The fluorescence intensity and lifetime of the 4,4′-difluoro-4-bora-5-(p-oxoalkyl)phenyl-3a,4a-diaza-s-indacene (1) show a strong correlation with the viscosity of the medium due to the viscosity-dependent twisting of the 5-phenyl group, which gives access to the dark nonemissive excited state. We propose a sensitive and versatile method for measuring the local microviscosity in biological systems, based on the determination of the fluorescence lifetime of 1. Fluorescence lifetime imaging (FLIM) performed on live cells incubated with 1 demonstrates the distinct intracellular lifetime of the molecular rotor of 1.6 ± 0.2 ns corresponding to the intracellular viscosity of ca. 140 cP. Time-resolved fluorescence anisotropy of 1 in cells confirms insignificant binding of the fluorophore. The viscosity value obtained in the present study is considerably higher than that of water and of cellular cytoplasm. The high viscosity of intracellular compartments is likely to play an important role in vital intracellular processes, including the rate of diffusion of reactive oxygen species, causing programmed cell destruction.

No abstract

This article describes an emerging method for quantitative measurement and spatial imaging of microviscosity within individual domains of live cells. The method is based on fluorescence detection from small synthetic molecules termed 'molecular rotors', which are characterised by a strong response of fluorescence lifetimes or spectra to the viscosity of their immediate environment. Alongside this new method, two complementary techniques are discussed, which provide further insights into diffusion controlled processes on a microscopic scale in a biological environment. These are time resolved fluorescence anisotropy and imaging of short-lived excited state of molecular oxygen, termed 'singlet oxygen'. It is possible to utilise all three approaches for the quantitative determination of viscosity in individual organelles of live cells. Finally, it is discussed how the major advantage of molecular rotor imaging, fast signal acquisition, can be used to monitor changing viscosity during dynamic biological processes within cells, such as photoinduced cell death.

The spatial control of optical absorption provided by twophoton excitation (TPE) has led to tremendous advances in microscopy 1 and microfabrication 2 . Medical applications of TPE in photodynamic therapy (PDT) 3,4 have often been suggested 5-18 , but have been made impractical by the low twophoton cross-sections of photosensitiser drugs (i.e. compounds taken up by living tissues that become toxic on absorption of light). The invention of efficient two-photon activated drugs will allow precise manipulation of treatment volumes in three dimensions, to a level unattainable with current techniques. Here we present a new family of PDT drugs designed for efficient TPE, and use one of them to demonstrate selective closure of blood vessels via TPE-PDT in vivo. These conjugated porphyrin dimers have two-photon cross-sections that are more than two orders of magnitude greater than those of clinical photosensitisers 17 . This is the first demonstration of in vivo PDT using a photosensitiser engineered for efficient two-photon excitation.Photodynamic therapy is used to treat diseases characterised by neoplastic growth including various cancers, age-related macular degeneration (AMD) and actinic keratosis 3,4 . Cell death is induced by photoexcitation of a sensitiser, generally via production of singlet oxygen. In the absence of light the photosensitiser is benign, so systemic toxicity is rare and treatment may be repeated without acquired resistance. Two-photon excitation of the photosensitiser should allow greater precision than is attainable by conventional one-photon excitation, as a consequence of the quadratic dependence of TPE on the local light intensity -the amount of TPE is inversely proportional to the fourth power of the distance from the focus. In addition, the longer wavelengths associated with TPE allow treatment deeper into tissue, by minimising absorption from endogenous chromophores.High instantaneous photon densities are essential for two-photon excitation. Early TPE-PDT studies used nanosecond lasers, but the dominant effect was photothermal damage [5][6][7] . The advent of commercial femtosecond tuneable Ti:sapphire lasers has greatly facilitated the investigation of TPE-PDT, and the limiting factor has become the availability of suitable photosensitisers. The majority of chromophores possess low two-photon cross-sections, of the order of 1-100 Goeppert-Mayer units (1 GM = 10 -50 cm 4 s photon -1 ). For example, the two FDA-approved PDT photosensitisers, verteporfin and Photofrin (cross sections 50 GM and 10 GM respectively) 17 , are unlikely to be suitable for TPE-PDT, as the high light intensities needed to achieve a therapeutic effect are close to the thresholds for photothermal or photomechanical damage 18 .Several design strategies for TPE-PDT photosensitisers have been reported recently [11][12][13][14][15][16] , but few of these compounds have yet been studied in vitro 15 , and, to date, none have progressed to in vivo testing. Porphyrin derivatives are often effective PDT agents, as exemplified ...

We report intracellular fluorescence lifetime imaging (FLIM) and fluorescence anisotropy measurements of two meso-substituted fluorophores based on the boron-dipyrrin (BODIPY) structure.Both dyes incorporate hydrophobic groups, which render them membrane-soluble. We have obtained quantum yields, radiative and non-radiative rate constants, fluorescence lifetimes and time-resolved fluorescence anisotropy of the dyes in homogeneous methanol/glycerol solutions of varying viscosity from 0.6 cP to 950 cP. We find that the fluorescence lifetimes and rotational correlation times for both dyes increase with increasing viscosity, as predicted by theory. These molecules can thus serve as fluorescent molecular rotors to report on local microviscosity, including that in live cells. The dyes are readily taken up by cells as imaged using confocal fluorescence microscopy. Using FLIM we have detected two distinct fluorescence lifetime populations for both dyes in live SK-OV-3 human ovarian carcinoma cells, corresponding to apparent viscosity values of 160 ± 20 cP and 260 ± 40 cP, each found in distinct intracellular domains. In both cellular domains, independent of the fluorophore used, the viscosity values significantly exceed that expected for the aqueous phase of cellular cytoplasm, suggesting slower diffusion and reaction rates in this hydrophobic microenvironment. FLIM measurements were complemented with time-resolved fluorescence anisotropy measurements, which confirm the high viscosity values in the immediate environment of both rotors. The present study highlights the power of FLIM to map heterogeneous microenvironments of complex biological systems and also the use of fluorescent molecular rotors as microviscosity sensors.3

Molecular rotors have emerged as versatile probes for microscopic viscosity in live cells, however, the exclusive localisation of rotors in the plasma membrane has remained elusive. We report the synthesis, spectroscopic characterisation and live cell imaging of a new BODIPY-based molecular rotor suitable for mapping viscosity in the cell plasma membrane.

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.

Guanine-rich oligonucleotides can fold into quadruple-stranded helical structures known as G-quadruplexes. Mounting experimental evidence has gathered suggesting that these non-canonical nucleic acid structures form in vivo and play essential biological roles. However, to date, there are no small-molecule optical probes to image G-quadruplexes in live cells. Herein, we report the design and development of a small fluorescent molecule, which can be used as an optical probe for G-quadruplexes. We demonstrate that the fluorescence lifetime of this new probe changes considerably upon interaction with different nucleic acid topologies. Specifically, longer fluorescence lifetimes are observed in vitro for G-quadruplexes than for double- and single-stranded nucleic acids. Cellular studies confirm that this molecule is cell permeable, has low cytotoxicity and localizes primarily in the cell nucleus. Furthermore, using fluorescence lifetime imaging microscopy, live-cell imaging suggests that the probe can be used to study the interaction of small molecules with G-quadruplexes in vivo.