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2.
Appl Opt ; 58(12): 3104-3114, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31044784

RESUMO

Modern microscopes are designed with functionalities that are tailored to enhance image contrast. Dark-field imaging, phase contrast, differential interference contrast, and other optical techniques enable biological cells and other phase-only objects to be visualized. Quantitative phase imaging refers to an emerging set of techniques that allow for the complex transmission function of the sample to be measured. With this quantitative phase image available, any optical technique can then be simulated; it is trivial to generate a phase contrast image or a differential interference contrast image. Rheinberg illumination, proposed almost a century ago, is an optical technique that applies color contrast to images of phase-only objects by introducing a type of optical staining via an amplitude filter placed in the illumination path that consists of two or more colors. In this paper, the complete theory of Rheinberg illumination is derived, from which an algorithm is proposed that can digitally simulate the technique. Results are shown for a number of quantitative phase images of diatom cells obtained via digital holographic microscopy. The results clearly demonstrate the potential of the technique for label-free color staining of subcellular features.


Assuntos
Diatomáceas/citologia , Holografia/métodos , Iluminação , Microscopia de Contraste de Fase/métodos , Coloração e Rotulagem/métodos , Algoritmos
3.
Eur Radiol ; 29(10): 5378-5385, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30937586

RESUMO

OBJECTIVE: To investigate the incidence, CT appearance, and implication for prognosis of the hollow adrenal gland sign (HAGS). METHODS: A total of 194 patients with septic shock and 24 patients with hemorrhagic shock (as control group) were retrospectively included in this study and the patients with septic shock were further divided into four subgroups (digestive tract diseases, DTD, n = 49; biliary and pancreatic diseases, BPD, n = 41; postsurgical infection, PI, n = 64; and other diseases, OD, n = 40). All patients underwent a dual-phase contrast-enhanced CT within 1 week after diagnosis. CT findings and clinical records were reviewed. If in the arterial phase the central zone of adrenal gland showed temporally much lower attenuation than the peripheral zone, it was defined as HAGS positive. The incidence of the HAGS in patients with septic shock and hemorrhagic shock, the demographic features, and mortality between HAGS-positive and HAGS-negative patients in each group were respectively compared. RESULTS: The incidence of the HAGS in the septic shock group was nearly 30%, while it was 0 in the hemorrhagic shock group. There was no significant difference in age or gender between HAGS-positive and HAGS-negative patients in all groups, while the mortality of HAGS-positive patients was significantly higher than that of HAGS-negative patients in each group (p < 0.05). The concordance correlation coefficient value showed excellent reproducibility of the two observers (κ = 0.977). CONCLUSION: The HAGS is specific and common on dual-phase contrast-enhanced CT in patients with septic shock and predicts a poor prognosis. KEY POINTS: • The hollow adrenal gland sign (HAGS) newly described in this study is a special enhancing pattern of adrenal gland on dual-phase contrast-enhanced CT in patients with septic shock. • The HAGS is characterized by the much lower-attenuated central zone of the adrenal gland in arterial phase and it showed excellent reproducibility between different observers. • The HAGS is specific and common on dual-phase contrast-enhanced CT in patients with septic shock and predicts a poor prognosis.


Assuntos
Glândulas Suprarrenais/diagnóstico por imagem , Choque Séptico/diagnóstico por imagem , Adulto , Idoso , Meios de Contraste , Feminino , Humanos , Masculino , Microscopia de Contraste de Fase/métodos , Pessoa de Meia-Idade , Variações Dependentes do Observador , Prognóstico , Reprodutibilidade dos Testes , Estudos Retrospectivos , Choque Séptico/mortalidade , Tomografia Computadorizada por Raios X/métodos
4.
Nat Commun ; 10(1): 1877, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015402

RESUMO

Many microorganisms have evolved chemotactic strategies to exploit the microscale heterogeneity that frequently characterizes microbial habitats. Chemotaxis has been primarily studied as an average characteristic of a population, with little regard for variability among individuals. Here, we adopt a classic tool from animal ecology - the T-maze - and implement it at the microscale by using microfluidics to expose bacteria to a sequence of decisions, each consisting of migration up or down a chemical gradient. Single-cell observations of clonal Escherichia coli in the maze, coupled with a mathematical model, reveal that strong heterogeneity in the chemotactic sensitivity coefficient exists even within clonal populations of bacteria. A comparison of different potential sources of heterogeneity reveals that heterogeneity in the T-maze originates primarily from the chemotactic sensitivity coefficient, arising from a distribution of pathway gains. This heterogeneity may have a functional role, for example in the context of migratory bet-hedging strategies.


Assuntos
Quimiotaxia/genética , Escherichia coli/fisiologia , Modelos Biológicos , Fenótipo , Dimetilpolisiloxanos/química , Microscopia Intravital/métodos , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Contraste de Fase/métodos , Análise de Célula Única/métodos
5.
Analyst ; 144(9): 3136-3143, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30941383

RESUMO

Nosema bombycis (Nb) is the pathogen that causes pebrine in silkworms. Aldehydes are effective disinfectants commonly used in sericulture. However, the precise mechanism of their action on Nb spores remains unclear. Here, we used laser tweezers Raman spectroscopy to investigate the effects of glutaraldehyde and formaldehyde on individual Nb spores, as well as phase contrast microscopy imaging to monitor the germination dynamics of individual treated spores, to acquire a deeper understanding of the mechanism of action of aldehydes and to provide a theoretical reference for establishing an effective strategy for disease control in sericulture. The positions of the Raman peaks remained constant during treatment. The Raman intensity was enhanced and the germination rate of the spores significantly decreased with treatment time. Tlag, the time when individual spores begin to germinate, and Tgerm, the time for complete germination, increased with enhanced treatment. The germination time (ΔTgerm) showed no significant difference from that for untreated spores. Heterogeneity was shown, which is relevant to the resistance of Nb spores to aldehydes. The results indicate that glutaraldehyde and formaldehyde do not destroy the spore wall and plasma membrane, do not cause the leakage of intracellular components, and might not damage the extrusion apparatus. The effects of aldehydes on Nb spores are mainly on the spore coat. They may block the external factors that stimulate spore germination. Single-cell analysis based on novel optical techniques reveals the action of chemical sporicides on microsporidia spores in real time and explains the heterogeneity of cell stress resistance. These applications of new techniques offer new insight into traditional disinfectants.


Assuntos
Desinfetantes/farmacologia , Formaldeído/farmacologia , Glutaral/farmacologia , Nosema/efeitos dos fármacos , Esporos Fúngicos/efeitos dos fármacos , Microscopia de Contraste de Fase/métodos , Pinças Ópticas , Análise de Célula Única/métodos , Análise Espectral Raman/métodos
6.
J Low Genit Tract Dis ; 23(2): 176-181, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30907778

RESUMO

OBJECTIVES: Wet mount microscopy (WMM) is an ancillary test in the diagnosis of vulvovaginitis. However, there are little data about the impact of the sampling site. Our main objective was to determine the agreement between samples collected from different vaginal sites. MATERIALS AND METHODS: Five vaginal samples (one from each of the fornices and one from the lower third) were collected from 50 consecutive women and evaluated using phase-contrast WMM, including the following: lactobacillary grade, presence of other bacteria, clue cells, inflammation, and atrophy. Global percentage of agreement, κ index [poor (<0), slight (0.01-0.20), fair (0.21-0.40), moderate (0.41-0.60), substantial (0.61-0.80), and almost perfect (0.81-1)], sensitivity and negative predictive value were calculated. RESULTS: Global percentage of agreement was high for all individual parameters (>75%), but low for the final diagnosis (57%). Agreement rate was substantial or almost perfect for the evaluation of normal versus abnormal flora, substantial for the presence of absence of bacteria other than lactobacilli and for the presence of clue cells, and moderate to almost perfect for the presence of dysbiosis and cytolysis. Agreement rates were worse for inflammation, atrophy, and the presence of Candida. The lower third of the vagina had the highest sensitivity for Candida (0.6, 95% CI = 0.41-0.86) and bacterial vaginosis (0.92, 95% CI = 0.73-1.00), whereas the anterior fornix performed better for cytolytic vaginosis (0.75, 95% CI = 0.43-0.93). CONCLUSIONS: There is some variation in the results according to the sampling site, which may be overcome by collecting 2 samples (anterior fornix and lower third of the vagina).


Assuntos
Microscopia de Contraste de Fase/métodos , Manejo de Espécimes/métodos , Vagina/patologia , Vulvovaginite/diagnóstico , Vulvovaginite/patologia , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Adulto Jovem
7.
Biotechniques ; 66(3): 128-133, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30869548

RESUMO

The need for technologies to monitor cell health is increasing with advancements in the field of cell therapy and regenerative medicine. In this study, we demonstrated unlabeled optical metabolic imaging of cultured living cells. This imaging technique is based on motion vector analysis with a block-matching algorithm to compare sequential time-lapse images. Motion vector analysis evaluates the movement of intracellular granules observed with a phase-contrast microscope. We demonstrated that the motion speed of intracellular movement reflects adenosine triphosphate (ATP)-dependent intracellular trafficking in cells. We also confirmed that intracellular motion speed is correlated with the ATP concentrations of the cells. This assay can measure cellular viability at a single-cell level without requiring any reagents.


Assuntos
Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Contraste de Fase/métodos , Análise de Célula Única/métodos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/isolamento & purificação , Algoritmos , Linhagem Celular , Movimento Celular/genética , Humanos
8.
Asian Pac J Cancer Prev ; 20(3): 757-765, 2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30909682

RESUMO

Aim: Newcastle disease virus (NDV) is a member of genus Avulavirus within the family Paramyxoviridae. Interest of using NDV as an anticancer agent has arisen from its ability to kill tumor cells with limited toxicity to normal cells. Methods: In this investigation, the proliferation of brain tumor cell line, glioblastoma multiform (DBTRG.05MG) induced by NDV strain AF2240 was evaluated in-vitro, by using MTT proliferation assay. Furthermore, Cytological observations were studied using fluorescence microscopy and transmission electron microscopy, DNA laddering in agarose gel electrophoresis assay used to detect the mode of cell death and analysis of the cellular DNA content by flowcytometery. Results: MTT proliferation assay, Cytological observations using fluorescence microscopy and transmission electron microscopy show the anti-proliferation effect and apoptogenic features of NDV on DBTRG.05MG. Furthermore, analysis of the cellular DNA content showed that there was a loss of treated cells in all cell cycle phases (G1, S and G2/M) accompanied with increasing in sub-G1 region (apoptosis peak). Conclusion: It could be concluded that NDV strain AF2240 is a potent antitumor agent that induce apoptosis and its cytotoxicity increasing while increasing of time and virus titer.


Assuntos
Apoptose , Proliferação de Células , Glioblastoma/patologia , Microscopia de Contraste de Fase/métodos , Vírus da Doença de Newcastle/fisiologia , Terapia Viral Oncolítica/métodos , Ciclo Celular , Glioblastoma/terapia , Humanos , Células Tumorais Cultivadas
9.
Appl Opt ; 58(5): A26-A31, 2019 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-30873988

RESUMO

While simultaneous phase-contrast and two-photon fluorescence imaging in microscopy can bring abundant biomedical information, it is difficult to retrieve phase information from conventional two-photon microscopes. To realize low-cost, in situ phase-contrast and two-photon fluorescence imaging, we propose Schlieren two-photon microscopy, a method that implements phase-contrast imaging on two-photon microscopes. This method involves spatially modulated fluorescence plates, which are made of two-photon fluorescence dyes or upconversion nanoparticles. We demonstrate that the fluorescence intensity fluctuation reflects the phase gradients of the specimen via theoretical analysis, simulations, and experiments. The proposed method is fully compatible with commercial two-photon microscopes, thus enabling widespread applications in live tissue imaging.


Assuntos
Células HeLa/citologia , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Contraste de Fase/métodos , Corantes Fluorescentes , Humanos
10.
PLoS One ; 14(1): e0210291, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30625220

RESUMO

BACKGROUND: The extent of intraductal carcinoma in situ (DCIS) is commonly underestimated due to the discontinuous growth and lack of microcalcifications. Specimen radiography has been established to reduce the rate of re-excision. However, the predictive value for margin assessment with conventional specimen radiography for DCIS is low. In this study we assessed the potential of grating-based phase-contrast computed tomography (GBPC-CT) at conventional X-ray sources for specimen tomography of DCIS containing samples. MATERIALS AND METHODS: GBPC-CT was performed on four ex-vivo breast specimens containing DCIS and invasive carcinoma of non-specific type. Phase-contrast and absorption-based datasets were manually matched with corresponding histological slices as the standard of reference. RESULTS: Matching of CT images and histology was successful. GBPC-CT showed an improved soft tissue contrast compared to absorption-based images revealing more histological details in the same sections. Non-calcifying DCIS exceeding the invasive tumor could be correlated to areas of dilated bright ducts around the tumor. CONCLUSIONS: GBPC-CT imaging at conventional X-ray sources offers improved depiction quality for the imaging of breast tissue samples compared to absorption-based imaging, allows the identification of diagnostically relevant tissue details, and provides full three-dimensional assessment of sample margins.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Carcinoma Intraductal não Infiltrante/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Calcinose/diagnóstico por imagem , Calcinose/patologia , Carcinoma Ductal de Mama/diagnóstico por imagem , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Intraductal não Infiltrante/cirurgia , Feminino , Humanos , Técnicas In Vitro , Mamografia/métodos , Microscopia de Contraste de Fase/métodos , Estudos Prospectivos
11.
Nano Lett ; 19(2): 793-804, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30616354

RESUMO

Understanding the uptake and transport dynamics of engineered nanomaterials (ENMs) by mammalian cells is an important step in designing next-generation drug delivery systems. However, to track these materials and their cellular interactions, current studies often depend on surface-bound fluorescent labels, which have the potential to alter native cellular recognition events. As a result, there is still a need to develop methods capable of monitoring ENM-cell interactions independent of surface modification. Addressing these concerns, here we show how scatter enhanced phase contrast (SEPC) microscopy can be extended to work as a generalized label-free approach for monitoring nanoparticle uptake and transport dynamics. To determine which materials can be studied using SEPC, we turn to Lorenz-Mie theory, which predicts that individual particles down to ∼35 nm can be observed. We confirm this experimentally, demonstrating that SEPC works for a variety of metal and metal oxides, including Au, Ag, TiO2, CeO2, Al2O3, and Fe2O3 nanoparticles. We then demonstrate that SEPC microscopy can be used in a quantitative, time-dependent fashion to discriminate between distinct modes of active cellular transport, including intracellular transport and membrane-assisted transport. Finally, we combine this technique with microcontact printing to normalize transport dynamics across multiple cells, allowing for a careful study of ensemble TiO2 nanoparticle uptake. This revealed three distinct regions of particle transport across the cell, indicating that membrane dynamics play an important role in regulating particle flow. By avoiding fluorescent labels, SEPC allows for a rational exploration of the surface properties of nanomaterials in their native state and their role in endocytosis and cellular transport.


Assuntos
Microscopia de Contraste de Fase/instrumentação , Nanopartículas/metabolismo , Transporte Biológico , Endocitose , Desenho de Equipamento , Células Endoteliais da Veia Umbilical Humana , Humanos , Metais/análise , Metais/metabolismo , Microscopia de Contraste de Fase/métodos , Nanopartículas/análise , Óxidos/análise , Óxidos/metabolismo , Propriedades de Superfície
12.
Eur Radiol ; 29(8): 4169-4176, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30617486

RESUMO

OBJECTIVES: To investigate the feasibility of fetal phase-contrast (PC)-MR angiography of the descending aorta (AoD) using an MR-compatible Doppler ultrasound sensor (DUS) for fetal cardiac gating and to compare velocimetry with Doppler ultrasound measurements. METHODS: In this prospective study, 2D PC-MR angiography was performed in 12 human fetuses (mean gestational age 32.8 weeks) using an MR-compatible DUS for gating of the fetal heart at 1.5 T. Peak flow velocities in the fetal AoD were compared with Doppler ultrasound measurements performed on the same day. Reproducibility of PC-MR measurements was tested by repeated PC-MR in five fetuses. RESULTS: Dynamic PC-MR angiography in the AoD was successfully performed in all fetuses using the DUS, with an average fetal heart rate of 140 bpm (range 129-163). Time-velocity curves revealed typical arterial blood flow patterns. PC-MR mean flow velocity and mean flux were 21.2 cm/s (range 8.6-36.8) and 8.4 ml/s (range 3.2-14.6), respectively. A positive association between PC-MR mean flux and stroke volume with gestational age was obtained (r = 0.66, p = 0.02 and r = 0.63, p = 0.03). PC-MR and Doppler ultrasound peak velocities revealed a highly significant correlation (r = 0.8, p < 0.002). Peak velocities were lower for PC-MR with 69.1 cm/s (range 39-125) compared with 96.7 cm/s (range 60-142) for Doppler ultrasound (p < 0.001). Reproducibility of PC-MR was high (p > 0.05). CONCLUSION: The MR-compatible DUS for fetal cardiac gating allows for PC-MR angiography in the fetal AoD. Comparison with Doppler ultrasound revealed a highly significant correlation of peak velocities with underestimation of PC-MR velocities. This new technique for direct fetal cardiac gating indicates the potential of PC-MR angiography for assessing fetal hemodynamics. KEY POINTS: • The developed MR-compatible Doppler ultrasound sensor allows direct fetal cardiac gating and can be used for prenatal dynamic cardiovascular MRI. • The MR-compatible Doppler ultrasound sensor was successfully applied to perform intrauterine phase-contrast MR angiography of the fetal aorta, which revealed a highly significant correlation with Doppler ultrasound measurements. • As fetal flow hemodynamics is an important parameter in the diagnosis and management of fetal pathologies, fetal phase-contrast MR angiography may offer an alternative imaging method in addition to Doppler ultrasound and develop as a second line tool in the evaluation of fetal flow hemodynamics.


Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Feto/fisiologia , Angiografia por Ressonância Magnética/métodos , Diagnóstico Pré-Natal/métodos , Adulto , Artérias/embriologia , Artérias/fisiologia , Feminino , Sangue Fetal/fisiologia , Idade Gestacional , Humanos , Angiografia por Ressonância Magnética/normas , Idade Materna , Microscopia de Contraste de Fase/métodos , Gravidez , Diagnóstico Pré-Natal/normas , Estudos Prospectivos , Reprodutibilidade dos Testes , Ultrassonografia Doppler/métodos , Ultrassonografia Pré-Natal/métodos
13.
Microscopy (Oxf) ; 68(2): 144-158, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30496508

RESUMO

Activated sludge (AS) is a biological treatment process that is employed in wastewater treatment plants. Filamentous bacteria in AS plays an important role in the settling ability of the sludge. Proper settling of the sludge is essential for normal functionality of the wastewater plants, where filamentous bulking is always a persistent problem preventing sludge from settling. The performance of AS plants is conventionally monitored by physico-chemical procedures. An alternative way of monitoring the AS in wastewater treatment process is to use image processing and analysis. Good performance of the image segmentation algorithms is important to quantify flocs and filaments in AS. In this article, an algorithm is proposed to perform segmentation of filaments in the phase contrast images using phase stretch transform. Different values of strength (S) and warp (W) are tested to obtain optimum segmentation results and decrease the halo and shade-off artefacts encountered in phase contrast microscopy. The performance of the algorithm is assessed using DICE coefficient, accuracy, false positive rate (FPR), false negative rate (FNR) and Rand index (RI). Sixty-one gold approximations of ground truth images were manually prepared to assess the segmentation results. Thirty-two of them were acquired at 10× magnification and 29 of them were acquired at 20× magnification. The proposed algorithm exhibits better segmentation performance with an average DICE coefficient equal to 52.25%, accuracy 99.74%, FNR 41.8% and FPR 0.14% and RI 99.49%, based on 61 images.


Assuntos
Algoritmos , Bactérias/classificação , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Contraste de Fase/métodos , Esgotos/microbiologia , Purificação da Água/métodos
14.
Phys Med Biol ; 63(24): 24NT03, 2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30524112

RESUMO

X-ray phase imaging has the potential to dramatically improve soft tissue contrast sensitivity, which is a crucial requirement in many diagnostic applications such as breast imaging. In this context, a program devoted to perform in vivo phase-contrast synchrotron radiation breast computed tomography is ongoing at the Elettra facility (Trieste, Italy). The used phase-contrast technique is the propagation-based configuration, which requires a spatially coherent source and a sufficient object-to-detector distance. In this work the effect of this distance on image quality is quantitatively investigated scanning a large breast surgical specimen at three object-to-detector distances (1.6, 3, 9 m) and comparing the images both before and after applying the phase-retrieval procedure. The sample is imaged at 30 keV with a [Formula: see text] pixel pitch CdTe single-photon-counting detector, positioned at a fixed distance of 31.6 m from the source. The detector fluence is kept constant for all acquisitions. The study shows that, at the largest distance, a 20-fold SNR increase can be obtained by applying the phase-retrieval procedure. Moreover, it is shown that, for phase-retrieved images, changing the object-to-detector distance does not affect spatial resolution while boosting SNR (four-fold increase going from the shortest to the largest distance). The experimental results are supported by a theoretical model proposed by other authors, whose salient results are presented in this paper.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Mama/anormalidades , Hipertrofia/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Contraste de Fase/métodos , Pontos Quânticos , Síncrotrons/instrumentação , Tomografia Computadorizada por Raios X/métodos , Mama/diagnóstico por imagem , Feminino , Humanos , Modelos Teóricos
15.
J Synchrotron Radiat ; 25(Pt 5): 1490-1504, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30179189

RESUMO

X-ray imaging allows biological cells to be examined at a higher resolution than possible with visible light and without some of the preparation difficulties associated with electron microscopy of thick samples. The most used and developed technique is absorption contrast imaging in the water window which exploits the contrast between carbon and oxygen at an energy of around 500 eV. A variety of phase contrast techniques are also being developed. In general these operate at a higher energy, enabling thicker cells to be examined and, in some cases, can be combined with X-ray fluorescence imaging to locate specific metals. The various methods are based on the differences between the complex refractive indices of the cellular components and the surrounding cytosol or nucleosol, the fluids present in the cellular cytoplasm and nucleus. The refractive indices can be calculated from the atomic composition and density of the components. These in turn can be obtained from published measurements using techniques such as chemical analysis, scanning electron microscopy and X-ray imaging at selected energies. As examples, the refractive indices of heterochromatin, inner mitochondrial membranes, the neutral core of lipid droplets, starch granules, cytosol and nucleosol are calculated. The refractive index calculations enable the required doses and fluences to be obtained to provide images with sufficient statistical significance, for X-ray energies between 200 and 4000 eV. The statistical significance (e.g. the Rose criterion) for various requirements is discussed. The calculations reveal why some cellular components are more visible by absorption contrast and why much greater exposure times are required to see some cellular components. A comparison of phase contrast as a function of photon energy with absorption contrast in the water window is provided and it is shown that much higher doses are generally required for the phase contrast measurements. This particularly applies to those components with a high carbon content but with a mass density similar to the surrounding cytosol or nucleosol. The results provide guidance for the most appropriate conditions for X-ray imaging of individual cellular components within cells of various thicknesses.


Assuntos
Núcleo Celular/química , Citosol/química , Heterocromatina/química , Lipídeos/química , Microscopia de Contraste de Fase/métodos , Membranas Mitocondriais/química , Amido/química , Microscopia Eletrônica de Varredura , Fótons , Doses de Radiação , Raios X
16.
Appl Opt ; 57(17): 4795-4801, 2018 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-30118100

RESUMO

We propose a differential interference contrast method for cells using hard x-ray Gabor holography and knife-edge filtering in the spatial frequency domain, without relying on beam shearing. A phase object is holographically recorded and reconstructed by computer. Interference between the wavefronts of zeroth order weighted by ejπ/2 in the positive frequency region produces a dark image. Similarly, interference between the wavefronts of the zeroth order weighted by ej3π/2 in the negative frequency region produces a bright image. By adding these two intensity distributions, good quality phase-contrast images of 8-µm-diameter polystyrene beads and human HeLa cells were obtained.


Assuntos
Holografia/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Contraste de Fase/métodos , Microesferas , Células HeLa/patologia , Humanos , Poliestirenos , Raios X
17.
Plast Reconstr Surg ; 142(5): 1240-1247, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30113440

RESUMO

BACKGROUND: In obtaining human tenocytes for tendon tissue engineering, a low proliferation rate and phenotype loss during passaging is a problem. It was the authors' aim to evaluate the influence of vascular endothelial growth factor (VEGF) on human tenocyte growth and gene expression. METHODS: Human tenocytes were exposed to human VEGF in various concentrations (5, 10, and 20 ng/ml) for 5 days. Cell proliferation was counted and expression of tendon-related genes was analyzed. RESULTS: Tenocyte count was 1.4 × 10(5)/ml, 2.7 × 10(5)/ml, 2.3 × 10(5)/ml, and 3.7 × 10(5)/ml for 0, 5, 10, and 20 ng/ml VEGF, respectively. Expression of Col1 was up-regulated 6.4 ± 4.2-fold, 60.1 ± 21.6-fold, and 15.8 ± 10.2-fold for 5, 10, and 20 ng/ml VEGF; all differences were significant with p < 0.05. Col3 was down-regulated to 0.2 ± 0.1-fold, 0.3 ± 0.1-fold, and 0.1 ± 0.03-fold for 5, 10, and 20 ng/ml VEGF; all differences were significant. Eln was up-regulated 2.3 ± 1.7-fold, 25.5 ± 10.9-fold, and 16.6 ± 9.0-fold for 5, 10, and 20 ng/ml VEGF; differences were significant for 10 and 20 ng/ml VEGF. TSC was down-regulated to 0.3 ± 0.1-fold and 0.3 ± 0.1-fold for 5 and 20 ng/ml VEGF; differences were significant for 5 and 20 ng/ml. SCX was up-regulated to 31.3 ± 8.5-fold, 49.1 ± 23.4-fold, and 20.9 ± 9.5-fold for 5, 10, and 20 ng/ml VEGF; all changes were significant. CONCLUSIONS: VEGF enhances proliferation and expression of tendon-related genes in human tenocytes. It could therefore be a useful addition for tenocyte cultivation.


Assuntos
Proliferação de Células/fisiologia , Expressão Gênica/efeitos dos fármacos , Tenócitos/citologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Contagem de Células , Técnicas de Cultura de Células , Proliferação de Células/genética , Células Cultivadas , Regulação para Baixo/fisiologia , Expressão Gênica/genética , Humanos , Microscopia de Contraste de Fase/métodos , Regulação para Cima/fisiologia
18.
Sci Rep ; 8(1): 11014, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-30030461

RESUMO

High-spatial-resolution histology of coronary artery autopsy samples play an important role for understanding heart disease such as myocardial infarction. Unfortunately, classical histology is often destructive, has thick slicing, requires extensive sample preparation, and is time-consuming. X-ray micro-CT provides fast nondestructive 3D imaging but absorption contrast is often insufficient, especially for observing soft-tissue features with high resolution. Here we show that propagation-based x-ray phase-contrast tomography has the resolution and contrast to image clinically relevant soft-tissue features in intact coronary artery autopsy samples with cellular resolution. We observe microscopic lipid-rich plaques, individual adipose cells, ensembles of few foam cells, and the thin fibrous cap. The method relies on a small-spot laboratory x-ray microfocus source, and provides high-spatial resolution in all three dimensions, fast data acquisition, minimum sample distortion and requires no sample preparation.


Assuntos
Vasos Coronários/diagnóstico por imagem , Vasos Coronários/patologia , Microtomografia por Raio-X/métodos , Aterosclerose , Cadáver , Técnicas Histológicas , Humanos , Imagem Tridimensional/métodos , Microscopia de Contraste de Fase/métodos , Tomografia por Raios X/métodos
19.
Opt Lett ; 43(14): 3365-3368, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30004507

RESUMO

We present a single-shot quantitative phase imaging (QPI) method based on color-multiplexed Fourier ptychographic microscopy (FPM). Three light-emitting diode (LED) elements with respective R/G/B channels in a programmable LED array illuminate the specimen simultaneously, providing triangle oblique illuminations matching the numerical aperture of the objective precisely. A color image sensor records the light transmitted through the specimen, and three monochromatic intensity images at each color channel are then separated and utilized to recover the phase of the specimen. After one-step deconvolution based on the phase contrast transfer function, the obtained initial phase map is further refined by the FPM-based iterative recovery algorithm to overcome pixel-aliasing and improve the phase recovery accuracy. The high-speed, high-throughput QPI capabilities of the proposed approach are demonstrated by imaging HeLa cells mitosis in vitro, achieving a half-pitch resolution of 388 nm across a wide field of view of 1.33 mm2 at camera-limited frame rates (50 fps).


Assuntos
Estruturas Celulares , Análise de Fourier , Microscopia de Contraste de Fase/métodos , Algoritmos , Calibragem , Células HeLa , Humanos
20.
Phys Med Biol ; 63(14): 14NT03, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-29938688

RESUMO

Clinical studies performed using computer simulation are inexpensive, flexible methods that can be used to study aspects of a proposed imaging technique prior to a full clinical study. Typically, lesions are simulated into (experimental) data to assess the clinical potential of new methods or algorithms. In grating-based phase-contrast imaging (GB-PCI), full wave simulations are, however, computationally expensive due to the high periodicity of the gratings and therefore not practically applicable when large data sets are required. This work describes the development of a hybrid modelling platform that combines analytical and empirical input data for a more rapid simulation of GB-PCI images with little loss of accuracy. Instead of an explicit implementation of grating details, measured summary metrics (i.e. visibility, flux, noise power spectra, presampling modulation transfer function) are applied in order to generate transmission and differential phase images with large fields of view. Realistic transmission and differential phase images were obtained with good quantitative accuracy. The different steps of the simulation framework, as well as the methods to measure the summary metrics, are discussed in detail such that the technique can be easily customized for a given system. The platform offers a fast, accurate alternative to full wave simulations when the focus switches from grating/system design and set up to the generation of GB-PCI images for an established system.


Assuntos
Algoritmos , Simulação por Computador , Coração/diagnóstico por imagem , Microscopia de Contraste de Fase/métodos , Modelos Teóricos , Imagens de Fantasmas , Humanos , Raios X
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