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1.
Eur J Dermatol ; 34(1): 55-58, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38557459

RESUMO

Lichen striatus (LS), linear psoriasis (LPs), linear cutaneous lupus erythematosus (LCLE) and linear lichen planus (LLP) often have similar clinical manifestations, which makes clinical diagnosis with the naked eye difficult; therefore, they are easily misdiagnosed. The purpose of this study was to determine whether reflectance confocal microscopy (RCM) is helpful in differentiating between these four linear dermatoses in children. This retrospective study included 14 patients with LS, nine with LPs, eight with LCLE and 12 with LLP. All patients were analysed using RCM, and biopsies were collected from lesions previously imaged by RCM. For LS, the dermal papillary rings were partially absent, but when present, manifested with small, homogeneously round, bright cells and occasionally highly refractive plump cellular structures, aggregated in clusters. LPs exhibited dark cyst-like structures with small, bright, round cells aggregated at the epidermal level; at the dermal-epidermal junction, homogeneously distributed, enlarged, faint dermal papillary rings and numerous enlarged low-refractive canalicular structures were observed in the superficial dermis. LCLE and LLP exhibited similar manifestations, including epidermal disarray, almost total absence of dermal papillary rings, and various sized refractive structures densely distributed in the dermis. The key distinguishing features of LCLE were the different sized structures mainly clustered around hair follicles, while LLP demonstrated dense structures with a scattered distribution. RCM may be used to distinguish between the key features of LS, LPs, LCLE and LLP in children.


Assuntos
Ceratose , Líquen Plano , Psoríase , Criança , Humanos , Estudos Retrospectivos , Lipopolissacarídeos , Epiderme/patologia , Líquen Plano/patologia , Ceratose/patologia , Psoríase/patologia , Prurido/patologia , Microscopia Confocal/métodos
2.
Molecules ; 29(5)2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38474655

RESUMO

In this research, we explore the synthesis of and characterize α-aminophosphonates derived from anthraquinone and benzanthrone, focusing on their fluorescence properties and potential applications in confocal laser scanning microscopy (CLSM). The synthesized compounds exhibit notable solvatochromic behavior, emitting fluorescence from green to red across various solvents. Spectroscopic analysis, including 1H-, 13C-, and 31P-NMR, FTIR, and mass spectrometry, confirms the chemical structures. The compounds' toxicity is evaluated using etiolated wheat sprouts, revealing varying degrees of impact on growth and oxidative damage. Furthermore, the study introduces these α-aminophosphonates for CLSM imaging of the parasitic flatworm Opisthorchis felineus, demonstrating their potential in visualizing biological specimens. Additionally, an X-ray crystallographic study of an anthraquinone α-aminophosphonate provides valuable structural insights.


Assuntos
Benzo(a)Antracenos , Opisthorchis , Organofosfonatos , Animais , Cristalografia por Raios X , Organofosfonatos/química , Espectroscopia de Ressonância Magnética , Microscopia Confocal/métodos , Antraquinonas
3.
Cells ; 13(5)2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38474343

RESUMO

PURPOSE: Chemical eye injury is an acute emergency that can result in vision loss. Neurotrophic keratitis (NK) is the most common long-term manifestation of chemical injury. NK due to alkali burn affects ocular surface health and is one of its most common causes. Here, we established a rabbit model of corneal alkali burns to evaluate the severity of NK-associated changes. MATERIAL METHODS: Alkali burns were induced in NZ rabbits by treating the cornea with (i) a 5 mm circular filter paper soaked in 0.75 N NaOH for 10 s (Mild NK) and (ii) trephination using a guarded trephine (5 mm diameter and 150-micron depth), followed by alkali burn, with a 5 mm circular filter paper soaked in 0.75 N NaOH for 10 s (a severe form of NK). Immediately after, the cornea was rinsed with 10 mL of normal saline to remove traces of NaOH. Clinical features were evaluated on Day 0, Day 1, Day 7, Day 15, and Day 21 post-alkali burn using a slit lamp, Pentacam, and anterior segment optical coherence tomography (AS-OCT). NK-like changes in epithelium, sub-basal nerve plexus, and stroma were observed using in vivo confocal microscopy (IVCM), and corneal sensation were measured using an aesthesiometer post alkali injury. After 21 days, pro-inflammatory cytokines were evaluated for inflammation through ELISA. RESULTS: Trephination followed by alkali burn resulted in the loss of epithelial layers (manifested using fluorescein stain), extensive edema, and increased corneal thickness (550 µm compared to 380 µm thickness of control) evaluated through AS-OCT and increased opacity score in alkali-treated rabbit (80 compared to 16 controls). IVCM images showed complete loss of nerve fibers, which failed to regenerate over 30 days, and loss of corneal sensation-conditions associated with NK. Cytokines evaluation of IL6, VEGF, and MMP9 indicated an increased angiogenic and pro-inflammatory milieu compared to the milder form of NK and the control. DISCUSSION: Using clinical parameters, we demonstrated that the alkali-treated rabbit model depicts features of NK. Using IVCM in the NaOH burn animal model, we demonstrated a complete loss of nerve fibers with poor self-healing capability associated with sub-basal nerve degeneration and compromised corneal sensation. This pre-clinical rabbit model has implications for future pre-clinical research in neurotrophic keratitis.


Assuntos
Queimaduras Químicas , Doenças da Córnea , Ceratite , Coelhos , Animais , Queimaduras Químicas/tratamento farmacológico , Álcalis , Hidróxido de Sódio/uso terapêutico , Córnea , Microscopia Confocal/métodos , Citocinas
4.
Crit Rev Oncog ; 29(2): 53-63, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38505881

RESUMO

The protocol for treating locally advanced rectal cancer consists of the application of chemoradiotherapy (neoCRT) followed by surgical intervention. One issue for clinical oncologists is predicting the efficacy of neoCRT in order to adjust the dosage and avoid treatment toxicity in cases when surgery should be conducted promptly. Biomarkers may be used for this purpose along with in vivo cell-level images of the colorectal mucosa obtained by probe-based confocal laser endomicroscopy (pCLE) during colonoscopy. The aim of this article is to report our experience with Motiro, a computational framework that we developed for machine learning (ML) based analysis of pCLE videos for predicting neoCRT response in locally advanced rectal cancer patients. pCLE videos were collected from 47 patients who were diagnosed with locally advanced rectal cancer (T3/T4, or N+). The patients received neoCRT. Response to treatment by all patients was assessed by endoscopy along with biopsy and magnetic resonance imaging (MRI). Thirty-seven patients were classified as non-responsive to neoCRT because they presented a visible macroscopic neoplastic lesion, as confirmed by pCLE examination. Ten remaining patients were considered responsive to neoCRT because they presented lesions as a scar or small ulcer with negative biopsy, at post-treatment follow-up. Motiro was used for batch mode analysis of pCLE videos. It automatically characterized the tumoral region and its surroundings. That enabled classifying a patient as responsive or non-responsive to neoCRT based on pre-neoCRT pCLE videos. Motiro classified patients as responsive or non-responsive to neoCRT with an accuracy of ~ 0.62 when using images of the tumor. When using images of regions surrounding the tumor, it reached an accuracy of ~ 0.70. Feature analysis showed that spatial heterogeneity in fluorescence distribution within regions surrounding the tumor was the main contributor to predicting response to neoCRT. We developed a computational framework to predict response to neoCRT by locally advanced rectal cancer patients based on pCLE images acquired pre-neoCRT. We demonstrate that the analysis of the mucosa of the region surrounding the tumor provides stronger predictive power.


Assuntos
Neoplasias Colorretais , Segunda Neoplasia Primária , Neoplasias Retais , Humanos , Terapia Neoadjuvante , Microscopia Confocal/métodos , Colonoscopia/métodos , Neoplasias Colorretais/diagnóstico , Neoplasias Retais/diagnóstico por imagem , Neoplasias Retais/terapia
5.
Sci Rep ; 14(1): 6784, 2024 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-38514670

RESUMO

In this multi-center, assessor-blinded pilot study, the diagnostic efficacy of cCeLL-Ex vivo, a second-generation confocal laser endomicroscopy (CLE), was compared against the gold standard frozen section analysis for intraoperative brain tumor diagnosis. The study was conducted across three tertiary medical institutions in the Republic of Korea. Biopsy samples from newly diagnosed brain tumor patients were categorized based on location and divided for permanent section analysis, frozen section analysis, and cCeLL-Ex vivo imaging. Of the 74 samples from 55 patients, the majority were from the tumor core (74.3%). cCeLL-Ex vivo exhibited a relatively higher diagnostic accuracy (89.2%) than frozen section analysis (86.5%), with both methods showing a sensitivity of 92.2%. cCeLL-Ex vivo also demonstrated higher specificity (70% vs. 50%), positive predictive value (PPV) (95.2% vs. 92.2%), and negative predictive value (NPV) (58.3% vs. 50%). Furthermore, the time from sample preparation to diagnosis was notably shorter with cCeLL-Ex vivo (13 min 17 s) compared to frozen section analysis (28 min 28 s) (p-value < 0.005). These findings underscore cCeLL-Ex vivo's potential as a supplementary tool for intraoperative brain tumor diagnosis, with future studies anticipated to further validate its clinical utility.


Assuntos
Neoplasias Encefálicas , Humanos , Projetos Piloto , Estudos Prospectivos , Microscopia Confocal/métodos , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/cirurgia , Lasers
6.
Eye Contact Lens ; 50(4): 163-170, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38350097

RESUMO

AIM: To characterize the regional variations in corneal epithelial cell density and morphology using in vivo confocal microscopy (IVCM). METHODS: Corneal imaging (IVCM) at 10 locations was performed; corneal apex (i.e., the center), immediately anterior to the corneal nerve whorl (i.e., slightly inferior to the apex), and four quadrants (superior, nasal, inferior, and temporal) both at 1.5 mm and 4.5 mm from the corneal apex (corresponding to 3 mm central and 9 mm peripheral diameter rings). The data of 21 young adults, aged 18 to 35 years, were analyzed. Cell morphometric parameters, including cell density, area, perimeter, Feret diameter, and circularity, were measured for basal and wing cells using Image J software. RESULTS: There was a significant difference in basal cell density (BCD) ( P <0.001) and wing cell density (WCD) ( P <0.001) for different corneal locations. The BCD (mean±SD: 8,839±416 cells/mm 2 ) and WCD (mean±SD: 5,932±310 cells/mm 2 ) were highest at the corneal nerve whorl compared with all other corneal locations. There were significant differences in wing cell area ( P <0.001), perimeter ( P <0.001), Feret diameter ( P <0.001), and circularity ( P <0.001) at varying corneal locations. CONCLUSION: There are significant regional variations in corneal epithelial cell density and morphology. The BCD and WCD was highest anterior to the corneal nerve whorl.


Assuntos
Epitélio Corneano , Adulto Jovem , Humanos , Córnea , Microscopia Confocal/métodos , Contagem de Células , Células Epiteliais
7.
J Biomed Opt ; 29(2): 026501, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38414657

RESUMO

Significance: The imaging depth of microscopy techniques is limited by the ability of light to penetrate biological tissue. Recent research has addressed this limitation by combining a reflectance confocal microscope with the NIR-II (or shortwave infrared) spectrum. This approach offers significant imaging depth, is straightforward in design, and remains cost-effective. However, the imaging system, which relies on intrinsic signals, could benefit from adjustments in its optical design and post-processing methods to differentiate cortical cells, such as neurons and small blood vessels. Aim: We implemented a phase contrast detection scheme to a reflectance confocal microscope using NIR-II spectral range as illumination. Approach: We analyzed the features retrieved in the images while testing the imaging depth. Moreover, we introduce an acquisition method for distinguishing dynamic signals from the background, allowing the creation of vascular maps similar to those produced by optical coherence tomography. Results: The phase contrast implementation is successful to retrieve deep images in the cortex up to 800 µm using a cranial window. Vascular maps were retrieved at similar cortical depth and the possibility of combining multiple images can provide a vessel network. Conclusions: Phase contrast reflectance confocal microscopy can improve the outlining of cortical cell bodies. With the presented framework, angiograms can be retrieved from the dynamic signal in the biological tissue. Our work presents an optical implementation and analysis techniques from a former microscope design.


Assuntos
Microscopia , Tomografia de Coerência Óptica , Microscopia de Contraste de Fase , Neuroimagem , Microscopia Confocal/métodos
8.
Eur Arch Otorhinolaryngol ; 281(4): 2115-2122, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38329525

RESUMO

PURPOSE: Confocal Laser Endomicroscopy (CLE) is an imaging tool, that has demonstrated potential for intraoperative, real-time, non-invasive, microscopical assessment of surgical margins of oropharyngeal squamous cell carcinoma (OPSCC). However, interpreting CLE images remains challenging. This study investigates the application of OpenAI's Generative Pretrained Transformer (GPT) 4.0 with Vision capabilities for automated classification of CLE images in OPSCC. METHODS: CLE Images of histological confirmed SCC or healthy mucosa from a database of 12 809 CLE images from 5 patients with OPSCC were retrieved and anonymized. Using a training data set of 16 images, a validation set of 139 images, comprising SCC (83 images, 59.7%) and healthy normal mucosa (56 images, 40.3%) was classified using the application programming interface (API) of GPT4.0. The same set of images was also classified by CLE experts (two surgeons and one pathologist), who were blinded to the histology. Diagnostic metrics, the reliability of GPT and inter-rater reliability were assessed. RESULTS: Overall accuracy of the GPT model was 71.2%, the intra-rater agreement was κ = 0.837, indicating an almost perfect agreement across the three runs of GPT-generated results. Human experts achieved an accuracy of 88.5% with a substantial level of agreement (κ = 0.773). CONCLUSIONS: Though limited to a specific clinical framework, patient and image set, this study sheds light on some previously unexplored diagnostic capabilities of large language models using few-shot prompting. It suggests the model`s ability to extrapolate information and classify CLE images with minimal example data. Whether future versions of the model can achieve clinically relevant diagnostic accuracy, especially in uncurated data sets, remains to be investigated.


Assuntos
Neoplasias de Cabeça e Pescoço , Humanos , Reprodutibilidade dos Testes , Microscopia Confocal/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Lasers
9.
Sci Rep ; 14(1): 2768, 2024 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-38307985

RESUMO

The accurate determination of the size and depth of infiltration is critical to the treatment and excision of melanoma and other skin cancers. However, current techniques, such as skin biopsy and histological examination, pose invasiveness, time-consumption, and have limitations in measuring at the deepest level. Non-invasive imaging techniques like dermoscopy and confocal microscopy also present limitations in accurately capturing contrast and depth information for various skin types and lesion locations. Thus, there is a pressing need for non-invasive devices capable of obtaining high-resolution 3D images of skin lesions. In this study, we introduce a novel device that combines 18 MHz ultrasound and photoacoustic tomography into a single unit, enabling the acquisition of colocalized 3D images of skin lesions. We performed in vivo measurements on 25 suspicious human skin nevi that were promptly excised following measurements. The combined ultrasound/photoacoustic tomography imaging technique exhibited a strong correlation with histological Breslow thickness between 0.2 and 3 mm, achieving a coefficient of determination (R[Formula: see text]) of 0.93, which is superior to the coefficients from the individual modalities. The results procured in our study underscore the potential of combined ultrasound and photoacoustic tomography as a promising non-invasive 3D imaging approach for evaluating human nevi and other skin lesions. Furthermore, the system allows for integration of other optical modalities such as optical coherence tomography, microscopy, or Raman spectroscopy in future applications.


Assuntos
Nevo , Neoplasias Cutâneas , Humanos , Projetos Piloto , Imageamento Tridimensional , Neoplasias Cutâneas/patologia , Tomografia de Coerência Óptica , Microscopia Confocal/métodos
10.
In Vivo ; 38(2): 855-863, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38418139

RESUMO

BACKGROUND/AIM: The need for instant histological evaluation of fresh tissue, especially in cancer treatment, remains paramount. The conventional frozen section technique has inherent limitations, prompting the exploration of alternative methods. A recently developed confocal laser endomicroscopic system provides real-time imaging of the tissue without the need for glass slide preparation. Herein, we evaluated its applicability in the histologic evaluation of gastric cancer tissues. MATERIALS AND METHODS: A confocal laser endomicroscopic system (CLES) with a Lissajous pattern laser scanning, was developed. Fourteen fresh gastric cancer tissues and the same number of normal gastric tissues were obtained from advanced gastric cancer patients. Fluorescein sodium was used for staining. Five pathologists interpreted 100 endomicroscopic images and decided their histologic location and the presence of cancer. Following the review of matched hematoxylin and eosin (H&E) slides, their performance was evaluated with another 100 images. RESULTS: CLES images mirrored gastric tissue histology. Pathologists were able to detect the histologic location of the images with 65.7% accuracy and differentiate cancer tissue from normal with 74.7% accuracy. The sensitivity and specificity of cancer detection were 71.9% and 76.1%. Following the review of matched H&E images, the accuracy of identifying the histologic location was increased to 92.8% (p<0.0001), and that of detecting cancer tissue was also increased to 90.9% (p<0.001). The sensitivity and specificity of cancer detection were enhanced to 89.1% and 93.2% (p<0.0001). CONCLUSION: High-quality histological images were immediately acquired by the CLES. The operator training enabled the accurate detection of cancer and histologic location raising its potential applicability as a real-time tissue imaging modality.


Assuntos
Neoplasias Gástricas , Humanos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patologia , Microscopia Confocal/métodos , Fluoresceína , Amarelo de Eosina-(YS) , Lasers
12.
Respiration ; 103(4): 171-176, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38387451

RESUMO

INTRODUCTION: Increasing numbers of cases of mild asymptomatic pulmonary alveolar proteinosis (PAP) are being reported with the recent increase in chest computed tomography (CT). Bronchoscopic diagnosis of mild PAP is challenging because of the patchy distribution of lesions, which makes it difficult to obtain sufficient biopsy samples. Additionally, the pathological findings of mild PAP, particularly those that differ from severe PAP, have not been fully elucidated. This study aimed to clarify the pathological findings of mild PAP and the usefulness of optical biopsy using probe-based confocal laser endomicroscopy (pCLE). METHODS: We performed bronchoscopic optical biopsy using pCLE and tissue biopsy in 5 consecutive patients with PAP (three with mild PAP and two with severe PAP). We compared the pCLE images of mild PAP with those of severe PAP by integrating clinical findings, tissue pathology, and chest CT images. RESULTS: pCLE images of PAP showed giant cells with strong fluorescence, amorphous substances, and thin alveolar walls. Images of affected lesions in mild PAP were equivalent to those obtained in arbitrary lung lesions in severe cases. All 3 patients with mild PAP spontaneously improved or remained stable after ≥3 years of follow-up. Serum autoantibodies to granulocyte-macrophage colony-stimulating factor were detected in all 5 cases. CONCLUSION: Optical biopsy using pCLE can yield specific diagnostic findings, even in patients with mild PAP. pCLE images of affected areas in mild and severe PAP showed similar findings, indicating that the dysfunction level of pathogenic alveolar macrophages in affected areas is similar between both disease intensities.


Assuntos
Doenças Autoimunes , Proteinose Alveolar Pulmonar , Humanos , Proteinose Alveolar Pulmonar/diagnóstico por imagem , Microscopia Confocal/métodos , Biópsia , Lasers
13.
J Control Release ; 368: 797-807, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38350493

RESUMO

Tracking drug disposition in the skin in a non-destructive and at least semi-quantitative fashion is a relevant objective for the assessment of local (cutaneous) bioavailability. Confocal Raman spectroscopy has been shown potentially useful in this regard and, importantly, recent advances have enabled the presence of applied chemicals in the viable epidermis below the stratum corneum (SC) to be determined without ambiguity and having addressed the challenges of (a) background signals from endogenous species and noise and (b) signal attenuation due to absorption and scattering. This study aimed to confirm these observations using a different vibrational spectroscopy approach - specifically, stimulated Raman scattering (SRS) microscopy - and the more conventional in vitro skin penetration test (IVPT). SRS is a nonlinear optical imaging technique which enables more precise location of the skin surface and enhanced skin depth resolution relative to confocal Raman microscopy. The method can also probe larger areas of the sample under investigation and identify the localization of the permeating chemical in specific structural components of the skin. Here, SRS was shown capable of tracking the uptake and distribution of 4-cyanophenol (CP), the same model compound used in the recent confocal Raman investigation, at depths beyond the SC following skin treatment with different vehicles and for different times. The SRS results correlated well with those from the confocal Raman experiments, and both were consistent with independent IVPT measurements. Acquired images clearly delineated CP preference for the intercellular lipid layers of the SC relative to the corneocytes. The stage is now set to apply these and other correlative techniques to examine commercial drug products.


Assuntos
Epiderme , Pele , Pele/metabolismo , Epiderme/metabolismo , Absorção Cutânea , Microscopia Confocal/métodos , Microscopia Óptica não Linear , Análise Espectral Raman/métodos
14.
Curr Eye Res ; 49(4): 354-361, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38165267

RESUMO

PURPOSE: Optic neuritis (ON), a demyelinating disease of the central nervous system, is often a precursor manifestation of neuromyelitis optica spectrum disorders (NMOSD) or multiple sclerosis (MS). Reduced corneal nerve fiber counts have been found in patients with NMOSD or MS. This study aimed to observe and compare the corneal subbasal nerve plexus in patients with three types of ON and controls without ON using in vivo corneal confocal microscopy (IVCM). METHODS: Data were analyzed for 77 eyes of 48 patients with ON, grouped according to seropositivity for anti-aquaporin-4 IgG, myelin oligodendrocyte glycoprotein antibody, or no seropositivity, and 35 healthy eyes in the control group. Corneal parameters were quantified based on IVCM images. Visual function indicators were recorded, following which their correlations with IVCM parameters were analyzed. RESULTS: Significant differences in IVCM parameters were detected among the different groups. Reductions in corneal nerve fiber counts were negatively correlated with visual acuity. Corneal nerve fibers were significantly more damaged in the affected eye than in the unaffected eye in patients with ON. CONCLUSION: IVCM revealed corneal nerve fiber loss of varying degrees, depending on the type of ON. This indicates that, although ON primarily affects the central nervous system, peripheral nerves, such as the trigeminal nerve, which innervates the corneal subbasal nerve plexus may also be damaged in affected patients.


Assuntos
Esclerose Múltipla , Neurite Óptica , Humanos , Estudos Transversais , Córnea/inervação , Fibras Nervosas , Neurite Óptica/diagnóstico , Microscopia Confocal/métodos
15.
J Biophotonics ; 17(4): e202300386, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38200691

RESUMO

Ex vivo confocal microscope (EVCM) rapidly images freshly excised tissue at a histopathological resolution. EVCM features of keratinocyte skin cancers are well-established, but those of benign clinical mimickers remain scarce. We describe EVCM features of common benign lesions and compare them with their malignant differentials. EVCM was used to image 14 benign and 3 cancer tissues. We compared EVCM features of benign lesions with corresponding histopathology and with those of keratinocyte cancers. Key features of benign lesions were identified and differentiated from malignant lesions. Elastin and fat appeared prominent in EVCM; while koilocytes and melanin were difficult to identify. Visualization of entire epidermis was challenging due to difficulty of tissue flattening during imaging. Benign lesions can be differentiated from keratinocyte cancers with EVCM. Using EVCM, a rapid, bedside diagnosis and management of skin neoplasms is possible, especially in a remote location without a histopathology lab.


Assuntos
Neoplasias Cutâneas , Humanos , Neoplasias Cutâneas/patologia , Epiderme/patologia , Microscopia Confocal/métodos , Melaninas , Queratinócitos/patologia
18.
Sensors (Basel) ; 24(2)2024 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-38276359

RESUMO

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.


Assuntos
Corantes Fluorescentes , Fótons , Corantes Fluorescentes/química , Microscopia Confocal/métodos , Lasers , Luz , Microscopia de Fluorescência por Excitação Multifotônica/métodos
19.
Appl Environ Microbiol ; 90(2): e0200723, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38265212

RESUMO

Bacterial biofilms have a complex and heterogeneous three-dimensional architecture that is characterized by chemically and structurally distinct microenvironments. Confocal microscopy-based pH ratiometry and fluorescence lectin-binding analysis (FLBA) are well-established methods to characterize pH developments and the carbohydrate matrix architecture of biofilms at the microscale. Here, we developed a combined analysis, pH-FLBA, to concomitantly map biofilm pH and the distribution of matrix carbohydrates in bacterial biofilms while preserving the biofilm microarchitecture. As a proof of principle, the relationship between pH and the presence of galactose- and fucose-containing matrix components was investigated in dental biofilms grown with and without sucrose. The pH response to a sucrose challenge was monitored in different areas at the biofilm base using the ratiometric pH-sensitive dye C-SNARF-4. Thereafter, the fucose- and galactose-specific fluorescently labeled lectins Aleuria aurantia lectin (AAL) and Morus nigra agglutinin G (MNA-G) were used to visualize carbohydrate matrix components in the same biofilm areas and their immediate surroundings. Sucrose during growth significantly decreased biofilm pH (P < 0.05) and increased the amounts of both MNA-G- and AAL-targeted matrix carbohydrates (P < 0.05). Moreover, it modulated the biofilm composition towards a less diverse community dominated by streptococci, as determined by 16S rRNA gene sequencing. Altogether, these results suggest that the production of galactose- and fucose-containing matrix carbohydrates is related to streptococcal metabolism and, thereby, pH profiles in dental biofilms. In conclusion, pH-FLBA using lectins with different carbohydrate specificities is a useful method to investigate the association between biofilm pH and the complex carbohydrate architecture of bacterial biofilms.IMPORTANCEBiofilm pH is a key regulating factor in several biological and biochemical processes in environmental, industrial, and medical biofilms. At the microscale, microbial biofilms are characterized by steep pH gradients and an extracellular matrix rich in carbohydrate components with diffusion-modifying properties that contribute to bacterial acid-base metabolism. Here, we propose a combined analysis of pH ratiometry and fluorescence lectin-binding analysis, pH-FLBA, to concomitantly investigate the matrix architecture and pH developments in microbial biofilms, using complex saliva-derived biofilms as an example. Spatiotemporal changes in biofilm pH are monitored non-invasively over time by pH ratiometry, while FLBA with lectins of different carbohydrate specificities allows mapping the distribution of multiple relevant matrix components in the same biofilm areas. As the biofilm structure is preserved, pH-FLBA can be used to investigate the in situ relationship between the biofilm matrix architecture and biofilm pH in complex multispecies biofilms.


Assuntos
Fucose , Galactose , Fucose/metabolismo , Galactose/metabolismo , RNA Ribossômico 16S/metabolismo , Carboidratos , Concentração de Íons de Hidrogênio , Streptococcus/metabolismo , Lectinas/metabolismo , Bactérias/metabolismo , Microscopia Confocal/métodos , Hexoses/metabolismo , Biofilmes , Sacarose/metabolismo
20.
Actas Dermosifiliogr ; 115(3): T258-T264, 2024 Mar.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-38244840

RESUMO

The terminology used to describe reflectance confocal microscopy (RCM) findings in both melanocytic and nonmelanocytic lesions has been standardized in English. We convened a panel of Spanish-speaking RCM experts and used the Delphi method to seek consensus on which Spanish terms best describe RCM findings in this setting. The experts agreed on 52 terms: 28 for melanocytic lesions and 24 for nonmelanocytic lesions. The resulting terminology will facilitate homogenization, leading to a better understanding of structures, more standardized descriptions in clinical registries, and easier interpretation of clinical reports exchanged between dermatologists.


Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Neoplasias Cutâneas/patologia , Melanoma/diagnóstico por imagem , Melanoma/patologia , Técnica Delfos , Microscopia Confocal/métodos , Consenso
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