RESUMO
Visualizing hydrated interfaces is of widespread interest across the physical sciences and is a particularly acute need for layered minerals, whose properties are governed by the structure of the electric double layer (EDL) where mineral and solution meet. Here, we show that cryo electron microscopy and tomography enable direct imaging of the EDL at montmorillonite interfaces in monovalent electrolytes with ångstrom resolution over micron length scales. A learning-based multiple-scattering reconstruction method for cryo electron tomography reveals ions bound asymmetrically on opposite sides of curved, exfoliated layers. We observe conserved ion-density asymmetry across stacks of interacting layers in cryo electron microscopy that is associated with configurations of inner- and outer-sphere ion-water-mineral complexes that we term complexation waves. Coherent X-ray scattering confirms that complexation waves propagate at room-temperature via a competition between ion dehydration and charge interactions that are coupled across opposing sides of a layer, driving dynamic transitions between stacked and aggregated states via layer exfoliation.
Assuntos
Eletrólitos , Minerais , Eletrólitos/química , Minerais/química , Água/químicaRESUMO
3D phase imaging recovers an object's volumetric refractive index from intensity and/or holographic measurements. Partially coherent methods, such as illumination-based differential phase contrast (DPC), are particularly simple to implement in a commercial brightfield microscope. 3D DPC acquires images at multiple focus positions and with different illumination source patterns in order to reconstruct 3D refractive index. Here, we present a practical extension of the 3D DPC method that does not require a precise motion stage for scanning the focus and uses optimized illumination patterns for improved performance. The user scans the focus by hand, using the microscope's focus knob, and the algorithm self-calibrates the axial position to solve for the 3D refractive index of the sample through a computational inverse problem. We further show that the illumination patterns can be optimized by an end-to-end learning procedure. Combining these two, we demonstrate improved 3D DPC with a commercial microscope whose only hardware modification is LED array illumination.
RESUMO
Fluorescence microscopy is a powerful tool for imaging biological samples with molecular specificity. In contrast, phase microscopy provides label-free measurement of the sample's refractive index (RI), which is an intrinsic optical property that quantitatively relates to cell morphology, mass, and stiffness. Conventional imaging techniques measure either the labeled fluorescence (functional) information or the label-free RI (structural) information, though it may be valuable to have both. For example, biological tissues have heterogeneous RI distributions, causing sample-induced scattering that degrades the fluorescence image quality. When both fluorescence and 3D RI are measured, one can use the RI information to digitally correct multiple-scattering effects in the fluorescence image. Here, we develop a new computational multi-modal imaging method based on epi-mode microscopy that reconstructs both 3D fluorescence and 3D RI from a single dataset. We acquire dozens of fluorescence images, each 'illuminated' by a single fluorophore, then solve an inverse problem with a multiple-scattering forward model. We experimentally demonstrate our method for epi-mode 3D RI imaging and digital correction of multiple-scattering effects in fluorescence images.
RESUMO
Next-generation nano- and quantum devices have increasingly complex 3D structure. As the dimensions of these devices shrink to the nanoscale, their performance is often governed by interface quality or precise chemical or dopant composition. Here, we present the first phase-sensitive extreme ultraviolet imaging reflectometer. It combines the excellent phase stability of coherent high-harmonic sources, the unique chemical sensitivity of extreme ultraviolet reflectometry, and state-of-the-art ptychography imaging algorithms. This tabletop microscope can nondestructively probe surface topography, layer thicknesses, and interface quality, as well as dopant concentrations and profiles. High-fidelity imaging was achieved by implementing variable-angle ptychographic imaging, by using total variation regularization to mitigate noise and artifacts in the reconstructed image, and by using a high-brightness, high-harmonic source with excellent intensity and wavefront stability. We validate our measurements through multiscale, multimodal imaging to show that this technique has unique advantages compared with other techniques based on electron and scanning probe microscopies.
RESUMO
Electron tomography is used in both materials science and structural biology to image features well below the optical resolution limit. Here, we present a new method for high-resolution 3D transmission electron microscopy (TEM) which approximately reconstructs the electrostatic potential of a sample at atomic resolution in all three dimensions. We use phase contrast images captured through-focus and at varying tilt angles, along with an implicit phase retrieval algorithm that accounts for dynamical and strong scattering, providing more accurate results with much lower electron doses than current atomic electron tomography methods. We test our algorithm using simulated images of a synthetic needle geometry dataset composed of an amorphous silicon dioxide shell around a silicon core. By simulating various levels of electron dose, tilt and defocus, missing projections, and regularization methods, we identify a configuration that allows us to accurately determine both atomic positions and species. We also test the ability of our method to recover randomly positioned vacancies in light elements such as silicon, and to accurately reconstruct strongly-scattering elements such as tungsten.
RESUMO
Optical diffraction tomography (ODT) reconstructs a sample's volumetric refractive index (RI) to create high-contrast, quantitative 3D visualizations of biological samples. However, standard implementations of ODT use interferometric systems, and so are sensitive to phase instabilities, complex mechanical design, and coherent noise. Furthermore, their reconstruction framework is typically limited to weakly scattering samples, and thus excludes a whole class of multiple-scattering samples. Here, we implement a new 3D RI microscopy technique that utilizes a computational multi-slice beam propagation method to invert the optical scattering process and reconstruct high-resolution (NA > 1.0) 3D RI distributions of multiple-scattering samples. The method acquires intensity-only measurements from different illumination angles and then solves a nonlinear optimization problem to recover the sample's 3D RI distribution. We experimentally demonstrate the reconstruction of samples with varying amounts of multiple-scattering: a 3T3 fibroblast cell, a cluster of C. elegans embryos, and a whole C. elegans worm, with lateral and axial resolutions of ≤ 240 nm and ≤ 900 nm, respectively. The results of this work lays groundwork for future studies into using optical wavelengths to probe 3D RI distributions of highly scattering biological organisms.
RESUMO
PURPOSE: The study had two purposes: to examine the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors on uveal melanoma cells and metastases arising from uveal melanoma and to determine the susceptibility of uveal melanoma cells to TRAIL-induced apoptosis. METHODS: Nine human uveal melanoma cell lines and three cell lines derived from uveal melanoma metastases were examined for TRAIL receptor expression by flow cytometry. In vitro apoptosis assays were performed to determine the relative susceptibility of uveal melanoma cells to TRAIL-induced apoptosis. Annexin V staining was also used to determine the capacity of either cycloheximide or interferon-beta to enhance TRAIL-induced apoptosis. RESULTS: Five of the nine uveal melanoma cell lines expressed TRAIL-R2 on more than 60% of the cells. All three of the cell lines derived from uveal melanoma metastases expressed TRAIL-R2 on more than 50% of the cells. Cycloheximide exerted a profound effect in enhancing TRAIL-induced apoptosis in all but two of the uveal melanoma cell lines and in all three of the metastases cell lines. Interferon-beta produced a similar enhancement of TRAIL-induced apoptosis, even in cell lines that were previously shown to be resistant. CONCLUSIONS: TRAIL is a potentially useful therapeutic modality for the management of uveal melanomas and their metastases. Moreover, pharmacological agents and biological response modifiers that independently display antineoplastic properties can enhance TRAIL-induced apoptosis in resistant uveal melanoma cells.
Assuntos
Apoptose/efeitos dos fármacos , Melanoma/metabolismo , Glicoproteínas de Membrana/farmacologia , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Neoplasias Uveais/metabolismo , Anexina A5/metabolismo , Proteínas Reguladoras de Apoptose , Cicloeximida/farmacologia , Sinergismo Farmacológico , Citometria de Fluxo , Proteínas Ligadas por GPI , Humanos , Interferon beta/farmacologia , Melanoma/secundário , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Membro 10c de Receptores do Fator de Necrose Tumoral , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/secundário , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Receptores Chamariz do Fator de Necrose Tumoral , Neoplasias Uveais/patologiaRESUMO
PURPOSE: To determine recovery time needed for the corneal epithelium to return to a normal proliferation rate following 24-hour rigid gas-permeable (RGP) lens wear. METHODS: An RGP lens (Dk/t = 10) was fitted at 9:00 am on one randomly chosen eye of each rabbit (N = 16) and removed 24 hours later while the other eye served as a control. Following contact lens removal, the rabbits were injected at four different time intervals (1,2, 4, and 7 days) with 5-bromo-deoxyuridine (BrdU) at 9:00 am to label dividing corneal epithelial cells. All animals were sacrificed 24 hours after BrdU injection, and corneas were stained for BrdU. A series of continuously adjacent digital images of the whole-mount epithelium was collected from superior limbus to central cornea, and all BrdU-labeled epithelial cell pairs on each digitized image were counted. RESULTS: On day 1 following lens removal, the number of BrdU-labeled corneal epithelium cells was reduced by 50% centrally (C) and 36% peripherally (P) (P < 0.001) compared with control eyes. At day 2, there was no statistically significant difference between control and experimental corneas (P = 0.675). At day 4, a marked significant increase in BrdU-labeled cells was noted, averaging 83% (C) and 96% (P) (P < 0.001). At day 7, BrdU-labeling returned to control values, although it was still slightly elevated (14% [C] and 5% [P], P = 0.045). CONCLUSIONS: The corneal epithelium in the rabbit required at least 1 full week to recover to its stable baseline proliferation rate following only 24 hours of RGP lens wear. Interestingly, hyperproliferation was noted within the limbal, peripheral, and mid-peripheral corneal epithelium 4 days after lens removal.
Assuntos
Lentes de Contato de Uso Prolongado , Epitélio Corneano/citologia , Epitélio Corneano/fisiologia , Hipóxia/fisiopatologia , Animais , Bromodesoxiuridina/metabolismo , Divisão Celular/fisiologia , Lentes de Contato de Uso Prolongado/efeitos adversos , Replicação do DNA/fisiologia , Remoção de Dispositivo , Células Epiteliais/citologia , Hipóxia/etiologia , Coelhos , Recuperação de Função Fisiológica , Fatores de TempoRESUMO
PURPOSE: To examine the rabbit corneal epithelial cell proliferation rate after extended wear of disposable or silicone hydrogel contact lenses or prolonged eyelid closure. METHODS: One randomly chosen eye of 40 New Zealand White rabbits was assigned to silicone hydrogel contact lens wear (n = 15, SH), disposable hydrogel contact lens wear (n = 6, DH), eyelid suturing (n = 15, SUT), or no intervention (n = 4). Contralateral eyes served as the control. After 24 hours or 1 week of lens wear, 5-bromo-2-deoxyuridine (BrdU) was injected intravenously to label dividing corneal epithelial cells, and animals were killed 24 hours after injection. Corneas were stained with monoclonal anti-BrdU antibody and FITC-conjugated secondary antibody. A series of continuous digital images of the wholemounted epithelium were collected from the superior to inferior limbus, and the number of BrdU-labeled cell pairs was counted. RESULTS: SH, DH, and SUT caused a significant decrease in BrdU-labeled pairs of cells over the entire corneal epithelium at day 2 compared with the number in contralateral control eyes (P < 0.001). One week of SUT or SH caused a significant increase centrally in BrdU-labeled cells (P < 0.01). BrdU labeling at the limbus in all groups was not significantly different from the control. Unexpectedly, the proliferation rate of the control corneas was also significantly affected by contralateral lens wear and suturing. CONCLUSIONS: Short-term overnight SH, DH, and SUT all significantly suppressed the cell proliferation rate in the rabbit corneal epithelium. However, adaptation, with central hyperproliferation of cells, appeared to occur at 8 days. The effects of lens wear and eyelid suturing on the cell proliferation rate in contralateral control eyes suggests a central mechanism that regulates corneal epithelial proliferation.
Assuntos
Lentes de Contato de Uso Prolongado , Epitélio Corneano/citologia , Pálpebras/fisiologia , Elastômeros de Silicone , Animais , Bromodesoxiuridina , Divisão Celular , Replicação do DNA , Equipamentos Descartáveis , Técnica Indireta de Fluorescência para Anticorpo , Microscopia de Fluorescência , Coelhos , Distribuição Aleatória , Técnicas de SuturaRESUMO
OBJECTIVE: To test prospectively a new biologic rationale for an advanced hyper oxygen-transmissible lens (HOTL) providing prospects for safer daily (DW) or extended (EW) contact lens wear. DESIGN: Prospective, randomized, double-masked, single-center, 13-month clinical trial. PARTICIPANTS: One hundred sixty-eight patients completed the DW study (1 month): control lens (n = 70); HOTL (n = 98). One hundred thirty-six patients finished 1 year of EW: controls (n = 56), HOTL (n = 25, 6 nights; n = 55, 30 nights). TESTING: Irrigation chamber to collect corneal surface cells, confocal microscopy, tear collection at baseline, 2, and 4, weeks of DW, and 24 hours, 1, 3, 6, 9, and 12 months of EW. MAIN OUTCOME MEASURES: (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness (CET); (3) superficial cell area (SCA); (4) epithelial surface cell exfoliation (DESQ); and (5) tear lactate dehydrogenase (LDH). RESULTS: Daily wear with control lens increased PA binding from 5.90 +/- 2.60 to 7.81 +/- 3.04 bacteria per cell (P < 0.01); HOTL wear increased PA binding significantly less (5.31 +/- 1.87-5.98 +/- 2.26; P < 0.01). Daily wear produced no significant changes in CET or SCA. Significant decreases in DESQ were seen with both lenses with no significant intergroup differences. Tear LDH increased significantly in DW with HOTL wear versus control (P = 0.0017), but not after 1 month of subsequent EW (P = 0.533). One to 3 months of EW with control lens showed significantly higher PA binding than HOTL wear (P < 0.01); binding adaptively decreased thereafter, returning to baseline at 9 to 12 months. Lens EW produced significantly enlarged SCA, thinning of CET (except 6-night HOTL wear), and decreased DESQ (P < 0.01). Some adaptive recovery was seen with CET and DESQ, but not SCA; importantly, the data indicated no significant difference between 6- or 30-night EW for all outcomes. CONCLUSIONS: Hyper oxygen-transmissible lens wear (DW or EW) produced significantly decreased PA binding compared with control lens wear, with no significant difference in wearing schedule (6 nights vs. 30 nights); additionally, there was a remarkable and unexpected adaptive recovery in the first 6 months of all soft lens wear, with a return to baseline PA binding levels and partial recovery for the other outcomes except SCA at 1 year. These results suggest that HOTL use should result in a decrease in the incidence of and risk(s) for lens-related microbial keratitis and that further epidemiologic studies should consider time in adapted EW in future risk and incidence analyses.
Assuntos
Aderência Bacteriana , Lentes de Contato de Uso Prolongado , Epitélio Corneano/microbiologia , Oxigênio/metabolismo , Pseudomonas aeruginosa/fisiologia , Método Duplo-Cego , Feminino , Humanos , L-Lactato Desidrogenase/metabolismo , Masculino , Estudos Prospectivos , Lágrimas/enzimologiaAssuntos
Núcleo do Cristalino/patologia , Subluxação do Cristalino/etiologia , Vitrectomia/efeitos adversos , Retinopatia Diabética/cirurgia , Humanos , Núcleo do Cristalino/diagnóstico por imagem , Subluxação do Cristalino/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Ultrassonografia , Hemorragia Vítrea/cirurgiaRESUMO
PURPOSE: To determine the effects of open or closed eye and overnight contact lens wear on rabbit corneal epithelial surface cell death, detected by annexin V binding to cell surface phosphatidylserine and propidium iodide (PI) double-labeling. METHOD: New Zealand white rabbits (n = 42) weighing 2.5 to 3.5 kg were divided into 7 study groups: hyper Dk/t rigid gas-permeable (RGP) lens; high Dk/t RGP lens; low Dk/t RGP lens; hyper Dk/t soft lens; high Dk/t soft lens; eyelid closure; and nictitating membranectomy study group (n = 6 rabbits for each group). Each rabbit was randomly chosen to have either one eye fitted with a test contact lens or both eyelids sutured closed; the contralateral eye served as a control. Rabbits were humanely sacrificed after 24 hours. Corneal buttons including the limbus were excised and stained with annexin V-FITC and PI to identify the number of nonviable epithelial surface cells. A series of sequential microscopic adjacent fields (200 microm X560 microm) from the inferior limbus to the central cornea were evaluated using epifluorescence microscopy, and the total number of cells stained with annexin V or PI, or both, was counted in each field. RESULTS: Nonviable cells (cells with positive staining) were lowest in density at the limbus and gradually increased in numbers towards the central cornea under normal open eye conditions (P<0.05). Eyelid closure (no lens) caused a significant decrease in the numbers of nonviable cells in the peripheral and central cornea (P<0.05) but not at the limbus (P>0.05). Overnight wear of hyper Dk/t or high Dk/t RGP soft contact test lenses caused significant decreases in the numbers of nonviable cells on the central corneal surface (P<0.05). Hyper Dk/t and high Dk/t soft lenses had similar suppressive effects on the number of surface nonviable cells, independent of lens oxygen transmissibility. By contrast, the number of nonviable cells was dependent upon RGP lens oxygen transmissibility; hyper Dk/t RGP lens wear produced significantly less suppression of the number of nonviable cells in the central cornea than high Dk/t RGP lens wear (P<0.05). Wear of a nonphysiologic low oxygen transmissible RGP test lens however, produced a significant increase in nonviable cells in the central cornea. The number of nonviable epithelial surface cells was not affected by nictitating membranectomy under open eye conditions (P>0.05). CONCLUSIONS: This study revealed in the rabbit model a gradient of nonviable surface epithelial cells increasing towards the central cornea under open eye conditions and with central suppression of surface cell death following closed eye (no lens) or physiologic contact lens wear. Taken together, the results suggest that eyelid closure or contact lens wear may protect the corneal epithelial cells against the shear stress forces exerted by eyelid blinking, which are believed to drive central epithelial surface cell death and exfoliation. However, under very low oxygen tensions combined with lens effect, such as in low Dk/t RGP lens wear, surface cell death may be accelerated.
Assuntos
Anexina A5/metabolismo , Lentes de Contato de Uso Prolongado , Epitélio Corneano/metabolismo , Pálpebras/cirurgia , Fosfatidilserinas/metabolismo , Animais , Apoptose , Piscadela , Contagem de Células , Epitélio Corneano/citologia , Fluoresceína-5-Isotiocianato , Propídio , Coelhos , Privação SensorialRESUMO
OBJECTIVE: To determine effects of lens type and oxygen transmissibility on human corneal epithelium during extended wear (EW). DESIGN: Prospective, randomized, double-masked, single-center, parallel treatment groups, 1-year clinical trial. PARTICIPANTS: One hundred seventy-eight patients completed the study: (1) high-O(2) soft lens (6-night [N] EW) (n = 27); (2) hyper-O(2) soft lens (6N-EW, n = 33) or (30N-EW, n = 66); and (3) hyper-O(2) rigid gas-permeable lens (RGP) (30N-EW, n = 52). INTERVENTION: Irrigation chamber to collect exfoliated corneal surface cells, confocal microscopy, and tear collection at baseline, 1, 3, 6, 9, 12 months of EW. MAIN OUTCOME MEASURES: (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness; (3) superficial epithelial cell area; (4) epithelial surface cell exfoliation; and (5) tear lactate dehydrogenase. RESULTS: Quantitative evidence demonstrated increased binding of PA to human exfoliated corneal epithelial cells during the first 3 months of soft lens EW; the control high-O(2) test lens showed significantly higher bacterial binding (P < 0.05). Binding activity gradually decreased thereafter and returned to baseline after 9 and 12 months. The corneal epithelium demonstrated enlargement of surface cell size, thinning of central epithelium, and a significant decrease in surface cell shedding (P < 0.05). Remarkably, there was subsequent partial adaptive recovery in cell shedding and epithelial thickness but not surface cell size. There was no significant difference between 6N and 30N continuous wear of the hyper-O(2) soft lens for all outcome measures. Importantly, hyper-O(2) RGP lens wear did not show significantly increased PA binding during 1 year. CONCLUSIONS: This study establishes three important new findings: (1) hyper-O(2) soft lens EW produces significantly less PA binding than the lower O(2) soft lens with no significant difference in PA binding with 6N versus 30N EW of the hyper-O(2) soft lens; (2) there is a remarkable adaptive recovery after 6 months with all soft lens wear with gradual return to prelens PA binding levels and partial recovery of other outcome measures for all test lenses EW except surface cell size; (3) 30N EW of the hyper-O(2) RGP lens produced no significant increases in PA binding over 1 year. Taken together, these results suggest that introduction of new hyper-O(2) transmissible lens materials into clinical use may offer safer EW, and future epidemiologic studies of ulcerative infectious keratitis should consider both lens type and time in lens EW in any incidence/risk analysis.
Assuntos
Aderência Bacteriana , Lentes de Contato de Uso Prolongado , Epitélio Corneano/microbiologia , Oxigênio/metabolismo , Pseudomonas aeruginosa/fisiologia , Adulto , Método Duplo-Cego , Epitélio Corneano/metabolismo , Feminino , Humanos , L-Lactato Desidrogenase/metabolismo , Masculino , Microscopia Confocal , Estudos Prospectivos , Lágrimas/enzimologia , Fatores de TempoRESUMO
PURPOSE: To determine the effects of open, closed eye, and overnight contact lens wear on homeostatic epithelial surface cell death in the rabbit cornea. METHODS: One eye of each rabbit was either closed by eyelid suture or fitted with one of the following test contact lenses: (1) low Dk/t rigid gas permeable (RGP) lens, (2) hyper Dk/t RGP lens, (3) hyper Dk/t soft lens. The other eye served as a control. After 24 hours, whole corneas were carefully excised and immediately stained with a calcein-acetoxymethyl ester-ethidium homodimer viability assay to quantify the number of nonviable surface epithelial cells. In addition, exfoliated corneal epithelial cells were collected with an eye irrigation chamber to determine cell viability. RESULTS: In the normal cornea, open-eye conditions showed significantly more nonviable surface cells in the central cornea than in the periphery (p < 0.05). Overnight wear of all test lenses and eyelid closure induced significant decreases in the number of nonviable cells on the central corneal surface compared with controls (p < 0.05). All exfoliated corneal epithelial cells collected by eye irrigation were nonviable. CONCLUSION: In the rabbit model, overnight contact lens wear significantly downregulated spontaneous epithelial surface cell death independent of lens rigidity or material oxygen transmissibility. These effects were similar to eyelid closure without lens wear. Taken together, these results suggest that eyelid closure and the physical presence of the contact lens may protect against the shear stress forces exerted by eyelid blinking, which are believed to cause central surface cell death and subsequent exfoliation.
Assuntos
Sobrevivência Celular/fisiologia , Lentes de Contato , Córnea/citologia , Células Epiteliais/fisiologia , Pálpebras/fisiologia , Animais , Piscadela/fisiologia , Fluoresceínas , Coelhos , Privação SensorialRESUMO
This report reviews and integrates our recent clinical and laboratory findings of the effects of daily (DW)/overnight (EW) contact lens wear on corneal epithelial homeostasis. In a prospective, double-masked human clinical study, three lens types (high and hyper Dk/t soft; hyper Dk/t RGP) were used to assess the effects of daily and overnight lens wear on corneal epithelial thickness, cell surface size, Pseudomonas aeruginosa (PA) binding to exfoliated epithelial cells and exfoliation rates. Concomitantly, we studied in a rabbit model, the effects of overnight lens wear on corneal epithelial surface cell viability (live/dead assay, TUNEL-labeling, Annexin-V staining, Bcl-2 expression), basal cell proliferation and migration patterns. The comprehensive results obtained yield important new insights on both the normal homeostasis of the corneal epithelium and the biological changes induced by contact lens wear.