Imaging Chromophores With Undetectable Fluorescence by Stimulated Emission Microscopy

Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging.Chemistry and Chemical Biolog

Fluorescence studies on new potential antitumoral benzothienopyran-1-ones in solution and in liposomes

Fluorescence properties of four new potential antitumoral compounds, 3-arylbenzothieno[2,3-c]pyran-1-ones, were studied in solution and in lipid membranes of dipalmitoyl phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (Egg-PC) and dioctadecyldimethylammonium bromide (DODAB). The 3-(4-methoxyphenyl)benzothieno[2,3-c]pyran-1-one (1c) exhibits the higher fluorescence quantum yields in all solvents studied. All compounds present a solvent sensitive emission, with significant red shifts in polar solvents for the methoxylated compounds. The results point to an ICT character of the excited state, more pronounced for compound 1c. Fluorescence (steady-state) anisotropy measurements of the compounds incorporated in liposomes of DPPC, DODAB and Egg-PC indicate that all compounds have two different locations, one due to a deep penetration in the lipid membrane and another corresponding to a more hydrated environment. In general, the methoxylated compounds prefer hydrated environments inside the liposomes. The 3-(4- fluorophenyl)benzothieno[2,3-c]pyran-1-one (1a) clearly prefers a hydrated environment, with some molecules located at the outer part of the liposome interface. On the contrary, the preferential location of 3-(2-fluorophenyl)benzothieno[2,3-c]pyran-1-one (1b) is in the region of lipid hydrophobic tails. Compounds with a planar geometry (1a and 1c) have higher mobility in the lipid membranes when phase transition occurs.Portugal and FEDER (Fundo Europeu de Desenvolvimento Regional), for financial support through Centro de Física (CFUM) and Centro de Química (CQ-UM) of University of Minho and through the Project PTDC/QUI/81238/2006. M.S.D. Carvalho and R.C. Calhelha acknowledge FCT for their PhD grants SFRH/BD/47052/2008 and SFRH/BD/29274/2006, respectively.Fundação para a Ciência e a Tecnologia (FCT

Charge Transport in DNA-Based Devices

Charge migration along DNA molecules has attracted scientific interest for over half a century. Reports on possible high rates of charge transfer between donor and acceptor through the DNA, obtained in the last decade from solution chemistry experiments on large numbers of molecules, triggered a series of direct electrical transport measurements through DNA single molecules, bundles and networks. These measurements are reviewed and presented here. From these experiments we conclude that electrical transport is feasible in short DNA molecules, in bundles and networks, but blocked in long single molecules that are attached to surfaces. The experimental background is complemented by an account of the theoretical/computational schemes that are applied to study the electronic and transport properties of DNA-based nanowires. Examples of selected applications are given, to show the capabilities and limits of current theoretical approaches to accurately describe the wires, interpret the transport measurements, and predict suitable strategies to enhance the conductivity of DNA nanostructures.Comment: A single pdf file of 52 pages, containing the text and 23 figures. Review about direct measurements of DNA conductivity and related theoretical studies. For higher-resolution figures contact the authors or retrieve the original publications cited in the caption

In quest of a systematic framework for unifying and defining nanoscience

This article proposes a systematic framework for unifying and defining nanoscience based on historic first principles and step logic that led to a “central paradigm” (i.e., unifying framework) for traditional elemental/small-molecule chemistry. As such, a Nanomaterials classification roadmap is proposed, which divides all nanomatter into Category I: discrete, well-defined and Category II: statistical, undefined nanoparticles. We consider only Category I, well-defined nanoparticles which are >90% monodisperse as a function of Critical Nanoscale Design Parameters (CNDPs) defined according to: (a) size, (b) shape, (c) surface chemistry, (d) flexibility, and (e) elemental composition. Classified as either hard (H) (i.e., inorganic-based) or soft (S) (i.e., organic-based) categories, these nanoparticles were found to manifest pervasive atom mimicry features that included: (1) a dominance of zero-dimensional (0D) core–shell nanoarchitectures, (2) the ability to self-assemble or chemically bond as discrete, quantized nanounits, and (3) exhibited well-defined nanoscale valencies and stoichiometries reminiscent of atom-based elements. These discrete nanoparticle categories are referred to as hard or soft particle nanoelements. Many examples describing chemical bonding/assembly of these nanoelements have been reported in the literature. We refer to these hard:hard (H-n:H-n), soft:soft (S-n:S-n), or hard:soft (H-n:S-n) nanoelement combinations as nanocompounds. Due to their quantized features, many nanoelement and nanocompound categories are reported to exhibit well-defined nanoperiodic property patterns. These periodic property patterns are dependent on their quantized nanofeatures (CNDPs) and dramatically influence intrinsic physicochemical properties (i.e., melting points, reactivity/self-assembly, sterics, and nanoencapsulation), as well as important functional/performance properties (i.e., magnetic, photonic, electronic, and toxicologic properties). We propose this perspective as a modest first step toward more clearly defining synthetic nanochemistry as well as providing a systematic framework for unifying nanoscience. With further progress, one should anticipate the evolution of future nanoperiodic table(s) suitable for predicting important risk/benefit boundaries in the field of nanoscience

EPR Detection of Guanine Radicals in a DNA Duplex under Biological Conditions: Selective Base Oxidation by Ru(phen)₂dppz³⁺ Using the Flash-Quench Technique

Continuous-wave X-band EPR spectroscopy has been employed in examining the guanine radical within a DNA duplex at ambient temperature using the flash-quench technique. Guanine was selectively oxidized by DNA-bound [Ru(phen)(2)dppz](3+) (dppz = dipyridophenazine, phen = 1,10-phenanthroline) generated in situ by photolysis in the presence of [Co(NH3)(5)Cl](2+) as the oxidative quencher. An EPR signal centered at g(iso) = 2.0048 is observed in experiments with poly(dG-dC) as substrate. Comparable signals are also detected with a 13-mer oligonucleotide duplex containing only one guanine base and with calf thymus DNA, but no signal is observed with poly(dA-dT) or poly(dI-dC). These observations reflect the base selectivity of the reaction in forming the guanine radical. With ruthenium hexaammine as oxidative quencher, no signal is observed, while, with methyl viologen, a strong signal with hyperfine pattern is seen, characteristic of the reduced viologen radical and indicating that [Ru(phen)(2)dppz](3+) was generated. The guanine radical signal, once formed upon continuous irradiation in argon-saturated aqueous buffer solution (pH 7), decays with a half-life of 30 s, but vanishes instantaneously in the dark or upon introduction of oxygen. Spin trapping experiments with N-tert-butyl-alpha-phenylnitrone substantiate the selectivity in generating the guanine radical; in the presence of poly(dG-dC), calf thymus DNA, the 13-mer oligonucleotide but not with poly(dA-dT) and poly(dI-dC), the detected nitroxide EPR signals are the same with g(iso) = 2.0059, [a(N)] = 15.05 G, and [a(H)] = 3.11 G. Upon titration of the ruthenium intercalator into poly(dG-dC), the signal intensity increases smoothly as the [base pair]/[intercalator] ratio decreases from 100 to 25, at which point the signal intensity decreases markedly; this result may be an indication of an antiferromagnetic exchange interaction between guanine radicals. indeed. using the flash-quench technique, EPR spectroscopy of guanine radicals within DNA now will permit the evaluation of how radicals within the DNA base stack may be coupled under biological conditions.</p

RADICAL IONS AND PHOTOCHEMICAL CHARGE-TRANSFER PHENOMENA .22. PRESSURE-INDUCED DIASTEREOSELECTIVITY IN PHOTOINDUCED DIELS-ALDER REACTIONS

CHUNG WS, TURRO NJ, MERTES J, Mattay J. RADICAL IONS AND PHOTOCHEMICAL CHARGE-TRANSFER PHENOMENA .22. PRESSURE-INDUCED DIASTEREOSELECTIVITY IN PHOTOINDUCED DIELS-ALDER REACTIONS. The Journal of Organic Chemistry. 1989;54(20):4881-4887