Klaus Suhling

Imaging intracellular viscosity of a single cell during photoinduced cell death.

Diffusion-mediated cellular processes, such as metabolism, signalling and transport, depend on the hydrodynamic properties of the intracellular matrix. Photodynamic therapy, used in the treatment of cancer, relies on the generation of short-lived cytotoxic agents within a cell on irradiation of a drug. The efficacy of this treatment depends on the viscosity of the medium through which the cytotoxic agent must diffuse. Here, spectrally resolved fluorescence measurements of a porphyrin-dimer-based molecular rotor are used to quantify intracellular viscosity changes in single cells. We show that there is a dramatic increase in the viscosity of the immediate environment of the rotor on photoinduced cell death. The effect of this viscosity increase is observed directly in the diffusion-dependent kinetics of the photosensitized formation and decay of a key cytotoxic agent, singlet molecular oxygen. Using these tools, we provide insight into the dynamics of diffusion in cells, which is pertinent to drug delivery, cell signalling and intracellular mass transport.

Phospholipid Encapsulated Semiconducting Polymer Nanoparticles: Their Use in Cell Imaging and Protein Attachment

Semiconducting polymer nanospheres (SPNs) have been synthesized and encapsulated in phospholipid micelles by a solvent evaporation technique. Four different conjugated polymers were used, yielding aqueous dispersions of nanoparticles which emit across the visible spectrum. The synthesis was simple and easily reproducible, and the resultant nanoparticle solutions exhibited high colloidal stability. As these encapsulated SPNs do not contain any toxic materials and show favorable optical properties, they appear to be a promising imaging agent in biomedical and imaging applications. The SPNs were used in simple fluorescence imaging experiments and showed uptake in SH-SY5Y neuroblastoma and live HeLa cells. Carboxylic acid functionalized SPNs were also synthesized and conjugated to bovine serum albumin (BSA) by carbodiimide-mediated chemistry, a key step in the realization of targeted imaging using conjugated polymers.

Imaging the environment of green fluorescent protein.

An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also probe their environment. Such a technique is demonstrated here using fluorescence lifetime imaging of green fluorescent protein (GFP). We first show, by time-correlated single-photon counting, that the fluorescence decay of GFP depends on the local refractive index. This is in agreement with the Strickler Berg formula, relating the Einstein A and B coefficients for absorption and spontaneous emission in molecules. We then quantitatively image, by wide-field time-gated fluorescence lifetime imaging, the refractive index of the environment of GFP. This novel approach paves the way for imaging the biophysical environment of specific GFP-tagged proteins in live cells.

Fluorescence lifetime and polarization-resolved imaging in cell biology

Fluorescence lifetime imaging (FLIM) and fluorescence polarization imaging are complementary techniques that can be used to extract information about macromolecules from biological samples. Owing to the sensitivity of fluorescence to the physicochemical environment, and nanometer-scale interactions via Förster resonance energy transfer (FRET), FLIM has been implemented in many laboratories for numerous applications in the life sciences and beyond. This review seeks to provide a brief overview of some of the recent advances in the techniques and more pertinently their applications in cell and tissue imaging. The particular merits of polarization-resolved fluorescence measurements are highlighted, including the unique ability to elucidate the occurrence of homo-FRET.

Effects of axial ligands on the photophysical properties of silicon octaphenoxyphthalocyanine

The photochemistry and photophysics of six axially substituted silicon phthalocyanines are reported and show the importance of the axial groups in the photochemistry of these compounds. The fluorescence quantum yields are especially affected by the axial ligand. A very good correlation was found for the experimentally determined fluorescence lifetimes and the theoretically determined lifetimes using the Strickler-Berg equation for the unaggregated molecules.

Photophysical properties and intracellular imaging of water-soluble porphyrin dimers for two-photon excited photodynamic therapy

We have investigated the photophysical properties and intracellular behaviour of a series of hydrophilic conjugated porphyrin dimers. All the dimers exhibit intense linear absorption at 650-800 nm and high singlet oxygen quantum yields (0.5-0.9 in methanol), as required for an efficient sensitiser for photodynamic therapy (PDT). They also exhibit fluorescence at 700-800 nm, with fluorescence quantum yields of up to 0.13 in methanol, and show extremely large two-photon absorption maxima of 8,000-17,000 GM in the near-IR. The dimers aggregate in aqueous solution, but aggregation is reduced by binding to bovine serum albumin (BSA), as manifested by an increase in fluorescence intensity and a sharpening in the emission bands. This process can be regarded as a model for the interaction with proteins under physiological conditions. Confocal fluorescence microscopy of live cells was used to monitor the rate of cellular uptake, intracellular localisation and photostability. Porphyrin dimers with positively charged substituents partition into cells more efficiently than the negatively charged dimers. The photostability of these dimers, in living cells, is significantly better than that of the clinical photosensitiser verteporfin. Analysis of the photophysical parameters and intracellular imaging data indicates that these dimers are promising candidates for one-photon and two-photon excited PDT.

Biosynthesis of luminescent quantum dots in an earthworm

The synthesis of designer solid-state materials by living organisms is an emerging field in bio-nanotechnology. Key examples include the use of engineered viruses as templates for cobalt oxide (Co(3)O(4)) particles, superparamagnetic cobalt-platinum alloy nanowires and gold-cobalt oxide nanowires for photovoltaic and battery-related applications. Here, we show that the earthworms metal detoxification pathway can be exploited to produce luminescent, water-soluble semiconductor cadmium telluride (CdTe) quantum dots that emit in the green region of the visible spectrum when excited in the ultraviolet region. Standard wild-type Lumbricus rubellus earthworms were exposed to soil spiked with CdCl(2) and Na(2)TeO(3) salts for 11 days. Luminescent quantum dots were isolated from chloragogenous tissues surrounding the gut of the worm, and were successfully used in live-cell imaging. The addition of polyethylene glycol on the surface of the quantum dots allowed for non-targeted, fluid-phase uptake by macrophage cells.

Time-resolved fluorescence anisotropy imaging applied to live cells

We have developed a wide-field time-resolved imaging system to image quantitatively both the fluorescence lifetime and the rotational correlation time of a fluorophore. Using a polarization-resolved imager, we simultaneously image orthogonal polarization components of the fluorescence emission onto a time-gated intensified CCD. We demonstrate imaging of solvent viscosity variations through the rotational correlation time of fluorescein in a multiwell plate and apply this technique to probe the microviscosity in live cells.

The interactions between a small molecule and G-quadruplexes are visualized by fluorescence lifetime imaging microscopy

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.

Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse

Fluorescence imaging of green fluorescent protein (GFP) may be used to locate proteins in live cells and fluorescence lifetime imaging (FLIM) may be employed to probe the local microenvironment of proteins. Here we apply FLIM to GFP‐tagged proteins at the cell surface and at an inhibitory natural killer (NK) cell immunological synapse (IS). We present a novel quantitative analysis of fluorescence lifetime images that we believe is useful to determine whether apparent FLIM heterogeneity is statistically significant. We observe that, although the variation of observed fluorescence lifetime of GFP‐tagged proteins at the cell surface is close to the expected statistical range, the lifetime of GFP‐tagged proteins in cells is shorter than recombinant GFP in solution. Furthermore the lifetime of GFP‐tagged major histocompatibility complex class I protein is shortened at the inhibitory NK cell IS compared with the unconjugated membrane. Following our previous work demonstrating the ability of FLIM to report the local refractive index of GFP in solution, we speculate that these lifetime variations may indicate local refractive index changes. This application of our method for detecting small but significant differences in fluorescence lifetimes shows how FLIM could be broadly useful in imaging discrete membrane environments for a given protein.