Maddalena Collini

Two-photon fluorescence excitation and related techniques in biological microscopy

This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation.

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor κB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca2+/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cγ2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

A Molecular Thermometer for Nanoparticles for Optical Hyperthermia

We developed an all-optical method to measure the temperature on gold (nanorods and nanostars) and magnetite nanoparticles under near-infrared and radiofrequency excitation by monitoring the excited state lifetime of Rhodamine B that lies within =/~20 nm from the nanoparticle surface. We reached high temperature sensitivity (0.029 ± 0.001 ns/°C) and low uncertainty (±0.3 °C). Gold nanostars are =/~3 and =/~100 times more efficient than gold nanorods and magnetite nanoparticles in inducing localized hyperthermia.

Competitive binding of fatty acids and the fluorescent probe 1‐8‐anilinonaphthalene sulfonate to bovine β‐lactoglobulin

The use of spectroscopy in the study of fatty acids binding to bovine β‐lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty‐acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS–BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid‐binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

Synthesis of branched Au nanoparticles with tunable near-infrared LSPR using a zwitterionic surfactant

Asymmetric branched gold nanoparticles are obtained using for the first time in the seed-growth approach a zwitterionic surfactant, laurylsulfobetaine, whose concentration in the growth solution allows to control both the length to base-width ratio of the branches and the LSPR position, that can be tuned in the 700-1100 nm near infrared range.

Evidence of heterogeneous 1-anilinonaphthalene-8-sulfonate binding to beta-lactoglobulin from fluorescence spectroscopy

Steady-state and dynamic fluorescence titrations show that: (a) the complex between beta-lactoglobulin (BLG) and 1-anilinonaphthalene-8-sulfonate (ANS) displays a heterogeneous equilibrium with large changes in the binding strength vs. pH and ion concentration; and (b) the fluorescence response of bound ANS reveals two separate lifetimes that suggest two different sites (or binding modes). While steady-state fluorescence titrations yield effective values of the binding constant and of the bound ANS quantum efficiency, it is shown that, by combining steady-state fluorescence and lifetime decay of ANS, it is possible to give quantitative estimates of the association constants for each site. When heading from the acid (pH approximately 2) to the native state (pH approximately 6) the main result is a very large reduction of the effective binding constant. This and the results of titrations vs. ionic strength suggest that electrostatic interactions are a major contribution to ANS binding to BLG.

A Luminescent Poly(amidoamine)-Iridium Complex as a New Singlet-Oxygen Sensitizer for Photodynamic Therapy

A polymer complex (1P) was synthesized by binding bis(cyclometalated) Ir(ppy)2(+) fragments (ppy = 2-phenylpyridyl) to phenanthroline (phen) pendants of a poly(amidoamine) copolymer (PhenISA, in which the phen pendants involved ∼6% of the repeating units). The corresponding molecular complex [Ir(ppy)2(bap)](+) (1M, bap = 4-(butyl-4-amino)-1,10-phenanthroline) was also prepared for comparison. In water solution 1P gives nanoaggregates with a hydrodynamic diameter of 30 nm in which the lipophilic metal centers are presumed to be segregated within polymer tasks to reduce their interaction with water. Such confinement, combined with the dilution of triplet emitters along the polymer chains, led to 1P having a photoluminescence quantum yield greater than that of 1M (0.061 vs 0.034, respectively, in an aerated water solution) with a longer lifetime of the (3)MLCT excited states and a blue-shifted emission (595 nm vs 604 nm, respectively). NMR data supported segregation of the metal centers. Photoreaction of O2 with 1,5-dihydroxynaphthalene showed that 1P is able to sensitize (1)O2 generation but with half the quantum yield of 1M. Cellular uptake experiments showed that both 1M and 1P are efficient cell staining agents endowed with two-photon excitation (TPE) imaging capability. TPE microscopy at 840 nm indicated that both complexes penetrate the cellular membrane of HeLa cells, localizing in the perinuclear region. Cellular photodynamic therapy tests showed that both 1M and 1P are able to induce cell apoptosis upon exposure to Xe lamp irradiation. The fraction of apoptotic cells for 1M was higher than that for 1P (74 and 38%, respectively) 6 h after being irradiated for 5 min, but cells incubated with 1P showed much lower levels of necrosis as well as lower toxicity in the absence of irradiation. More generally, the results indicate that cell damage induced by 1M was avoided by binding the iridium sensitizers to the poly(amidoamine).

IL-15 cis Presentation Is Required for Optimal NK Cell Activation in Lipopolysaccharide-Mediated Inflammatory Conditions

Natural killer (NK) cells have antitumor, antiviral, and antibacterial functions, and efforts are being made to manipulate them in immunotherapeutic approaches. However, their activation mechanisms remain poorly defined, particularly during bacterial infections. Here, we show that upon lipopolysaccharide or E. coli exposure, dendritic cells (DCs) produce three cytokines-interleukin 2 (IL-2), IL-18, and interferon β (IFN-β)-necessary and sufficient for NK cell activation. IFN-β enhances NK cell activation by inducing IL-15 and IL-15 receptor α not only in DCs but, surprisingly, also in NK cells. This process allows the transfer of IL-15 on NK cell surface and its cis presentation. cis-presented NK cell-derived and trans-presented DC-derived IL-15 contribute equally to optimal NK cell activation.

Similarities and differences of innate immune responses elicited by smooth and rough LPS.

The lipopolysaccharide is the major component of Gram-negative bacteria outer membrane. LPS comprises three covalently linked regions: the lipid A, the rough core oligosaccharide, and the O-antigenic side chain determining serotype specificity. Wild-type LPS (sLPS) contains the O-antigenic side chain and is referred to as smooth. Rough LPS (rLPS) does not contain the O-side chain. Most wt bacteria and especially wt Enterobacteriaceae express prevalently the sLPS form although some truncated rLPS molecules always reach the external membrane. The two sLPS and rLPS forms are used almost indistinctly to study the effects on innate immune cells. Nevertheless, there is evidence that their mechanism of action may be different. For instance, while sLPS requires CD14 for the initiation of both MyD88-dependent and independent signal transduction pathways at least at low doses, rLPS leads to MyD88-dependent responses in the absence of CD14 even at low doses. Here we have identified additional differences in the signaling capacity of the two LPS species in the mouse. We have found that rLPS, diversely from sLPS, is capable of activating in dendritic cells (DCs) the Ca(2+)/calcineurin and NFAT pathway in a CD14-independent manner, moreover it is also capable per se of activating the inflammasome and eliciting IL-1β secretion independent of the presence of additional stimuli required instead for sLPS. The ability of rLPS of activating the inflammasome in vitro has as a direct consequence a higher efficiency of rLPS-exposed DCs in activating natural killer (NK) cells compared to sLPS-exposed DCs. However, diversely from possible predictions, we found that the different efficiencies of the two LPS species in eliciting innate responses are almost nullified in vivo. Therefore, sLPS and rLPS induce nearly similar in vivo innate responses but with different mechanisms of signaling.

Thermal and Chemical Stability of Thiol Bonding on Gold Nanostars.

The stability of thiol bonding on the surface of star-shaped gold nanoparticles was studied as a function of temperature in water and in a set of biologically relevant conditions. The stability was evaluated by monitoring the release of a model fluorescent dye, Bodipy-thiol (BDP-SH), from gold nanostars (GNSs) cocoated with poly(ethylene glycol) thiol (PEG-SH). The increase in the BDP-SH fluorescence emission, quenched when bound to the GNSs, was exploited to this purpose. A maximum 15% dye release in aqueous solution was found when the bulk temperature of gold nanostars solutions was increased to T = 42 °C, the maximum physiological temperature. This fraction reduces 3-5% for temperatures lower than 40 °C. Similar results were found when the temperature increase was obtained by laser excitation of the near-infrared (NIR) localized surface plasmon resonance of the GNSs, which are photothermally responsive. Besides the direct impact of temperature, an increased BDP-SH release was observed upon changing the chemical composition of the solvent from pure water to phosphate-buffered saline and culture media solutions. Moreover, also a significant fraction of PEG-SH was released from the GNS surface due to the increase in temperature. We monitored it with a different approach, that is, by using a coating of α-mercapto-ω-amino PEG labeled with tetramethylrhodamine isothiocyanate on the amino group, that after heating was separated from GNS by ultracentrifugation and the released PEG was determined by spectrofluorimetric techniques on the supernatant solution. These results suggest some specific limitations in the use of the gold-thiolate bond for coating of nanomaterials with organic compounds in biological environments. These limitations come from the duration and the intensity of the thermal treatment and from the medium composition and could also be exploited in biological media to modulate the in vivo release of drugs.