Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

University of Perugia

The alpha relaxation process of the fragile glass former salol is investigated in the T-P domain by means of photon correlation spectroscopy. We find that a time-pressure superposition principle is obeyed for the relaxation function in addition to the time-temperature superposition. The behavior of the relaxation time is studied by using an extended version of the Adam-Gibbs model including the pressure dependence. The excellent conformity of the equation describing the bidimensional surface tau(T,P) to the experimental data provides a positive check for this model, here verified on photon correlation measurements. The same model gives a rationale of the phenomenological expressions recently introduced to describe the changes in the slow dynamics induced by varying both temperature and pressure. These findings suggest that the reduction of configurational entropy actually guides the liquid toward the glass transition.

Temperature and pressure dependence of the alpha relaxation and configurational entropy of a prototype glass former.

Glass formation is usually viewed in terms of physical vitrification: a liquid in a metastable state is cooled or compressed so as to avoid crystallization. However, glasses may also be formed by chemical vitrification, a process involving progressive polymerization of the constituent molecules via the formation of irreversible chemical bonds. The formation of most of the materials used in engineering plastics and the hardening of natural and synthetic resins are based on chemical vitrification. Despite the differences in the molecular processes involved in chemical and physical vitrification, surprising similarities are observed in the slowing down of the dynamics and in the thermodynamical properties of the resulting glasses. Explaining such similarities would improve general understanding of the glass transition and may disclose its universal nature. Here we report dielectric and photon-correlation measurements that reveal the origin of the similarity in the dynamical behaviour of physical and chemical glass formers. We find that the evolution of their configurational restrictions proceeds in a similar manner. In particular, we make a connection between the reduction in configurational entropy and the number of chemical bonds, a quantity that can be controlled in experiments.

Bond-controlled configurational entropy reduction in chemical vitrification.

The Cauchy-like relation M(infinity) = A + BG(infinity) has recently been found to hold for the high frequency limit values of the longitudinal modulus M(infinity) and transverse modulus G(infinity) of viscoelastic liquids, with B approximately 3 in all the investigated systems. The Brillouin scattering results here reported for curing epoxy systems and thermal glass formers give evidence for the validity of a Cauchy-like relation M(') = A + BG(') for the real part of the elastic moduli measured at finite frequencies. Our results suggest as well the validity of a pure Cauchy relation DeltaM = 3 DeltaG for the relaxation strengths of longitudinal and shear moduli in relaxing liquids.

Cauchy relation in relaxing liquids.

&#x57FA;&#x4E8E;&#x540C;&#x6B65;&#x8F90;&#x5C04; x &#x5C04;&#x7EBF;&#x8367;&#x5149; (S-XRF&#xFF09; &#x662F;&#x6210;&#x50CF;&#x5143;&#x7D20;&#x5206;&#x5E03;&#x7684;&#x8BB8;&#x591A;&#x751F;&#x7269;&#x76F8;&#x5173;&#x4EE5;&#x53CA;&#x6545;&#x610F;&#x5F15;&#x5165;&#x7EC4;&#x7EC7;&#x548C;&#x7EC6;&#x80DE;&#xFF0C;&#x4F8B;&#x5982;&#xFF0C;&#x901A;&#x8FC7;&#x529F;&#x80FD;&#x6027;&#x7EB3;&#x7C73;&#x7684;&quot;&#x4EBA;&#x5DE5;&quot;&#x5143;&#x7D20;&#x8D77;&#x5F3A;&#x5927;&#x7684;&#x6280;&#x672F;&#x3002;&#x5728;&#x6B64;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x63A2;&#x8BA8;&#x4E86;&#x7EC6;&#x80DE;&#x901A;&#x8FC7;&#x529F;&#x80FD;&#x6027; CdSe/ZnS &#x91CF;&#x5B50;&#x70B9; &#xFF08;&#x91CF;&#x5B50;&#xFF09; &#x4F7F;&#x7528;&#x7684;&#x5316;&#x5B66; nanoimaging 100 nm &#x7A7A;&#x95F4;&#x5206;&#x8FA8;&#x7387; nanoXRF&#xFF09; &#x7684; S XRF &#x6F5C;&#x529B;&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x5546;&#x4E1A;&#x4E0A;&#x53EF;&#x7528; QD &#x4E2D;&#x5B66;&#x6297;&#x4F53;&#x5171;&#x8F6D;&#x5230;&#x6807;&#x7B7E;&#x7684;&#x764C;&#x75C7;&#x6807;&#x8BB0; HER2 (&#x4EBA;&#x7C7B;&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x53D7;&#x4F53; 2) SKOV3 &#x7EC6;&#x80DE;&#x548C; &beta;-&#x5FAE;&#x7BA1;&#x86CB;&#x767D;&#x4E0E;&#x7EC6;&#x80DE;&#x9AA8;&#x67B6;&#x5FAE;&#x7BA1;&#x76F8;&#x5173;&#x86CB;&#x767D;&#x7684;&#x8868;&#x9762;&#x4E0A;&#x3002;&#x6211;&#x4EEC;&#x6210;&#x7ACB;&#x4E86;&#x73AF;&#x6C27;&#x5217;&#x5165;&#x4E0E;&#x7EC6;&#x80DE;&#x5185;&#x6807;&#x8BB0;&#x7684;&#x6837;&#x672C;&#xFF0C;&#x4EE5;&#x53CA;&#x6837;&#x54C1;&#x65E0;&#x73AF;&#x6C27;&#x5217;&#x5165;&#xFF0C;&#x8868;&#x9762;&#x8D34;&#x6807;&#x3002;&#x73AF;&#x6C27;&#x5217;&#x5165;&#xFF0C;&#x4E5F;&#x7528;&#x4E8E;&#x7535;&#x5B50;&#x663E;&#x5FAE;&#x955C;&#xFF0C;&#x6709;&#x7EF4;&#x6301;&#x7EC6;&#x80DE;&#x5F62;&#x6001;&#x548C;&#x4FDD;&#x8BC1;&#x957F;&#x671F;&#x7A33;&#x5B9A;&#x7684;&#x4F18;&#x52BF;&#x3002;&#x91CF;&#x5B50;&#x88AB;&#x8BC1;&#x660E;&#x662F;&#x9002;&#x5408;&#x63A2;&#x5934; nanoXRF Se &#x6392;&#x653E;&#x4E50;&#x961F;&#xFF0C;&#x4E0D;&#x662F;&#x4EFB;&#x4F55;&#x5176;&#x4ED6;&#x6CE2;&#x6BB5;&#x3001; &#x6392;&#x653E;&#x63A5;&#x8FD1;&#xFF0C;&#x548C;&#x4FE1;&#x53F7;&#x7684;&#x7279;&#x5F02;&#x6027;&#xFF0C;&#x800C;&#x4FDD;&#x5B58;&#x5728;&#x8FD9;&#x4E24;&#x79CD;&#x7C7B;&#x578B;&#x7684;&#x6807;&#x7B7E;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x4F7F;&#x7528; QD &#x57FA;&#x4E8E;&#x6807;&#x8BB0;&#x7684; nanoXRF &#x53EF;&#x4EE5;&#x975E;&#x5E38;&#x6709;&#x6548;&#x5730;&#x5728; colocalizing &#x80DE;&#x5185;&#x4E0E;&#x81EA;&#x7136;&#x5B58;&#x5728;&#x7684;&#x5355;&#x5143;&#x683C;&#x4E2D;&#x7684;&#x5185;&#x5BB9;&#x7684;&#x5177;&#x4F53;&#x76EE;&#x6807;&#x548C;&#x53EF;&#x8865;&#x5145;&#x4EE5;&#x534F;&#x540C;&#x7684;&#x65B9;&#x5F0F;&#x5171;&#x7126;&#x8367;&#x5149;&#x663E;&#x5FAE;&#x955C;&#x3002;

&#x57FA;&#x4E8E;&#x540C;&#x6B65;&#x8F90;&#x5C04; x &#x5C04;&#x7EBF;&#x8367;&#x5149;&#x6210;&#x50CF; CdSe &#x91CF;&#x5B50;&#x70B9;&#x6807;&#x8BB0;&#x7684;&#x4EBA;&#x7C7B;&#x7EC6;&#x80DE;&#x3002;

Synchrotron-based X-ray fluorescence (S-XRF) is a powerful technique for imaging the distribution of many biologically relevant elements as well as of "artificial" elements deliberately introduced into tissues and cells, for example, through functionalized nanoparticles. In this study, we explored the potential of S-XRF for chemical nanoimaging (100 nm spatial resolution, nanoXRF) of human cells through the use of functionalized CdSe/ZnS quantum dots (QDs). We used a commercially available QD-secondary antibody conjugate to label the cancer marker HER2 (human epidermal growth factor receptor 2) on the surface of SKOV3 cancer cells and beta-tubulin, a protein associated with cytoskeleton microtubules. We set up samples with epoxy inclusion and intracellular labeling as well as samples without epoxy inclusion and with surface labeling. Epoxy inclusion, also used in electron microscopy, has the advantage of preserving cell morphology and guaranteeing long-term stability. QDs proved to be suitable probes for nanoXRF due to the Se emission band, which is not in close proximity to any other emission band, and the signal specificity, which is preserved in both types of labeling. Therefore, nanoXRF using QD-based markers can be very effective at colocalizing specific intracellular targets with elements naturally present in the cell and may complement confocal fluorescence microscopy in a synergistic fashion.

Synchrotron-based X-ray fluorescence imaging of human cells labeled with CdSe quantum dots.

Synchrotron bas&#xE9; sur X-ray fluorescence (S-XRF) est une technique puissante pour l&#x26;#39;imagerie de la distribution de nombreux &#xE9;l&#xE9;ments biologiquement pertinentes ainsi que des &#xAB;artificielle&#xBB; des &#xE9;l&#xE9;ments d&#xE9;lib&#xE9;r&#xE9;ment introduites dans les tissus et les cellules, par exemple, par le biais des nanoparticules fonctionnalis&#xE9;es. Dans cette &#xE9;tude, nous avons explor&#xE9; le potentiel de S-XRF pour les produits chimiques NanoImaging (100 nm de r&#xE9;solution spatiale, nanoXRF) des cellules humaines par l&#x26;#39;utilisation de CdSe fonctionnalis&#xE9; / ZnS quantum dots (QD). On a utilis&#xE9; un conjugu&#xE9; d&#x26;#39;anticorps disponible dans le commerce QD-secondaire &#xE0; &#xE9;tiqueter le marqueur du cancer HER2 (r&#xE9;cepteur humain de facteur de croissance &#xE9;pidermique 2) sur la surface de cellules canc&#xE9;reuses et SKOV3 b&#xEA;ta-tubuline, une prot&#xE9;ine associ&#xE9;e aux microtubules cytosquelette. Nous mettons en place des &#xE9;chantillons avec l&#x26;#39;inclusion &#xE9;poxy et marquage intracellulaire ainsi que des &#xE9;chantillons sans l&#x26;#39;inclusion &#xE9;poxy et &#xE0; l&#x26;#39;&#xE9;tiquetage de surface. L&#x26;#39;inclusion &#xE9;poxy, &#xE9;galement utilis&#xE9; en microscopie &#xE9;lectronique, a l&#x26;#39;avantage de pr&#xE9;server la morphologie des cellules et de garantir la stabilit&#xE9; &#xE0; long terme. BQs r&#xE9;v&#xE9;l&#xE9;e sondes appropri&#xE9;es pour nanoXRF dues &#xE0; la bande d&#x26;#39;&#xE9;mission de Se, qui n&#x26;#39;est pas &#xE0; proximit&#xE9; imm&#xE9;diate de toute autre bande d&#x26;#39;&#xE9;mission, et la sp&#xE9;cificit&#xE9; de signal, qui est conserv&#xE9; dans les deux types de marquage. Par cons&#xE9;quent, en utilisant nanoXRF QD-marqueurs &#xE0; base peuvent &#xEA;tre tr&#xE8;s efficaces &#xE0; colocalisent sp&#xE9;cifiques des cibles intracellulaires avec des &#xE9;l&#xE9;ments naturellement pr&#xE9;sents dans la cellule et peut compl&#xE9;ter la microscopie confocale de fluorescence d&#x26;#39;une mani&#xE8;re synergique.

Synchrotron bas&#xE9; sur X-ray d&#x26;#39;imagerie par fluorescence de cellules humaines marqu&#xE9;es avec des points quantiques de CdSe

Synchrotron-basierte R&ouml;ntgenfluoreszenz (S-XRF) ist eine leistungsf&auml;hige Technik f&uuml;r die Verteilung der vielen biologisch relevanten Elemente sowie ab &quot;k&uuml;nstliche&quot; Elemente absichtlich eingef&uuml;hrt in Gewebe und Zellen, z. B. durch funktionalisierten Nanopartikeln imaging. In dieser Studie erforschten wir das Potenzial von S-XRF f&uuml;r chemische Nanoimaging (100 nm r&auml;umlicher Aufl&ouml;sung, NanoXRF) menschlicher Zellen durch den Einsatz von funktionalisierten CdSe/ZnS-Quantenpunkte (QDs). Wir verwendeten eine handels&uuml;bliche QD-sekund&auml;re Antik&ouml;rper konjugiert Label den Krebs-Marker HER2 (humanen epidermalen Wachstumsfaktor-Rezeptor 2) auf der Oberfl&auml;che der Krebszellen SKOV3 und Beta-Tubulin, ein Protein mit Mikrotubuli des Zytoskeletts verbunden. Wir Proben mit Epoxy-Aufnahme und intrazellul&auml;re Beschriftung sowie Proben ohne Epoxy-Aufnahme und mit Oberfl&auml;che beschriften eingerichtet. Epoxy-Aufnahme, auch verwendet in der Elektronenmikroskopie, hat den Vorteil, Erhaltung der Zellmorphologie und langfristigen Stabilit&auml;t zu gew&auml;hrleisten. QDs erwies sich geeignete Sonden f&uuml;r NanoXRF durch die Se-Emission-Band, die nicht in unmittelbarer N&auml;he eine andere Emission-Band ist, und die Signal-Besonderheit, die bei beiden Typen der Bezeichnung beibehalten wird. NanoXRF mit QD-basierten Markern kann deshalb sehr effektiv bei colocalizing intrazellul&auml;re Einzelziele mit Elementen in der Zelle nat&uuml;rlich vorkommt und konfokale Fluoreszenzmikroskopie in synergistischer Weise erg&auml;nzen kann.

Synchrotron-basierte R&ouml;ntgen Fluoreszenz Bildgebung menschlicher Zellen mit CdSe Quantenpunkte bezeichnet.

Sincrotrone-basato fluorescenza x (XRF-S) &egrave; una tecnica potente per la distribuzione di molti elementi biologicamente rilevanti anche a partire da elementi &quot;artificiali&quot; deliberatamente introdotti in tessuti e cellule, per esempio, attraverso le nanoparticelle funzionalizzate di imaging. In questo studio, abbiamo esplorato il potenziale di S-XRF per chimica nanoimaging (risoluzione spaziale di 100 nm, nanoXRF) di cellule umane attraverso l&#39;utilizzo di punti quantici di CdSe/ZnS funzionalizzate (QDs). Abbiamo usato un commercialmente disponibile secondaria QD-anticorpo coniugato all&#39;etichetta il marcatore tumorale HER2 (human epidermal growth factor receptor 2) sulla superficie delle cellule tumorali SKOV3 e beta-tubulina, una proteina associata microtubuli del citoscheletro. Abbiamo istituito campioni con resina epossidica inclusione ed etichettatura intracellulare, nonch&eacute; campioni senza l&#39;inclusione di resina epossidica e con superficie di etichettatura. L&#39;inclusione di resina epossidica, anche usato in microscopia elettronica, ha il vantaggio di preservare la morfologia cellulare e garantendo stabilit&agrave; a lungo termine. QDs si dimostr&ograve; adatte sonde per nanoXRF a causa della banda di emissione Se, che non &egrave; in prossimit&agrave; di qualsiasi altra banda di emissione e la specificit&agrave; di segnale, che &egrave; conservata in entrambi i tipi di etichettatura. Pertanto, utilizzando gli indicatori basati su QD nanoXRF pu&ograve; essere molto efficace a colocalizing target intracellulari specifici con elementi naturalmente presenti nella cella e pu&ograve; integrare la microscopia di fluorescenza confocale in maniera sinergica.

Sincrotrone-basato fluorescenza imaging a raggi x di cellule umane marcato con CdSe quantum dots.

&#x30B7;&#x30F3;&#x30AF;&#x30ED;&#x30C8;&#x30ED;&#x30F3;&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x86CD;&#x5149;X&#x7DDA;&#xFF08;S-XRF&#xFF09;&#x306F;&#x3001;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x591A;&#x304F;&#x306E;&#x751F;&#x7269;&#x5B66;&#x7684;&#x306B;&#x95A2;&#x9023;&#x3057;&#x305F;&#x5143;&#x7D20;&#x306E;&#x5206;&#x5E03;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x3001;&#x610F;&#x56F3;&#x7684;&#x306B;&#x5B98;&#x80FD;&#x5316;&#x3055;&#x308C;&#x305F;&#x30CA;&#x30CE;&#x7C92;&#x5B50;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x3001;&#x4F8B;&#x3048;&#x3070;&#x3001;&#x7D44;&#x7E54;&#x3084;&#x7D30;&#x80DE;&#x306B;&#x5C0E;&#x5165;&#x3055;&#x308C;&#x305F; &#x26;quot;&#x4EBA;&#x5DE5;&#x7684;&#x26;quot;&#x8981;&#x7D20;&#x306E;&#x3088;&#x3046;&#x306B;&#x5F37;&#x529B;&#x306A;&#x624B;&#x6CD5;&#x3067;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x5B98;&#x80FD;&#x5316;&#x3055;&#x308C;&#x305F;CdSe / ZnS&#x91CF;&#x5B50;&#x30C9;&#x30C3;&#x30C8;&#xFF08;QD&#xFF09;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x30D2;&#x30C8;&#x7D30;&#x80DE;&#x306E;&#x5316;&#x5B66;nanoimaging&#xFF08;100 nm&#x306E;&#x7A7A;&#x9593;&#x5206;&#x89E3;&#x80FD;&#x3001;nanoXRF&#xFF09;&#x306E;S-XRF&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x691C;&#x8A0E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;SKOV3&#x764C;&#x7D30;&#x80DE;&#x3068;&#x3B2;-&#x30C1;&#x30E5;&#x30FC;&#x30D6;&#x30EA;&#x30F3;&#x306E;&#x8868;&#x9762;&#x306B;&#x764C;&#x30DE;&#x30FC;&#x30AB;&#x30FC;HER2&#xFF08;&#x30D2;&#x30C8;&#x4E0A;&#x76AE;&#x6210;&#x9577;&#x56E0;&#x5B50;&#x53D7;&#x5BB9;&#x4F53;2&#xFF09;&#x3001;&#x7D30;&#x80DE;&#x9AA8;&#x683C;&#x306E;&#x5FAE;&#x5C0F;&#x7BA1;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3092;&#x6A19;&#x8B58;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5E02;&#x8CA9;&#x306E;QD-&#x4E8C;&#x6B21;&#x6297;&#x4F53;&#x30B3;&#x30F3;&#x30B8;&#x30E5;&#x30B2;&#x30FC;&#x30C8;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3044;&#x307E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;&#x30A8;&#x30DD;&#x30AD;&#x30B7;&#x6A39;&#x8102;&#x5C01;&#x5165;&#x3057;&#x3001;&#x7D30;&#x80DE;&#x5185;&#x30E9;&#x30D9;&#x30EA;&#x30F3;&#x30B0;&#x3068;&#x540C;&#x69D8;&#x306B;&#x30A8;&#x30DD;&#x30AD;&#x30B7;&#x6A39;&#x8102;&#x5C01;&#x5165;&#x305B;&#x305A;&#x306B;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x3067;&#x3001;&#x8868;&#x9762;&#x306E;&#x30E9;&#x30D9;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x3092;&#x8A2D;&#x5B9A;&#x3057;&#x307E;&#x3059;&#x3002;&#x307E;&#x305F;&#x3001;&#x96FB;&#x5B50;&#x9855;&#x5FAE;&#x93E1;&#x3067;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x308B;&#x30A8;&#x30DD;&#x30AD;&#x30B7;&#x6A39;&#x8102;&#x5C01;&#x5165;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x306E;&#x5F62;&#x614B;&#x3092;&#x7DAD;&#x6301;&#x3057;&#x3001;&#x9577;&#x671F;&#x7684;&#x306A;&#x5B89;&#x5B9A;&#x6027;&#x3092;&#x4FDD;&#x8A3C;&#x3059;&#x308B;&#x3068;&#x3044;&#x3046;&#x5229;&#x70B9;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x91CF;&#x5B50;&#x30C9;&#x30C3;&#x30C8;&#x306F;&#x3001;&#x4ED6;&#x306E;&#x767A;&#x5149;&#x5E2F;&#x306B;&#x8FD1;&#x63A5;&#x3057;&#x3066;&#x3044;&#x306A;&#x3044;Se&#x306E;&#x767A;&#x5149;&#x5E2F;&#x3001;&#x304A;&#x3088;&#x3073;&#x30E9;&#x30D9;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x4E21;&#x65B9;&#x306E;&#x30BF;&#x30A4;&#x30D7;&#x3067;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x4FE1;&#x53F7;&#x306E;&#x7279;&#x7570;&#x6027;&#x306B;&#x8D77;&#x56E0;&#x3059;&#x308B;nanoXRF&#x306B;&#x9069;&#x3057;&#x305F;&#x30D7;&#x30ED;&#x30FC;&#x30D6;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x5224;&#x660E;&#x3057;&#x305F;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;QD&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;nanoXRF&#x306F;&#x3001;&#x7D30;&#x80DE;&#x5185;&#x306E;&#x5929;&#x7136;&#x306B;&#x5B58;&#x5728;&#x3059;&#x308B;&#x8981;&#x7D20;&#x3092;&#x6301;&#x3064;&#x7279;&#x5B9A;&#x306E;&#x7D30;&#x80DE;&#x5185;&#x6A19;&#x7684;&#x3092;&#x5171;&#x5C40;&#x5728;&#x3067;&#x975E;&#x5E38;&#x306B;&#x6709;&#x52B9;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3068;&#x76F8;&#x4E57;&#x52B9;&#x679C;&#x306E;&#x65B9;&#x6CD5;&#x3067;&#x5171;&#x7126;&#x70B9;&#x86CD;&#x5149;&#x9855;&#x5FAE;&#x93E1;&#x3092;&#x88DC;&#x5B8C;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;

CdSe&#x91CF;&#x5B50;&#x30C9;&#x30C3;&#x30C8;&#x3067;&#x6A19;&#x8B58;&#x3057;&#x305F;&#x30D2;&#x30C8;&#x7D30;&#x80DE;&#x306E;&#x30B7;&#x30F3;&#x30AF;&#x30ED;&#x30C8;&#x30ED;&#x30F3;&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x86CD;&#x5149;X&#x7DDA;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;

X-&#xC120; &#xD615;&#xAD11; synchrotron &#xAE30;&#xBC18; (S-x &#xC120; &#xD615;&#xAD11;) &#xC774;&#xBBF8;&#xC9D5; &#xBFD0;&#xB9CC; &#xAE30;&#xB2A5;&#xC131;&#xB41C; &#xB098;&#xB178; &#xC785;&#xC790;&#xB97C; &#xD1B5;&#xD574; &#xC870;&#xC9C1; &#xBC0F; &#xC138;&#xD3EC;, &#xC608;&#xB97C; &#xB4E4;&#xC5B4;, &#xC2E0;&#xC911; &#xD558; &#xAC8C; &#xB3C4;&#xC785; &quot;&#xC778;&#xACF5;&quot; &#xC694;&#xC18C; &#xC911; &#xD558;&#xB098;&#xB85C; &#xC11C; &#xB9CE;&#xC740; &#xC0DD;&#xBB3C;&#xD559; &#xAD00;&#xB828; &#xC694;&#xC18C; &#xBD84;&#xD3EC;&#xC5D0; &#xB300; &#xD55C; &#xAC15;&#xB825;&#xD55C; &#xAE30;&#xC220; &#xC774;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; &#xACF5;&#xC5C5;&#xD654; CdSe/ZnS &#xC591;&#xC790; &#xC810; (QDs)&#xC758; &#xC0AC;&#xC6A9;&#xC744; &#xD1B5;&#xD574; &#xC778;&#xAC04; &#xC138;&#xD3EC;&#xC758; &#xD654;&#xD559; nanoimaging (100 nm &#xACF5;&#xAC04; &#xD574;&#xC0C1;&#xB3C4;, nanoXRF)&#xC5D0; &#xB300; &#xD55C; S XRF&#xC758; &#xC7A0;&#xC7AC;&#xB825;&#xC744; &#xD0D0;&#xD5D8;. SKOV3 &#xC554; &#xC138;&#xD3EC;&#xC758; cytoskeleton microtubules &#xC5F0;&#xAD00; &#xB2E8;&#xBC31;&#xC9C8; &#xBCA0;&#xD0C0; tubulin &#xD45C;&#xBA74;&#xC5D0;&#xB294; &#xC0C1;&#xC6A9; qd&#xB294; 2 &#xCC28; &#xD56D; &#xCCB4; &#xACF5;&#xC561; &#xB808;&#xC774;&#xBE14;&#xC5D0; HER2 &#xC554; &#xD45C;&#xC2DD; (&#xC778;&#xAC04; &#xD45C; &#xD53C; &#xC131;&#xC7A5; &#xC778;&#xC790; &#xC218;&#xC6A9; &#xCCB4; 2)&#xB97C; &#xC0AC;&#xC6A9; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xC5D0;&#xD3ED;&#xC2DC;&#xB97C; &#xD3EC;&#xD568; &#xD558;&#xC9C0; &#xC54A;&#xACE0; &#xACE0; &#xD45C;&#xBA74; &#xB77C;&#xBCA8; &#xC0D8;&#xD50C;&#xC73C;&#xB85C; &#xC11C; &#xC5D0;&#xD3ED;&#xC2DC; &#xD3EC;&#xD568; &#xBC0F; &#xC138;&#xD3EC;&#xB0B4; &#xB77C;&#xBCA8; &#xC0D8;&#xD50C;&#xC744; &#xC124;&#xC815;&#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C; &#xC804;&#xC790; &#xD604;&#xBBF8;&#xACBD;&#xC5D0; &#xC0AC;&#xC6A9; &#xB418;&#xB294; &#xC5D0;&#xD3ED;&#xC2DC; &#xD3EC;&#xD568; &#xC140; &#xD615;&#xD0DC;&#xB97C; &#xBCF4;&#xC874; &#xD558; &#xACE0; &#xC7A5;&#xAE30;&#xAC04;&#xC5D0; &#xAC78;&#xCE5C; &#xC548;&#xC815;&#xC131;&#xC744; &#xBCF4;&#xC7A5;&#xC758; &#xC7A5;&#xC810;&#xC774; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. QDs nanoXRF &#xB2E4;&#xB978; &#xBC29;&#xCD9C; &#xBC34;&#xB4DC;&#xC5D0; &#xAC00;&#xAE4C;&#xC6B4; &#xADFC;&#xC811;&#xC5D0; &#xC788;&#xC9C0;, Se &#xBC29;&#xCD9C; &#xBC34;&#xB4DC;&#xC640; &#xB450; &#xAC00;&#xC9C0; &#xC720;&#xD615;&#xC758; &#xB77C;&#xBCA8;&#xC5D0; &#xBCF4;&#xC874; &#xC2E0;&#xD638; &#xD2B9;&#xC774;&#xC131;&#xC5D0; &#xB300; &#xD55C; &#xC801;&#xD569; &#xD55C; &#xD504;&#xB85C;&#xBE0C; &#xC785;&#xC99D; &#xD588;&#xB2E4;. &#xB530;&#xB77C;&#xC11C; nanoXRF qd&#xB294;-&#xAE30;&#xBC18; &#xB9C8;&#xCEE4;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC140;&#xC5D0; &#xC790;&#xC5F0;&#xC801;&#xC73C;&#xB85C; &#xC874;&#xC7AC; &#xD558;&#xB294; &#xC131;&#xBD84;&#xC73C;&#xB85C; &#xC138;&#xD3EC;&#xB0B4; &#xD2B9;&#xC815; &#xB300;&#xC0C1; colocalizing&#xC5D0; &#xB9E4;&#xC6B0; &#xD6A8;&#xACFC;&#xC801;&#xC77C; &#xC218; &#xC788;&#xB2E4; &#xACE0; &#xC2DC;&#xB108;&#xC9C0; &#xBC29;&#xC2DD;&#xC5D0;&#xC11C; confocal &#xD615;&#xAD11; &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95;&#xC744; &#xBCF4;&#xC644; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

Synchrotron &#xAE30;&#xBC18;&#xC758; x-&#xC120; &#xD615;&#xAD11; &#xC774;&#xBBF8;&#xC9D5; CdSe &#xC591;&#xC790; &#xC810; &#xB4E4;&#xC73C;&#xB85C; &#xD45C;&#xC2DC; &#xB41C; &#xC778;&#xAC04; &#xC138;&#xD3EC;&#xC758;.

Sincrotr&#xF3;n basado en fluorescencia de rayos X (S-FRX) es una potente t&#xE9;cnica para obtener im&#xE1;genes de la distribuci&#xF3;n de muchos elementos biol&#xF3;gicamente relevantes, as&#xED; como de &#x26;quot;artificial&#x26;quot; elementos deliberadamente introducidos en las c&#xE9;lulas y tejidos, por ejemplo, a trav&#xE9;s de nanopart&#xED;culas funcionalizadas. En este estudio, hemos explorado el potencial de la S-fluorescencia de rayos X para la industria qu&#xED;mica nanoimaging (100 nm de resoluci&#xF3;n espacial, nanoXRF) de las c&#xE9;lulas humanas a trav&#xE9;s del uso de CdSe funcionalizado / ZnS puntos cu&#xE1;nticos (puntos cu&#xE1;nticos). Se utiliz&#xF3; un comercialmente disponible QD secundario conjugado anticuerpo para etiquetar el marcador del c&#xE1;ncer HER2 (humano receptor de factor de crecimiento epid&#xE9;rmico 2) sobre la superficie de las c&#xE9;lulas cancerosas y SKOV3 tubulina beta, una prote&#xED;na asociada con los microt&#xFA;bulos del citoesqueleto. Hemos creado las muestras con la inclusi&#xF3;n de epoxy y etiquetado intracelular, as&#xED; como muestras sin la inclusi&#xF3;n de epoxy y con el etiquetado de la superficie. Inclusi&#xF3;n epoxi, tambi&#xE9;n utilizado en microscop&#xED;a electr&#xF3;nica, tiene la ventaja de preservar la morfolog&#xED;a celular y garantizando estabilidad a largo plazo. Cu&#xE1;nticos demostrado ser sondas adecuadas para nanoXRF debido a la banda de emisi&#xF3;n de Se, que no est&#xE1; en estrecha proximidad a cualquier otra banda de emisi&#xF3;n, y la especificidad de la se&#xF1;al, que se conserva en ambos tipos de etiquetado. Por lo tanto, nanoXRF con QD basados &#x200B;&#x200B;en marcadores pueden ser muy eficaces en el colocalizing objetivos espec&#xED;ficos intracelulares con elementos naturales presentes en la c&#xE9;lula y puede complementar la microscopia de fluorescencia confocal de un modo sin&#xE9;rgico.

Sincrotr&#xF3;n basado en fluorescencia de rayos X de imagen de las c&#xE9;lulas humanas Etiquetada con CdSe Puntos Cu&#xE1;nticos

A broadband depolarized light scattering (DLS) study is performed on diluted lysozyme aqueous solutions as a function of temperature and concentration. The dynamical susceptibility, obtained in a wide spectral range (0.6-36000 GHz) through the coupled use of interferometric and dispersive devices, is interpreted and compared with neutron scattering and Raman-induced optical Kerr-effect literature data, thus giving a general picture of relaxation phenomena. We show that the proposed approach represents a suitable tool for investigating the hydration dynamics of protein-water solutions. A detailed analysis of the quasi-elastic scattering region evidences the existence of two distinct relaxational processes at picosecond time scales. The fast process (fractions of picosecond) is attributed to bulk water dynamics, while the slow one (few picoseconds) is attributed to dynamical rearrangements of water molecules strongly influenced by the protein (hydration water). The retardation effect here estimated of about 6-7 can be regarded as a direct measure of the increased protein-water and water-water hydrogen bond stability of the water molecules within the protein hydration shell. Interestingly, a similar effect was previously observed on small hydrophilic sugar molecules. Moreover, backbone and side chains torsional motions of the protein in the 600-5300 GHz frequency range are found to be insensitive to thermal variations and to eventual changes occurring in the premelting zone.

Broadband depolarized light scattering study of diluted protein aqueous solutions.

We have studied the influence of the amphiphilic model peptide N-acetyl-leucine-methylamide (NALMA) on the dynamics of water using extended frequency range depolarized light scattering (EDLS), between 0.3 GHz and 36 THz. This technique allowed us to separate solute from solvent dynamics and bulk from hydration water, providing both characteristic times and relative fractions. In the temperature range 5-65 °C, a retardation factor from 9 to 7 is found for water hydrating NALMA. Moreover, in the same range, a hydration number from 62 to 50 is observed, corresponding to more than two hydration layers. This strong perturbation suggests the existence of a collective effect of amphiphilic molecules on surrounding water molecules.

Extended frequency range depolarized light scattering study of N-acetyl-leucine-methylamide-water solutions.

Extended depolarized light scattering (EDLS) measurements have been recently employed to investigate the dynamics of water solvating biological molecules, giving evidence of the presence of two different dynamical regimes among water molecules. An interpretation of EDLS has been proposed that provides an independent estimate of the retardation factor of slowdown with respect to fast water molecules and of the number of solvent molecules affected by this slowing down. Nevertheless this measure is an inherently complex one, due to the collective nature of the physical property probed. In the present work a molecular dynamics (MD) approach has been used to more deeply understand experimental results. Time correlation functions of the collective polarizability anisotropy have been calculated for the prototype disaccharide trehalose in aqueous solutions as a function of concentration. The unique capability of MD to disentangle the contributions to the dynamics arising from solute, solvent, and cross terms between the two allowed us to check the reliability of an interpretation that assumes a spectral separation of water and sugar dynamics, as well as to highlight the very presence of two distinct relaxation processes in water. The two processes have been attributed to the dynamics of bulk and hydration water, respectively. A retardation factor of ~5 and concentration dependent hydration numbers have been observed, in good agreement with experimental results [Paolantoni, M.; et al. J. Phys. Chem. B 2009, 113, 7874-7878].

Dynamics of biological water: insights from molecular modeling of light scattering in aqueous trehalose solutions.

The scaling behavior of the so-called boson peak in glass-formers and its relation to the elastic properties of the system remains a source of controversy. Here the boson peak in a binary reactive mixture is measured by Raman scattering (i) on cooling the unreacted mixture well below its glass-transition temperature and (ii) after quenching to very low temperature the mixture at different times during isothermal polymerization. We find that the scaling behavior of the boson peak with the properties of the elastic medium - as measured by the Debye frequency - holds for states in which the elastic moduli follow a generalized Cauchy-like relationship, and breaks down in coincidence with the departure from this relation. A possible explanation is given in terms of the development of long-range stresses in glasses. The present study provides new insight into the boson peak behavior and is able to reconcile the apparently conflicting results presented in literature.

Stress-induced modification of the boson peak scaling behavior.

The low frequency depolarized Raman spectra of 100 mg/ml aqueous solutions of hen egg white lysozyme (HEWL) have been collected in the 25-85 °C range. Short and long exposures to high temperatures have been used to modulate the competition between the thermally induced reversible and irreversible denaturation processes. A peculiar temperature evolution of spectra is evidenced under prolonged exposure of the protein solution at temperatures higher than 65 °C. This result is connected to the self-assembling of polypeptide chains and testifies the sensitivity of the technique to the properties of both protein molecule and its surrounding. Solvent free spectra have been obtained after subtraction of elastic and solvent components and assigned to a genuine vibrational contribution of hydrated HEWL. A straight similarity is observed between the solvent-free THz Raman feature and the vibrational density of states as obtained by molecular dynamics simulations; according to this, we verify the relation between this spectroscopic observable and the effective protein volume, and distinguish the properties of this latter respect to those of the hydration shell in the pre-melting region.

Volume properties and spectroscopy: a terahertz Raman investigation of hen egg white lysozyme.

The molecular dynamics of aqueous solutions of a model amphiphilic peptide is studied as a function of concentration by broad-band light scattering experiments. Similarly to protein aqueous solutions, a considerable retardation, of about a factor 6-8, of hydration water dynamics with respect to bulk water is found, showing a slight dependence on solute concentration. Conversely, the average number of water molecules perturbed by the presence of peptide, i.e. the hydration number, appears to be strongly modified by adding solute. Its behaviour, decreasing upon increasing concentration, can be interpreted considering the random close-to-contact condition experienced by solute particles. Overall, the present findings support the view of a "long range" effect of peptides on the surrounding water, extending beyond the first two hydration shells.

Concentration dependence of hydration water in a model peptide.

Brillouin light scattering (BLS) spectroscopy is a technique that is able to detect thermally excited phonons within a material. The speed of propagation of these phonons can be determined from the magnitude of the Brillouin frequency shift between incident and scattered light, thereby providing a measure of the mechanical properties of the material in the gigahertz range. The mechanical properties of the extracellular matrices of biological tissues and their constituent biopolymers are important for normal tissue function and disturbances in these properties are widely implicated in disease. BLS offers the prospect of measuring mechanical properties on a microscopic scale in living tissues, thereby providing insights into structure-function relationships under normal and pathological conditions. In this study, we investigated BLS in collagen and elastin-the fibrous proteins of the extracellular matrix (ECM). Measurements were made on type I collagen in rat tail tendon, type II collagen in articular cartilage and nuchal ligament elastin. The dependence of the BLS spectrum on fibre orientation was investigated in a backscattering geometry using a reflective substrate. Two peaks, a bulk mode arising from phonon propagation along a quasi-radial direction to the fibre axis and a mode parallel to the surface, depending on sample orientation relative to the fibre axis, could be distinguished. The latter peak was fitted to a model of wave propagation through a hexagonally symmetric elastic solid, and the five components of the elasticity tensor were combined to give axial and transverse Young's, shear and bulk moduli of the fibres. These were 10.2, 8.3, 3.2 and 10.9 GPa, and 6.1, 5.3, 1.9 and 8 GPa for dehydrated type I collagen and elastin, respectively. The former values are close to those previously reported. A microfocused BLS approach was also applied providing selection of single fibres. The moduli of collagen and elastin are much higher than those measured at lower frequency using macroscopic strains, and the difference between them is much less. We therefore believe, like previous investigators, that molecular-scale viscoelastic effects are responsible for the frequency dependence of the fibre biomechanics. Combining BLS with larger-scale mechanical testing methods therefore should, in the future, provide a means of following the evolution of mechanical properties in the formation of the complex structures found in the ECM.

Biomechanics of fibrous proteins of the extracellular matrix studied by Brillouin scattering.

In this Letter, we present a simple method to avoid the well-known spurious contributions in the Brillouin light scattering (BLS) spectrum arising from the finite aperture of collection optics. The method relies on the use of special spatial filters able to select the scattered light with arbitrary precision around a given value of the momentum transfer (Q). We demonstrate the effectiveness of such filters by analyzing the BLS spectra of a reference sample as a function of scattering angle. This practical and inexpensive method could be an extremely useful tool to fully exploit the potentiality of Brillouin acoustic spectroscopy, as it will easily allow for effective Q-variable experiments with unparalleled luminosity and resolution.

Practical way to avoid spurious geometrical contributions in Brillouin light scattering experiments at variable scattering angles.

The density and orientational relaxation of bulk water can be separately studied by depolarized light scattering (DLS) and dielectric spectroscopy (DS), respectively. Here, we ask the question of what are the leading collective modes responsible for polarization anisotropy relaxation (DLS) and dipole moment relaxation (DS) of solutions involving mostly hydrophobic solute-water interfaces. We study, by atomistic molecular dynamics simulations, the dynamics and structure of hydration water interfacing N-Acetyl-leucine-methylamide dipeptide. The DLS response of the solution is consistent with three relaxation processes: bulk water, rotations of single solutes, and collective dipole-induced-dipole polarizability of the solutes, with the time-scale of 130-200 ps. No separate DLS response of the hydration shell has been identified by our simulations. Density fluctuations of the hydration layer, which largely contribute to the response, do not produce a dynamical process distinct from bulk water. We find that the structural perturbation of the orientational distribution of hydration waters by the dipeptide solute is quite significant and propagates ∼3-5 hydration layers into the bulk. This perturbation is still below that produced by hydrated globular proteins. Despite this structural perturbation, there is little change in the orientational dynamics of the hydration layers, compared to the bulk, as probed by both single-particle orientational dynamics and collective dynamics of the dipole moment of the shells. There is a clear distinction between the perturbation of the interfacial structure by the solute-solvent interaction potential and the perturbation of the interfacial dynamics by the corresponding forces.

Depolarized light scattering and dielectric response of a peptide dissolved in water.

Biological interfaces characterized by a complex mixture of hydrophobic, hydrophilic, or charged moieties interfere with the cooperative rearrangement of the hydrogen-bond network of water. In the present study, this solute-induced dynamical perturbation is investigated by extended frequency range depolarized light scattering experiments on an aqueous solution of a variety of systems of different nature and complexity such as small hydrophobic and hydrophilic molecules, amino acids, dipeptides, and proteins. Our results suggest that a reductionist approach is not adequate to describe the rearrangement of hydration water because a significant increase of the dynamical retardation and extension of the perturbation occurs when increasing the chemical complexity of the solute.

More Is Different: Experimental Results on the Effect of Biomolecules on the Dynamics of Hydration Water.

Barrett's oesophagus is a condition characterized by a change in the lining of the oesophagus that markedly increases the risk of adenocarcinoma. We demonstrate the first site-matched application of Brillouin microscopy, Raman microscopy and FTIR micro-spectroscopic imaging to ex-vivo epithelial tissue - Barrett's oesophagus. The mechanical and chemical characters of the epithelium were assessed in histological sections from a patient subjected to endoscopic oesophageal biopsy. Previous studies have shown that both these properties change within the oesophageal wall, owing to the presence of distinct cellular and extracellular constituents which are putatively affected by oesophageal cancer. Brillouin microscopy enables maps of elasticity of the epithelium to be obtained, whilst Raman and FTIR imaging provide 'chemical images' without the need for labelling or staining. This site-matched approach provides a valuable platform for investigating the structure, biomechanics and composition of complex heterogeneous systems. A combined Brillouin-Raman device has potential for in-vivo diagnosis of pathology. First application of site-matched micro Brillouin, Raman and FTIR spectroscopic imaging to epithelial tissue in Barrett's oesophagus.

Daniele Fioretto

Department of Physics and Geology

University of Perugia

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Daniele Fioretto

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physics and Geology

University of Perugia

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Department of Physics and Geology