á Fluorescence Á Fluorescence lifetime imaging microscopy Á Imaging Á Iridium Á Metal complex Á Microscopy Á Oxygen sensing Á Phosphorescence Á Phosphorescence lifetime imaging microscopy Á Platinum Á Ruthenium Á Time-resolved emission imaging microscopy Á Time-resolved fluorescence Á Time-resolved luminescence microscopy Á Tissue imaging

Towards Water Soluble Mitochondria-Targeting Theranostic Osmium(II) Triazole-Based Complexes

“…Of recent and growing interest has been the use of luminescent complexes in biological applications [26][27][28]. Since phosphorescence is longer-lived than the autofluorescence from biological organic compounds, emissive complexes are amenable to use in time-gated imaging microscopy [29][30][31]. Osmium(II) complexes, however, often exhibit lower energy spin-forbidden direct 3 MLCT optical absorption bands of moderate extinction coefficient due to the high spin-orbit coupling constant for the osmium centre [32,33].…”

Dinuclear Design of a Pt(II) Complex Affording Highly Efficient Red Emission: Photophysical Properties and Application in Solution-Processible OLEDs

“…Meanwhile, such molecular metal complexes are proving to be of interest in other areas where intense emission is required, for example, as probes suitable for time-resolved detection in bio-imaging and sensing. 9,10,11,12 The vast majority of complexes explored to date are mononuclear, i.e., they feature one metal ion per molecule. However, multinuclear metal complexes -featuring two or more metal ions linked via rigid bridging ligands -have begun to emerge as a distinct class of phosphorescent molecules.…”

Water-soluble cyclometalated platinum(ii) and iridium(iii) complexes: synthesis, tuning of the photophysical properties, and in vitro and in vivo phosphorescence lifetime imaging

“…This considerably increases image resolution in luminescence microscopy and lowers detection threshold in sensing experiments. 1,4,11,12 As a result, advanced sensing platforms were developed to recognize various biologically relevant analytes, including (but not limited to) detection of cyanide ions, 13 biothiols 14 (including thiourea 15 ), b-galactosidase, 16 dopamine receptor, 17 or inhibitor of tumor necrosis factor-a. 18 Moreover, the sensitivity of emission parameters (intensity and lifetime) to oxygen concentration makes possible mapping of oxygen concentration in in vitro and in vivo experiments, 19,20 including quantitative measurements with the use not only emission intensity but also phosphorescence lifetime imaging (PLIM) techniques, [21][22][23][24][25] which provides considerably higher precision in oxygen biodistribution mapping.…”