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1.
Eur. j. anat ; 24(1): 1-7, ene. 2020. ilus, tab
Artigo em Inglês | IBECS | ID: ibc-186059

RESUMO

The aim of this study is to demonstrate that the denervation of the pancreas may affect the enteric neuronal plexus, which controls both the endocrine and exocrine parts of the pancreas. By using the light microscope, the histological changes of the islets of Langerhans and the pancreatic acini in the rat pancreas were studied two and three weeks after sympathectomy and truncal vagotomy. More-over, measurements of the changes infasting blood glucose levels and glucose tolerance tests in the control and experimental animals were recorded. Atrophic changes and degeneration of the pancreatic acinar cells and islets of Langerhans cells were observed after both sympathectomy and vagotomy. Biochemical measurements of fasting blood, and the glucose tolerance tests after sympathectomy and vagotomy were increased significantly, which is consistent with the histological results. The results of this study explain that the exocrine and endocrine parts of the pancreas are dependent on both sympathetic and parasympathetic innervation via the enteric plexuses of the rat pancreas. These results establish a firm correlation between the autonomic innervation and the enteric plexus, which controls the function of the endocrine and exocrine parts of the pancreas


No disponible


Assuntos
Animais , Ratos , Pâncreas/anatomia & histologia , Pâncreas/diagnóstico por imagem , Microscopia/métodos , Microscopia/veterinária , Denervação/veterinária , Simpatectomia/métodos , Simpatectomia/veterinária , Vagotomia Troncular/métodos , Vagotomia Troncular/veterinária , Ilhotas Pancreáticas/anatomia & histologia
3.
Ophthalmic Res ; 63(1): 34-40, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31352453

RESUMO

AIM: To report the outcomes of ab externo surgery using a surgical microscope, wide-angle viewing system, and chandelier endoilluminator (microscope-assisted ab externo surgery) for rhegmatogenous retinal detachment (RRD). METHODS: This was a retrospective study. Consecutive charts of patients with RRD who underwent microscope-assisted ab externo surgery were analyzed. The following demographic parameters were analyzed: age (years), gender (male/female), and eye (right/left). Clinical parameters were axial length (AL) measured in millimeters (mm), preoperative best-corrected visual acuity (BCVA) measured in logarithm of minimum angle of resolution (logMAR), intraocular pressure (IOP), and lens status (phakic/pseudophakic). The parameters of RRD were number and type of retinal breaks, location of retinal breaks, extent of retinal detachment (RD) (number of detached quadrants), and macular detachment (MD), as well as retinal breaks not detected preoperatively. Use of cryopexy, circumferential or segmental scleral buckle, drainage of subretinal fluid, injection of air or gas, and duration of surgery were recorded. The postoperative parameters analyzed were BCVA, IOP and recurrence of RD and postoperative complications. Follow-up was established at 3 months. RESULTS: A total of 213 eyes (97 right, 116 left) of 205 patients (114 males, 91 females) affected by primary RRD were included. Fifty-two eyes (24.4%) were affected by high myopia (AL >26.5 mm), and 160 patients (75.1%) were affected by RRD caused by a single retinal break and involving only one quadrant. The superior quadrant was the most frequently involved (49.3%). Forty-two eyes (19.7%) were affected by MD. In 13 eyes (11.3%), retinal breaks were not detected preoperatively. The duration of surgery was 75.5 ± 42 min. No significant BCVA changes were observed in the whole group, whereas a significant improvement of BCVA from the baseline (2.83 ± 0.87 logMAR) to each time point of follow-up was observed in the subgroup of patients affected by MD. Six eyes (2.8%) developed a recurrent RD, secondary to proliferative vitreoretinopathy (3 eyes) and secondary to a new retinal break (3 eyes). Two eyes developed a persistent vitreous hemorrhage, and one eye developed a macular hole after 1 week. PPV was performed for both. CONCLUSION: Microscope-assisted ab externo surgery is effective and safe, it reduces discomfort, it allows the surgeon to work with both hands free, and provides an adequate visualization of each step being performed.


Assuntos
Crioterapia/métodos , Tamponamento Interno/métodos , Procedimentos Cirúrgicos Oftalmológicos/métodos , Descolamento Retiniano/cirurgia , Adulto , Idoso , Feminino , Humanos , Masculino , Microscopia/métodos , Pessoa de Meia-Idade , Perfurações Retinianas/cirurgia , Estudos Retrospectivos , Acuidade Visual
4.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117386, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31336320

RESUMO

Non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups such as O26, O45, O103, O111, O121 and O145 often cause illness to people in the United States and the conventional identification of these "Big-Six" are complex. The label-free hyperspectral microscope imaging (HMI) method, which provides spectral "fingerprints" information of bacterial cells, was employed to classify serogroups at the cellular level. In spectral analysis, principal component analysis (PCA) method and stacked auto-encoder (SAE) method were conducted to extract principal spectral features for classification task. Based on these features, multiple classifiers including linear discriminant analysis (LDA), support vector machine (SVM) and soft-max regression (SR) methods were evaluated. Different sizes of datasets were also tested in search for the suitable classification models. Among the results, SAE-based classification models performed better than PCA-based models, achieving classification accuracy of SAE-LDA (93.5%), SAE-SVM (94.9%) and SAE-SR (94.6%), respectively. In contrast, classification results of PCA-based methods such as PCA-LDA, PCA-SVM and PCA-SR were only 75.5%, 85.7% and 77.1%, respectively. The results also suggested the increasing number of training samples have positive effects on classification models. Taking advantage of increasing dataset, the SAE-SR classification model finally performed better than others with average accuracy of 94.9% in classifying STEC serogroups. Specifically, O103 serogroup was classified with the highest accuracy of 97.4%, followed by O111 (96.5%), O26 (95.3%), O121 (95%), O145 (92.9%) and O45 (92.4%), respectively. Thus, the HMI technology coupled with SAE-SR classification model has the potential for "Big-Six" identification.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Escherichia coli Shiga Toxigênica , Algoritmos , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Imagem Óptica/métodos , Análise de Componente Principal , Escherichia coli Shiga Toxigênica/química , Escherichia coli Shiga Toxigênica/classificação
5.
PLoS Pathog ; 15(12): e1008209, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790506

RESUMO

The processes of cell attachment and membrane fusion of Herpes Simplex Virus 1 involve many different envelope glycoproteins. Viral proteins gC and gD bind to cellular receptors. Upon binding, gD activates the gH/gL complex which in turn activates gB to trigger membrane fusion. Thus, these proteins must be located at the point of contact between cellular and viral envelopes to interact and allow fusion. Using super-resolution microscopy, we show that gB, gH/gL and most of gC are distributed evenly round purified virions. In contrast, gD localizes essentially as clusters which are distinct from gB and gH/gL. Upon cell binding, we observe that all glycoproteins, including gD, have a similar ring-like pattern, but the diameter of these rings was significantly smaller than those observed on cell-free viruses. We also observe that contrary to cell-free particles, gD mostly colocalizes with other glycoproteins on cell-bound particles. The differing patterns of localization of gD between cell-free and cell-bound viruses indicates that gD can be reorganized on the viral envelope following either a possible maturation of the viral particle or its adsorption to the cell. This redistribution of glycoproteins upon cell attachment could contribute to initiate the cascade of activations leading to membrane fusion.


Assuntos
Herpesvirus Humano 1/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Linhagem Celular , Glicoproteínas/metabolismo , Glicoproteínas/ultraestrutura , Herpesvirus Humano 1/ultraestrutura , Humanos , Microscopia/métodos , Proteínas do Envelope Viral/ultraestrutura , Vírion/ultraestrutura , Ligação Viral , Internalização do Vírus
6.
BMC Infect Dis ; 19(1): 1058, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842773

RESUMO

BACKGROUND: Xpert® MTB/RIF (Xpert) has high sensitivity for diagnosing tuberculosis (TB) compared to sputum-smear microscopy (smear) and can reduce time-to-diagnosis, time-to-treatment and potentially unfavorable patient-level treatment outcome. METHODS: People living with HIV (PLHIV) initiating antiretroviral therapy at 22 HIV clinics were enrolled and underwent systematic screening for TB (August 2012-November 2014). GeneXpert instruments were deployed following a stepped-wedge design at 13 centers from October 2012-June 2013. Treatment outcomes classified as an unfavorable outcome (died, treatment failure or loss-to-follow-up) or favorable outcome (cured and treatment completed). To determine outcome, smear was performed at month 5 or 6. Empiric treatment was defined as initiating treatment without/before receiving TB-positive results. Adjusting for intra-facility correlation, we compared patient-level treatment outcomes between patients screened using smear (smear arm)- and Xpert-based algorithms (Xpert arm). RESULTS: Among 6041 patients enrolled (smear arm, 1816; Xpert arm, 4225), 256 (199 per 2985 and 57 per 1582 person-years of follow-up in Xpert and smear arms, respectively; adjusted incidence rate ratio, 9.07; 95% confidence interval [CI]: 4.70-17.48; p < 0.001) received TB diagnosis and were treated. TB treatment outcomes were available for 203 patients (79.3%; Xpert, 157; smear, 46). Unfavorable outcomes were reported for 21.7% (10/46) in the smear and 13.4% (21/157) in Xpert arm (adjusted hazard ratio, 1.40; 95% CI: 0.75-2.26; p = 0.268). Compared to smear, in Xpert arm median days from sputum collection to TB treatment was 6 days (interquartile range [IQR] 2-17 versus 22 days [IQR] 3-51), p = 0.005; patients with available sputum test result had microbiologically confirmed TB in 59.0% (102/173) versus 41.9% (18/43), adjusted Odds Ratio [aOR], 2.00, 95% CI: 1.01-3.96, p = 0.048). In smear arm empiric treatment was 68.4% (39/57) versus 48.7% (97/199), aOR, 2.28, 95% CI: 1.24-4.20, p = 0.011), compared to Xpert arm. CONCLUSIONS: TB treatment outcomes were similar between the smear and Xpert arms. However, compared to the smear arm, more patients in the Xpert arm received a TB diagnosis, had a microbiologically confirmed TB, and had a shorter time-to-treatment, and had a lower empiric treatment. Further research is recommended to identify potential gaps in the Botswana health system and similar settings. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02538952. Retrospectively registered on 2 September 2015.


Assuntos
Infecções por HIV/complicações , Microscopia/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Escarro/microbiologia , Tuberculose/complicações , Tuberculose/tratamento farmacológico , Adulto , Botsuana , Confiabilidade dos Dados , Feminino , Seguimentos , Humanos , Perda de Seguimento , Masculino , Programas de Rastreamento , Estudos Prospectivos , Sensibilidade e Especificidade , Tempo para o Tratamento , Resultado do Tratamento , Tuberculose/diagnóstico , Tuberculose/microbiologia
7.
Int. j. morphol ; 37(4): 1416-1421, Dec. 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1040147

RESUMO

The indiscriminate use of anabolic steroids in gyms has been growing in a generalized way, among which, the most common is growth hormone (GH). In the short term GH may potentiate muscle growth, especially when taken in combination with resistance training. However, the effects of this hormone are not yet fully understood in the literature, especially in relation to collagen properties. The objective of this study was to evaluate the effect of the application of growth hormone (GH) and resistance training (RT) on the collagen properties of femoral bone tissue using Raman Spectroscopy. In this study 40 male rats were randomly distributed into four groups (n=10): control (C), control and GH application (GH), resistance training (T), and resistance training and GH application (GHT). The training consisted of four series of 10 water jumps, performed three times a week, with an overload corresponding to 50 % of body weight and duration of four weeks. GH was applied at a dosage of 0.2 IU/Kg (0.067 mg/kg) to each animal, three times a week, every other day. The animals were euthanized and the right femurs were collected for analysis of bone structure. Raman spectroscopy (RS) was used to observe the following compounds from their respective bands: type I collagen (662 cm-1), amide III (1243 cm-1), proteins including type I collagen (1278 cm-1), woven collagen (1322 cm-1), association of collagen, phospholipids, nucleic acid, and phosphate (1330 cm-1), and collagen and protein deformation (1448 cm-1). The results demonstrated an increase in the collagen properties in all analyzed variables, however, the T group presented a statistically significant difference (p<0.05). It is possible to conclude that isolated physical training was shown to be more efficient than when combined with the application of GH to increase the collagen properties of the femoral bone tissue.


El uso indiscriminado de anabolizantes en los gimnasios ha aumentado de forma generalizada, entre éstos la hormona de crecimiento (HC) es una de las más utilizadas, y a corto plazo puede potencializar el crecimiento muscular, principalmente cuando es realizado en combinación con el entrenamiento de fuerza. Sin embargo, los efectos de esta hormona aún no están totalmente esclarecidos en la literatura, especialmente en relación a las propiedades colágenas. El objetivo del estudio fue evaluar el efecto de la aplicación del HC y entrenamiento de fuerza (E) en las propiedades colágenas del tejido óseo femoral a partir de la utilización de la espectroscopía Raman. Se usaron 40 ratas Wistar distribuidos en cuatro grupos (n=10): control (C), control y aplicación del HC (HCC), entrenamiento de fuerza (E) y entrenamiento de fuerza y aplicación del HC (THC). El entrenamiento fue compuesto por cuatro series de 10 saltos acuáticos, realizados tres veces por semana, con sobrecarga correspondiente a 50 % del peso corporal y duración de cuatro semanas. El HC fue aplicado en una dosificación de 0,2 UI/Kg (0,067 mg/kg) en cada animal, tres veces por semana, en días no consecutivos. Los animales fueran eutanasiados y se retiró el fémur derecho para realización del análisis de la estructura ósea. La espectroscopía Raman (ER) fue utilizada para observar los siguientes compuestos a partir de las respectivas bandas: colágeno tipo I (662 cm-1), amida III (1243 cm1), proteínas, incluido colágeno tipo I (1278 cm-1), colágeno retorcido (1322 cm-1), asociación de colágeno, fosfolípidos, ácidos nucleicos y fosfato (1330 cm-1), deformación de colágeno y proteína (1448 cm-1). Hubo aumento en las propiedades colágenas en todas las variables analizadas, sin embargo, solamente el grupo E demostró una diferencia estadísticamente significativa (p<0,05). En conclusión, para el aumento de las propiedades colágenas del tejido óseo femoral, el entrenamiento físico aislado es más eficiente que el entrenamiento combinado con el uso de HC.


Assuntos
Animais , Masculino , Ratos , Resistência Física/fisiologia , Hormônio do Crescimento/farmacologia , Fêmur/efeitos dos fármacos , Fêmur/fisiologia , Hormônio do Crescimento/administração & dosagem , Exercício/fisiologia , Colágeno/efeitos dos fármacos , Ratos Wistar , Microscopia/métodos
8.
Malar J ; 18(1): 354, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690321

RESUMO

BACKGROUND: CareStart™ malaria HRP2/pLDH (Pf/pan) combo test is one of the several rapid diagnostic tests (RDT) approved for diagnosis of malaria at the point of care in Tanzania. However, there are limited studies on the diagnostic performance of RDT after wide scale use in primary health care facilities in Tanzania. Therefore, this study was carried out to determine the diagnostic performance of RDT when compared with blood smear (BS) microscopy as a reference standard. METHODS: A cross-sectional study was conducted between March and August 2019 at Kibiti Health Centre, Pwani region, Tanzania. Blood samples for malaria tests were collected from patients with malaria symptoms. Diagnostic performance parameters of RDT, i.e. sensitivity, specificity, positive and negative likelihood ratios (LR+/-), diagnostic accuracy and predictive values were determined using contingency table. An agreement between RDT and microscopy was statistically determined by Cohen's kappa test. RESULTS: Of 980 patients screened, 567 (57.9%) were found to be malaria positive by RDT, whereas 510 patients (52%) were positive by microscopy. Of the 510 microscopy-positive patients, 487 (95.5%) were infected with Plasmodium falciparum. The geometric mean parasite density was 2921parasites/µl, whereas majority (68.6%) of patients had parasite density greater than 10,000/µl. The sensitivity, specificity, positive and negative predictive values of CareStart™ were 99.8%, 87.6%, 89.8%, and 99.8%, respectively. The LR+ and LR- were 8.0 and 0.002, respectively. The diagnostic accuracy was 0.5. There was a strong agreement between the results obtained using CareStart™ and BS microscopy (kappa = 0.863, P < 0.0001). CONCLUSION: CareStart™ malaria HRP2/pLDH (Pf/pan) had high sensitivity and strong agreement with microscopy results. However, moderate specificity of RDT resulted in a substantial number of patients with false positive malaria test. Wherever available, microscopy should be used to confirm RDT test results.


Assuntos
Testes Diagnósticos de Rotina/métodos , Malária Falciparum/diagnóstico , Microscopia/métodos , Adolescente , Criança , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Lactente , Masculino , Plasmodium falciparum/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , Tanzânia , Adulto Jovem
9.
Nat Methods ; 16(12): 1202, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31780828
10.
Eur. j. anat ; 23(6): 483-485, nov. 2019.
Artigo em Inglês | IBECS | ID: ibc-185093

RESUMO

We appreciate the time and attention paid to our paper by Prof. Mestres-Ventura and similarly appreciate the opportunity to respond to his concerns. We would like to address what we believe are several fundamental misunderstandings in his commentary.1. Scale: The most significant misunderstanding is one of scale. The schematic (Fig. 1) provided by Prof. Mestres-Ventura is (per personal communication) at the nano scale, while in vivo microscopy of extrahepatic bile duct and dermis shows that the collagen bundles we report are at the micron scale, each containing many individual fibrils at the nanometer scale. Indeed, examining the tissues described in our paper - submucosae, dermis and subcutaneous fascia - fresh in resected specimens or intraoperatively, we find that the structures we describe are visible at the macroscopic level (if one leans in closely enough). In other words, they are macroscopic, not microscopic. Prof. Mestres-Ventura, in summarizing the prior pCLE work of Wallace and Fockens, which he notes is similar to ours, states "the 'holes' shown under intravital microscopy and in cryofixed samples are huge (over 20 µm)" This is exactly our point - we were surprised as well at the scale of these structures, as this has not been well appreciated in the past


No disponible


Assuntos
Humanos , Ducto Colédoco/anatomia & histologia , Ducto Colédoco/ultraestrutura , Derme/ultraestrutura , Fáscia/ultraestrutura , Microscopia/métodos , Derme/anatomia & histologia , Fáscia/anatomia & histologia , Antígenos CD34 , Proteoglicanas
11.
Adv Clin Chem ; 93: 263-290, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31655732

RESUMO

Periodontal disease entails the inflammatory destruction of the tooth supporting (periodontal) tissues as a result of polymicrobial colonization of the tooth surface in the form of biofilms. Extensive data collected over the past decades on this chronic disease demonstrate that its progression is infrequent and episodic, and the susceptibility to it can vary among individuals. Physical assessments of previously occurring damage to periodontal tissues remain the cornerstone of detection and diagnosis, whereas traditionally used diagnostic procedures do neither identify susceptible individuals nor distinguish between disease-active and disease-inactive periodontal sites. Thus, more sensitive and accurate "measurable biological indicators" of periodontal diseases are needed in order to place diagnosis (e.g., the presence or stage) and management of the disease on a more rational less empirical basis. Contemporary "omics" technologies may help unlock the path to this quest. High throughput nucleic acid sequencing technologies have enabled us to examine the taxonomic distribution of microbial communities in oral health and disease, whereas proteomic technologies allowed us to decipher the molecular state of the host in disease, as well as the interactive cross-talk of the host with the microbiome. The newly established field of metaproteomics has enabled the identification of the repertoire of proteins that oral microorganisms use to compete or co-operate with each other. Vast such data is derived from oral biological fluids, including gingival crevicular fluid and saliva, which is progressively completed and catalogued as the analytical technologies and bioinformatics tools progressively advance. This chapter covers the current "omics"-derived knowledge on the microbiome, the host and their "interactome" with regard to periodontal diseases, and addresses challenges and opportunities ahead.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microscopia/métodos , Doenças Periodontais/metabolismo , Proteômica/métodos , Líquido do Sulco Gengival/metabolismo , Humanos , Doenças Periodontais/etiologia , Doenças Periodontais/patologia
12.
Virchows Arch ; 475(6): 735-744, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31588959

RESUMO

Focal or non-focal/extensive extraprostatic extension of prostate carcinoma is an important pathologic prognostic parameter to be reported after radical prostatectomy. Currently, there is no agreement on how to measure and what are the best cutoff points to be used in practice. We hypothesized that digital microscopy would potentially provide more objective measurements of extraprostatic extension, thus better defining its clinical significance. To further our knowledge on digital prostate pathology, we evaluated the status of extraprostatic extension in 107 consecutive laparoscopic radical prostatectomy samples, using digital and conventional light microscopy. Mean linear and radial measurements of extraprostatic extension by digital microscopy significantly correlated to pT status (p = 0.022 and p = 0.050, respectively) but only radial measurements correlated to biochemical recurrence (p = 0.042) and grade groups (p = 0.022). None of the measurements, whether conventional or digital, were associated with lymph node status. Receiving operating characteristic analysis showed a potential cutoff point to assess linear measurements by conventional (< vs. > 24.21 mm) or digital microscopy (< vs. > 15 mm) or by radial measurement (< vs. > 1.6 mm). Finally, we observed an association between the number of paraffin blocks bearing EPE with pT (p = 0.041) status (digital microscopy), and linear measurements by conventional (p = 0.044) or digital microscopy (p = 0.045) with lymph node status. Reporting EPE measurements by digital microscopy, both linear and radial, and the number of paraffin blocks with EPE, might provide additional prognostic features after radical prostatectomy.


Assuntos
Invasividade Neoplásica/patologia , Recidiva Local de Neoplasia/patologia , Próstata/patologia , Neoplasias da Próstata/patologia , Idoso , Intervalo Livre de Doença , Humanos , Masculino , Microscopia/métodos , Pessoa de Meia-Idade , Invasividade Neoplásica/diagnóstico , Recidiva Local de Neoplasia/diagnóstico , Prognóstico , Antígeno Prostático Específico/metabolismo , Prostatectomia/métodos , Neoplasias da Próstata/diagnóstico
13.
Nat Commun ; 10(1): 4483, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578369

RESUMO

Among optical imaging techniques light sheet fluorescence microscopy is one of the most attractive for capturing high-speed biological dynamics unfolding in three dimensions. The technique is potentially millions of times faster than point-scanning techniques such as two-photon microscopy. However light sheet microscopes are limited by volume scanning rate and/or camera speed. We present speed-optimized Objective Coupled Planar Illumination (OCPI) microscopy, a fast light sheet technique that avoids compromising image quality or photon efficiency. Our fast scan system supports 40 Hz imaging of 700 µm-thick volumes if camera speed is sufficient. We also address the camera speed limitation by introducing Distributed Planar Imaging (DPI), a scaleable technique that parallelizes image acquisition across cameras. Finally, we demonstrate fast calcium imaging of the larval zebrafish brain and find a heartbeat-induced artifact, removable when the imaging rate exceeds 15 Hz. These advances extend the reach of fluorescence microscopy for monitoring fast processes in large volumes.


Assuntos
Encéfalo/diagnóstico por imagem , Diagnóstico por Imagem/instrumentação , Processamento de Imagem Assistida por Computador/instrumentação , Medições Luminescentes/instrumentação , Microscopia/instrumentação , Animais , Diagnóstico por Imagem/métodos , Processamento de Imagem Assistida por Computador/métodos , Larva , Medições Luminescentes/métodos , Microscopia/métodos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Reprodutibilidade dos Testes , Peixe-Zebra
14.
PLoS Biol ; 17(10): e3000268, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31622337

RESUMO

Imaging dense and diverse microbial communities has broad applications in basic microbiology and medicine, but remains a grand challenge due to the fact that many species adopt similar morphologies. While prior studies have relied on techniques involving spectral labeling, we have developed an expansion microscopy method (µExM) in which bacterial cells are physically expanded prior to imaging. We find that expansion patterns depend on the structural and mechanical properties of the cell wall, which vary across species and conditions. We use this phenomenon as a quantitative and sensitive phenotypic imaging contrast orthogonal to spectral separation to resolve bacterial cells of different species or in distinct physiological states. Focusing on host-microbe interactions that are difficult to quantify through fluorescence alone, we demonstrate the ability of µExM to distinguish species through an in vitro defined community of human gut commensals and in vivo imaging of a model gut microbiota, and to sensitively detect cell-envelope damage caused by antibiotics or previously unrecognized cell-to-cell phenotypic heterogeneity among pathogenic bacteria as they infect macrophages.


Assuntos
Acetobacter/ultraestrutura , Escherichia coli/ultraestrutura , Lactobacillus plantarum/ultraestrutura , Microscopia/métodos , Muramidase/farmacologia , Acetobacter/efeitos dos fármacos , Acidaminococcus/efeitos dos fármacos , Acidaminococcus/ultraestrutura , Animais , Antibacterianos/farmacologia , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Drosophila melanogaster/microbiologia , Escherichia coli/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Humanos , Hidrólise , Lactobacillus plantarum/efeitos dos fármacos , Camundongos , Microscopia/instrumentação , Muramidase/química , Platelmintos/microbiologia , Células RAW 264.7 , Estresse Mecânico , Simbiose/fisiologia , Vancomicina/farmacologia
15.
Clin Lab ; 65(9)2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31532092

RESUMO

BACKGROUND: The profile of leukocytes in bronchoalveolar lavage (BAL) fluid provides important information for diagnosing various lung diseases. A differential cell count of BAL is conventionally performed by evaluating centrifuged samples under a light microscope and enumerating the stained cells. Another rarely used method to identify BAL leukocytes is flow cytometry (FCM). However, there are no guidelines for standardizing this method and related literature is limited. This study aimed to evaluate the accuracy of FCM for identifying BAL leukocytes. METHODS: The BAL samples accepted to the hematology laboratory between 2014 - 2018 were retrospectively evaluated via light microscopy (LM) by a hematologist; while flow cytometric analyses with a monoclonal antibody panel composed of CD45/CD14/CD16 were noted by another doctor. The percentages of macrophages, lymphocytes, neutrophils and eosinophils determined by both methods were recorded for analysis. Correlations between the results from LM and FCM were investigated. In addition, compatibility between LM and FCM for denoting pathological values for each cell type was checked. RESULTS: Among 140 reviewed BAL samples, 76 were included for further analysis. Comparisons revealed strong correlations between FCM and LM for identifying macrophages, lymphocytes, neutrophils, and eosinophils. In addition, regarding the normal cutoff values for each leukocyte type, FCM and LM were similar in the identification of pathological changes of all cell types except eosinophils. CONCLUSIONS: Flow cytometry was found to be feasible for use instead of LM and might become a more widely used technique to analyze BAL fluid in the future.


Assuntos
Anticorpos Monoclonais/análise , Líquido da Lavagem Broncoalveolar/citologia , Citometria de Fluxo/métodos , Pneumopatias/diagnóstico , Neoplasias Pulmonares/diagnóstico , Anticorpos Monoclonais/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Eosinófilos/citologia , Eosinófilos/imunologia , Estudos de Viabilidade , Feminino , Humanos , Contagem de Leucócitos , Leucócitos/citologia , Leucócitos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Microscopia/métodos , Pessoa de Meia-Idade , Neutrófilos/citologia , Neutrófilos/imunologia , Patologia Clínica/instrumentação , Patologia Clínica/métodos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade
16.
Nat Methods ; 16(10): 1037-1044, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501548

RESUMO

Rapid developments in live-cell three-dimensional (3D) microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, tools to systematically measure and visualize the intricate relationships between intracellular signaling, cytoskeletal organization and downstream cell morphological outputs do not exist. Here, we introduce u-shape3D, a computer graphics and machine-learning pipeline to probe molecular mechanisms underlying 3D cell morphogenesis and to test the intriguing possibility that morphogenesis itself affects intracellular signaling. We demonstrate a generic morphological motif detector that automatically finds lamellipodia, filopodia, blebs and other motifs. Combining motif detection with molecular localization, we measure the differential association of PIP2 and KrasV12 with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP2 is enhanced on blebs. This indicates that subcellular signaling processes are differentially modulated by local morphological motifs. Overall, our computational workflow enables the objective, 3D analysis of the coupling of cell shape and signaling.


Assuntos
Imagem Tridimensional/métodos , Microscopia/métodos , Frações Subcelulares/metabolismo , Linhagem Celular Tumoral , Forma Celular , Gráficos por Computador , Humanos , Aprendizado de Máquina , Transdução de Sinais
17.
Nat Methods ; 16(10): 1054-1062, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31562489

RESUMO

The limited per-pixel bandwidth of most microscopy methods requires compromises between field of view, sampling density and imaging speed. This limitation constrains studies involving complex motion or fast cellular signaling, and presents a major bottleneck for high-throughput structural imaging. Here, we combine high-speed intensified camera technology with a versatile, reconfigurable and dramatically improved Swept, Confocally Aligned Planar Excitation (SCAPE) microscope design that can achieve high-resolution volumetric imaging at over 300 volumes per second and over 1.2 GHz pixel rates. We demonstrate near-isotropic sampling in freely moving Caenorhabditis elegans, and analyze real-time blood flow and calcium dynamics in the beating zebrafish heart. The same system also permits high-throughput structural imaging of mounted, intact, cleared and expanded samples. SCAPE 2.0's significantly lower photodamage compared to point-scanning techniques is also confirmed. Our results demonstrate that SCAPE 2.0 is a powerful, yet accessible imaging platform for myriad emerging high-speed dynamic and high-throughput volumetric microscopy applications.


Assuntos
Microscopia/métodos , Animais , Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Coração/embriologia , Coração/fisiologia , Fótons , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia
18.
BMC Bioinformatics ; 20(1): 472, 2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31521104

RESUMO

BACKGROUND: Nucleus is a fundamental task in microscopy image analysis and supports many other quantitative studies such as object counting, segmentation, tracking, etc. Deep neural networks are emerging as a powerful tool for biomedical image computing; in particular, convolutional neural networks have been widely applied to nucleus/cell detection in microscopy images. However, almost all models are tailored for specific datasets and their applicability to other microscopy image data remains unknown. Some existing studies casually learn and evaluate deep neural networks on multiple microscopy datasets, but there are still several critical, open questions to be addressed. RESULTS: We analyze the applicability of deep models specifically for nucleus detection across a wide variety of microscopy image data. More specifically, we present a fully convolutional network-based regression model and extensively evaluate it on large-scale digital pathology and microscopy image datasets, which consist of 23 organs (or cancer diseases) and come from multiple institutions. We demonstrate that for a specific target dataset, training with images from the same types of organs might be usually necessary for nucleus detection. Although the images can be visually similar due to the same staining technique and imaging protocol, deep models learned with images from different organs might not deliver desirable results and would require model fine-tuning to be on a par with those trained with target data. We also observe that training with a mixture of target and other/non-target data does not always mean a higher accuracy of nucleus detection, and it might require proper data manipulation during model training to achieve good performance. CONCLUSIONS: We conduct a systematic case study on deep models for nucleus detection in a wide variety of microscopy images, aiming to address several important but previously understudied questions. We present and extensively evaluate an end-to-end, pixel-to-pixel fully convolutional regression network and report a few significant findings, some of which might have not been reported in previous studies. The model performance analysis and observations would be helpful to nucleus detection in microscopy images.


Assuntos
Interpretação de Imagem Assistida por Computador/métodos , Microscopia/métodos , Humanos
19.
World J Gastroenterol ; 25(35): 5334-5343, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31558877

RESUMO

BACKGROUND: Although pathological response is a common endpoint used to assess the efficacy of neoadjuvant chemotherapy (NAC) for gastric cancer, the problem of a low rate of concordance from evaluations among pathologists remains unresolved. Moreover, there is no globally accepted consensus regarding the optimal evaluation. A previous study based on a clinical trial suggested that pathological response measured using digitally captured virtual microscopic slides predicted patients' survival well. However, the pathological concordance rate of this approach and its usefulness in clinical practice were unknown. AIM: To investigate the prognostic utility of pathological response measured using digital microscopic slides in clinical practice. METHODS: We retrospectively evaluated pathological specimens of gastric cancer patients who underwent NAC followed by surgery and achieved R0 resection between March 2009 and May 2015. Residual tumor area and primary tumor beds were measured in one captured image slide, which contained the largest diameter of the resected specimens. We classified patients with < 10% residual tumor relative to the primary tumorous area as responders, and the rest as non-responders; we then compared overall survival (OS) and relapse-free survival (RFS) between these two groups. Next, we compared the prognostic utility of this method using conventional Japanese criteria. RESULTS: Fifty-four patients were evaluated. The concordance rate between two evaluators was 96.2%. Median RFS of 25 responders and 29 non-responders was not reached (NR) vs 18.2 mo [hazard ratio (HR) = 0.35, P = 0.023], and median OS was NR vs 40.7 mo (HR = 0.3, P = 0.016), respectively. This prognostic value was statistically significant even after adjustment for age, eastern cooperative oncology group performance status, macroscopic type, reason for NAC, and T- and N-classification (HR = 0.23, P = 0.018). This result was also observed even in subgroup analyses for different macroscopic types (Borrmann type 4/non-type 4) and histological types (differentiated/undifferentiated). Moreover, the adjusted HR for OS between responders and non-responders was lower in this method than that in the conventional histological evaluation of Japanese Classification of Gastric Carcinoma criteria (0.23 vs 0.39, respectively). CONCLUSION: The measurement of pathological response using digitally captured virtual microscopic slides may be useful in clinical practice.


Assuntos
Antineoplásicos/uso terapêutico , Interpretação de Imagem Assistida por Computador/métodos , Terapia Neoadjuvante , Neoplasias Gástricas/terapia , Estômago/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Gastrectomia , Humanos , Masculino , Microscopia/métodos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasia Residual , Prognóstico , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Estômago/patologia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Resultado do Tratamento , Adulto Jovem
20.
PLoS Comput Biol ; 15(9): e1007348, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31479439

RESUMO

Cellular microscopy images contain rich insights about biology. To extract this information, researchers use features, or measurements of the patterns of interest in the images. Here, we introduce a convolutional neural network (CNN) to automatically design features for fluorescence microscopy. We use a self-supervised method to learn feature representations of single cells in microscopy images without labelled training data. We train CNNs on a simple task that leverages the inherent structure of microscopy images and controls for variation in cell morphology and imaging: given one cell from an image, the CNN is asked to predict the fluorescence pattern in a second different cell from the same image. We show that our method learns high-quality features that describe protein expression patterns in single cells both yeast and human microscopy datasets. Moreover, we demonstrate that our features are useful for exploratory biological analysis, by capturing high-resolution cellular components in a proteome-wide cluster analysis of human proteins, and by quantifying multi-localized proteins and single-cell variability. We believe paired cell inpainting is a generalizable method to obtain feature representations of single cells in multichannel microscopy images.


Assuntos
Microscopia/métodos , Análise de Célula Única/métodos , Aprendizado de Máquina não Supervisionado , Células Cultivadas , Biologia Computacional , Humanos , Processamento de Imagem Assistida por Computador/métodos , Leveduras/citologia
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