RESUMEN

This chapter introduces protocols for culturing and maintaining Dictyostelium discoideum and methods for conducting virulence assays in this organism to study bacterial pathogenicity. It outlines advanced techniques, such as automated microscopy and flow cytometry, for detailed cellular analysis and traditional microbiological approaches. These comprehensive protocols will enable researchers to probe the virulence factors of pathogens like Klebsiella pneumoniae and to elucidate the details of host-pathogen interactions within a cost-effective and adaptable laboratory framework.

Asunto(s)

Dictyostelium , Citometría de Flujo , Klebsiella pneumoniae , Dictyostelium/microbiología , Citometría de Flujo/métodos , Klebsiella pneumoniae/patogenicidad , Fagocitosis , Virulencia , Interacciones Huésped-Patógeno , Microscopía/métodos

RESUMEN

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Asunto(s)

Técnicas Histológicas , Microscopía , Animales , Citometría de Flujo , Procesamiento de Imagen Asistido por Computador

RESUMEN

Estimating the post-mortem interval (PMI) of human remains based on the histomorphology of dental pulp parameters is promising, but available evidence is scarce and sometimes contradictory without a scientific model. The aim of the study is to characterise the histomorphological changes of dental pulp associated with the decomposition of human remains by a qualitative and quantitative approach. The main aim is to establish a correlation based on post-mortem (PM) dental pulp histomorphology and the PMI, and whether pulp degradation could be an available medico-legal tool for PMI estimation beyond the first week after death (late PMI). The eligible sample consisted of 27 sound teeth from 16 healthy patients aged 16 to 72 years due to orthodontic or oral surgery treatment, to create PMI's simulating the death of the subject as the time elapsed from tooth avulsion. Data collected from patients (sex, date of birth, tooth position, date and hour of the avulsion, date and hour of pulp extraction) were anonymised in accordance with the requirements of Faculty of Dental Medicine of the University of Lisbon. The sample was divided into 9 groups of 3 teeth according to different PMI sets from T0 (baseline) up to 2 weeks (T0, 7, 12, 24, 36, 48, and 72 hours, 1 and 2 weeks). All the dental samples were stored at room temperature up to the time of pulp extraction and then prepared with haematoxylin and eosin stain. High-resolution microscopy was performed to obtain histological images. An operator performed the qualitative evaluation of blood vessels, collagen fibres, and the extra-cellular matrix (ECM) in PM pulps and measured the variation in cells/nuclei density by counting 6 different ROIs (Regions of Interest) for each pulp manually and automatically (quantitative analysis). Qualitative results showed that the degeneration of dental pulp appears 7 hours after death but histological changes in vessels, fibres, and ECM in PM dental pulp are characterised by high variability, consequently it is not possible to generalise the results for early PMIs. Quantitative measurements proved that cell count cannot be standardised due to the presence of superimposed layers of cells and nuclei fragmentation. Odontoblasts did not demonstrate evidence of cellular or nuclear lysis up to 14 PM suggesting their applicability in late PMIs. Future research will focus on late PMIs and different techniques of tooth preparation.

Asunto(s)

Pulpa Dental , Cambios Post Mortem , Humanos , Pulpa Dental/patología , Adulto , Femenino , Adolescente , Masculino , Persona de Mediana Edad , Adulto Joven , Anciano , Odontología Forense/métodos , Odontoblastos/patología , Microscopía , Colágeno/análisis

RESUMEN

Hyperspectral microscope imaging (HMI) technique was employed to assess the changes in physicochemical parameters and microstructure of 'Golden Delicious' apples flesh during storage. Four regions of interest (ROIs), including whole-cell ROI, intercellular space ROI, cytoplasm ROI, and cell wall ROI were investigated to assess their relationships with physicochemical parameters. Different ROIs presented similar vibrational profiles, but with slight differences in spectral intensity, especially in the range of 800-1000 nm. Spectral angle mapper (SAM) was applied to the HMI of apple tissues at different storage stages to clearly show the structural changes of parenchyma cells, while principal component analysis (PCA) could highlight the distribution of sugars, water and pigments in apple flesh at the cellular scale. Simultaneously with the degradation of acid-soluble pectin (ASP), middle lamella dissolution and increased intercellular space were observed using SEM and TEM. Single feature variables were used to construct linear models based on pearson correlation analysis, with R2 of 0.96 for moisture at 982 nm, 0.85 for water-soluble pectin (WSP) at 420 nm, 0.82 for L* at 946 nm, 0.77 for soluble solids content (SSC) at 484 nm, and 0.66 for firmness at 490 nm. This work demonstrated the great potential of HMI technology as a fast, accurate and efficient solution for assessing the quality of 'Golden Delicious' apples.

Asunto(s)

Frutas , Imágenes Hiperespectrales , Malus , Pectinas , Malus/química , Frutas/química , Imágenes Hiperespectrales/métodos , Pectinas/química , Pectinas/análisis , Análisis de Componente Principal , Microscopía/métodos , Almacenamiento de Alimentos/métodos , Microscopía Electrónica de Rastreo , Pared Celular/química

RESUMEN

Significance: The emergence of label-free microscopy techniques has significantly improved our ability to precisely characterize biochemical targets, enabling non-invasive visualization of cellular organelles and tissue organization. However, understanding each label-free method with respect to the specific benefits, drawbacks, and varied sensitivities under measurement conditions across different types of specimens remains a challenge. Aim: We link all of these disparate label-free optical interactions together and compare the detection sensitivity within the framework of statistical estimation theory. Approach: To achieve this goal, we introduce a comprehensive unified framework for evaluating the bounds for signal detection with label-free microscopy methods, including second-harmonic generation, third-harmonic generation, coherent anti-Stokes Raman scattering, coherent Stokes Raman scattering, stimulated Raman loss, stimulated Raman gain, stimulated emission, impulsive stimulated Raman scattering, transient absorption, and photothermal effect. A general model for signal generation induced by optical scattering is developed. Results: Based on this model, the information obtained is quantitatively analyzed using Fisher information, and the fundamental constraints on estimation precision are evaluated through the Cramér-Rao lower bound, offering guidance for optimal experimental design and interpretation. Conclusions: We provide valuable insights for researchers seeking to leverage label-free techniques for non-invasive imaging applications for biomedical research and clinical practice.

Asunto(s)

Espectrometría Raman , Espectrometría Raman/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodos , Algoritmos , Modelos Estadísticos , Humanos

Asunto(s)

Paracoccidioides , Paracoccidioidomicosis , Paracoccidioidomicosis/diagnóstico , Paracoccidioidomicosis/microbiología , Paracoccidioidomicosis/patología , Humanos , Paracoccidioides/aislamiento & purificación , Paracoccidioides/genética , Masculino , Enfermedades de la Boca/microbiología , Enfermedades de la Boca/diagnóstico , Enfermedades de la Boca/patología , Antifúngicos/uso terapéutico , Histocitoquímica , Microscopía , Persona de Mediana Edad

RESUMEN

Rationale: Azoospermia is a significant reproductive challenge. Differentiating between non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) is crucial as each type requires distinct management strategies. Testicular microcirculation plays a profound role in spermatogenic functions. However, current diagnostic methods are limited in their ability to effectively elucidate this crucial connection. Methods: We employed ultrasound localization microscopy (ULM) to visualize testicular microcirculation in NOA and OA patients and quantified the testicular hemodynamic parameters. Pearson correlation analysis was conducted to investigate the inner connection between parameters of testicular microcirculation and clinical spermatogenic functions. We conducted multiple logistic regression analysis to establish a new diagnostic model that integrates follicle-stimulating hormone (FSH) and mean vascular diameter to distinguish NOA from OA. Results: Our findings demonstrated significant differences in vascular parameters between NOA and OA, with NOA characterized by lower mean vascular diameter (p < 0.001), vessel density (p < 0.001), and fractal number (p < 0.001). Testicular volume showed a moderate positive correlation with mean vascular diameter (r = 0.419, p < 0.01) and vessel density (r = 0.415, p < 0.01); Mean vascular diameter exhibited negative correlations with both FSH (r = -0.214, p < 0.05) and age (r = -0.240, p < 0.05); FSH (r = -0.202, p < 0.05) and luteinizing hormone (LH) (r = -0.235, p < 0.05) were negatively correlated with mean blood flow velocity. The diagnostic model demonstrated an area under the curve (AUC) of 0.968. We also reported a method to map the vascular pressure distribution derived from the blood flow velocity generated by ULM. Conclusions: ULM provides a non-invasive and detailed assessment of testicular microvascular dynamics. The ULM-derived vascular parameters are able to connect testicular microcirculation to spermatogenic functions. The combination of FSH and mean vascular diameter enhances diagnostic precision and holds potential for distinguishing NOA from OA.

Asunto(s)

Azoospermia , Hormona Folículo Estimulante , Microcirculación , Espermatogénesis , Testículo , Masculino , Humanos , Testículo/irrigación sanguínea , Testículo/diagnóstico por imagen , Azoospermia/diagnóstico por imagen , Azoospermia/fisiopatología , Adulto , Hormona Folículo Estimulante/sangre , Ultrasonografía/métodos , Persona de Mediana Edad , Microscopía/métodos

RESUMEN

Intraluminal epithelial abnormalities, potential precursors to significant conditions like cancer, necessitate early detection for improved prognosis. We present a motor-free telerobotic optical coherence tomography (OCT) endoscope that offers high-resolution intraluminal imaging and overcomes the limitations of traditional systems in navigating curved lumens. This system incorporates a compact magnetic rotor with a rotatable diametrically magnetized cylinder permanent magnet (RDPM) and a reflector, effectively mitigating thermal and electrical risks by utilizing an external magnetic field to maintain temperature increases below 0.5 °C and generated voltage under 0.02 mV. Additionally, a learning-based method corrects imaging distortions resulting from nonuniform rotational speeds. Demonstrating superior maneuverability, the device achieves steerable angles up to 110° and operates effectively in vivo, providing distortion-free 3D programmable imaging in mouse colons. This advancement represents a significant step towards guidewire-independent endomicroscopy, enhancing both safety and potential patient outcomes.

Asunto(s)

Tomografía de Coherencia Óptica , Animales , Tomografía de Coherencia Óptica/métodos , Tomografía de Coherencia Óptica/instrumentación , Ratones , Imagenología Tridimensional/métodos , Imagenología Tridimensional/instrumentación , Endoscopios , Humanos , Diseño de Equipo , Microscopía/métodos , Microscopía/instrumentación , Endoscopía/métodos , Endoscopía/instrumentación

RESUMEN

Absolute quantity imaging of biomolecules on a single cell level is critical for measurement assurance in biosciences and bioindustries. While infrared (IR) transmission microscopy is a powerful label-free imaging modality capable of chemical quantification, its applicability to hydrated biological samples remains challenging due to the strong IR absorption by water. Traditional IR imaging of hydrated cells relies on powerful light sources, such as synchrotrons, to mitigate the light absorption by water. However, we overcome this challenge by applying a solvent absorption compensation (SAC) technique to a home-built benchtop IR microscope based on an external-cavity quantum cascade laser. SAC-IR microscopy adjusts the incident light using a pair of polarizers to precompensate the IR absorption by water while retaining the full dynamic range. Integrating the IR absorbance over a cell yields the total mass of biomolecules per cell. We monitor the total mass of the biomolecules of live fibroblast cells over 12 h, demonstrating promise for advancing our understanding of the biomolecular processes occurring in live cells on the single-cell level.

Asunto(s)

Análisis de la Célula Individual , Animales , Ratones , Fibroblastos/citología , Fibroblastos/química , Espectrofotometría Infrarroja/métodos , Microscopía/métodos , Rayos Infrarrojos , Células 3T3 NIH

RESUMEN

Tumor cells undergo an epithelial-mesenchymal transition (EMT) accompanied by a reduction in elasticity to initiate metastasis. However, in vivo, tumor cells typically exhibit partial EMT rather than fully EMT. Whether cell mechanics can accurately identify the status of partial EMT, especially the dynamic process, remains unclear. To elucidate the relationship between cell mechanics and partial EMT, we employed scanning ion conductance microscopy (SICM) to analyze the dynamic changes in cell mechanics during the TGFß-induced partial EMT of HCT116 colon cancer cells. Cells undergoing partial EMT, characterized by increased expression of EMT transcription factors, Snai1 and Zeb1, and EMT-related genes, Fn1 and MMP9, while retaining the expression of the epithelial markers E-cadherin (E-cad) and EpCAM, did not exhibit significant changes in cell morphology, suggesting that morphological changes alone were inadequate for identifying partial EMT status. However, cell elasticity markedly decreased in partial EMT cells, and this reduction was reversed with the reversible transition of partial EMT. These findings suggest a strong correlation between cell mechanics and the dynamic process of partial EMT, indicating that cell mechanics could serve as a valuable label-free marker for identifying the status of partial EMT while preserving the physiological characteristics of tumor cells.

Asunto(s)

Transición Epitelial-Mesenquimal , Humanos , Células HCT116 , Factor de Crecimiento Transformador beta/metabolismo , Microscopía/métodos , Elasticidad

RESUMEN

The digitization of pathology departments in hospitals around the world is now a reality. The current commercial solutions applied to digitize histopathological samples consist of a robotic microscope with an RGB-type camera attached to it. This technology is very limited in terms of information captured, as it only works with three spectral bands of the visible electromagnetic spectrum. Therefore, we present an automated system that combines RGB and hyperspectral technology. Throughout this work, the hardware of the system and its components are described along with the developed software and a working methodology to ensure the correct capture of histopathological samples. The software is integrated by the controller of the microscope, which features an autofocus functionality, whole slide scanning with a stitching algorithm, and hyperspectral scanning functionality. As a reference, the time to capture and process a complete sample with 20 regions of high biological interest using the proposed method is estimated at a maximum of 79 min, reducing the time required by a manual operator by at least three times. Both hardware and software can be easily adapted to other systems that might benefit from the advantages of hyperspectral technology.

Asunto(s)

Algoritmos , Procesamiento de Imagen Asistido por Computador , Microscopía , Programas Informáticos , Microscopía/métodos , Microscopía/instrumentación , Procesamiento de Imagen Asistido por Computador/métodos , Humanos , Bases de Datos Factuales , Imágenes Hiperespectrales/métodos , Imágenes Hiperespectrales/instrumentación

RESUMEN

OBJECTIVE: To evaluate the diagnostic accuracy of a commercial real-time polymerase chain reaction (PCR) kit targeting 18S rRNA against Giemsa-stained tissue slides in patients clinically suspected of cutaneous leishmaniasis (CL). STUDY DESIGN: Cross-sectional analytical study. Place and Duration of the Study: Department of Microbiology, Armed Forces Institute of Pathology / National University of Medical Sciences, Rawalpindi, Pakistan, from July to December 2022. METHODOLOGY: Samples of skin tissue in 98 patients suspected of CL were evaluated. These samples were subjected to Giemsa-staining for microscopy and real-time PCR. Sensitivity, specificity, and accuracy of the PCR were calculated keeping Giemsa-stained tissue slide microscopy as gold standard. RESULTS: Out of the 98 tissue samples, 37 were found positive for leishmaniasis on PCR while 13 were found Leishmania positive on microscopy of Giemsa-stained slides. The sensitivity, specificity, and accuracy of the PCR for the detection of Leishmania species were 100%, 71.8%, and 91.8%, respectively with 100% negative predictive value. CONCLUSION: This study demonstrates that the commercial PCR is a reliable diagnostic test for the diagnosis of CL. The ease, rapidity, and reliability of the PCR make it a dependable tool in diagnostic repertoire of CL. KEY WORDS: Giemsa stain, Leishmania spp., Polymerase chain reaction, Viasure.

Asunto(s)

Colorantes Azulados , Leishmaniasis Cutánea , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Humanos , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , Estudios Transversales , Masculino , Femenino , Pakistán , Reproducibilidad de los Resultados , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adulto , Biopsia/métodos , Coloración y Etiquetado/métodos , Adolescente , Leishmania/aislamiento & purificación , Leishmania/genética , Persona de Mediana Edad , Piel/parasitología , Piel/patología , Adulto Joven , Niño , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 18S/genética , Microscopía/métodos

RESUMEN

With recent advances in multi-color super-resolution light microscopy, it is possible to simultaneously visualize multiple subunits within biological structures at nanometer resolution. To optimally evaluate and interpret spatial proximity of stainings on such an image, colocalization analysis tools have to be able to integrate prior knowledge on the local geometry of the recorded biological complex. We present MultiMatch to analyze the abundance and location of chain-like particle arrangements in multi-color microscopy based on multi-marginal optimal unbalanced transport methodology. Our object-based colocalization model statistically addresses the effect of incomplete labeling efficiencies enabling inference on existent, but not fully observable particle chains. We showcase that MultiMatch is able to consistently recover existing chain structures in three-color STED images of DNA origami nanorulers and outperforms geometry-uninformed triplet colocalization methods in this task. MultiMatch generalizes to an arbitrary number of color channels and is provided as a user-friendly Python package comprising colocalization visualizations.

Asunto(s)

Procesamiento de Imagen Asistido por Computador , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Color , Algoritmos , ADN/química , ADN/metabolismo , Microscopía/métodos , Programas Informáticos

RESUMEN

In scenarios where yeast and bacterial cells coexist, it is of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared, mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries, and analyzed using bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter Counter (at 1:1 and 100:1 ratios). We observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter Counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (p > .05). In contrast, the Coulter Counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (p < .05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasn't possible due to an overlap between yeast cell debris and bacterial cells. We conclude that each method has limitations, advantages, and disadvantages. ONE-SENTENCE SUMMARY: This study compares methods for simultaneously quantifying yeast and bacterial cells in a mixed sample, highlighting that in different cell proportions, some methods cannot quantify both cell types and present distinct advantages and limitations regarding time, cost, and precision.

Asunto(s)

Microbiología Industrial , Saccharomyces cerevisiae , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/citología , Microbiología Industrial/métodos , Citometría de Flujo/métodos , Recuento de Colonia Microbiana/métodos , Carga Bacteriana/métodos , Saccharum/microbiología , Microscopía/métodos

RESUMEN

BACKGROUND: Malaria is the parasitic disease with the highest morbimortality worldwide. The World Health Organization (WHO) estimates that there were approximately 249 million cases in 2022, of which 3.4% were in Angola. Diagnosis is based on parasite identification by microscopy examination, antigen detection, and/or molecular tests, such as polymerase chain reaction (PCR). This study aimed to evaluate the usefulness of real-time PCR as a diagnostic method for malaria in an endemic area (Cubal, Angola). METHODS: A cross-sectional study was carried out at the Hospital Nossa Senhora da Paz in Cubal, Angola, including 200 patients who consulted for febrile syndrome between May and July 2022. From each patient, a capillary blood sample was obtained by finger prick for malaria field diagnosis [microscopy and rapid diagnostic test (RDT)] and venous blood sample for real-time PCR performed at the Hospital Universitario Vall d'Hebron in Barcelona, Spain. Any participant with a positive result from at least one of these three methods was diagnosed with malaria. RESULTS: Of the 200 participants included, 54% were female and the median age was 7 years. Malaria was diagnosed by at least one of the three techniques (microscopy, RDT, and/or real-time PCR) in 58% of the participants, with RDT having the highest percentage of positivity (49%), followed by real-time PCR (39.5%) and microscopy (33.5%). Of the 61 discordant samples, 4 were only positive by microscopy, 13 by real-time PCR, and 26 by RDT. Plasmodium falciparum was the most frequent species detected (90.63%), followed by P. malariae (17.19%) and P. ovale (9.38%). Coinfections were detected in ten participants (15.63%): six (60%) were caused by P. falciparum and P. malariae, three (30%) by P. falciparum and P. ovale, and one (10%) triple infection with these three species. In addition, it was observed that P. falciparum and P. malariae coinfection significantly increased the parasite density of the latter. CONCLUSIONS: RDT was the technique with the highest positivity rate, followed by real-time PCR and microscopy. The results of the real-time PCR may have been underestimated due to suboptimal storage conditions during the transportation of the DNA eluates. However, real-time PCR techniques have an important role in the surveillance of circulating Plasmodium species, given the epidemiological importance of the increase in non-falciparum species in the country, and can provide an estimate of the intensity of infection.

Asunto(s)

Fiebre , Malaria , Plasmodium , Reacción en Cadena en Tiempo Real de la Polimerasa , Humanos , Angola/epidemiología , Femenino , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Masculino , Estudios Transversales , Malaria/diagnóstico , Malaria/parasitología , Malaria/epidemiología , Niño , Fiebre/parasitología , Preescolar , Plasmodium/aislamiento & purificación , Plasmodium/genética , Plasmodium/clasificación , Adolescente , Adulto , Microscopía/métodos , Adulto Joven , Lactante , Sensibilidad y Especificidad , Persona de Mediana Edad , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Pruebas Diagnósticas de Rutina/métodos

RESUMEN

Introduction: Ehrlich-Ziehl-Neelsen (EZN) staining and culture methods are often used to diagnose tuberculosis. This study aimed to determine the acidfast bacteria (AFS) positivity rates in various clinical samples sent to our laboratory over five years and the growth and resistance rates in two different (solid and liquid) cultures and compare them with the data from Türkiye and the world. Materials and Methods: A total of 62.456 clinic samples were accepted in the microbiology laboratory between 2019 and 2024. The mycobacterial culture was performed by searching for acid-resistant bacilli microscopically and parallel inoculation media [solid Löwenstein-Jensen (L-J) and MGIT 960 liquid]. Those growing in the MGIT 960 system were identified using BD MGIT TBC Identification test kits that detect the MPT64 antigen. AFS and MPT64 antigenpositive samples were identified as Mycobacterium tuberculosis complex (MTBC) while AFS-positive samples and MPT64 antigen-negative results were classified as non-tuberculous mycobacteria (NTM). Drug susceptibility testing was performed with the BACTEC MGIT 960 SIRE kit. Susceptibility to NTM samples was not performed. Result: Out of a total of 120.829 samples, 95.101 were lung samples and 25.728 were extrapulmonary samples. AFS positivity was detected in 2961 (2.4%) samples. MTBC grew in 6854 (5.6%) samples, and NTM grew in 1506 (1.24%) samples. Contamination was detected in 7171 (5.9%) media. Two thousand one hundred and sixty-nine susceptibility tests were performed. Considering antibiotic resistance rates, isoniazid resistance was detected in 154 (7%), rifampicin resistance in 140 (6.4%), ethambutol resistance in 18 (0.8%), and streptomycin resistance in 120 (0.5%) samples. All four-drug resistance was observed in 91 (4.1%) samples. AFP positivity and resistance rates for rifampicin have decreased significantly, while there have been no significant changes in NTM rates over the years. Conclusions: When our data was determined, the sensitivity of microscopy was low. It is understood that mycobacterial culture and microscopy must be evaluated together to exclude tuberculosis infection. The high mycobacterial culture positivity rate, which is 5.6%, is due to the high number of follow-up patients and new referrals. It is seen that the change in sensitivity rates is due to the period of the COVID-19 epidemic, and it is similar to World Health Organization (WHO) data.

Asunto(s)

Mycobacterium tuberculosis , Humanos , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/microbiología , Tuberculosis/diagnóstico , Pruebas de Sensibilidad Microbiana , Turquía/epidemiología , Microscopía/métodos , Micobacterias no Tuberculosas/aislamiento & purificación , Micobacterias no Tuberculosas/efectos de los fármacos , Antituberculosos/uso terapéutico , Antituberculosos/farmacología , Técnicas Bacteriológicas/métodos , Farmacorresistencia Bacteriana

RESUMEN

Biophotonic nanostructures in butterfly wing scales remain fascinating examples of biological functional materials, with intriguing open questions with regard to formation and evolutionary function. One particularly interesting butterfly species, Erora opisena (Lycaenidae: Theclinae), develops wing scales that contain three-dimensional photonic crystals that closely resemble a single gyroid geometry. Unlike most other gyroid-forming butterflies, E. opisena develops discrete gyroid crystallites with a pronounced size gradient hinting at a developmental sequence frozen in time. Here, we present a novel application of a hyperspectral (wavelength-resolved) microscopy technique to investigate the ultrastructural organization of these gyroid crystallites in dry, adult wing scales. We show that reflectance corresponds to crystallite size, where larger crystallites reflect green wavelengths more intensely; this relationship could be used to infer size from the optical signal. We further successfully resolve the red-shifted reflectance signal from wing scales immersed in refractive index liquids with varying refractive index, including values similar to water or cytosol. Such photonic crystals with lower refractive index contrast may be similar to the hypothesized nanostructural forms in the developing butterfly scales. The ability to resolve these fainter signals hints at the potential of this facile light microscopy method for in vivo analysis of nanostructure formation in developing butterflies.

Asunto(s)

Mariposas Diurnas , Microscopía , Alas de Animales , Animales , Alas de Animales/ultraestructura , Microscopía/métodos , Nanoestructuras , Fotones

RESUMEN

Glaucoma is a blinding disease where the retinal ganglion cells and their axons degenerate. Degradation of axonal microtubules is thought to play a critical role in the pathogenesis, but the mechanism is unknown. Here we investigate whether microtubule disruption in glaucoma can be alleviated by metabolic rescue. The integrity of axonal microtubules and the morphology of the retinal nerve fibers were evaluated by second-harmonic generation microscopy in a mouse model of glaucoma, DBA/2J, which received a dietary supplement of nicotinamide (NAM) for reducing metabolic stress. It was compared with control DBA/2J, which did not receive NAM, and non-glaucomatous DBA/2J-Gpnmb+. We found that the morphology of the retinal nerve fibers, but not axonal microtubules, are significantly protected by NAM. The decoupling is analogous to microtubule deficit, a glaucoma pathology in which axonal microtubules exhibit rapid degradation compared to the morphology of the retinal nerve fibers. Understanding microtubule deficit could provide insights into the divergent responses to NAM. From co-registered images of second-harmonic generation and immunofluorescence, it was determined that microtubule deficit was not due to a shortage of tubulins. Furthermore, microtubule deficit colocalized with the sectors in which the retinal ganglion cells were disconnected from the brain, suggesting that microtubule disruption is associated with axonal transport deficit in glaucoma. Together, our data suggests significant role axonal microtubules play in glaucomatous degeneration, offering a new opportunity for neuroprotection.

Asunto(s)

Modelos Animales de Enfermedad , Glaucoma , Ratones Endogámicos DBA , Microtúbulos , Niacinamida , Células Ganglionares de la Retina , Animales , Glaucoma/patología , Glaucoma/metabolismo , Glaucoma/tratamiento farmacológico , Niacinamida/farmacología , Niacinamida/uso terapéutico , Ratones , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Axones/efectos de los fármacos , Axones/metabolismo , Axones/patología , Microscopía/métodos , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/patología , Fibras Nerviosas/metabolismo

RESUMEN

A deep understanding of neuron structure and function is crucial for elucidating brain mechanisms, diagnosing and treating diseases. Optical microscopy, pivotal in neuroscience, illuminates neuronal shapes, projections, and electrical activities. To explore the projection of specific functional neurons, scientists have been developing optical-based multimodal imaging strategies to simultaneously capture dynamic in vivo signals and static ex vivo structures from the same neuron. However, the original position of neurons is highly susceptible to displacement during ex vivo imaging, presenting a significant challenge for integrating multimodal information at the single-neuron level. This study introduces a graph-model-based approach for cell image matching, facilitating precise and automated pairing of sparsely labeled neurons across different optical microscopic images. It has been shown that utilizing neuron distribution as a matching feature can mitigate modal differences, the high-order graph model can address scale inconsistency, and the nonlinear iteration can resolve discrepancies in neuron density. This strategy was applied to the connectivity study of the mouse visual cortex, performing cell matching between the two-photon calcium image and the HD-fMOST brain-wide anatomical image sets. Experimental results demonstrate 96.67% precision, 85.29% recall rate, and 90.63% F1 Score, comparable to expert technicians. This study builds a bridge between functional and structural imaging, offering crucial technical support for neuron classification and circuitry analysis.

Asunto(s)

Neuronas , Animales , Ratones , Corteza Visual/diagnóstico por imagen , Corteza Visual/fisiología , Microscopía/métodos , Reconocimiento de Normas Patrones Automatizadas , Algoritmos , Imagen Multimodal/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Encéfalo/diagnóstico por imagen

RESUMEN

Digital pathology has become increasingly popular for research and clinical applications. Using high-quality microscopes to produce Whole Slide Images of tumor tissue enables the discovery of insights into biological aspects invisible to the human eye. These are acquired through downstream analyses using spatial statistics and artificial intelligence. Determination of the quality and consistency of these images is needed to ensure accurate outcomes when identifying clinical and subclinical image features. Additionally, the time-intensive process of generating high-volume images results in a trade-off that needs to be carefully balanced. This study aims to determine optimal instrument settings to generate representative images of pathological tissue using digital microscopy. Using various settings, an H&E stained sample was scanned using the ZEISS Axio Scan.Z1. Next, nucleus segmentation was performed on resulting images using StarDist. Subsequently, detections were compared between scans using a matching algorithm. Finally, nucleus-level information was compared between scans. Results indicated that while general matching percentages were high, similarity between information from replicates was relatively low. Additionally, settings resulting in longer scanning times and increased data volume did not increase similarity between replicates. In conclusion, the scan setting ultimately deemed optimal combined consistent and qualitative performance with low throughput time.

Asunto(s)

Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Procesamiento de Imagen Asistido por Computador/métodos , Algoritmos , Microscopía/métodos , Núcleo Celular