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"Otimização de pulsos ultracurtos via absorção de dois fótons" ; Ultrashort pulse optimization via two-photon absorption

Silva, Daniel Luiz da
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 31/03/2005 PT
Relevância na Pesquisa
88.65706%
Este trabalho teve como objetivo a montagem de um sistema de otimização de pulsos ultracurtos (oscilador laser modelocked de 15 fs), através de uma técnica de formatação de pulsos via absorção de dois fótons em compostos orgânicos. Está técnica utiliza uma estratégia evolucionária baseada em um algoritmo genético, onde se controla o formato do pulso pela deformação imposta a um espelho deformável, conjuntamente com o monitoramento de um sinal de realimentação. Desta forma, este sistema permite tanto a otimização do processo de absorção de dois fótons, quanto a otimização do próprio pulso do sistema laser. Após a montagem inicial do sistema de formatação de pulsos, foram implementados três métodos de otimização via monitoramento do processo de absorção de dois fótons, sendo que dois deles foram desenvolvidos nesta dissertação. Os métodos diferem entre si pelo emprego de distintos sinais de realimentação para o processo de otimização: (i) intensidade da fluorescência excitada por dois fótons; (ii) variação da transmitância não linear dos compostos orgânicos devido à absorção de dois fótons; e (iii) intensidade do efeito de lente térmica apresentada pelos compostos orgânicos após a absorção de dois fótons. Os três métodos de otimização apresentaram resultados similares e satisfatórios...

Controle coerente do processo de absorção de dois fótons em compostos orgânicos; Coherent control of two-photon absorption process in organic compounds

Silva, Daniel Luiz da
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 23/10/2009 PT
Relevância na Pesquisa
88.50041%
A larga banda espectral, característica de pulsos ultracurtos de luz laser, tem possibilitado o controle coerente da interação da luz com a matéria através da manipulação das componentes espectrais da fase do pulso. Esta nova área de pesquisa tem sido responsável por avanços no entendimento e controle de fenômenos foto-induzidos, especialmente no que diz respeito a processos ópticos não lineares. Nesta tese de doutorado, estudamos o controle coerente da absorção de dois fótons (A2F) em compostos orgânicos usando pulsos de femtossegundos. O processo de A2F em derivados de perilenos foi investigado utilizando pulsos com chirp linear (máscara de fase quadrática), a partir do monitoramento da fluorescência excitada por dois fótons. A otimização da A2F desses compostos, através da formatação da fase do pulso via algorítmo genético, revelou que pulsos limitados por transformada de Fourier induzem maior A2F. Cálculos de Química Quântica, empregando o formalismo da teoria do funcional densidade, foram utilizados para caracterizar a estrutura eletrônica e determinar as transições permitidas por dois fótons nos derivados de perilenos, fundamentando nossos resultados experimentais. Além disso, estudamos também o controle coerente da A2F de compostos orgânicos aplicando uma máscara de fase senoidal ao pulso. Neste caso...

Controle da fluorescência excitada por dois fótons no polímero conjugado MEH-PPV através da formatação pulsos ultracurtos; Control of the two-photon excited fluorescence in the conjugated polymer MEH-PPV by pulse shaping

Ferreira, Paulo Henrique Dias
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 17/08/2007 PT
Relevância na Pesquisa
99.10744%
Neste trabalho foi investigado o controle do processo de fluorescência excitada por absorção de dois fótons no polímero conjugado MEH-PPV, utilizando um sistema de formatação espectral da fase dos pulsos ultracurtos. Para tal estudo, foi utilizado um oscilador laser de Ti:Safira (15 fs, ~ 800 nm e largura de banda de 60 nm). Através de dois distintos métodos de formatação, observa-se a influência destes no processo de fotodegradação do MEHPPV, inferido pela diminuição da intensidade do sinal de fluorescência. No primeiro método de formatação, é estudada a influência de diferentes chirps impostos aos pulsos no processo de fluorescência do MEH-PPV. Observa-se uma menor taxa de fotodegradação para pulsos com maiores chirps, independente do sinal, em comparação a pulsos no limite de transformada. Esse efeito foi relacionado ao acréscimo na duração temporal dos pulsos com chirp, com consequente diminuição da intensidade. Numa segunda etapa, através do uso de um espelho deformável, a fase espectral do pulso é formatada usando uma máscara de fase senoidal. Neste caso, a intensidade de fluorescência foi modulada em aproximadamente 25%, num claro processo de controle coerente, sem nenhuma diferença apreciável no processo de fotodegradação. Desta forma...

Microscopias de óptica não linear : fluorescência excitada por absorção de dois fótons, geração de segundo harmônico e geração de terceiro harmônico; Non linear optical microscopies : two photon excited fluorescence, second harmonic generation and third harmonic generation

Vitor Bianchin Pelegati
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 21/12/2010 PT
Relevância na Pesquisa
129.29073%
Biologia celular é um novo mundo promissor com enorme impacto social, econômico e na saúde. Organismos vivos são capazes de, produzir a própria energia a partir da luz do sol, se reproduzir, de se auto-reparar, sinalizar e navegar por sinais bioquímicos, biomecânicos, luminosos, térmicos, e outros, e produzir materiais à temperatura ambiente. As possibilidades abertas por essa área incluem, desde bactérias e protozoários usados para destruir células de câncer, regeneração de órgãos inteiros, produção de etanol a partir de algas, e outros. Entretanto, para o entendimento da biologia em seu nível mais profundo, ferramentas de observação não destrutivas fazem-se necessária para seguir os processos celulares durante seu tempo de vida. A óptica tem a única onda não destrutiva capaz de fornecer informações em tempo real com suficiente resolução espacial de eventos acontecendo internamente à célula. Ademais, porque feixes de luz não colidem, a óptica permite a integração de diferentes técnicas capazes de reunir informações simultâneas de processos celulares. Óptica não linear é especialmente adequada para tal, pois não requer marcação ou processamentos especiais de amostras que poderiam destruir...

Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence

Zoumi, Aikaterini; Yeh, Alvin; Tromberg, Bruce J.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
88.67586%
Multiphoton microscopy relies on nonlinear light–matter interactions to provide contrast and optical sectioning capability for high-resolution imaging. Most multiphoton microscopy studies in biological systems have relied on two-photon excited fluorescence (TPEF) to produce images. With increasing applications of multiphoton microscopy to thick-tissue “intravital” imaging, second-harmonic generation (SHG) from structural proteins has emerged as a potentially important new contrast mechanism. However, SHG is typically detected in transmission mode, thus limiting TPEF/SHG coregistration and its practical utility for in vivo thick-tissue applications. In this study, we use a broad range of excitation wavelengths (730–880 nm) to demonstrate that TPEF/SHG coregistration can easily be achieved in unstained tissues by using a simple backscattering geometry. The combined TPEF/SHG technique was applied to imaging a three-dimensional organotypic tissue model (RAFT). The structural and molecular origin of the image-forming signal from the various tissue constituents was determined by simultaneous spectroscopic measurements and confirming immunofluorescence staining. Our results show that at shorter excitation wavelengths (<800 nm), the signal emitted from the extracellular matrix (ECM) is a combination of SHG and TPEF from collagen...

One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins.

Volkmer, A; Subramaniam, V; Birch, D J; Jovin, T M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/2000 EN
Relevância na Pesquisa
69.48859%
We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 +/- 1 ns). The approximately 1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional approximately 13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*...

Imaging Coronary Artery Microstructure Using Second-Harmonic and Two-Photon Fluorescence Microscopy

Zoumi, Aikaterini; Lu, Xiao; Kassab, Ghassan S.; Tromberg, Bruce J.
Fonte: Biophysical Society Publicador: Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em /10/2004 EN
Relevância na Pesquisa
68.95116%
The microstructural basis for the mechanical properties of blood vessels has not been directly determined because of the lack of a nondestructive method that yields a three-dimensional view of these vascular wall constituents. Here, we demonstrate that multiphoton microscopy can be used to visualize the microstructural basis of blood vessel mechanical properties, by combining mechanical testing (distension) of excised porcine coronary arteries with simultaneous two-photon excited fluorescence and second-harmonic generation microscopy. Our results show that second-harmonic generation signals derived from collagen can be spectrally isolated from elastin and smooth muscle cell two-photon fluorescence. Two-photon fluorescence signals can be further characterized by emission maxima at 495 nm and 520 nm, corresponding to elastin and cellular contributions, respectively. Two-dimensional reconstructions of spectrally fused images permit high-resolution visualization of collagen and elastin fibrils and smooth muscle cells from intima to adventitia. These structural features are confirmed by coregistration of multiphoton microscopy images with conventional histology. Significant changes in mean fibril thickness and overall wall dimension were observed when comparing no load (zero transmural pressure) and zero-stress conditions to 30 and 180 mmHg distension pressures. Overall...

Detection and Imaging of Non-Contractile Inclusions and Sarcomeric Anomalies in Skeletal Muscle by Second Harmonic Generation Combined with Two-Photon Excited Fluorescence

Ralston, E.; Swaim, B.; Czapiga, M.; Hwu, W.-L.; Chien, Y.-H.; Pittis, M.G.; Bembi, B.; Schwartz, O.; Plotz, P.; Raben, N.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
88.8857%
The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provide detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe Disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.

Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy

Hunter, Jennifer J.; Masella, Benjamin; Dubra, Alfredo; Sharma, Robin; Yin, Lu; Merigan, William H.; Palczewska, Grazyna; Palczewski, Krzysztof; Williams, David R.
Fonte: Optical Society of America Publicador: Optical Society of America
Tipo: Artigo de Revista Científica
Publicado em 17/12/2010 EN
Relevância na Pesquisa
69.311226%
In vivo two-photon imaging through the pupil of the primate eye has the potential to become a useful tool for functional imaging of the retina. Two-photon excited fluorescence images of the macaque cone mosaic were obtained using a fluorescence adaptive optics scanning laser ophthalmoscope, overcoming the challenges of a low numerical aperture, imperfect optics of the eye, high required light levels, and eye motion. Although the specific fluorophores are as yet unknown, strong in vivo intrinsic fluorescence allowed images of the cone mosaic. Imaging intact ex vivo retina revealed that the strongest two-photon excited fluorescence signal comes from the cone inner segments. The fluorescence response increased following light stimulation, which could provide a functional measure of the effects of light on photoreceptors.

Two-photon excited hemoglobin fluorescence

Zheng, Wei; Li, Dong; Zeng, Yan; Luo, Yi; Qu, Jianan Y.
Fonte: Optical Society of America Publicador: Optical Society of America
Tipo: Artigo de Revista Científica
Publicado em 06/12/2010 EN
Relevância na Pesquisa
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We discovered that hemoglobin emits high energy Soret fluorescence when two-photon excited by the visible femtosecond light sources. The unique spectral and temporal characteristics of hemoglobin fluorescence were measured by using a time-resolved spectroscopic detection system. The high energy Soret fluorescence of hemoglobin shows the spectral peak at 438 nm with extremely short lifetime. This discovery enables two-photon excitation fluorescence microscopy to become a potentially powerful tool for in vivo label-free imaging of blood cells and vessels.

Measurement and correction of in vivo sample aberrations employing a nonlinear guide-star in two-photon excited fluorescence microscopy

Aviles-Espinosa, Rodrigo; Andilla, Jordi; Porcar-Guezenec, Rafael; Olarte, Omar E.; Nieto, Marta; Levecq, Xavier; Artigas, David; Loza-Alvarez, Pablo
Fonte: Optical Society of America Publicador: Optical Society of America
Tipo: Artigo de Revista Científica
Publicado em 25/10/2011 EN
Relevância na Pesquisa
88.67586%
We demonstrate that sample induced aberrations can be measured in a nonlinear microscope. This uses the fact that two-photon excited fluorescence naturally produces a localized point source inside the sample: the nonlinear guide-star (NL-GS). The wavefront emitted from the NL-GS can then be recorded using a Shack-Hartmann sensor. Compensation of the recorded sample aberrations is performed by the deformable mirror in a single-step. This technique is applied to fixed and in vivo biological samples, showing, in some cases, more than one order of magnitude improvement in the total collected signal intensity.

In vivo two-photon excited fluorescence microscopy reveals cardiac- and respiration-dependent pulsatile blood flow in cortical blood vessels in mice

Santisakultarm, Thom P.; Cornelius, Nathan R.; Nishimura, Nozomi; Schafer, Andrew I.; Silver, Richard T.; Doerschuk, Peter C.; Olbricht, William L.; Schaffer, Chris B.
Fonte: American Physiological Society Publicador: American Physiological Society
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
88.67586%
Subtle alterations in cerebral blood flow can impact the health and function of brain cells and are linked to cognitive decline and dementia. To understand hemodynamics in the three-dimensional vascular network of the cerebral cortex, we applied two-photon excited fluorescence microscopy to measure the motion of red blood cells (RBCs) in individual microvessels throughout the vascular hierarchy in anesthetized mice. To resolve heartbeat- and respiration-dependent flow dynamics, we simultaneously recorded the electrocardiogram and respiratory waveform. We found that centerline RBC speed decreased with decreasing vessel diameter in arterioles, slowed further through the capillary bed, and then increased with increasing vessel diameter in venules. RBC flow was pulsatile in nearly all cortical vessels, including capillaries and venules. Heartbeat-induced speed modulation decreased through the vascular network, while the delay between heartbeat and the time of maximum speed increased. Capillary tube hematocrit was 0.21 and did not vary with centerline RBC speed or topological position. Spatial RBC flow profiles in surface vessels were blunted compared with a parabola and could be measured at vascular junctions. Finally, we observed a transient decrease in RBC speed in surface vessels before inspiration. In conclusion...

Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging

Wei, Lu; Chen, Zhixing; Min, Wei
Fonte: Optical Society of America Publicador: Optical Society of America
Tipo: Artigo de Revista Científica
Publicado em 22/05/2012 EN
Relevância na Pesquisa
69.435205%
Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8.

Highly sensitive detection of cancer cells using femtosecond dual-wavelength near-IR two-photon imaging

Starkey, Jean R.; Makarov, Nikolay S.; Drobizhev, Mikhail; Rebane, Aleksander
Fonte: Optical Society of America Publicador: Optical Society of America
Tipo: Artigo de Revista Científica
Publicado em 06/06/2012 EN
Relevância na Pesquisa
68.885703%
We describe novel imaging protocols that allow detection of small cancer cell colonies deep inside tissue phantoms with high sensitivity and specificity. We compare fluorescence excited in Styryl-9M molecules by femtosecond pulses at near IR wavelengths, where Styryl-9M shows the largest dependence of the two-photon absorption (2PA) cross section on the local environment. We show that by calculating the normalized ratio of the two-photon excited fluorescence (2PEF) intensity at 1200 nm and 1100 nm excitation wavelengths we can achieve high sensitivity and specificity for determining the location of cancer cells surrounded by normal cells. The 2PEF results showed a positive correlation with the levels of MDR1 proteins expressed by the cells, and, for high MDR1 expressors, as few as ten cancer cells could be detected. Similar high sensitivity is also demonstrated for tumor colonies induced in mouse external ears. This technique could be useful in early cancer detection, and, perhaps, also in monitoring dormant cancer deposits.

Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo

Krasieva, Tatiana B.; Stringari, Chiara; Liu, Feng; Sun, Chung-Ho; Kong, Yu; Balu, Mihaela; Meyskens, Frank L.; Gratton, Enrico; Tromberg, Bruce J.
Fonte: Society of Photo-Optical Instrumentation Engineers Publicador: Society of Photo-Optical Instrumentation Engineers
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
88.97226%
Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λex=1000  nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6±0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5±0.05 and 0.17±0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.

PROBING THE IMPACT OF GAMMA-IRRADIATION ON THE METABOLIC STATE OF NEURAL STEM AND PRECURSOR CELLS USING DUAL-WAVELENGTH INTRINSIC SIGNAL TWO-PHOTON EXCITED FLUORESCENCE

KRASIEVA, TATIANA B.; GIEDZINSKI, ERICH; TRAN, KATHERINE; LAN, MARY; LIMOLI, CHARLES L.; TROMBERG, BRUCE J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/07/2011 EN
Relevância na Pesquisa
88.911455%
Two-photon excited fluorescence (TPEF) spectroscopy and imaging were used to investigate the effects of gamma-irradiation on neural stem and precursor cells (NSPCs). While the observed signal from reduced nicotinamide adenine dinucleotide (NADH) was localized to the mitochondria, the signal typically associated with oxidized flavoproteins (Fp) was distributed diffusely throughout the cell. The measured TPEF emission and excitation spectra were similar to the established spectra of NAD(P)H and Fp. Fp fluorescence intensity was markedly increased by addition of the electron transport chain (ETC) modulator menadione to the medium, along with a concomitant decrease in the NAD(P)H signal. Three-dimensional (3D) neurospheres were imaged to obtain the cellular metabolic index (CMI), calculated as the ratio of Fp to NAD(P)H fluorescence intensity. Radiation effects were found to differ between low-dose (≤ 50 cGy) and high-dose (≥ 50 cGy) exposures. Low-dose irradiation caused a marked drop in CMI values accompanied by increased cellular proliferation. At higher doses, both NAD(P)H and Fp signals increased, leading to an overall elevation in CMI values. These findings underscore the complex relationship between radiation dose, metabolic state...

In vivo micro-vascular imaging and flow cytometry in zebrafish using two-photon excited endogenous fluorescence

Zeng, Yan; Yan, Bo; Sun, Qiqi; He, Sicong; Jiang, Jun; Wen, Zilong; Qu, Jianan Y.
Fonte: Optical Society of America Publicador: Optical Society of America
Tipo: Artigo de Revista Científica
Publicado em 04/02/2014 EN
Relevância na Pesquisa
69.200986%
Zebrafish has rapidly evolved as a powerful vertebrate model organism for studying human diseases. Here we first demonstrate a new label-free approach for in vivo imaging of microvasculature, based on the recent discovery and detailed characterization of the two-photon excited endogenous fluorescence in the blood plasma of zebrafish. In particular, three-dimensional reconstruction of the microvascular networks was achieved with the depth-resolved two-photon excitation fluorescence (TPEF) imaging. Secondly, the blood flow images, obtained by perpendicularly scanning the focal point across the blood vessel, provided accurate information for characterizing the hemodynamics of the circulatory system. The endogenous fluorescent signals of reduced nicotinamide adenine dinucleotide (NADH) enabled visualization of the circulating granulocytes (neutrophils) in the blood vessel. The development of acute sterile inflammation could be detected by the quantitative counting of circulating neutrophils. Finally, we found that by utilizing a short wavelength excitation at 650 nm, the commonly used fluorescent proteins, such as GFP and DsRed, could be efficiently excited together with the endogenous fluorophores to achieve four-color TPEF imaging of the vascular structures and blood cells. The results demonstrated that the multi-color imaging could potentially yield multiple view angles of important processes in living biological systems.

Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord

Uckermann, Ortrud; Galli, Roberta; Beiermeister, Rudolf; Sitoci-Ficici, Kerim-Hakan; Later, Robert; Leipnitz, Elke; Neuwirth, Ales; Chavakis, Triantafyllos; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
88.73394%
Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.

Surface recombination and charged exciton in nanocrystal quantum dots on photonic crystals under two-photon excitation

Xu, Xingsheng
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em 06/06/2014 EN
Relevância na Pesquisa
68.885703%
In this study, the two-photon excited fluorescence spectra from cadmium selenide quantum dots (QDs) on a silicon nitride photonic crystal (PhC) membrane under femtosecond laser irradiation were investigated. These spectra can be fit to a tri-Gaussian function in which one component is negative in amplitude, and in which the Gaussian components with positive amplitude are assigned to exciton emission and charged-exciton emission and that with negative amplitude is assigned to absorption from surface recombination. The photonic crystal enhance the charged-exciton emission and exciton emission and, at the same time, also the absorption from surface recombination. Both the charged-exciton emission and the surface recombination are related to Auger recombination; therefore, the photonic crystal controls both radiative recombination and non-radiative recombination. The asymmetries of the two-photon excited fluorescence spectra are due to not only the location of the resonant guide mode of the PhC slab but also the enhancement of the absorption from surface recombination by PhC.

Molecular heterogeneity of O-acetylserine sulfhydrylase by two-photon excited fluorescence fluctuation spectroscopy.

Chirico, G; Bettati, S; Mozzarelli, A; Chen, Y; Müller, J D; Gratton, E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/2001 EN
Relevância na Pesquisa
69.84109%
O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation. We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon two-photon excitation. We sampled the fluorescence intensity from a small number of molecules (approximately 10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules. The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine. Their relative abundance is estimated to be 4:1, whereas the ratio of their two-photon cross sections is reversed with respect to the single-photon excitation case. Consistent results are obtained from the measurement of the lifetime decays...