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1.
Artigo em Inglês | MEDLINE | ID: mdl-32048796

RESUMO

We have proposed a label-free method for measuring intracellular temperature using a Raman image of a cell in the O-H stretching band. Raman spectra of cultured cells and the medium were first measured at various temperatures using a Raman microscope and the intensity ratio of the two regions of the O-H stretching band was calculated. The intensity ratio varies linearly with temperature in both the medium and cells, and the resulting calibration line allows simultaneous visualization of both intracellular and extracellular temperatures in a label-free manner. We applied this method to the measurement of temperature changes after the introduction of FCCP (carbonyl cyanide- p -trifluoromethoxyphenylhydrazone) in living cells. We observed a temperature rise in the cytoplasm and succeeded in obtaining an image of the change in intracellular temperature after the FCCP treatment.

2.
Nat Commun ; 10(1): 2186, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097703

RESUMO

The increasing availability of X-ray free-electron lasers (XFELs) has catalyzed the development of single-object structural determination and of structural dynamics tracking in real-time. Disentangling the molecular-level reactions triggered by the interaction with an XFEL pulse is a fundamental step towards developing such applications. Here we report real-time observations of XFEL-induced electronic decay via short-lived transient electronic states in the diiodomethane molecule, using a femtosecond near-infrared probe laser. We determine the lifetimes of the transient states populated during the XFEL-induced Auger cascades and find that multiply charged iodine ions are issued from short-lived (∼20 fs) transient states, whereas the singly charged ones originate from significantly longer-lived states (∼100 fs). We identify the mechanisms behind these different time scales: contrary to the short-lived transient states which relax by molecular Auger decay, the long-lived ones decay by an interatomic Coulombic decay between two iodine atoms, during the molecular fragmentation.

3.
J Phys Chem B ; 122(51): 12375-12385, 2018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30507183

RESUMO

Phase separation dynamics of a water/2-butoxyethanol (2BE) mixture was studied with newly developed time-resolved structured illumination microscopy (SIM). Interestingly, an employed hydrophobic fluorescent probe for SIM showed spectral shifts up to 500 ns after a laser-induced temperature jump, which suggests 2BE micellar-like aggregates become more hydrophobic at the initial stage of phase separation. This hydrophobic environment in 2BE aggregates, probably due to the ejection of water molecules, continued up to at least 10 µs. Time-resolved SIM and previously reported light scattering data clearly showed that the size of a periodic structure remained constant (ca. 300 nm) from 3 to 10 µs, and then the growth of periodic structures having the self-similarity started. We think that the former and the latter processes correspond to "early stage" (concentration growth) and "late stage" (size growth), respectively, in phase separation dynamics. Here we suggest that, in the early stage, the entity to bear 2BE phase be water-poor 2BE aggregates, and the number density of these aggregates would simply increase in time.

4.
J Phys Chem B ; 122(24): 6364-6370, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29775068

RESUMO

Peptide-lipid interactions are widely involved with biologically significant phenomena, including the pathogenic mechanisms of protein misfolding diseases and transmembrane protein folding. In this paper, the interaction of the cysteine/tryptophan (Cys/Trp) motif, which is a metal-binding motif of copper transporter (Ctr) proteins, with a lipid bilayer was studied using fluorescence and circular dichroism (CD) spectroscopy. The binding of Cu(I) to the Cys/Trp motif induced a large red-edge excitation shift in the Trp fluorescence, indicating that the Trp residue is located inside the lipid bilayer following complexation of Cu(I) with the Cys/Trp motif. The Stern-Volmer quenching of the Trp fluorescence also supported the Cu(I) binding peptide embedding in the lipid bilayer. The measurement of the CD spectra indicated the increase in ß-sheet content of the Cys/Trp motif peptide as a result of Cu(I) binding. These results lead to the conclusion that complexation with Cu(I) induces the change in the secondary structure of the Cys/Trp motif, which results in the peptide embedding in the lipid bilayer. Cu(I)-induced enhancement of the lipid affinity is discussed in terms of the mechanism for copper transport by Ctr.


Assuntos
Cobre/química , Bicamadas Lipídicas/química , Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Cobre/metabolismo , Cisteína/química , Bicamadas Lipídicas/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta , Espectrometria de Fluorescência , Triptofano/química
5.
J Phys Chem Lett ; 8(21): 5241-5245, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29022721

RESUMO

We report direct observation of a spatial distribution of water molecules inside of a living cell using Raman images of the O-H stretching band of water. The O-H Raman intensity of the nucleus was higher than that of the cytoplasm, indicating that the water density is higher in the nucleus than that in the cytoplasm. The shape of the O-H stretching band of the nucleus differed from that of the cytoplasm but was similar to that of the balanced salt solution surrounding cells, indicating less crowded environments in the nucleus. The concentration of biomolecules having C-H bonds was also estimated to be lower in the nucleus than that in the cytoplasm. These results indicate that the nucleus is less crowded with biomolecules than the cytoplasm.


Assuntos
Citoplasma/química , Análise Espectral Raman/métodos , Água/química , Núcleo Celular/química , Ligações de Hidrogênio , Microscopia
6.
Adv Exp Med Biol ; 1035: 163-172, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29080138

RESUMO

Raman imaging microscopy is a powerful tool for label-free imaging of biological samples. It has the advantage of measuring the spatial distribution of endogenous proteins and lipids in cells, as well as obtaining chemical information on these endogenous molecules, such as hydrogen bonding and electrostatic interactions. However, because Raman intensity is very weak compared with fluorescence intensity, obtaining a reliable Raman image requires fast acquisition of a Raman image and rejection of background fluorescence. In this chapter, we describe the procedure for obtaining images of the Raman band of interest using a multipoint technique, which is the fast acquisition method for obtaining an image.


Assuntos
Aminoácidos/análise , Lipídeos/análise , Microscopia Óptica não Linear/métodos , Nucleotídeos/análise , Imagem Óptica/métodos , Proteínas/análise , Aminoácidos/metabolismo , Meios de Cultura/química , Células HeLa , Humanos , Ligações de Hidrogênio , Lipídeos/química , Microscopia Óptica não Linear/instrumentação , Nucleotídeos/metabolismo , Imagem Óptica/instrumentação , Proteínas/metabolismo , Eletricidade Estática , Fatores de Tempo , Vibração
7.
J Biochem ; 161(3): 291-296, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28003430

RESUMO

Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína bcl-X/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Sítios de Ligação , Células CHO , Cricetulus , Células HEK293 , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Proteína bcl-X/química
8.
Phys Rev E ; 94(5-1): 052601, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27967026

RESUMO

We propose a model molecule to investigate microscopic properties of a binary mixture with a closed-loop coexistence region. The molecule is comprised of a Lennard-Jones particle and a uniaxial quadrupole. Gibbs ensemble Monte Carlo simulations demonstrate that the high-density binary fluid of the molecules with the quadrupoles of the same magnitude but of the opposite signs can show closed-loop immiscibility. We find that an increase in the magnitude of the quadrupoles causes a shrinkage of the coexistence region. Molecular dynamics simulations also reveal that aggregates with two types of molecules arranged alternatively are formed in the stable one-phase region both above and below the coexistence region. String structures are dominant below the lower critical solution temperature, while branched aggregates are observed above the upper critical solution temperature. We conclude that the anisotropic interaction between the quadrupoles of the opposite signs plays a crucial role in controlling these properties of the phase behavior.

9.
Chemphyschem ; 15(17): 3832-8, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25212489

RESUMO

The effect of solute affinity on solute diffusion in binary liquids well below the lower critical solution temperature (LCST) was studied by using fluorescence correlation spectroscopy. We measured the hydrodynamic radii of a hydrophobic and an amphiphilic fluorescent dye under systematic variation of the relative molar fractions of water/2-butoxyethanol and, for comparison, of water/methanol mixtures, which do not show phase separation. We found that the apparent hydrodynamic radius of the hydrophobic dye almost doubled in water/2-butoxyethanol, whereas it remained largely unchanged for the amphiphilic dye and in water/methanol mixtures. Our results indicate that the translational diffusion of solutes is influenced by transient local solution structures, even at temperatures well below the LCST. We conclude that, even far below LCST, different solutes can experience different environments in binary liquid mixtures depending on both the solute and solvent properties, all of which impact their reactivity.

10.
Chem Commun (Camb) ; 50(69): 9862-4, 2014 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-25026098

RESUMO

Gold surfaces were modified by benzyl-mercaptan (BM) and then partly replaced with benzenethiol (BT), which formed binary self-assembled monolayers (SAM). Initially BT randomly replaced BM in the monolayer, but at long exchange times >15 nm radius domains were observed with specific relative composition of BT and BM.

11.
Chem Commun (Camb) ; 50(69): 9839-41, 2014 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-24956261

RESUMO

A chemically synthesized silver nanowire was used for atomic-resolution STM imaging and tip-enhanced Raman scattering (TERS) spectroscopy, yielding excellent reproducibility. This TERS tip will open a new venue to surface analysis, such as molecular finger printing at nanoscales.

12.
Photochem Photobiol Sci ; 13(6): 891-7, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24777199

RESUMO

The earliest stages of phase separation in a liquid triethylamine (TEA)-water mixture were observed using a picosecond IR laser pulse to produce a temperature jump and ultrafast Raman spectroscopy. Raman spectral changes in the water OH stretching region showed that the temperature rise induced by IR pulses equilibrated within a few tens of picoseconds. Amplitude changes in the TEA CH-stretching region of difference Raman spectra consisted of an initial faster and a subsequent slower process. The faster process within 100 ps is attributed to hydrogen bond weakening caused by the temperature rise. The slower process attributed to phase separation was observed for several nanoseconds, showing the number of hydrogen bond between TEA and water gradually decreased with time. The kinetics of hydrogen bond scission during phase separation indicated a linear growth of the phase-separated component, as observed previously on the nanosecond time scale, rather than the more usual exponential growth. A peak blueshift was observed in the difference Raman spectra during phase separation. This shift implies that hydrogen bond scission of TEA-water aggregates involving very few water molecules took place in the initial stage of phase separation (up to 2 ns), and then was followed by the breaking of TEA-water pairs surrounded by water molecules. This effect may be a result from spatial inhomogeneities associated with the phase separation process: aggregates or clusters existing naturally in solution even below the lower critical soluble temperature.

13.
Langmuir ; 29(19): 5889-95, 2013 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-23586696

RESUMO

Ultrafast phase separation of water and 2-butoxyethanol mixture was induced by nanosecond IR laser pulse irradiation. After a certain delay time, a UV laser pulse was introduced to induce photoreduction of aurate ions, which led to the formation of gold nanoparticles in dynamic phase-separating media. The structure and size of the nanoparticles varied depending on the delay time between the IR and UV pulses. For a delay time of 5 and 6 µs, gold square plates having edge lengths of 150 and 100 nm were selectively obtained, respectively. With a delay time of 3 µs, on the other hand, the size of the square plates varied widely from 100 nm to a few micrometers. The size of the gold square plates was also varied by varying the total irradiation time of the IR and UV pulses. The size distribution of the square plates obtained under different conditions suggests that the growth process of the square plates was affected by the size of the nanophases during phase separation. Electron diffraction patterns of the synthesized square plates showed that the square plates were highly crystalline with a Au(100) surface. These results showed that the nanophases formed during laser-induced phase separation can provide detergent-free reaction fields for size-controlled nanomaterial synthesis.

14.
Biointerphases ; 5(3): FA105-9, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21171703

RESUMO

Two-dimensional condensation was initiated in a self-assembled mixed monolayer of spiropyran and octadecanol by a nanosecond laser pulse. The dynamics of the process were monitored using nanosecond pump-probe Brewster angle microscopy. Domain growth followed a power law with a growth exponent of 0.47 at a velocity approaching 20 ms(-1). This represents a limit for the rate of longitudinal signaling of pressure waves through a self-assembled amphiphilic layer at an interface and adds to our understanding of signal transmission rates in biomimetic membranes where morphological change in one region can be signaled to a more remote region.


Assuntos
Benzopiranos/metabolismo , Materiais Biomiméticos/metabolismo , Indóis/metabolismo , Lasers , Nitrocompostos/metabolismo , Ácidos Oleicos/metabolismo , Microscopia/métodos
15.
Photochem Photobiol Sci ; 9(2): 208-12, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20126796

RESUMO

Photo induced phase separation of a mixture of water and 2-butoxyethanol, in which spiropyran was dissolved as a photoresponsive molecule, was investigated. It was found that the phase separation temperature of the merocyanine (MC) form solution was higher than that of the spiropyrane (SP) form; therefore phase separation was induced by visible light irradiation to the solution which caused the photoisomerization from the MC form to the SP form. The system also exhibits reversible photoinduced phase mixing by irradiation of UV light. The photo-chemical phase separation was also induced by the nanosecond laser pulse irradiation and the dynamics of the phase domain growth were studied.

16.
J Nanosci Nanotechnol ; 9(1): 59-68, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19441279

RESUMO

Transient events in thin films and interfaces have been studied using the technique of time resolved pump-probe nanosecond Brewster angle microscopy. For p-polarized light there is a minimum reflectivity at the Brewster angle. When the interface is viewed with light that is both incident and reflected at the Brewster angle the resulting image is dark. Subsequent small changes is refractive index will then cause an increase in the reflectivity in affected regions providing high contrast images of an altered interface with a dark background level. This is the basis of Brewster angle microscopy. In the present work two synchronized nanosecond pulsed lasers were used in the pump-probe configuration in order to induce changes at an air-liquid interface and to monitor the resulting morphology changes over a range of time delays from nanosecond to milliseconds after laser-excitation. This method can be used to observe morphological changes in phase altering thin-films and molecular monolayers. Further it can be used to obtain information about transient photochemistry even in optically thin materials and nano-films. In the current work the method is used to monitor laser induced processes in phase separating binary liquid mixtures as well as in monolayers of photo-responsive amphiphilic molecules derived from spiropyran on water. The two systems are quite different but provide valuable comparisons.

17.
Phys Chem Chem Phys ; 10(34): 5256-63, 2008 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-18728868

RESUMO

A nanosecond pulsed IR (1.9 microm) laser rapidly heated water, in an open vessel, to temperatures well below the boiling point. The subsequent dynamics of volume expansion were monitored using time-resolved interferometry in order to measure the increase in the water level in the heated area. The water expanded at less than the speed of sound, taking just less than 100 ns to increase its height by approximately 500 nm at surface temperature jumps of 20 K. The initial expansion was followed by an apparent contraction and then a re-expansion. The first expansion phase occurred more slowly than the timescale for bulk H-bond re-structuring of the water, as determined from vibrational bands in the Raman spectra, and represents the limit to the rate at which the overpressure caused by sudden heating can be released. The second phase of the expansion was caused by hydrodynamic effects and is accompanied by morphological changes resulting in light scattering as well as droplet spallation.

18.
Phys Rev E Stat Nonlin Soft Matter Phys ; 73(1 Pt 1): 011502, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16486149

RESUMO

Spinodal demixing was initiated in two systems, with critical and off-critical compositions, using nanosecond pulsed laser-induced temperature jumps (T-jumps) of various magnitude. In this way, deep quenches could be imposed on the systems. One system was the simple triethylamine (TEA)/water mixture and the other was the ionic mixture of 2-butoxyethanol (2BE)/water/KCl. The demixing process was followed using the technique of nanosecond time-resolved microscopic shadowgraphy. The growth of the evolving phase-separated domains followed a simple power law with respect to time in every case. For a given composition, the magnitude of the T-jump had little effect on the growth exponent, however the composition was found to influence the rate of domain growth. At off-critical mole fractions of 0.2 with respect to TEA, the domains grew according to the following expression: L(t)=t(0.70) (where L(t)= the domain size) whereas at the critical TEA mole fraction of 0.08 the domains grew as L(t)=t(0.52). 2BE/water/KCl mixtures quenched at the just off-critical composition of fraction with respect to 2BE evolved as L(t)=t(0.63). These results will be compared to theoretical models and simulations and discussed in terms of estimated Reynolds numbers as well as the consumption and conversion of the available surface energy that fuels the demixing process.

19.
Photochem Photobiol Sci ; 4(1): 113-8, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15616701

RESUMO

The dynamics of Re(2,2'-bipyridine)(CO)3Cl MLCT state formation and decay were determined after femtosecond UV laser excitation and picosecond pulsed X-ray excitation, in an N,N-dimethylformamide (DMF) solution as well as in its solid form. At room temperature, after UV excitation, this MLCT excited state emits both in DMF solution and in the solid form. Transient absorption spectra were measured in solution at various delay times following excitation by a 160 fs, 390 nm laser pulse. There was a prompt absorption increase at around 460 nm occurring within the pump probe convolution (<1 ps), which was assigned to the formation of the 3MLCT state. This transient absorbance was constant over 100 ps. In contrast to the solution state, in the solid state, the emission maximum slightly red-shifts with increasing time after laser excitation. In both solid and solution the emission rises within the system response time. The solid sample exhibited a 1.4 ns emission decay that was not observed for the solution sample. The emission rise from a solid sample after 20 ps pulsed X-ray excitation was significantly slower than the system's time resolution. It is proposed that kinetically energetic electrons are ejected following X-ray induced ionisation, creating ionised tracks in which energetic cations and electrons take time to recombine yielding delayed 3MLCT states that emit.


Assuntos
Compostos Organometálicos/química , Compostos Organometálicos/efeitos da radiação , Rênio/química , Monóxido de Carbono/química , Cloretos/química , Estrutura Molecular , Compostos Organometálicos/síntese química , Fotoquímica , Fatores de Tempo , Raios Ultravioleta , Raios X
20.
Phys Rev E Stat Nonlin Soft Matter Phys ; 68(2 Pt 1): 020501, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-14524941

RESUMO

We initiated morphological and molecular level changes in the spinodal decomposition (SD) of H(2)O/2-butoxyethanol/KCl with a pulsed ir laser. Transient Raman spectra gave us a molecular level view of the early stage of this process that could be linked to later morphological events. Chemical changes during SD, such as reorganization of H bonds and forced hydrophobic interactions, ended after 1 micros; however, phase domains continued to grow with self-similarity after 30 micros. The growth of the phase domains satisfied the power law L(t) approximately t(0.55) and was consistent with the late stage of SD. The time scale for the onset of late stage SD is many orders of magnitude faster than previously reported in ionic and nonionic conditions.

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