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1.
J Biophotonics ; : e201900242, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804752

RESUMO

Development of label-free methods for accurate classification of cells with high throughput can yield powerful tools for biological research and clinical applications. We have developed a deep neural network of DINet for extracting features from cross-polarized diffraction image (p-DI) pairs on multiple pixel scales to accurately classify cells in five types. A total of 6185 cells were measured by a polarization diffraction imaging flow cytometry (p-DIFC) method followed by cell classification with DINet on p-DI data. The averaged value and SD of classification accuracy were found to be 98.9% ± 1.00% on test data sets for 5-fold training and test. The invariance of DINet to image translation, rotation, and blurring has been verified with an expanded p-DI data set. To study feature-based classification by DINet, two sets of correctly and incorrectly classified cells were selected and compared for each of two prostate cell types. It has been found that the signature features of large dissimilarities between p-DI data of correctly and incorrectly classified cell sets increase markedly from convolutional layers 1 and 2 to layers 3 and 4. These results clearly demonstrate the importance of high-order correlations extracted at the deep layers for accurate cell classification.

2.
J Biophotonics ; 12(4): e201800287, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30447049

RESUMO

Methods for rapid and label-free cell assay are highly desired in life science. Single-shot diffraction imaging presents strong potentials to achieve this goal as evidenced by past experimental results using methods such as polarization diffraction imaging flow cytometry. We present here a platform of methods toward solving these problems and results of optical cell model (OCM) evaluations by calculations and analysis of cross-polarized diffraction image (p-DI) pairs. Four types of realistic OCMs have been developed with two prostate cell structures and adjustable refractive index (RI) parameters to investigate the effects of cell morphology and index distribution on calculated p-DI pairs. Image patterns have been characterized by a gray-level co-occurrence matrix (GLCM) algorithm and four GLCM parameters and linear depolarization ratio δL have been selected to compare calculated against measured data of prostate cells. Our results show that the irregular shapes of and heterogeneity in RI distributions for organelles play significant roles in the spatial distribution of scattered light by cells in comparison to the average RI values and their differences among the organelles. Discrepancies in GLCM and δL parameters between calculated and measured p-DI data provide useful insight for understanding light scattering by single cells and improving OCM.

3.
Biomed Opt Express ; 9(5): 2081-2094, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29760971

RESUMO

A new and noncontact approach of multispectral reflectance imaging has been developed to inversely determine the absorption coefficient of µ a , the scattering coefficient of µs and the anisotropy factor g of a turbid target from one measured reflectance image. The incident beam was profiled with a diffuse reflectance standard for deriving both measured and calculated reflectance images. A GPU implemented Monte Carlo code was developed to determine the parameters with a conjugate gradient descent algorithm and the existence of unique solutions was shown. We noninvasively determined embedded region thickness in heterogeneous targets and estimated in vivo optical parameters of nevi from 4 patients between 500 and 950nm for melanoma diagnosis to demonstrate the potentials of quantitative reflectance imaging.

4.
PLoS One ; 12(9): e0184726, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28886199

RESUMO

Morphological changes in apoptotic cells provide essential markers for defining and detection of apoptosis as a fundamental mechanism of cell death. Among these changes, the nuclear fragmentation and condensation have been regarded as the important markers but quantitative characterization of these changes is yet to be achieved. We have acquired confocal image stacks of 206 viable and apoptotic MCF-7 cells stained by three fluorescent dyes. Three-dimensional (3D) parameters were extracted to quantify and compare their differences in morphology. To analyze nuclear fragmentation, a new method has been developed to determine clustering of nuclear voxels in the reconstructed cells due to fluorescence intensity changes in nuclei of apoptotic cells. The results of these studies reveal that the 3D morphological changes in cytoplasm and nuclear membranes in apoptotic cells provide sensitive targets for label-free detection and staging of apoptosis. Furthermore, the clustering analysis and morphological data on nuclear fragmentation are highly useful for derivation of optical cell models and simulation of diffraction images to investigate light scattering by early apoptotic cells, which can lead to future development of label-free and rapid methods of apoptosis assay based on cell morphology.


Assuntos
Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Apoptose/genética , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Fragmentação do DNA , Humanos , Células MCF-7 , Mitocôndrias/metabolismo
5.
Appl Opt ; 55(8): 2079-85, 2016 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-26974805

RESUMO

Spectrophotometric quantification of turbidity by multiple optical parameters has wide-ranging applications in material analysis and life sciences. A robust system design needs to combine hardware for precise measurement of light signals with software to accurately model measurement configuration and rapidly solve a sequence of challenging inverse problems. We have developed and validated a design approach and performed system validation based on radiative transfer theory for determination of absorption coefficient, scattering coefficient, and anisotropy factor without using an integrating sphere. Accurate and rapid determination of parameters and spectra is achieved for microsphere suspension samples by combining photodiode-based measurement of four signals with the Monte Carlo simulation and perturbation-based inverse calculations. The three parameters of microsphere suspension samples have been determined from the measured signals as functions of wavelength from 400 to 800 nm and agree with calculated results based on the Mie theory. It has been shown that the inverse problems in the cases of microsphere suspension samples are well posed with convex cost functions to yield unique solutions, and it takes about 1 min to obtain the three parameters per wavelength.


Assuntos
Microesferas , Fenômenos Ópticos , Espectrofotometria/métodos , Método de Monte Carlo , Nefelometria e Turbidimetria
6.
Opt Express ; 24(1): 366-77, 2016 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-26832267

RESUMO

Coherent light scattering presents complex spatial patterns that depend on morphological and molecular features of biological cells. We present a numerical approach to establish realistic optical cell models for generating virtual cells and accurate simulation of diffraction images that are comparable to measured data of prostate cells. With a contourlet transform algorithm, it has been shown that the simulated images and extracted parameters can be used to distinguish virtual cells of different nuclear volumes and refractive indices against the orientation variation. These results demonstrate significance of the new approach for development of rapid cell assay methods through diffraction imaging.


Assuntos
Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Refratometria/métodos , Tamanho Celular , Células Cultivadas , Simulação por Computador , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
Appl Opt ; 54(16): 5223-8, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-26192687

RESUMO

Blurred diffraction images acquired from flowing particles affect the measurement of fringe patterns and subsequent analysis. An imaging unit with one time-delay-integration (TDI) camera has been developed to acquire two cross-polarized diffraction images. It was shown that selected elements of Mueller matrix of single scatters can be imaged with pixel matching precision in this configuration. With the TDI camera, the effect of blurring on imaging of scattered light propagating along the side directions was found to be much more significant for biological cells than microspheres. Despite blurring, classification of MCF-7 and K562 cells is feasible since the effect has similar influence on extracted image parameters. Furthermore, image blurring can be useful for analysis of the correlations among texture parameters for characterization of diffraction images from single cells. The results demonstrate that with one TDI camera the polarization diffraction imaging flow cytometry can be significantly improved and angular distribution of selected Mueller matrix elements can be accurately measured for rapid and morphology-based assay of particles and cells without fluorescent labeling.


Assuntos
Citometria de Fluxo/instrumentação , Aumento da Imagem/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Contraste de Fase/instrumentação , Refratometria/instrumentação , Frações Subcelulares/ultraestrutura , Rastreamento de Células/instrumentação , Rastreamento de Células/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Citometria de Fluxo/métodos , Humanos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/instrumentação , Interpretação de Imagem Assistida por Computador/métodos , Células K562 , Células MCF-7 , Microscopia de Fluorescência/métodos , Microscopia de Contraste de Fase/métodos , Refratometria/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Integração de Sistemas
8.
Cytometry A ; 85(9): 817-26, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25044756

RESUMO

Label-free and rapid classification of cells can have awide range of applications in biology. We report a robust method of polarization diffraction imaging flow cytometry (p-DIFC) for achieving this goal. Coherently scattered light signals are acquired from single cells excited by a polarized laser beam in the form of two cross-polarized diffraction images. Image texture and intensity parameters are extracted with a gray level co-occurrence matrix (GLCM) algorithm to obtain an optimized set of feature parameters as the morphological "fingerprints" for automated cell classification. We selected the Jurkat T cells and Ramos B cells to test the p-DIFC method's capacity for cell classification. After detailed statistical analysis, we found that the optimized feature vectors yield accuracies of classification between the Jurkat and Ramos ranging from 97.8% to 100% among different cell data sets. Confocal imaging and three-dimensional reconstruction were applied to gain insights on the ability of p-DIFC method for classifying the two cell lines of highly similar morphology. Based on these results we conclude that the p-DIFC method has the capacity to discriminate cells of high similarity in their morphology with "fingerprints" features extracted from the diffraction images, which may be attributed to subtle but statistically significant differences in the nucleus-to-cell volume ratio in the case of Jurkat and Ramos cells.


Assuntos
Linfócitos B/citologia , Citometria de Fluxo/métodos , Citometria por Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Reconhecimento Automatizado de Padrão/métodos , Algoritmos , Linhagem Celular Tumoral , Humanos , Aumento da Imagem/métodos , Imagem Tridimensional/métodos , Células Jurkat , Microscopia Confocal , Microscopia de Polarização
9.
Opt Express ; 22(25): 31568-74, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25607106

RESUMO

Diffraction imaging of scattered light allows extraction of information on scatterer's morphology. We present a method for accurate simulation of diffraction imaging of single particles by combining rigorous light scattering model with ray-tracing software. The new method has been validated by comparison to measured images of single microspheres. Dependence of fringe patterns on translation of an objective based imager to off-focus positions has been analyzed to clearly understand diffraction imaging with multiple optical elements. The calculated and measured results establish unambiguously that diffraction imaging should be pursued in non-conjugate configurations to ensure accurate sampling of coherent light distribution from the scatterer.

10.
Opt Express ; 21(21): 24819-28, 2013 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-24150325

RESUMO

It was found that the diffraction images acquired along the side scattering directions with objects in a cell sample contain pattern variations at both the global and local scales. We show here that the global pattern variation is associated with the categorical size and morphological heterogeneity of the imaged objects. An automated image processing method has been developed to separate the acquired diffraction images into three types of global patterns. Combined with previously developed method for quantifying local texture pattern variations, the new method allows fully automated analysis of diffraction images for rapid and label-free classification of cells according to their 3D morphology.


Assuntos
Fenômenos Fisiológicos Celulares , Separação Celular/métodos , Citometria de Fluxo/métodos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imagem Tridimensional/métodos , Refratometria/métodos , Algoritmos
11.
Opt Lett ; 38(12): 2095-7, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23938988

RESUMO

Solving inverse problems requires multiple forward calculations of measured signals. We present a fast method combining graphic processing unit-accelerated Monte Carlo simulations of individual photons and a new perturbation scheme for a 300-fold speedup in comparison to conventional CPU-based approaches. The method allows rapid calculations of the diffuse reflectance and transmittance signals from a turbid sample of absorption coefficient µ(a), scattering coefficient µ(s), and anisotropy factor g based on the principle of correlated sampling. To demonstrate its strong utility, we have applied the method for determining the optical parameters of diluted intralipid samples with satisfactory results.


Assuntos
Método de Monte Carlo , Fenômenos Ópticos , Absorção , Algoritmos , Fatores de Tempo
12.
Cytometry A ; 83(11): 1027-33, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23839922

RESUMO

Achieving effective hydrodynamic focusing and flow stability at low speed presents a challenging design task in flow cytometry for studying phenomena such as cell adhesion and diffraction imaging of cells with low-cost cameras. We have developed different designs of flow chamber and sheath nozzle to accomplish the above goal. A 3D computational model of the chambers has been established to simulate the fluid dynamics in different chamber designs and measurements have been performed to determine the velocity and size distributions of the core fluid from the nozzle. Comparison of the simulation data with experimental results shows good agreement. With the computational model significant insights were gained for optimization of the chamber design and improvement of the cell positioning accuracy for study of slow moving cells. The benefit of low flow speed has been demonstrated also by reduced blurring in the diffraction images of single cells. Based on these results, we concluded that the new designs of chamber and sheath nozzle produce stable hydrodynamic focusing of the core fluid at low speed and allow detailed study of cellular morphology under various rheological conditions using the diffraction imaging method.


Assuntos
Citometria de Fluxo/métodos , Microscopia Eletrônica de Transmissão , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Reologia/instrumentação
13.
Integr Biol (Camb) ; 4(11): 1428-36, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23064132

RESUMO

Quantification of 3D morphology and measurement of cellular functions were performed on the mouse melanoma cell lines of B16F10 to investigate the intriguing problem of structure-function relations in the genetically engineered cells with GPR4 overexpression. Results of 3D analysis of cells in suspension and phase contrast imaging of adherent cells yield consistent evidence that stimulation of the proton-sensing GPR4 receptor in these cells may modify significantly their morphology with diminishing ability to produce membrane protrusions and to migrate. Examination of the 3D parameters of mitochondria provide further insights on the measured variation of the maximal capacity of oxygen consumption rate among the genetically modified cells, indicating that the proton-sensing receptor may regulate cancer cell metabolism with increased mitochondrial surface area. Our study demonstrates clearly the significant benefits of quantitative 3D morphological study in illuminating cellular functions and development of novel morphology based cell assay methods.


Assuntos
Melanoma Experimental/patologia , Melanoma Experimental/fisiopatologia , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Engenharia Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imagem Tridimensional , Melanoma Experimental/genética , Camundongos , Microscopia Confocal , Mitocôndrias/metabolismo , Consumo de Oxigênio , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
14.
Opt Express ; 20(20): 22245-51, 2012 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-23037372

RESUMO

We report a novel method of diffraction imaging flow cytometry to measure and analyze size distribution of microspheres. An automated and robust image processing software based on the short-time-Fourier-transform algorithm has been developed to analyze the characteristic and spatially varying oscillations of side scatters recorded as a diffraction image. Our results demonstrate that the new method allows accurate and rapid determination of single microspheres' diameters ranging from 1 to 100 µm. The capacity for analysis of light scattering by two-sphere aggregates has been demonstrated but analytical tools for characterization of aggregates by multiple microspheres remain to be developed.


Assuntos
Inteligência Artificial , Citometria de Fluxo/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microesferas , Nefelometria e Turbidimetria/métodos , Tamanho da Partícula , Reconhecimento Automatizado de Padrão/métodos , Algoritmos
15.
Photochem Photobiol ; 88(4): 969-77, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22443292

RESUMO

Photodynamic therapy (PDT) provides an effective option for treatment of tumors and other diseases in superficial tissues and attracts attention for in vitro study with cells. In this study, we present a significantly improved model of in vitro cell killing through Type-II PDT for simulation of the molecular interactions and cell killing in time domain in the presence of oxygen transport within a spherical cell. The self-consistency of the approach is examined by determination of conditions for obtaining positive definitive solutions of molecular concentrations. Decay constants of photosensitizers and unoxidized receptors are extracted as the key indices of molecular kinetics with different oxygen diffusion constants and permeability at the cell membrane. By coupling the molecular kinetics to cell killing, we develop a modeling method of PDT cytotoxicity caused by singlet oxygen and obtain the cell survival ratio as a function of light fluence or initial photosensitizer concentration with different photon density or irradiance of incident light and other parameters of oxygen transport. The results show that the present model of Type-II PDT yields a powerful tool to quantitate various events underlying PDT at the molecular and cellular levels and to interpret experimental results of in vitro cell studies.


Assuntos
Modelos Biológicos , Neoplasias/tratamento farmacológico , Oxigênio/química , Fotoquimioterapia , Fótons , Oxigênio Singlete/química , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/efeitos da radiação , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Permeabilidade da Membrana Celular , Simulação por Computador , Difusão , Humanos , Cinética , Luz , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia
16.
Biomed Opt Express ; 2(6): 1717-26, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21698031

RESUMO

Automated classification of biological cells according to their 3D morphology is highly desired in a flow cytometer setting. We have investigated this possibility experimentally and numerically using a diffraction imaging approach. A fast image analysis software based on the gray level co-occurrence matrix (GLCM) algorithm has been developed to extract feature parameters from measured diffraction images. The results of GLCM analysis and subsequent classification demonstrate the potential for rapid classification among six types of cultured cells. Combined with numerical results we show that the method of diffraction imaging flow cytometry has the capacity as a platform for high-throughput and label-free classification of biological cells.

17.
Opt Lett ; 34(19): 2985-7, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19794790

RESUMO

Diffraction images record angle-resolved distribution of scattered light from a particle excited by coherent light and can correlate highly with the 3D morphology of a particle. We present a jet-in-fluid design of flow chamber for acquisition of clear diffraction images in a laminar flow. Diffraction images of polystyrene spheres of different diameters were acquired and found to correlate highly with the calculated ones based on the Mie theory. Fast Fourier transform analysis indicated that the measured images can be used to extract sphere diameter values. These results demonstrate the significant potentials of high-throughput diffraction imaging flow cytometry for extracting 3D morphological features of cells.


Assuntos
Citometria de Fluxo/métodos , Óptica e Fotônica , Diagnóstico por Imagem/instrumentação , Elasticidade , Desenho de Equipamento/instrumentação , Análise de Fourier , Luz , Distribuição Normal , Poliestirenos/química , Espalhamento de Radiação , Água/química
18.
J Biophotonics ; 2(8-9): 521-7, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19593764

RESUMO

Diffraction imaging of polystyrene spheres and B16F10 mouse melanoma cells embedded in gel has been investigated with a microscope objective. The diffraction images acquired with the objective from a sphere have been shown to be comparable to the Mie theory based projection images of the scattered light if the objective is translated to defocused positions towards the sphere. Using a confocal imaging based method to reconstruct and analyze the 3D structure, we demonstrated that genetic modifications in these cells can induce morphological changes and the modified cells can be used as an experimental model for study of the correlation between 3D morphology features and diffraction image data.


Assuntos
Melanoma/patologia , Poliestirenos/química , Espalhamento de Radiação , Animais , Linhagem Celular Tumoral , Imagem Tridimensional , Luz , Melanoma/genética , Camundongos , Microscopia
19.
Med Phys ; 35(9): 3979-87, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18841849

RESUMO

Determination of optical parameters of turbid media from reflectance image data is an important class of inverse problems due to its potential for noninvasive characterization of materials and biological tissues, which demands rapid modeling tools to generate calculated images. We treat the problem of reflectance imaging with homogeneous semi-infinite turbid media as a boundary-value problem of diffusion type in the P1 approximation to the radiative transfer equation. A closed-form solution has been obtained for an oblique incident beam of arbitrary profile and its accuracy has been examined against a Monte Carlo method and measured data. We find that the diffusion solution provides a sufficiently accurate tool to rapidly calculate reflectance images for samples of large or moderate scattering albedo illuminated by a beam of arbitrary profile as long as the anisotropy factor remains less than 0.7 and single scattering albedo larger than 0.8. The closed-form solution can thus be used as a part of a forward modeling toolbox to determine optical parameters from reflectance image data in combination with other method such as the Monte Carlo simulation.


Assuntos
Modelos Teóricos , Método de Monte Carlo , Difusão , Espalhamento de Radiação
20.
Med Phys ; 34(7): 2939-48, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17822002

RESUMO

Reflectance imaging of biological tissues with visible and near-infrared light has the significant potential to provide a noninvasive and safe imaging modality for diagnosis of dysplastic and malignant lesions in the superficial tissue layers. The difficulty in the extraction of optical and structural parameters lies in the lack of efficient methods for accurate modeling of light scattering in biological tissues of turbid nature. We present a parallel Monte Carlo method for accurate and efficient modeling of reflectance images from turbid tissue phantoms. A parallel Monte Carlo code has been developed with the message passing interface and evaluated on a computing cluster with 16 processing elements. The code was validated against the solutions of the radiative transfer equation on the bidirectional reflection and transmission functions. With this code we investigated numerically the dependence of reflectance image on the imaging system and phantom parameters. The contrasts of reflectance images were found to be nearly independent of the numerical aperture (NA) of the imaging camera despite the fact that reflectance depends on the NA. This enables efficient simulations of the reflectance images using an NA at 1.00. Using heterogeneous tissue phantoms with an embedded region simulating a lesion, we investigated the correlation between the reflectance image profile or contrast and the phantom parameters. It has been shown that the image contrast approaches 0 when the single-scattering albedos of the two regions in the heterogeneous phantoms become matched. Furthermore, a zone of detection has been demonstrated for determination of the thickness of the embedded region and optical parameters from the reflectance image profile and contrast. Therefore, the utility of the reflectance imaging method with visible and near-infrared light has been firmly established. We conclude from these results that the optical parameters of the embedded region can be determined inversely from reflectance images acquired with full-field illumination at multiple incident angles or multiple wavelengths.


Assuntos
Método de Monte Carlo , Imagens de Fantasmas , Diagnóstico por Imagem , Humanos , Luz , Espalhamento de Radiação
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