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2.
Nat Commun ; 11(1): 4248, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843623

RESUMO

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time. We find that the photocycle kinetics and structural dynamics of PYP in the crystalline form deviate from those in solution from the very first steps following photon absorption. This illustrates that ultrafast TRC results cannot be uncritically extrapolated to in vivo function, and that comparative spectroscopic experiments on proteins in crystalline and solution states can help identify structural intermediates under native conditions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X/métodos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Proteínas de Bactérias/efeitos da radiação , Cinética , Luz , Estrutura Molecular , Processos Fotoquímicos , Fotorreceptores Microbianos/efeitos da radiação , Conformação Proteica , Análise Espectral
3.
Wound Repair Regen ; 28(5): 666-675, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32570295

RESUMO

The treatment of burn wounds by enzymatic debridement using bromelain has shown promising results in our burn center. However, inadequate debridement occurred in a few cases in which the etiology of the burn was attributed to relatively low temperature burns. We hypothesized that bromelain is ineffective in burns in which collagen denaturation, which occurs approximately at 65°C, has not taken place. Our objective was to assess whether there is a relationship between the denaturation of collagen and the ability of bromelain to debride acute scald burn wounds of different temperatures. Ex vivo human skin from four different donors was cut into 1x1 cm samples, and scald burns were produced by immersion in water at temperatures of 40°C, 50°C, 60°C, 70°C, and 100°C for 20 minutes. Denaturation of collagen was assessed with histology, using hematoxylin and eosin (H&E) staining and a fluorescently labeled collagen hybridizing peptide (CHP), and with second harmonic generation (SHG) microscopy. Burned samples and one control sample (room temperature) were weighed before and after application of enzymatic debridement to assess the efficacy of enzymatic debridement. After enzymatic debridement, a weight reduction of 80% was seen in the samples heated to 70°C and 100°C, whereas the other samples showed a reduction of 20%. Unfolding of collagen, loss of basket-weave arrangement, and necrosis was seen in samples heated to 60°C or higher. Evident CHP fluorescence, indicative of collagen denaturation, was seen in samples of 60°C, 70°C and 100°C. SHG intensity, signifying intact collagen, was significantly lower in the 70°C and 100°C group (P <.05) compared to the lower temperatures. In conclusion, denaturation of collagen in skin samples occurred between 60°C and 70°C and strongly correlated with the efficacy of enzymatic debridement. Therefore, enzymatic debridement with the use of bromelain is ineffective in scald burns lower than 60°C.


Assuntos
Bromelaínas/farmacologia , Queimaduras/tratamento farmacológico , Desbridamento/métodos , Colágeno , Humanos , Técnicas In Vitro , Cicatrização/fisiologia
4.
J Biophotonics ; 13(5): e201960197, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32049417

RESUMO

Millions of women worldwide have silicone breast implants. It has been reported that implant failure occurs in approximately a tenth of patients within 10 years, and the consequences of dissemination of silicone debris are poorly understood. Currently, silicone detection in histopathological slides is based on morphological features as no specific immunohistochemical technique is available. Here, we show the feasibility and sensitivity of stimulated Raman scattering (SRS) imaging to specifically detect silicone material in stained histopathological slides, without additional sample treatment. Histology slides of four periprosthetic capsules from different implant types were obtained after explantation, as well as an enlarged axillary lymph node from a patient with a ruptured implant. SRS images coregistered with bright-field images revealed the distribution and quantity of silicone material in the tissue. Fast and high-resolution imaging of histology slides with molecular specificity using SRS provides an opportunity to investigate the role of silicone debris in the pathophysiology of implant-linked diseases.


Assuntos
Implantes de Mama , Diagnóstico por Imagem , Feminino , Humanos , Linfonodos , Silicones , Análise Espectral Raman
5.
Transl Biophotonics ; 2(4): e202000009, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34341777

RESUMO

During lung cancer operations a rapid and reliable assessment of tumor tissue can reduce operation time and potentially improve patient outcomes. We show that third harmonic generation (THG), second harmonic generation (SHG) and two-photon excited autofluorescence (2PEF) microscopy reveals relevant, histopathological information within seconds in fresh unprocessed human lung samples. We used a compact, portable microscope and recorded images within 1 to 3 seconds using a power of 5 mW. The generated THG/SHG/2PEF images of tumorous and nontumorous tissues are compared with the corresponding standard histology images, to identify alveolar structures and histopathological hallmarks. Cellular structures (tumor cells, macrophages and lymphocytes) (THG), collagen (SHG) and elastin (2PEF) are differentiated and allowed for rapid identification of carcinoid with solid growth pattern, minimally enlarged monomorphic cell nuclei with salt-and-pepper chromatin pattern, and adenocarcinoma with lipidic and micropapillary growth patterns. THG/SHG/2PEF imaging is thus a promising tool for clinical intraoperative assessment of lung tumor tissue.

6.
Adv Sci (Weinh) ; 6(11): 1900163, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31179222

RESUMO

Distinguishing tumors from normal brain cells is important but challenging in glioma surgery due to the lack of clear interfaces between the two. The ability of label-free third harmonic generation (THG) microscopy in combination with automated image analysis to quantitatively detect glioma infiltration in fresh, unprocessed tissue in real time is assessed. The THG images reveal increased cellularity in grades II-IV glioma samples from 23 patients, as confirmed by subsequent hematoxylin and eosin histology. An automated image quantification workflow is presented for quantitative assessment of the imaged cellularity as a reflection of the degree of glioma invasion. The cellularity is validated in three ways: 1) Quantitative comparison of THG imaging with fluorescence microscopy of nucleus-stained samples demonstrates that THG reflects the true tissue cellularity. 2) Thresholding of THG cellularity differentiates normal brain from glioma infiltration, with 96.6% sensitivity and 95.5% specificity, in nearly perfect (93%) agreement with pathologists. 3) In one patient, a good correlation between THG cellularity and preoperative magnetic resonance and positron emission tomography imaging is demonstrated. In conclusion, quantitative real-time THG microscopy accurately assesses glioma infiltration in ex vivo human brain samples, and therefore holds strong potential for improving the accuracy of surgical resection.

7.
J Biophotonics ; 12(6): e201800297, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30684312

RESUMO

Real-time assessment of excised tissue may help to improve surgical results in breast tumor surgeries. Here, as a step towards this purpose, the potential of second and third harmonic generation (SHG, THG) microscopy is explored. SHG and THG are nonlinear optical microscopic techniques that do not require labeling of tissue to generate 3D images with intrinsic depth-sectioning at sub-cellular resolution. Until now, this technique had been applied on fixated breast tissue or to visualize the stroma only, whereas most tumors start in the lobules and ducts. Here, SHG/THG images of freshly excised unprocessed healthy human tissue are shown to reveal key breast components-lobules, ducts, fat tissue, connective tissue and blood vessels, in good agreement with hematoxylin and eosin histology. DNA staining of fresh unprocessed mouse breast tissue was performed to aid in the identification of cell nuclei in label-free THG images. Furthermore, 2- and 3-photon excited auto-fluorescence images of mouse and human tissue are collected for comparison. The SHG/THG imaging modalities generate high quality images of freshly excised tissue in less than a minute with an information content comparable to that of the gold standard, histopathology. Therefore, SHG/THG microscopy is a promising tool for real-time assessment of excised tissue during surgery.


Assuntos
Mama/diagnóstico por imagem , Microscopia de Geração do Segundo Harmônico/métodos , Animais , Mama/citologia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Camundongos
8.
J Am Chem Soc ; 141(1): 520-530, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30511841

RESUMO

The orange carotenoid protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV-visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 ms. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid ß1-ring in picoseconds occurs at a low yield of <1%, whereby the ß1-ring retains a trans configuration with respect to the conjugated π-electron chain. This event initiates structural changes at the N-terminal domain in 1 µs, which allow the carotenoid to translocate into the N-terminal domain in 10 µs. We identified infrared signatures of helical elements that dock on the C-terminal domain ß-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer time scales, lagging carotenoid translocation by at least 2 decades of time.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Luz , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína
9.
Plast Reconstr Surg Glob Open ; 6(1): e1610, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29464156

RESUMO

Background: In the human ear and nose, cartilage plays a key role in establishing its form and function. Interestingly, there is a noticeable paucity on biochemical, structural, and mechanical studies focused on facial cartilage. Such studies are needed to provide elementary knowledge that is fundamental to tissue engineering of cartilage. Therefore, in this study, a comparison is made of the biochemical, structural, and mechanical differences between ear, ala nasi, and septum on the extracellular matrix (ECM) level. Methods: Cartilage samples were harvested from 10 cadaveric donors. Each sample was indented 10 times with a nanoindenter to determine the effective Young's modulus. Structural information of the cartilage was obtained by multiple-photon laser scanning microscopy capable of revealing matrix components at subcellular resolution. Biochemistry was performed to measure glycosaminoglycan (GAG), DNA, elastin, and collagen content. Results: Significant differences were seen in stiffness between ear and septal cartilage (P = 0.011) and between ala nasi and septal cartilage (P = 0.005). Elastin content was significantly higher in ear cartilage. Per cartilage subtype, effective Young's modulus was not significantly correlated with cell density, GAG, or collagen content. However, in septal cartilage, low elastin content was associated with higher stiffness. Laser microscopy showed a distinct difference between ear cartilage and cartilage of nasal origin. Conclusion: Proposed methods to investigate cartilage on the ECM level provided good results. Significant differences were seen not only between ear and nasal cartilage but also between the ala nasi and septal cartilage. Albeit its structural similarity to septal cartilage, the ala nasi has a matrix stiffness comparable to ear cartilage.

10.
J Biophotonics ; 11(1)2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28464543

RESUMO

Third harmonic generation (THG) microscopy is a label-free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all-nuclei-highlighted fluorescence images, acquired simultaneously from the same tissue area. For quantitative comparison of a substantial pair of images, we present here a segmentation workflow that is applicable for both THG and fluorescence images, with a precision of 91.3 % and 95.8 % achieved respectively. We find that the correspondence between the main features of the two imaging modalities amounts to 88.9 %, providing quantitative evidence of the interpretation of dark holes as brain cells. Moreover, 80 % bright objects in THG images overlap with nuclei highlighted in the fluorescence images, and they are 2 times smaller than the dark holes, showing that cells of different morphologies can be recognized in THG images. We expect that the described quantitative comparison is applicable to other types of brain tissue and with more specific staining experiments for cell type identification.


Assuntos
Imageamento Tridimensional , Microscopia de Fluorescência/métodos , Microscopia de Geração do Segundo Harmônico/métodos , Animais , Encéfalo/diagnóstico por imagem , Camundongos , Razão Sinal-Ruído
11.
Bioinformatics ; 33(11): 1712-1720, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28130231

RESUMO

Motivation: The morphologies contained in 3D third harmonic generation (THG) images of human brain tissue can report on the pathological state of the tissue. However, the complexity of THG brain images makes the usage of modern image processing tools, especially those of image filtering, segmentation and validation, to extract this information challenging. Results: We developed a salient edge-enhancing model of anisotropic diffusion for image filtering, based on higher order statistics. We split the intrinsic 3-phase segmentation problem into two 2-phase segmentation problems, each of which we solved with a dedicated model, active contour weighted by prior extreme. We applied the novel proposed algorithms to THG images of structurally normal ex-vivo human brain tissue, revealing key tissue components-brain cells, microvessels and neuropil, enabling statistical characterization of these components. Comprehensive comparison to manually delineated ground truth validated the proposed algorithms. Quantitative comparison to second harmonic generation/auto-fluorescence images, acquired simultaneously from the same tissue area, confirmed the correctness of the main THG features detected. Availability and Implementation: The software and test datasets are available from the authors. Contact: z.zhang@vu.nl. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Encéfalo/anatomia & histologia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia de Geração do Segundo Harmônico/métodos , Software , Algoritmos , Encéfalo/patologia , Humanos
12.
J Phys Chem Lett ; 7(17): 3472-6, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27537211

RESUMO

Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal Jα helix that is docked on a ß-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds. In D2O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation of the covalent cysteinyl-FMN adduct in 10 µs. We observe a two-step unfolding of the Jα helix: The first phase occurs concomitantly with Cys-FMN covalent adduct formation in 10 µs, along with hydrogen-bond rupture of the FMN C4═O with Gln-513, motion of the ß-sheet, and an additional helical element. The second phase occurs in approximately 240 µs. The final spectrum at 500 µs is essentially identical to the steady-state light-minus-dark Fourier transform infrared spectrum, indicating that Jα helix unfolding is complete on that time scale.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Ligação a DNA/química , Fotorreceptores Microbianos/química , Análise Espectral/métodos , Ligação de Hidrogênio , Modelos Moleculares , Desdobramento de Proteína , Vibração
13.
Tissue Eng Part C Methods ; 22(6): 573-84, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27089896

RESUMO

Scaffold contraction is a common but underestimated problem in the field of tissue engineering. It becomes particularly problematic when creating anatomically complex shapes such as the ear. The aim of this study was to develop a contraction-free biocompatible scaffold construct for ear cartilage tissue engineering. To address this aim, we used three constructs: (i) a fibrin/hyaluronic acid (FB/HA) hydrogel, (ii) a FB/HA hydrogel combined with a collagen I/III scaffold, and (iii) a cage construct containing (ii) surrounded by a 3D-printed poly-ɛ-caprolactone mold. A wide range of different cell types were tested within these constructs, including chondrocytes, perichondrocytes, adipose-derived mesenchymal stem cells, and their combinations. After in vitro culturing for 1, 14, and 28 days, all constructs were analyzed. Macroscopic observation showed severe contraction of the cell-seeded hydrogel (i). This could be prevented, in part, by combining the hydrogel with the collagen scaffold (ii) and prevented in total using the 3D-printed cage construct (iii). (Immuno)histological analysis, multiphoton laser scanning microscopy, and biomechanical analysis showed extracellular matrix deposition and increased Young's modulus and thereby the feasibility of ear cartilage engineering. These results demonstrated that the 3D-printed cage construct is an adequate model for contraction-free ear cartilage engineering using a range of cell combinations.


Assuntos
Cartilagem/citologia , Matriz Extracelular/química , Polímeros/química , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Tecidos Suporte/química , Tecido Adiposo/citologia , Animais , Células Cultivadas , Condrócitos/citologia , Condrogênese , Cabras , Células-Tronco Mesenquimais/citologia , Impressão Tridimensional
14.
Biochim Biophys Acta ; 1847(1): 2-11, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24973600

RESUMO

In recent years visible pump/mid-infrared (IR) probe spectroscopy has established itself as a key technology to unravel structure-function relationships underlying the photo-dynamics of complex molecular systems. In this contribution we review the most important applications of mid-infrared absorption difference spectroscopy with sub-picosecond time-resolution to photosynthetic complexes. Considering several examples, such as energy transfer in photosynthetic antennas and electron transfer in reaction centers and even more intact structures, we show that the acquisition of ultrafast time resolved mid-IR spectra has led to new insights into the photo-dynamics of the considered systems and allows establishing a direct link between dynamics and structure, further strengthened by the possibility of investigating the protein response signal to the energy or electron transfer processes. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.


Assuntos
Fotossíntese/fisiologia , Espectrofotometria Infravermelho/métodos , Transporte de Elétrons , Transferência de Energia , Modelos Moleculares , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo
15.
J Phys Chem B ; 119(6): 2372-83, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25144816

RESUMO

Biological signal transduction by photoactive yellow protein (PYP) in halophilic purple sulfur bacteria is initiated by trans-to-cis isomerization of the p-coumaric acid chromophore (pCa) of PYP. pCa is engaged in two short hydrogen bonds with protein residues E46 and Y42, and it is negatively charged at the phenolate oxygen. We investigated the role in the isomerization process of the E46 short hydrogen bond and that of the negative charge on the anionic phenolate moiety of the chromophore. We used wild-type PYP and the mutant E46A, in protonated and deprotonated states (referred to as pE46A and dpE46A, respectively), to reduce the number of hydrogen bond interactions between the pCa phenolate oxygen and the protein and to vary the negative charge density in the chromophore-binding pocket. Their effects on the yield and rate of chromophore isomerization were determined by ultrafast spectroscopy. Molecular dynamics simulations were used to relate these results to structural changes in the mutant protein. We found that deprotonated pCa in E46A has a slower isomerization rate as the main part of this reaction was associated with time constants of 1 and 6 ps, significantly slower than the 0.6 ps time constant in wild-type PYP. The quantum yield of isomerization in dpE46A was estimated to be 30 ± 2%, and that of pE46A was 32 ± 3%, very close to the value determined for wtPYP of 32 ± 2%. Relaxation of the isomerized product state I0 to I1 was faster in dpE46A. We conclude that the negative charge on pCa stabilized by the short hydrogen bonds with E46 and Y42 affects the rate of isomerization but not the quantum yield of isomerization. With this information, we propose a scheme for the potential energy surfaces involved in the isomerization and suggest protein motions near the pCa backbone as key events in successful isomerization.


Assuntos
Proteínas de Bactérias/química , Fotorreceptores Microbianos/química , Ligação de Hidrogênio , Isomerismo , Cinética , Simulação de Dinâmica Molecular
16.
J Phys Chem B ; 117(38): 11260-71, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-23837465

RESUMO

Bacteriochlorophyll a with Ni(2+) replacing the central Mg(2+) ion was used as an ultrafast excitation energy dissipation center in reconstituted bacterial LH1 complexes. B870, a carotenoid-less LH1 complex, and B880, an LH1 complex containing spheroidene, were obtained via reconstitution from the subunits isolated from chromatophores of Rhodospirillum rubrum . Ni-substituted bacteriochlorophyll a added to the reconstitution mixture partially substituted the native pigment in both forms of LH1. The excited-state dynamics of the reconstituted LH1 complexes were probed by femtosecond pump-probe transient absorption spectroscopy in the visible and near-infrared spectral region. Spheroidene-binding B880 containing no excitation dissipation centers displayed complex dynamics in the time range of 0.1-10 ps, reflecting internal conversion and intersystem crossing in the carotenoid, exciton relaxation in BChl complement, and energy transfer from carotenoid to the latter. In B870, some aggregation-induced excitation energy quenching was present. The binding of Ni-BChl a to both B870 and B880 resulted in strong quenching of the excited states with main deexcitation lifetime of ca. 2 ps. The LH1 excited-state lifetime could be modeled with an intrinsic decay time constant in Ni-substituted bacteriochlorophyll a of 160 fs. The presence of carotenoid in LH1 did not influence the kinetics of energy trapping by Ni-BChl unless the carotenoid was directly excited, in which case the kinetics was limited by a slower carotenoid S1 to bacteriochlorophyll energy transfer.


Assuntos
Proteínas de Bactérias/química , Bacterioclorofila A/química , Complexos de Proteínas Captadores de Luz/química , Níquel/química , Rhodospirillum rubrum/metabolismo , Proteínas de Bactérias/metabolismo , Carotenoides/química , Transferência de Energia , Íons/química , Complexos de Proteínas Captadores de Luz/metabolismo , Microscopia de Força Atômica , Protoclorifilida/química , Protoclorifilida/metabolismo , Espectrometria de Fluorescência , Fatores de Tempo
17.
Biophys J ; 104(11): 2493-502, 2013 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-23746522

RESUMO

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on BA and HA via the appearance of the BA and HA anion bands. We observed an unexpectedly early rise of the HA⁻ band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between BA and HA, and the effect of protein conformation on the electron transfer rate.


Assuntos
Feofitinas/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/enzimologia , Transporte de Elétrons , Modelos Moleculares , Conformação Molecular , Feofitinas/química , Rhodobacter sphaeroides/metabolismo
18.
J Phys Chem B ; 117(38): 11042-8, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-23477674

RESUMO

Photoinduced ionization of the chromophore inside photoactive yellow protein (PYP) was investigated by ultrafast spectroscopy in the visible and near-infrared spectral regions. An absorption band that extended from around 550 to 850 nm was observed and ascribed to solvated electrons, ejected from the p-hydroxycinnamic acid anion chromophore upon the absorption of two 400 nm photons. Global kinetic analysis showed that the solvated electron absorption decayed in two stages: a shorter phase of around 10 ps and a longer phase of more than 3 ns. From a simulation based on a diffusion model we conclude that the diffusion rate of the electron is about 0.8 Å(2)/ps in wild type PYP, and that the electron is ejected to a short distance of only several angstroms away from the chromophore. The chromophore-protein pocket appears to provide a water-similar local environment for the electron. Because mutations at different places around the chromophore have different effect on the electron recombination dynamics, we suggest that solvated electrons could provide a new method to investigate the local dielectric environment inside PYP and thus help to understand the role of the protein in the photoisomerization process.


Assuntos
Proteínas de Bactérias/química , Fotorreceptores Microbianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Difusão , Elétrons , Halorhodospira halophila/metabolismo , Cinética , Mutação , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Espectrofotometria , Espectrofotometria Infravermelho
19.
Biomed Opt Express ; 3(9): 2184-9, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23024912

RESUMO

We demonstrate a single-shot holographic phase microscope that combines short-coherence laser pulses with an off-axis geometry. By introducing a controlled pulse front tilt, ultrashort pulses are made to interfere over a large field-of-view without loss of fringe contrast. With this microscope, quantitative phase images of live cells can be recorded in a full-field geometry without moving parts. We perform phase imaging of HEK293 cells, to study the dynamics of cell volume regulation in response to an osmotic shock.

20.
Opt Express ; 20(10): 10562-71, 2012 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-22565682

RESUMO

Broadband femtosecond mid-infrared pulses can be converted into the visible spectral region by chirped pulse upconversion. We report here the upconversion of pump probe transient signals in the frequency region below 1800cm(-1), using the nonlinear optical crystal AgGaGeS4, realizing an important expansion of the application range of this method. Experiments were demonstrated with a slab of GaAs, in which the upconverted signals cover a window of 120cm(-1), with 1.5cm(-1) resolution. In experiments on the BLUF photoreceptor Slr1694, signals below 1 milliOD were well resolved after baseline correction. Possibilities for further optimization of the method are discussed. We conclude that this method is an attractive alternative for the traditional MCT arrays used in most mid-infrared pump probe experiments.


Assuntos
Proteínas de Bactérias/química , Fotorreceptores Microbianos/química , Processamento de Sinais Assistido por Computador , Espectrofotometria Infravermelho/instrumentação , Espectrofotometria Infravermelho/métodos , Arsenicais/química , Cristalização , Desenho de Equipamento , Gálio/química , Raios Infravermelhos , Distribuição Normal , Óptica e Fotônica , Espalhamento de Radiação , Análise Espectral , Fatores de Tempo
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