Relevant bibliographies by topics / Confocal fluorescence microscopy

Academic literature on the topic 'Confocal fluorescence microscopy'

Author: Grafiati
Published: 4 June 2021
Last updated: 24 January 2023

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  1. Journal articles
  2. Dissertations / Theses
  3. Books
  4. Book chapters
  5. Conference papers
  6. Reports

Journal articles on the topic "Confocal fluorescence microscopy":

1

Zhibin Wang, Zhibin Wang, Guohua Shi Guohua Shi, and Yudong Zhang Yudong Zhang. "Adaptive aberration correction in confocal scanning fluorescence microscopy." Chinese Optics Letters 12, s1 (2014): S11103–311105. http://dx.doi.org/10.3788/col201412.s11103.

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Volkov, I. A., N. V. Frigo, L. F. Znamenskaya, and O. R. Katunina. "Application of Confocal Laser Scanning Microscopy in Biology and Medicine." Vestnik dermatologii i venerologii 90, no. 1 (February 24, 2014): 17–24. http://dx.doi.org/10.25208/0042-4609-2014-90-1-17-24.

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Fluorescence confocal laser scanning microscopy and reflectance confocal laser scanning microscopy are up-to-date highend study methods. Confocal microscopy is used in cell biology and medicine. By using confocal microscopy, it is possible to study bioplasts and localization of protein molecules and other compounds relative to cell or tissue structures, and to monitor dynamic cell processes. Confocal microscopes enable layer-by-layer scanning of test items to create demonstrable 3D models. As compared to usual fluorescent microscopes, confocal microscopes are characterized by a higher contrast ratio and image definition.
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Wright, S. J., J. S. Walker, H. Schatten, C. Simerly, J. J. McCarthy, and G. Schatten. "Confocal fluorescence microscopy with the tandem scanning light microscope." Journal of Cell Science 94, no. 4 (December 1, 1989): 617–24. http://dx.doi.org/10.1242/jcs.94.4.617.

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Applications of the tandem scanning confocal microscope (TSM) to fluorescence microscopy and its ability to resolve fluorescent biological structures are described. The TSM, in conjunction with a cooled charge-coupled device (cooled CCD) and conventional epifluorescence light source and filter sets, provided high-resolution, confocal data, so that different fluorescent cellular components were distinguished in three dimensions within the same cell. One of the unique features of the TSM is the ability to image fluorochromes excited by ultraviolet light (e.g. Hoechst, DAPI) in addition to fluorescein and rhodamine. Since the illumination is dim, photobleaching is insignificant and prolonged viewing of living specimens is possible. Series of optical sections taken in the Z-axis with the TSM were reproduced as stereo images and three-dimensional reconstructions. These data show that the TSM is potentially a powerful tool in fluorescence microscopy for determining three-dimensional relationships of complex structures within cells labeled with multiple fluorochromes.
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Welzel, J., Raphaela Kästle, and Elke C. Sattler. "Fluorescence (Multiwave) Confocal Microscopy." Dermatologic Clinics 34, no. 4 (October 2016): 527–33. http://dx.doi.org/10.1016/j.det.2016.06.002.

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Nie, Shuming, Daniel T. Chiu, and Richard N. Zare. "Real-time observation of single molecules by confocal fluorescence microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 53 (August 13, 1995): 60–61. http://dx.doi.org/10.1017/s0424820100136672.

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The ability to detect, identify, and manipulate individual molecules offer exciting possibilities in many fields, including chemical analysis, materials research, and the biological sciences. A particularly powerful approach is to combine the exquisite sensitivity of laser-induced fluorescence and the spatial localization and imaging capabilities of diffraction-limited or near-field optical microscopes. Unlike scanning tunneling microscopy (STM) and atomic force microscopy (AFM), which lack molecular specificity, optical spectroscopy and microscopy techniques can be used for real-time monitoring and molecular identification at nanometer dimensions or in ultrasmall volumes.We report the use of confocal fluorescence microscopy coupled with a diffraction-limit laser beam and a high-efficiency photodiode for real-time detection of single fluorescent molecules in solution at room temperature. Rigler and Eigen have also demonstrated single-molecule detection with a confocal microscope and fluorescence correlation spectroscopy. The probe (or sampling) volume is effectively an elongated cylinder, with its radius being determined by optical diffraction and length by spherical aberration.
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Jason Kirk. "Beyond the Hype - Is 2-Photon Microscopy Right for You?" Microscopy Today 11, no. 2 (April 2003): 26–29. http://dx.doi.org/10.1017/s1551929500052469.

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Confocal microscopes have come a long way in the past decade. Not only are they more stable and easier to use than ever before, but their cost has dropped enough that multi-user facilities are finding competition from individual labs using the new breed of "personal" confocals. In fact it has, in some cases, become the de facto standard for fluorescence imaging regardless of whether the user actually has requirements for it or not.But, researchers always have an ear out for something better. Enter 2-photon microscopy (2PLSM). The “bigger & badder” cousin of the confocal microscope has become a new weapon in the arsenal of a microscopy industry that caters to researchers who can't wait to fill their labs with the latest and greatest imaging systems. Advertised by the industry and researchers alike as a technique that seems to solve most of the problems that plague confocal, “2-photon” has become the new buzzword in the vocabulary of researchers eager to enhance their fluorescence applications.
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Cheng, P. C., S. J. Pan, A. Shih, W. S. Liou, M. S. Park, T. Watson, J. Bhawalkar, and P. Prasard. "Two-Photon Laser Scanning Confocal Microscopy." Microscopy and Microanalysis 3, S2 (August 1997): 847–48. http://dx.doi.org/10.1017/s1431927600011120.

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Two-photon fluorescence microscopy has become an important research tool in both biological and material sciences. The technique uses long wavelength, typically in the near IR, as the excitation light to obtain shorter wavelength fluorescence (e.g. visible light). Because of the low linear absorption coefficient of most biological and polymeric specimens, this technique allows deeper penetration of the excitation beam, achieving optical sectioning to a depth of 250μm or more into the specimen. As a result of the quadratic dependency of the two-photon induced fluorescence to the excitation intensity, the fluorescent emission and photobleaching are limited to the vicinity of focal spot. This capability of addressing a specimen’s 3D space allows exciting possibilities in biological researches, such as 3D photobleaching recovery experiment.Two-photon confocal fluorescence microscopy is ideal for the study of thick biological and material specimen in 3D. For example, Figure 1 shows a three-dimensional isosurface rendered image of a vascular bundle from a maize stem.
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Oostveldt, P., and S. Bauwens. "Quantitative fluorescence in confocal microscopy." Journal of Microscopy 158, no. 2 (May 1990): 121–32. http://dx.doi.org/10.1111/j.1365-2818.1990.tb02985.x.

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VISSCHER, K., G. J. BRAKENHOFF, and T. D. VISSER. "Fluorescence saturation in confocal microscopy." Journal of Microscopy 175, no. 2 (August 1994): 162–65. http://dx.doi.org/10.1111/j.1365-2818.1994.tb03479.x.

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Ragazzi, Moira, Simonetta Piana, Caterina Longo, Fabio Castagnetti, Monica Foroni, Guglielmo Ferrari, Giorgio Gardini, and Giovanni Pellacani. "Fluorescence confocal microscopy for pathologists." Modern Pathology 27, no. 3 (September 13, 2013): 460–71. http://dx.doi.org/10.1038/modpathol.2013.158.

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Journal articles Dissertations / Theses Books

Dissertations / Theses on the topic "Confocal fluorescence microscopy":

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Eigenbrot, Ilya Vladimirovich. "A time-resolved confocal fluorescence microscope." Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342331.

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Alawadhi, Fahimah. "Statistical image analysis and confocal microscopy." Thesis, University of Bath, 2001. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341639.

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Jiang, Shihong. "Non-scanning fluorescence confocal microscopy using laser speckle illumination." Thesis, University of Nottingham, 2005. http://eprints.nottingham.ac.uk/10139/.

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Confocal scanning microscopy (CSM) is a much used and advantageous form of microscopy. Although CSM is superior to conventional microscopy in many respects, a major disadvantage is the complexity of the scanning process and the sometimes long time to perform the scan. In this thesis a novel non-scanning fluorescence confocal microscopy is investigated. The method uses a random time-varying speckle pattern to illuminate the specimen, recording a large number of independent full-field frames without the need for a scanning system. The recorded frames are then processed in a suitable way to give a confocal image. The goal of this research project is to confirm the effectiveness and practicality of speckle-illumination microscopy and to develop this proposal into a functioning microscope system. The issues to be addressed include modelling of the system performance, setting up experiments, computer control and image processing. This work makes the following contributions to knowledge: * The development of criteria for system performance evaluation * The development of methods for speckle processing, whereby the number of frames required for an image of acceptable quality can be reduced * The implementation of non-scanning fluorescence confocal microscopy based upon separate recording of the speckle patterns and the fluorescence frames, demonstrating the practicality and effectiveness of this method * The realisation of real-time image processing by optically addressed spatial light modulator, showing how this new form of optical arrangement may be used in practice The thesis is organised into three main segments. Chapters 1-2 review related work and introduce the concepts of fluorescence confocal microscopy. Chapters 3-5 discuss system modelling and present results of performance evaluation. Chapters 6-8 present experimental results based upon the separate recording scheme and the spatial light modulation scheme, draw conclusions and offer some speculative suggestions for future research.
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Wang, Xiao. "Confocal angle resolved linear dichroism microscopy for structural fluorescence imaging." Ecole centrale de Marseille, 2013. http://tel.archives-ouvertes.fr/docs/00/87/10/10/PDF/Wang-Thesis.pdf.

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La microscopie de fluorescence a récemment été complétée par une technique appelée dichroïsme linéaire résolu angulairement, basé sur le fait que l'absorption de la lumière est un processus sensible à l'orientation moléculaire. En analysant la réponse d'émission de fluorescence en fonction de l'orientation de la polarisation de la lumière excitatrice, cette technique permet de remonter à l'information d'orientation sur un ensemble de molécules fluorescentes, plus précisément son angle d'orientation moyenne et l'amplitude de ses fluctuations angulaires autour de cette moyenne. Dans cette thèse, nous mettons en oeuvre de nouvelle méthodes et instrumentations capables d'améliorer la robustesse et la rapidité de l'analyse de données de réponses résolues en polarisation, la vitesse de l'acquisition de données, et d'explorer la possibilité de mesurer l'orientation 3D de molécules. Nous proposons une méthode capable de mesurer les propriétés d'orientation de sondes lipidiques fluorescentes par l'utilisation d'un disque de Nipkow couplé à une imagerie par caméra, et combiné avec la modulation rapide de la polarisation par modulateur électro-optique. Une nouvelle méthode de traitement de données est développée pour considérablement améliorer la rapidité et la précision de l'information par une étude des sources de bruit et d'incertitude, dues au bruit et aux facteurs instrumentaux. Cette technique a été testée avec succés sur des vésicules géantes uni-lamellaires et sur cellules vivantes, marquées par les sondes lipidiques DiIC18 et di-8-ANEPPQ. Cette méthode est capable d'acquérir une information précise sur l'orientation moléculaire à une cadence d'une image par seconde. Enfin, afin de sonder de manière non ambiguë l'orientation 3D d'un ensemble de molécules, une nouvelle méthode est proposée, supportée par des simulations numériques, basée sur la variation hors plan de la polarisation d'excitation dans le volume focal par une somme cohérente de champs polarisés linéairement et radialement
Based on the fact that the absorption of light is a molecular-orientation sensitive process, fluorescence microscopy has been recently completed by a technique called angle-resolved linear dichroism. By analyzing the fluorescence emission response with respect to the polarization orientation of the exciting light, this technique allows retrieving orientation information of an ensemble of fluorescent molecules, namely the average orientation angle and the amplitude of the angular fluctuations around this average. In this PhD thesis, we implement new methods and instrumentation tools able to improve the robustness and speed of the polarization resolved data analysis, the rate of the data acquisition, and at last to explore the possibility to record molecular 3D orientation information. A scheme able to monitor the real-time orientation properties of fluorescent lipid probes is proposed using a high-speed spinning disk coupled to camera imaging, combined with fast switching of the polarization state by an electro optical modulator. A new data processing method is developed which considerably improves the speed and the precision of the retrieved information by investigating the sources of bias and uncertainty due to noise and instrumentation factors. The technique has been successfully tested on giant unilamellar vesicles and on living cells labeled with different fluorescent lipid probes, DiIC18 and di-8-ANEPPQ. It was able to acquire precise molecular orientation images at full frame rates in the range of one frame per second. At last in order to probe unambiguously the 3D orientation information of an ensemble of molecules, a new method is proposed and supported by simulations, based on the out-of-plane tuning of the excitation polarization realized in the focusing volume by coherently summing linearly and radially polarized fields
5

Gösch, Michael. "Microfluidic analysis and parallel confocal detection of single molecules /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-663-4/.

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Risi, Matthew D. "Advances In Combined Endoscopic Fluorescence Confocal Microscopy And Optical Coherence Tomography." Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/332772.

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Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure. Results from an ongoing clinical study to detect ovarian cancer with a novel confocal fluorescent microendoscope are presented. As an imaging modality, confocal fluorescence microendoscopy typically requires exogenous fluorophores, has a relatively limited penetration depth (100μm), and often employs specialized aperture configurations to achieve real-time imaging in vivo. Two primary research directions designed to overcome these limitations and improve diagnostic capability are presented. Ideal confocal imaging performance is obtained with a scanning point illumination and confocal aperture, but this approach is often unsuitable for real-time, in vivo biomedical imaging. By scanning a slit aperture in one direction, image acquisition speeds are greatly increased, but at the cost of a reduction in image quality. The design, implementation, and experimental verification of a custom multi-point-scanning modification to a slit-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution while maintaining real-time imaging rates. In addition, the multi-point aperture geometry greatly reduces the effects of tissue scatter on imaging performance. Optical coherence tomography (OCT) has seen wide acceptance and FDA approval as a technique for ophthalmic retinal imaging, and has been adapted for endoscopic use. As a minimally invasive imaging technique, it provides morphological characteristics of tissues at a cellular level without requiring the use of exogenous fluorophores. OCT is capable of imaging deeper into biological tissue (~1-2 mm) than confocal fluorescence microscopy. A theoretical analysis of the use of a fiber-bundle in spectral-domain OCT systems is presented. The fiber-bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the optical coherence tomography data. However, the multi-mode characteristic of the fibers in the fiber-bundle affects the depth sensitivity of the imaging system. A description of light interference in a multi-mode fiber is presented along with numerical simulations and experimental studies to illustrate the theoretical analysis.
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Slimani, Amel. "Photonic approach for the study of dental hard tissues and carious lesion detection." Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT125.

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Les propriétés photoniques des tissus durs dentaires nous ont permis d’étudier l’email et la dentine a un niveau moléculaire (in vitro) en utilisant des techniques de microscopie optique non linéaires. La microscopie confocale Raman est technique d’imagine de haute résolution permettant d’analyse d’échantillon sans préparation spécifique ni marquage. Cette méthode nous a permis de reconstituer une cartographie de la réticulation du collagène et de la cristallinité au niveau de la jonction émail-dentine et cela avec une résolution spatiale non atteinte jusque-là. Cette analyse chimique de la jonction émail-dentine a permis de redéfinir la largeur de cette zone de transition. Cette largeur est nettement supérieure à celles proposées par les études précédentes. Par ailleurs, l’étude portant sur les changements de fluorescence intrinsèque entre les tissues dentaires sains et cariés suggèrent l’implication de la protoporphyrin IX et de la pentosidine dans l’expression de la fluorescence rouge des tissus cariés. La microscopie multiphotonique quant à elle nous a permis de détecter la lésion carieuse et de suivre son développement en utilisant la génération de seconde harmonique (SHG) et la fluorescence par excitation à deux photons (2PEF). Nos études ont démontré la validité du ratio SHG/2PEF comme paramètre fiable pour la détection de la lésion carieuse. Les études proposées par cette thèse montrent le potentiel des propriétés photoniques de l’émail et de la dentine en utilisant les microscopies Raman et multiphotoniques dans l’étude de ces tissus au niveau moléculaire. Cela offre de nouvelles perspectives en recherche et en applications cliniques
Photonic properties of dental hard tissues allowed us to proceed to in vitro analysis of enamel and dentin on a molecular level. Confocal Raman microscopy has been used to produce a mapping of collagen cross-link and crystallinity of human dentin–enamel junction (DEJ) with a spatial resolution not achieved up to now. The method is a non-invasive, label-free and a high spatial resolution imaging technique. This chemical analysis of DEJ led us to redefine a wider width of this transition zone and advance our understanding of dental histology. A study on the intrinsic fluorescence changes of sound and carious tissues using conventional fluorescence microscopy suggests the involvement of protoporphyrin IX and pentosidine in the fluorescence red-shift observed in carious tissues. Multiphoton microscopy allowed to detect nonlinear optical signal changes during caries process using second harmonic generation (SHG) and two-photon excitation fluorescence (2PEF). Our studies led us to propose the ratio SHG/2PEF as valuable parameter to monitor caries lesion. Collectively, advances described in this thesis show the potential of photonic properties of enamel and dentin using Raman and multiphoton microcopies for molecular investigations on sound as much as on carious tissues. It opens new perspective in dental research and clinical applications
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Tsutae, Fernando Massayuki. "Espectroscopia de correlação de fluorescência aplicada em estudos de sistemas moleculares, biológicos e celulares." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-14102016-101124/.

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A espectroscopia de correlação de fluorescência (FCS) é uma das diferentes técnicas de análise por imagens de alta resolução espacial e temporal de biomoléculas em concentrações extremamente baixas. Ela se tornou uma técnica extremamente poderosa e sensível em áreas como bioquímica e biofísica. Como uma técnica bem estabelecida, ela é utilizada para medir concentrações locais de biomoléculas, através da marcação com moléculas fluorescentes. Coeficientes de difusão e constantes cinéticas também podem ser medidos através de FCS assim como detecção de molécula única. Ela também pode dar informação precisa sobre interações de antígeno-anticorpo, ácidos nucleicos e proteínas. Através de uma combinação de marcadores de alto rendimento quântico, fontes de luz estável (lasers), detecção ultrassensível e microscopia confocal, é possível realizar medidas de FCS em volumes de fentolitros (fL) e em concentrações de nanomolar (nM) em soluções aquosas ou em células vivas. Em contraste com outras técnicas de fluorescência, a sensibilidade da FCS aumenta com a diminuição da concentração do fluoróforo marcador, porque o parâmetro de interesse não é a intensidade de emissão de fluorescência, mas sim as flutuações espontâneas da fluorescência. Durante o tempo em que a partícula ou molécula atravessa o volume de medida pode ocorrer mudanças conformacionais e reações químicas e fotofísicas que alteram as características de emissão do fluoróforo e causam flutuações no sinal detectado. Estas flutuações são então monitoradas e transformadas em uma curva de autocorrelação, por intermédio de um software comercial que emprega um modelo físico apropriado para FCS. Em nosso estudo, utilizamos um marcador comercial (ALEXA 488®) para marcar proteínas. Primeiramente utilizamos a técnica de FCS para medir concentrações extremamente baixas de marcadores fluorescentes. Também realizamos um experimento testando a influência da viscosidade do meio na difusão livre do fluoróforo, assim como as melhores condições em que temos um melhor sinal de FCS. Por fim, estudamos a difusão de proteínas marcadas (PUC II e IV) em meio aquoso (PBS) e no interior de células.
Fluorescence correlation spectroscopy (FCS) is one of the many different modes of high-resolution spatial and temporal analysis of extremely low concentrated biomolecules. It has become a powerful and sensitive tool in fields like biochemistry and biophysics. As a well established technique, it is used to measure local concentrations of fluorescently labeled biomolecules, diffusion coefficients, kinetic constants and single molecule studies. Through a combination of high quantum yield fluorescent dyes, stable light sources (lasers), ultrasensitive detection and confocal microscopy is possible to perform FCS measurements in femtoliters volumes and nanomolar concentrations in aquous solution or in live cells. Unlike with other fluorescence technics, its sensibility increases with the decrease of dye concentrarion, because the main factor is not the emission intensity itself. Instead this, spontaneous statistical fluctuation of fluorescence becomes the main factor in FCS analisys. During the time that the conjugated-dye cross the volume detection can occur conformational changes, chemical reaction and photophysical processes that can change the emission properties of the dye and, then, change the detected sinal. This fluctuations are tracked and changed into a autocorrelation curve, by a specific software, appropriate to perform FCS analisys. In our study, we use comercial dye (Alexa 488) to label proteins. Firstly, we applied FCS to measure extremally diluted concentrations of dyes (~1 nM). We have performed experiments testing the influence of the viscosity medium in the free difusion of the dyes and the optical apparatus and conditions that result in the best FCS signal. We also have studied protein diffusion (PUC II e IV) in aquous medium (PBS) and toward the inner of the cells.
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Kakade, Rohan. "Improved resolution and signal-to-noise ratio performance of a confocal fluorescence microscope." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33699/.

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A culmination of theory, techniques and devices stemming from a wide variety of sources and disciplines, optical microscopy presents vast possibilities for visualisation of small structures. One of the most fundamental yet significant optical microscopy techniques is Confocal Fluorescence Microscopy (CFM). CFM is studied here by analysing its performance with respect to the two most important metrics - Signal-to-noise ratio and 3D optical resolution. Several authors have commented on the inherent inefficiency of imaging systems such as CFM to utilise the available light when providing resolution beyond the well-known diffraction limit, primarily due to the precise mechanisms that help realise the resolution gain in the first place. In CFM, the detection pinhole is the key mechanism that helps realise up to 1.4 times resolution improvement over conventional wide-field microscopy techniques by trading off SNR. First, an investigation of the inherent SNR-resolution trade-off in a CFM system is studied; the impact of the detection pinhole geometry on the performance of a CFM is examined by means of an effective trade-off curve. Using alternative pinhole geometries in conjunction with new detection schemes, it is next shown how performance gains are realised in both the lateral and axial directions. Examined next is a recently developed detection scheme called subtractive imaging; wherein a special annular pinhole is used to divide the confocal point spread function signal into two detectors. By using fast point detectors in place of CCD arrays, it is shown how using numerical optimisation yields an optimum “differential pinhole” to achieve considerable 3D resolution gains over conventional (circular pinhole based) CFM systems. By examining the trade-off curves it is also shown that the proposed design is able to offer simultaneous and maximum performance gains up to a considerably high SNR in comparison to conventional (circular pinhole) based CFM systems. Lastly, the work will propose the use of a deconvolution technique and an alternative detection scheme to demonstrate substantially higher improvements in the quality of images acquired by a CFM system. Image reconstruction is a tried and tested image post processing strategy to realise super resolution. An image reconstruction technique, based on an expectation maximisation maximum likelihood (EM-ML) algorithm is used in conjunction with array detectors to demonstrate enhanced resolution and noise performance of a CFM system. The point scan method used here renders the algorithm slow with long run times. To mitigate this, structured illumination is used to show how similar resolution gains in the array detector based CFM systems could be realised but in a much shorter time.
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Ferro, Daniela Peixoto 1981. "Aplicação da biofotônica para o estudo de cicatrizes." [s.n.], 2015. http://repositorio.unicamp.br/jspui/handle/REPOSIP/312786.

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Orientador: Konradin Metze
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A aplicação integrada de técnicas modernas, como a Geração do Segundo Harmônico (SHG) e os tempos de vida da fluorescência (FLIM), com análise de imagens matemáticas nos permitem visualizar detalhes não vistos por microscopia de luz convencional. O objetivo deste estudo foi investigar se isto também pode ser aplicado para a investigação de tecido cicatricial. Foram estudados 28 casos de preparações histológicas de rotina, de quelóides, cicatrizes hipertróficas e normais. A Fluorescência de dois fótons e SHG foram obtidas por um microscópio multifóton (LSM 780 NLO-Zeiss), em objetiva de 40X e excitados por um laser Mai Tai de Ti: Safira (comprimento de onda de 940 nm). Foram adquiridas imagens em 3D e foram criadas imagens justapostas a fim de comparar diferentes cicatrizes ou várias regiões no interior da mesma cicatriz com análise de imagens informatizadas. Variáveis de Textura derivadas a partir da matriz de coocorrência das imagens de fluorescência mostraram diferenças significativas entre as cicatrizes normais, cicatrizes hipertróficas e quelóides. Para a análise do FLIM, foi utilizado um sistema composto por um microscópio confocal (LSM780-NLO- Zeiss), com objetiva de 40x e um sistema FLIM acoplado. As amostras foram excitadas por um laser de diodo a 405nm. Estudamos secções não coradas de 32 casos processados rotineiramente de tecido cicatricial incluídos em parafina. As áreas das regiões centrais e periféricas foram selecionadas aleatoriamente e comparadas. Os tempos de vida de fluorescência das hemácias serviram como padrão interno. Os tempos de vida do colágeno em áreas centrais em todos os tipos de cicatrizes foram significativamente mais longo do que em áreas periféricas. Houve correlação positiva entre os tempos de vida de fluorescência das hemácias e as fibras de colágeno entre os casos. Em resumo, o SHG e a técnica Flim revelam em cicatrizes rotineiramente processadas, características morfológicas dos tecidos, que não podem ser detectadas por microscopia de luz convencional
Abstract: The integrated application of modern techniques such as Second Harmonic Generation (SHG) and fluorescence lifetime imaging (FLIM) with mathematical image analysis enable us to visualize details not seen by conventional light microscopy. The aim of this study was to investigate whether this could also be true for the investigation of scar tissue. 28 routine histological preparations of keloids, hypertrophic and normal scars were studied. Two-photon fluorescence and SHG was obtained by a multiphoton microscope (LSM 780 NLO-Zeiss (at 40X objective magnification) and a Mai Tai Ti: Sapphire laser with excitation at 940 nm wavelength. 3D reconstructed patchwork images were created in order to compare different scars or various regions inside the same scar with computerized image analysis. Texture variables derived from the co- occurrence matrix of the fluorescence images showed significant differences between normal scars, hypertrophic scars and keloids. For FLIM analysis we used a system composed of a confocal microscope Zeiss LSM780 Upright-NLO with the 40x objective and a FLIM detection system. The samples were excited by a laser diode at 405nm. We studied unstained sections of 32 routinely processed and paraffin-embedded cases of scar tissue. Randomly selected areas of the central and peripheral regions were compared. The fluorescence lifetimes of red blood cells served as internal standard. Lifetimes of collagen in central areas of all scar types were significantly longer than in the periphery. There was a significant positive correlation between the fluorescence lifetimes of red blood cells and collagen fibers among the cases. In summary, SHG and FLIM techniques reveal in routinely processed scar tissue morphological characteristics, which cannot be detected by conventional light microscopy
Doutorado
Biologia Estrutural, Celular, Molecular e do Desenvolvimento
Doutora em Fisiopatologia Médica
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Books on the topic "Confocal fluorescence microscopy":

1

Muller, Michiel. Introduction to confocal fluorescence microscopy. 2nd ed. Bellingham, Wash: SPIE Press, 2006.

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Brian, Matsumoto, and American Society for Cell Biology., eds. Cell biological applications of confocal microscopy. 2nd ed. Amsterdam: Academic Press, 2002.

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Periasamy, Ammasi, and Wilson Tony. Confocal, multiphoton, and nonlinear microscopic imaging III: 17-18 June 2007, Munich, Germany. Edited by SPIE (Society), Optical Society of America, European Optical Society, Wissenschaftliche Gesellschaft Lasertechnik, and Deutsche Gesellschaft für Lasermedizin. Bellingham, Wash., USA: SPIE, 2007.

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Kevin, Foskett J., and Grinstein Sergio 1950-, eds. Noninvasive techniques in cell biology. New York: Wiley-Liss, 1990.

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David, Shotton, ed. Electronic light microscopy: The principles and practice of video-enhanced contrast, digital intensified fluorescence, and confocal scanning light microscopy. New York: Wiley-Liss, 1993.

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T, Mason W., ed. Fluorescent and luminescent probes for biological activity: A practical guide to technology for quantitative real-time analysis. London: Academic Press, 1993.

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Conference on Multidimensional Spectroscopy: Acquisition, Interpretation, and Automation (1998 San Jose, Calif.). Proceedings of three-dimensional and multidimensional microscopy: Image acquisition and processing V : 27-29 January 1998, San Jose, California. Edited by Cogswell Carol J, Society of Photo-optical Instrumentation Engineers., and International Biomedical Optics Society. Bellingham, Wash., USA: SPIE, 1998.

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name, No. Three-dimensional and multidimensional microscopy: Image acquisition and processing X : 28-29 January 2003, San Jose, California, USA. Bellingham, WA: SPIE, 2003.

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R, José-Angel Conchello, Carol J. Cogswell, and Wilson Tony. Three-dimensional and multidimensional microscopy: Image acquisition and processing XIII : 24-26 January 2006, San Jose, California, USA. Edited by Society of Photo-optical Instrumentation Engineers. Bellingham, Wash: SPIE, 2006.

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José-Angel, Conchello R., Cogswell Carol J, Wilson Tony, and Society of Photo-optical Instrumentation Engineers., eds. Three-dimensional and multidimensional microscopy: Image acquisition and processing XII : 25-27 January 2005, San Jose, California, USA. Bellingham, Wash., USA: SPIE, 2005.

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Book chapters on the topic "Confocal fluorescence microscopy":

1

Naredi-Rainer, Nikolaus, Jens Prescher, Achim Hartschuh, and Don C. Lamb. "Confocal Microscopy." In Fluorescence Microscopy, 165–202. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2017. http://dx.doi.org/10.1002/9783527687732.ch5.

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Naredi-Rainer, Nikolaus, Jens Prescher, Achim Hartschuh, and Don C. Lamb. "Confocal Microscopy." In Fluorescence Microscopy, 175–213. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2013. http://dx.doi.org/10.1002/9783527671595.ch5.

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Jerome, W. Gray, and Robert L. Price. "Fluorescence Microscopy." In Basic Confocal Microscopy, 37–71. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-97454-5_3.

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Jerome, W. Gray (Jay), and Robert L. Price. "Fluorescence Microscopy." In Basic Confocal Microscopy, 29–59. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-0-387-78175-4_3.

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Gerritsen, Hans C. "Confocal Fluorescence Lifetime Imaging." In Fluorescence Microscopy and Fluorescent Probes, 35–46. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4899-1866-6_3.

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Demandolx, Denis, and Jean Davoust. "Subcellular Cytofluorometry in Confocal Microscopy." In Fluorescence Microscopy and Fluorescent Probes, 279–83. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4899-1866-6_43.

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Jerome, W. Gray. "The Theory of Fluorescence." In Basic Confocal Microscopy, 21–36. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-97454-5_2.

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Jerome, W. Gray (Jay). "The Theory of Fluorescence." In Basic Confocal Microscopy, 17–28. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-0-387-78175-4_2.

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Hibbs, Alan R. "Fluorescence Immunolabelling." In Confocal Microscopy for Biologists, 259–77. Boston, MA: Springer US, 2004. http://dx.doi.org/10.1007/978-0-306-48565-7_11.

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Gomez-Lazaro, M., A. Freitas, and C. C. Ribeiro. "Confocal Raman microscopy." In Fluorescence Imaging and Biological Quantification, 65–83. Boca Raton : Taylor & Francis, 2017.: CRC Press, 2017. http://dx.doi.org/10.1201/9781315121017-5.

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Conference papers on the topic "Confocal fluorescence microscopy":

1

Rüttinger, Steffen, Peter Kapusta, Volker Völlkopf, Felix Koberling, Rainer Erdmann, and Rainer Macdonald. "Fluorescence performance standards for confocal microscopy." In BiOS, edited by Ammasi Periasamy, Peter T. C. So, and Karsten König. SPIE, 2010. http://dx.doi.org/10.1117/12.840501.

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Doglia, Silvia M., L. Bianchi, Roberto Colombo, N. Allam, Hamid Morjani, Michel Manfait, and A. M. Villa. "Confocal fluorescence microscopy of living cells." In Laser Spectroscopy of Biomolecules: 4th International Conference on Laser Applications in Life Sciences, edited by Jouko E. Korppi-Tommola. SPIE, 1993. http://dx.doi.org/10.1117/12.146189.

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Stelzer, Ernst H. K., and Robert Bacallao. "Confocal Fluorescence Microscopy Of Epithelial Cells." In 1988 International Congress on Optical Science and Engineering. SPIE, 1989. http://dx.doi.org/10.1117/12.950336.

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Wang, Yu, Konstantin Maslov, Chulhong Kim, Song Hu, and Lihong V. Wang. "Integrated photoacoustic and fluorescence confocal microscopy." In SPIE BiOS, edited by Alexander A. Oraevsky and Lihong V. Wang. SPIE, 2011. http://dx.doi.org/10.1117/12.874888.

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Luo, Yuan, Chou-Min Chia, Hung-Chun Wang, and Yu-hsin Chia. "Multi-focal holographic slit confocal fluorescence microscopy." In Biomedical Imaging and Sensing Conference, edited by Osamu Matoba, Yasuhiro Awatsuji, Toyohiko Yatagai, and Yoshihisa Aizu. SPIE, 2018. http://dx.doi.org/10.1117/12.2316615.

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Bertero, M., P. Boccacci, and E. R. Pike. "Inverse Problems In Fluorescence Confocal Scanning Microscopy." In 1988 International Congress on Optical Science and Engineering. SPIE, 1989. http://dx.doi.org/10.1117/12.950302.

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Rafeq, S., A. Ernst, A. Majid, G. Michaud, C. Reddy, and F. Herth. "Bronchoscopic Imaging Using Fibered Confocal Fluorescence Microscopy." In American Thoracic Society 2009 International Conference, May 15-20, 2009 • San Diego, California. American Thoracic Society, 2009. http://dx.doi.org/10.1164/ajrccm-conference.2009.179.1_meetingabstracts.a5772.

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Loterie, Damien, Demetri Psaltis, and Christophe Moser. "Confocal microscopy via multimode fibers: fluorescence bandwidth." In SPIE BiOS, edited by Thomas G. Bifano, Joel Kubby, and Sylvain Gigan. SPIE, 2016. http://dx.doi.org/10.1117/12.2208017.

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Rodrigues, Isabel, Joao Xavier, and Joao Sanches. "Fluorescence Confocal Microscopy Imaging denoising with photobleaching." In 2008 30th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE, 2008. http://dx.doi.org/10.1109/iembs.2008.4649633.

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Fersch, Daniel, Pavel Malý, Jessica Rühe, Victor Lisinetskii, Matthias Hensen, Frank Würthner, and Tobias Brixner. "Single-Molecule Ultrafast Fluorescence-Detected Pump–Probe Microscopy." In International Conference on Ultrafast Phenomena. Washington, D.C.: Optica Publishing Group, 2022. http://dx.doi.org/10.1364/up.2022.m4a.3.

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Reports on the topic "Confocal fluorescence microscopy":

1

Hoffmeyer, Michaela. In Vivo Fluorescence Confocal Microscopy to Investigate the Role of RhoC in Inflammatory Breast Cancer. Fort Belvoir, VA: Defense Technical Information Center, April 2005. http://dx.doi.org/10.21236/ada435616.

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Wickramaratne, Chathuri, Emily Sappington, and Hanadi Rifai. Confocal Laser Fluorescence Microscopy to Measure Oil Concentration in Produced Water: Analyzing Accuracy as a Function of Optical Settings. Journal of Young Investigators, June 2018. http://dx.doi.org/10.22186/jyi.34.6.39-47.

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Morales García, María Dolores. Uso de la fluorescencia y la microscopía confocal en la investigación científica. Sociedad Española de Bioquímica y Biología Molecular (SEBBM), July 2012. http://dx.doi.org/10.18567/sebbmdiv_rpc.2012.07.1.

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Darrow, C., T. Huser, C. Campos, M. Yan, S. Lane, and R. Balhorn. Single Fluorescent Molecule Confocal Microscopy: A New Tool for Molecular Biology Research and Biosensor Development. Office of Scientific and Technical Information (OSTI), March 2000. http://dx.doi.org/10.2172/792442.

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