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1.
Sci Adv ; 9(27): eadh5435, 2023 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-37418534

RESUMO

The penetration depth of optical coherence tomography (OCT) reaches well beyond conventional microscopy; however, signal reduction with depth leads to rapid degradation of the signal below the noise level. The pursuit of imaging at depth has been largely approached by extinguishing multiple scattering. However, in OCT, multiple scattering substantially contributes to image formation at depth. Here, we investigate the role of multiple scattering in OCT image contrast and postulate that, in OCT, multiple scattering can enhance image contrast at depth. We introduce an original geometry that completely decouples the incident and collection fields by introducing a spatial offset between them, leading to preferential collection of multiply scattered light. A wave optics-based theoretical framework supports our experimentally demonstrated improvement in contrast. The effective signal attenuation can be reduced by more than 24 decibels. Notably, a ninefold enhancement in image contrast at depth is observed in scattering biological samples. This geometry enables a powerful capacity to dynamically tune for contrast at depth.


Assuntos
Microscopia , Tomografia de Coerência Óptica , Tomografia de Coerência Óptica/métodos , Óptica e Fotônica , Espalhamento de Radiação
2.
Appl Opt ; 61(17): 5315-5319, 2022 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-36256216

RESUMO

We derive analytical expressions for the length, thickness, and curvature of an Airy light sheet in terms of basic parameters of the cubic phase and the paraxially defined focusing optics that form the beam. The length and thickness are defined analogously to the Rayleigh range and beam waist of a Gaussian beam, hence providing a direct and quantitative comparison between the two beam types. The analytical results are confirmed via numerical Fresnel propagation simulations and discussed within the context of light-sheet microscopy, providing a comprehensive guide for the design of the illumination unit.

3.
Opt Express ; 29(8): 11819-11832, 2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33984955

RESUMO

We present the use of the Douglas-Gunn Alternating Direction Implicit finite difference method for computationally efficient simulation of the electric field propagation through a wide variety of optical fiber geometries. The method can accommodate refractive index profiles of arbitrary shape and is implemented in a tool called BPM-Matlab. We validate BPM-Matlab by comparing it to published experimental, numerical, and theoretical data and to commercially available state-of-the-art software. It is user-friendly, fast, and is available open-source. BPM-Matlab has a broad scope of applications in modeling a variety of optical fibers for diverse fields such as imaging, communication, material processing, and remote sensing.

5.
J Biomed Opt ; 25(7): 1-13, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32638570

RESUMO

SIGNIFICANCE: In multiphoton microscopy, two-photon excited fluorescence (TPEF) spectra carry valuable information on morphological and functional biological features. For measuring these biomarkers, separation of different parts of the fluorescence spectrum into channels is typically achieved by the use of optical band pass filters. However, spectra from different biomarkers can be unknown or overlapping, creating a crosstalk in between the channels. Previously, establishing these channels relied on prior knowledge or heuristic testing. AIM: The presented method aims to provide spectral bands with optimal separation between groups of specimens expressing different biomarkers. APPROACH: We have developed a system capable of resolving TPEF with high spectral resolution for the characterization of biomarkers. In addition, an algorithm is created to simulate and optimize optical band pass filters for fluorescence detection channels. To demonstrate the potential improvements in cell and tissue classification using these optimized channels, we recorded spectrally resolved images of cancerous (HT29) and normal epithelial colon cells (FHC), cultivated in 2D layers and in 3D to form spheroids. To provide an example of an application, we relate the results with the widely used redox ratio. RESULTS: We show that in the case of two detection channels, our system and algorithm enable the selection of optimized band pass filters without the need of knowing involved fluorophores. An improvement of 31,5% in separating different 2D cell cultures is achieved, compared to using established spectral bands that assume NAD(P)H and FAD as main contributors of autofluorescence. The compromise is a reduced SNR in the images. CONCLUSIONS: We show that the presented method has the ability to improve imaging contrast and can be used to tailor a given label-free optical imaging system using optical band pass filters targeting a specific biomarker or application.


Assuntos
Microscopia , Imagem Óptica , Biomarcadores , Corantes Fluorescentes , Microscopia de Fluorescência por Excitação Multifotônica , Fótons
6.
Sci Rep ; 10(1): 8090, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415135

RESUMO

Attenuation of optical fields owing to scattering and absorption limits the penetration depth for imaging. Whilst aberration correction may be used, this is difficult to implement over a large field-of-view in heterogeneous tissue. Attenuation-compensation allows tailoring of the maximum lobe of a propagation-invariant light field and promises an increase in depth penetration for imaging. Here we show this promising approach may be implemented in multi-photon (two-photon) light-sheet fluorescence microscopy and, furthermore, can be achieved in a facile manner utilizing a graded neutral density filter, circumventing the need for complex beam shaping apparatus. A "gold standard" system utilizing a spatial light modulator for beam shaping is used to benchmark our implementation. The approach will open up enhanced depth penetration in light-sheet imaging to a wide range of end users.

7.
Opt Express ; 28(4): 5758-5767, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32121791

RESUMO

In laser lighting, a major benefit over other lighting techniques is the possibility to achieve very high luminous exitance. Focusing the exciting laser to a very small spot size on the phosphor, however, does not necessarily provide a very small emitting area for the white light. In this study we investigate experimentally and numerically the relationship between the white light spot size and the incident blue laser spot size. We show that the specific phosphor material properties have significant impact on this relationship and on the achievable minimum spot size. This constitutes a limitation on the minimum spot size achievable in laser lighting and has important implications in applications.

8.
J Biophotonics ; 13(2): e201960025, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31617683

RESUMO

Existing approaches for early-stage bladder tumor diagnosis largely depend on invasive and time-consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real-time tumor diagnosis can enable immediate laser-based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real-time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe-based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low- and high-grade tumor.


Assuntos
Análise Espectral Raman , Neoplasias da Bexiga Urinária , Humanos , Análise Multivariada , Gradação de Tumores , Neoplasias da Bexiga Urinária/diagnóstico
9.
Phys Chem Chem Phys ; 21(35): 19554-19566, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31464321

RESUMO

Microthermometric measurements of a synthetic high-density (984 kg m-3) water inclusion in quartz revealed that only part of the super-cooled liquid water (L) transforms to solid ice Ih upon ice nucleation (L → ice Ih + L). While ice nucleation occurs in the ice Ih stability field at -41 °C and 28 MPa the pressure increases instantaneously to 315 MPa into the ice II stability field. At this point, both phases, liquid water and ice Ih are metastable. The coexistence of these two phases was confirmed by Raman spectroscopy and could be traced down to -80 °C. The pressure along this low-temperature metastable extension of the ice Ih melting curve was determined by means of the frequency shift of the ice Ih peak position using both the O-H stretching band around 3100 cm-1 and the lattice translational band around 220 cm-1. At -80 °C and 466 MPa the super-cooled ice Ih melting curve encounters the homogeneous nucleation limit (TH) and the remaining liquid water transformed either to metastable ice IV (ice Ih + L → ice Ih + ice IV) or occasionally to metastable ice III (ice Ih + L → ice Ih + ice III). The nucleation of ice IV resulted in a pressure drop of about 180 MPa. Upon subsequent heating the pressure develops along a slightly negatively sloped ice Ih-ice IV equilibrium line terminating in a triple point at -32.7 °C and 273 MPa, where ice IV melts to liquid water (ice Ih + ice IV → ice Ih + L). Hitherto existing experimental data of the ice IV melting curve (ice IV → L) were found to be in line with the observed ice Ih-ice IV-liquid triple point. If, on the other hand, ice III nucleated at -80 °C (instead of ice IV) the associated pressure drop was about 260 MPa. The ice Ih-ice III-liquid triple point was determined at -22.0 °C and 207 MPa (ice Ih + ice III → ice Ih + L), which is in agreement with previous experimental data.

10.
J Biomed Opt ; 23(12): 1-6, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30554503

RESUMO

While there exist many Monte Carlo (MC) programs for solving the radiative transfer equation (RTE) in biological tissues, we have identified a need for an open-source MC program that is sufficiently user-friendly for use in an education environment, in which detailed knowledge of compiling or UNIX command-line cannot be assumed. Therefore, we introduce MCmatlab, an open-source codebase thus far consisting of (a) a fast three-dimensional MC RTE solver and (b) a finite-element heat diffusion and Arrhenius-based thermal tissue damage simulator, both run in MATLAB. The kernel for both of these solvers is written in parallelized C and implemented as MATLAB MEX functions, combining the speed of C with the familiarity and versatility of MATLAB. We compare the RTE solver to Steven Jacques' mcxyz, which it is inspired by, and present example results generated by the thermal model. MCmatlab is easy to install and use and can be used by students and experienced researchers alike for simulating tissue light propagation and, optionally, thermal damage.


Assuntos
Imageamento Tridimensional/métodos , Modelos Biológicos , Método de Monte Carlo , Software , Vasos Sanguíneos/diagnóstico por imagem , Vasos Sanguíneos/efeitos da radiação , Análise de Elementos Finitos , Temperatura Alta , Humanos , Luz , Óptica e Fotônica , Espalhamento de Radiação , Pele/diagnóstico por imagem , Pele/efeitos da radiação
11.
Sci Rep ; 7(1): 1435, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28469191

RESUMO

We demonstrate the first light sheet microscope using propagation invariant, accelerating Airy beams that operates both in single- and two-photon modes. The use of the Airy beam permits us to develop an ultra compact, high resolution light sheet system without beam scanning. In two-photon mode, an increase in the field of view over the use of a standard Gaussian beam by a factor of six is demonstrated. This implementation for light sheet microscopy opens up new possibilities across a wide range of biomedical applications, especially for the study of neuronal processes.

12.
Development ; 142(11): 1992-2001, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25953346

RESUMO

The spatial arrangement of leaves and flowers around the stem, known as phyllotaxis, is controlled by an auxin-dependent reiterative mechanism that leads to regular spacing of the organs and thereby to remarkably precise phyllotactic patterns. The mechanism is based on the active cellular transport of the phytohormone auxin by cellular influx and efflux carriers, such as AUX1 and PIN1. Their important role in phyllotaxis is evident from mutant phenotypes, but their exact roles in space and time are difficult to address due to the strong pleiotropic phenotypes of most mutants in phyllotaxis. Models of phyllotaxis invoke the accumulation of auxin at leaf initials and removal of auxin through their developing vascular strand, the midvein. We have developed a precise microsurgical tool to ablate the midvein at high spatial and temporal resolution in order to test its function in leaf formation and phyllotaxis. Using amplified femtosecond laser pulses, we ablated the internal tissues in young leaf primordia of tomato (Solanum lycopersicum) without damaging the overlying L1 and L2 layers. Our results show that ablation of the future midvein leads to a transient accumulation of auxin in the primordia and to an increase in their width. Phyllotaxis was transiently affected after midvein ablations, but readjusted after two plastochrons. These results indicate that the developing midvein is involved in the basipetal transport of auxin through young primordia, which contributes to phyllotactic spacing and stability.


Assuntos
Ácidos Indolacéticos/metabolismo , Folhas de Planta/anatomia & histologia , Folhas de Planta/embriologia , Solanum lycopersicum/anatomia & histologia , Solanum lycopersicum/embriologia , Proteínas de Fluorescência Verde/metabolismo , Solanum lycopersicum/genética , Tamanho do Órgão , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/metabolismo
13.
J Biomed Opt ; 13(4): 044021, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19021349

RESUMO

Theileria annulata is an intracellular parasite that infects and transforms bovine leukocytes, inducing continuous proliferation of its host cell both in vivo and in vitro. Theileria-infected cells can easily be propagated in the laboratory and serve as a good model for laser ablation studies. Using single pulses from an amplified ultrashort pulse laser system, we developed a technique to introduce submicrometer holes in the plasma membrane of the intracellular schizont stage of Theileria annulata. This was achieved without compromising either the viability of the organisms or that of the host cell that harbors the parasite in its cytoplasm. Multiphoton microscopy was used to generate image stacks of the parasite before and after ablation. The high axial resolution allowed precise selection of the region of the membrane that was ablated. It also allowed observation of the size of the holes generated (in fixed, stained cells) and determination of the structural changes in the parasite resulting from the laser pulses (in living cells in vitro). This technique opens a new possibility for the transfection of Theileria or delivery of molecules to the schizont that may prove useful in the study of this special host-parasite relationship.


Assuntos
Terapia a Laser/instrumentação , Macrófagos/parasitologia , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microcirurgia/instrumentação , Cirurgia Assistida por Computador/instrumentação , Theileria/isolamento & purificação , Animais , Linhagem Celular , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Terapia a Laser/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Microcirurgia/métodos , Cirurgia Assistida por Computador/métodos
14.
PLoS One ; 3(4): e1871, 2008 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-18392149

RESUMO

BACKGROUND: Normal cell division is coordinated by a bipolar mitotic spindle, ensuring symmetrical segregation of chromosomes. Cancer cells, however, occasionally divide into three or more directions. Such multipolar mitoses have been proposed to generate genetic diversity and thereby contribute to clonal evolution. However, this notion has been little validated experimentally. PRINCIPAL FINDINGS: Chromosome segregation and DNA content in daughter cells from multipolar mitoses were assessed by multiphoton cross sectioning and fluorescence in situ hybridization in cancer cells and non-neoplastic transformed cells. The DNA distribution resulting from multipolar cell division was found to be highly variable, with frequent nullisomies in the daughter cells. Time-lapse imaging of H2B/GFP-labelled multipolar mitoses revealed that the time from the initiation of metaphase to the beginning of anaphase was prolonged and that the metaphase plates often switched polarity several times before metaphase-anaphase transition. The multipolar metaphase-anaphase transition was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the normal separase activity cycle. Centromeric AURKB and MAD2 foci were observed frequently to remain on the centromeres of multipolar ana-telophase chromosomes, indicating that multipolar mitoses were able to circumvent the spindle assembly checkpoint with some sister chromatids remaining unseparated after anaphase. Accordingly, scoring the distribution of individual chromosomes in multipolar daughter nuclei revealed a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister chromatids to the daughter cells. CONCLUSION: The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells. Spindle multipolarity could thus be a highly efficient generator of genetically diverse minority clones in transformed cell populations.


Assuntos
Genoma , Mitose , Evolução Biológica , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Cromátides/química , Cromátides/ultraestrutura , Mapeamento Cromossômico , Segregação de Cromossomos , Fibroblastos/metabolismo , Variação Genética , Humanos , Hibridização in Situ Fluorescente , Modelos Genéticos , Fatores de Tempo
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