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.

We report quantitative, real-time, online observations of microscopic viscosity changes in aerosol particles of atmospherically relevant composition, using fluorescence lifetime imaging (FLIM) of viscosity.

Microviscosity is a key parameter controlling the rate of diffusion and reactions on the microscale. One of the most convenient tools for measuring microviscosity is by fluorescent viscosity sensors termed 'molecular rotors'. BODIPY-based molecular rotors in particular proved extremely useful in combination with fluorescence lifetime imaging microscopy, for providing quantitative viscosity maps of living cells as well as measuring dynamic changes in viscosity over time. In this work, we investigate several new BODIPY-based molecular rotors with the aim of improving on the current viscosity sensing capabilities and understanding how the structure of the fluorophore is related to its function. We demonstrate that due to subtle structural changes, BODIPY-based molecular rotors may become sensitive to temperature and polarity of their environment, as well as to viscosity, and provide a photophysical model explaining the nature of this sensitivity. Our data suggests that a thorough understanding of the photophysics of any new molecular rotor, in environments of different viscosity, temperature and polarity, is a must before moving on to applications in viscosity sensing.

Using fluorescent probes, we demonstrate that the plasma membrane of porcine eye lens fiber cells displays an unprecedentedly high degree of lipid ordering.

We examine the effect of temperature on three viscosity-sensitive fluorophores termed ‘molecular rotors’. In the case of the conjugated porphyrin dimer, it can be used for measuring both viscosity and temperature concurrently.

Microviscosity is of paramount importance in materials and bio-sciences. Fluorescence imaging using molecular rotors has emerged as a versatile tool to measure microviscosity, either using a fluorescence lifetime or a ratiometric signal of the rotor; however, only a limited number of blue -to-green-emitting fluorophores with both the lifetime and the ratiometric signal sensitivity to viscosity have been reported to date. Here we report a deep red emitting dual viscosity sensor, which allows both the ratiometric and the lifetime imaging of viscosity. We study viscosity in a range of lipid-based systems and conclude that in complex dynamic systems dual detection is preferable in order to independently verify the results of the measurements as well as perform rapid detection of changing viscosity .

No abstract

Molecular rotors have emerged as versatile probes of microscopic viscosity in lipid bilayers, although it has proved difficult to find probes that stain both phases equally in phase-separated bilayers. Here, we investigate the use of a membrane-targeting viscositysensitive fluorophore based on a thiophene moiety with equal affinity for ordered and disordered lipid domains to probe ordering and viscosity within artificial lipid bilayers and live cell plasma membranes.As the physical boundary separating the interior of a cell from the external environment, the plasma membrane plays a vital role in the function of a cell, by controlling access to and from its interior. The plasma membrane is a complex mixture of lipids and membrane proteins. The lipids were once thought to play a purely passive role, 1 but it is now believed that they are integral to the normal functioning of the cell, affecting processes such as cell division, signal transduction and protein aggregation.Despite the apparent importance of the plasma membrane lipids, their supramolecular structures and lateral organisation are not yet well understood. Plasma membrane lipids are widely believed to transiently phase separate into coexisting, sub-micron ordered (Lo) and disordered (Ld) liquid domains, the so-called "lipid raft hypothesis", 2 where cholesterol rich Lo domains form platforms that function in membrane signalling and trafficking. 3 Despite growing evidence for phase separation within the plasma membrane and the demonstration of Lo-Ld phase separation in artificial membranes made up of plasma membrane extracts, [3][4][5][6] it is very difficult to directly observe this phase separation in vivo, mainly owing to the fact that the Lo domains are believed to have dimensions that are smaller than the diffraction limit of visible light, 6 with lifetimes on the microsecond timescale. 5,7,8 Fluorescence techniques provide a non-invasive way to investigate plasma membrane structure and organisation, and have found widespread application in recent years. 9 Various fluorescence studies have found evidence for sub-resolution ordered regions within cell membranes, 6 with a recent study suggesting that up to 76% of the plasma membrane lipids may be in an Lo phase at any one time. 10 An emerging fluorescence-based technique for investigating order and structure within biological systems is the use of molecular rotors, which are environmentally responsive fluorophores that show increased quantum yield and fluorescence lifetime within more viscous, ordered environments. 11 In general, after optical excitation, a molecular rotor can undergo either radiative or nonradiative decay. The rate of nonradiative decay, which typically occurs via an intramolecular rotation pathway, is directly affected by the friction imparted on the rotor by the surrounding environment. The competition between radiative and nonradiative decay leads to the environmentally sensitive fluorescence properties of the rotor. 11 Viscosity can be related to fluorescence quantum yield using t...