Elizabeth Baggaley

Dinuclear Ruthenium(II) Complexes as Two‐Photon, Time‐Resolved Emission Microscopy Probes for Cellular DNA

The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging.

Long-lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to PLIM and beyond

Lifetime imaging microscopy with sub-micron resolution provides essential understanding of living systems by allowing both the visualisation of their structure, and the sensing of bio-relevant analytes in vivo using external probes. Chemistry is pivotal for the development of the next generation of bio-tools, where contrast, sensitivity, and molecular specificity facilitate observation of processes fundamental to life. A fundamental limitation at present is the nanosecond lifetime of conventional fluorescent probes which typically confines the sensitivity to sub-nanosecond changes, whilst nanosecond background autofluorescence compromises the contrast. High-resolution visualization with complete background rejection and simultaneous mapping of bio-relevant analytes including oxygen – with sensitivity orders of magnitude higher than that currently attainable – can be achieved using time-resolved emission imaging microscopy (TREM) in conjunction with probes with microsecond (or longer) lifetimes. Yet the microsecond timescale has so far been incompatible with available multiphoton excitation/detection technologies. Here we realize for the first time microsecond-imaging with multiphoton excitation whilst maintaining the essential sub-micron spatial resolution. The new method is background-free and expands available imaging and sensing timescales 1000-fold. Exploiting the first engineered water-soluble member of a family of remarkably emissive platinum-based, microsecond-lived probes amongst others, we demonstrate (i) the first instance of background-free multiphoton-excited microsecond depth imaging of live cells and histological tissues, (ii) over an order-of-magnitude variation in the probe lifetime in vivo in response to the local microenvironment. The concept of two-photon TREM can be seen as “FLIM + PLIM” as it can be used on any timescale, from ultrafast fluorescence of organic molecules to slower emission of transition metal complexes or lanthanides/actinides, and combinations thereof. It brings together transition metal complexes as versatile emissive probes with the new multiphoton-excitation/microsecond-detection approach to create a transformative framework for multiphoton imaging and sensing across biological, medicinal and material sciences.

Metal Complexes for Two-Photon Photodynamic Therapy: A Cyclometallated Iridium Complex Induces Two-Photon Photosensitization of Cancer Cells under Near-IR Light

Abstract Photodynamic therapy (PDT) uses photosensitizers (PS) which only become cytotoxic upon light‐irradiation. Transition‐metal complexes are highly promising PS due to long excited‐state lifetimes, and high photo‐stabilities. However, these complexes usually absorb higher‐energy UV/Vis light, whereas the optimal tissue transparency is in the lower‐energy NIR region. Two‐photon excitation (TPE) can overcome this dichotomy, with simultaneous absorption of two lower‐energy NIR‐photons populating the same PS‐active excited state as one higher‐energy photon. We introduce two low‐molecular weight, long‐lived and photo‐stable iridium complexes of the [Ir(N^C)2(N^N)]+ family with high TP‐absorption, which localise to mitochondria and lysosomal structures in live cells. The compounds are efficient PS under 1‐photon irradiation (405 nm) resulting in apoptotic cell death in diverse cancer cell lines at low light doses (3.6 J cm−2), low concentrations, and photo‐indexes greater than 555. Remarkably 1 also displays high PS activity killing cancer cells under NIR two‐photon excitation (760 nm), which along with its photo‐stability indicates potential future clinical application.

Combined Two-Photon Excitation and d→f Energy Transfer in a Water-Soluble Ir(III) /Eu(III) Dyad: Two Luminescence Components from One Molecule for Cellular Imaging.

The first example of cell imaging using two independent emission components from a dinuclear d/f complex is reported. A water-stable, cell-permeable IrIII/EuIII dyad undergoes partial Ir→Eu energy transfer following two-photon excitation of the Ir unit at 780 nm. Excitation in the near-IR region generated simultaneously green Ir-based emission and red Eu-based emission from the same probe. The orders-of-magnitude difference in their timescales (Ir ca. μs; Eu ca. 0.5 ms) allowed them to be identified by time-gated detection. Phosphorescence lifetime imaging microscopy (PLIM) allowed the lifetime of the Ir-based emission to be measured in different parts of the cell. At the same time, the cells are simultaneously imaged by using the Eu-based emission component at longer timescales. This new approach to cellular imaging by using dual d/f emitters should therefore enable autofluorescence-free sensing of two different analytes, independently, simultaneously and in the same regions of a cell.

Two-photon phosphorescence lifetime imaging of cells and tissues using a long-lived cyclometallated Npyridyl^Cphenyl^Npyridyl Pt(II) complex

Using a combination of multiphoton excitation, confocal scanning, TCSPC and beam blanking in conjunction with a cyclometallated Npyridyl^Cphenyl^Npyridyl Pt(II) complex (1) with a long luminescence lifetime, we demonstrate lifetime mapping of living cells and histological tissue sections over a time-frame of 50 microseconds, using a laser on/off “beam blanking” approach. This method of performing phosphorescence lifetime imaging microscopy (PLIM) represents an order of magnitude enhancement of the two-photon time-resolved emission imaging microscopy (TP-TREM) method, where in order to achieve a longer imaging window, the excitation laser repetition rate was reduced by cavity dumping [Chem. Sci., 2014, 5, 879]. The method complements and expands other existing imaging methodologies by enabling simultaneous PLIM and FLIM (fluorescence lifetime imaging microscopy – recorded between beam blanking), whilst maintaining essential sub-micron spatial resolution. We demonstrate how the Pt(II) complex can be used to distinguish between cell nuclei and matrix proteins on the basis of emission lifetime, in both structured and homogeneous tissue sections; whilst also revealing how the Pt(II) emission lifetime varies with tissue matrix composition. The proposed imaging approach can be used in conjunction with any biocompatible emissive probe with a long emission lifetime – exemplified here by (1) – and for an array of fluorescent/phosphorescent labels, where discrimination is lifetime-based.

Photodynamic killing of cancer cells by a Platinum(II) complex with cyclometallating ligand.

Photodynamic therapy that uses photosensitizers which only become toxic upon light-irradiation provides a strong alternative to conventional cancer treatment due to its ability to selectively target tumour material without affecting healthy tissue. Transition metal complexes are highly promising PDT agents due to intense visible light absorption, yet the majority are toxic even without light. This study introduces a small, photostable, charge-neutral platinum-based compound, Pt(II) 2,6-dipyrido-4-methyl-benzenechloride, complex 1, as a photosensitizer, which works under visible light. Activation of the new photosensitizer at low concentrations (0.1–1 μM) by comparatively low dose of 405 nm light (3.6 J cm−2) causes significant cell death of cervical, colorectal and bladder cancer cell lines, and, importantly, a cisplatin resistant cell line EJ-R. The photo-index of the complex is 8. We demonstrate that complex 1 induces irreversible DNA single strand breaks following irradiation, and that oxygen is essential for the photoinduced action. Neither light, nor compound alone led to cell death. The key advantages of the new drug include a remarkably fast accumulation time (diffusion-controlled, minutes), and photostability. This study demonstrates a highly promising new agent for photodynamic therapy, and attracts attention to photostable metal complexes as viable alternatives to conventional chemotherapeutics, such as cisplatin.

Time-resolved emission imaging microscopy using phosphorescent metal complexes : taking FLIM and PLIM to new lengths.

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 terminology and acronyms have not developed in a systematic way. We review the distinction between time-gating (to eliminate background emission) and true time-resolved 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.

Heteronuclear Ir(III)–Ln(III) luminescent complexes: small-molecule probes for dual modal imaging and oxygen sensing

Luminescent, mixed metal d-f complexes have the potential to be used for dual (magnetic resonance imaging (MRI) and luminescence) in vivo imaging. Here, we present dinuclear and trinuclear d-f complexes, comprising a rigid framework linking a luminescent Ir center to one (Ir·Ln) or two (Ir·Ln2) lanthanide metal centers (where Ln = Eu(III) and Gd(III), respectively). A range of physical, spectroscopic, and imaging-based properties including relaxivity arising from the Gd(III) units and the occurrence of Ir(III) → Eu(III) photoinduced energy-transfer are presented. The rigidity imposed by the ligand facilitates high relaxivities for the Gd(III) complexes, while the luminescence from the Ir(III) and Eu(III) centers provide luminescence imaging capabilities. Dinuclear (Ir·Ln) complexes performed best in cellular studies, exhibiting good solubility in aqueous solutions, low toxicity after 4 and 18 h, respectively, and punctate lysosomal staining. We also demonstrate the first example of oxygen sensing in fixed cells using the dyad Ir·Gd, via two-photon phosphorescence lifetime imaging (PLIM).

Porphyrin/Platinum(II) C^N^N Acetylide Complexes: Synthesis, Photophysical Properties, and Singlet Oxygen Generation.

A new class of substituted porphyrins has been developed in which a different number of cyclometalated Pt(II) C^N^N acetylides and polyethylene glycol (PEG) chains are attached to the meso positions of the porphyrin core, which are meant for photophysical, electrochemical, and in vitro light-induced singlet oxygen ((1)O2) generation studies. All of these Zn(II) porphyrin-Pt(II) C^N^N acetylide conjugates show moderate to high (ΦΔ =0.55 to 0.63) singlet oxygen generation efficiency. The complexes are soluble in organic solvents but, despite the PEG substituents, slowly aggregate in aqueous solvent systems. These conjugates also exhibit interesting photophysical properties, including near-complete photoinduced energy transfer (PEnT) through the rigid acetylenic bond(s) from the Pt(II) C^N^N antenna units to the Zn(II) porphyrin core, which shows sensitized luminescence, as shown by quenching of Pt(II) C^N^N-based luminescence. Electrochemical measurements show a set of redox processes that are approximately the sum of what is observed for the Pt(II) C^N^N acetylide and Zn(II) porphyrin units. UV/Vis spectroscopic properties are supported by DFT calculations.

Oxygen Mapping of Melanoma Spheroids using Small Molecule Platinum Probe and Phosphorescence Lifetime Imaging Microscopy

Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations. Oxygen is an efficient quencher of emission of many phosphorescent compounds, thus oxygen concentration could in many cases be derived directly from relative emission intensity and lifetime. In this study, we extend our previous work on phosphorescent, low molecular weight platinum(II) complex as an oxygen sensing probe to study the variation in oxygen concentration in a viable multicellular 3D human tumour model. The data shows one of the first examples of non-invasive, real-time oxygen mapping across a melanoma tumour spheroid using one-photon phosphorescence lifetime imaging microscopy (PLIM) and a small molecule oxygen sensitive probe. These measurements were quantitative and enabled real time oxygen mapping with high spatial resolution. This combination presents as a valuable tool for optical detection of both physiological and pathological oxygen levels in a live tissue mass and we suggest has the potential for broader clinical application.