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1.
Eur Radiol Exp ; 8(1): 57, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38724831

RESUMO

BACKGROUND: We compared computed tomography (CT) images and holograms (HG) to assess the number of arteries of the lung lobes undergoing lobectomy and assessed easiness in interpretation by radiologists and thoracic surgeons with both techniques. METHODS: Patients scheduled for lobectomy for lung cancer were prospectively included and underwent CT for staging. A patient-specific three-dimensional model was generated and visualized in an augmented reality setting. One radiologist and one thoracic surgeon evaluated CT images and holograms to count lobar arteries, having as reference standard the number of arteries recorded at surgery. The easiness of vessel identification was graded according to a Likert scale. Wilcoxon signed-rank test and κ statistics were used. RESULTS: Fifty-two patients were prospectively included. The two doctors detected the same number of arteries in 44/52 images (85%) and in 51/52 holograms (98%). The mean difference between the number of artery branches detected by surgery and CT images was 0.31 ± 0.98, whereas it was 0.09 ± 0.37 between surgery and HGs (p = 0.433). In particular, the mean difference in the number of arteries detected in the upper lobes was 0.67 ± 1.08 between surgery and CT images and 0.17 ± 0.46 between surgery and holograms (p = 0.029). Both radiologist and surgeon showed a higher agreement for holograms (κ = 0.99) than for CT (κ = 0.81) and found holograms easier to evaluate than CTs (p < 0.001). CONCLUSIONS: Augmented reality by holograms is an effective tool for preoperative vascular anatomy assessment of lungs, especially when evaluating the upper lobes, more prone to anatomical variations. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04227444 RELEVANCE STATEMENT: Preoperative evaluation of the lung lobe arteries through augmented reality may help the thoracic surgeons to carefully plan a lobectomy, thus contributing to optimize patients' outcomes. KEY POINTS: • Preoperative assessment of the lung arteries may help surgical planning. • Lung artery detection by augmented reality was more accurate than that by CT images, particularly for the upper lobes. • The assessment of the lung arterial vessels was easier by using holograms than CT images.


Assuntos
Realidade Aumentada , Holografia , Neoplasias Pulmonares , Artéria Pulmonar , Tomografia Computadorizada por Raios X , Humanos , Feminino , Masculino , Tomografia Computadorizada por Raios X/métodos , Idoso , Estudos Prospectivos , Neoplasias Pulmonares/cirurgia , Neoplasias Pulmonares/diagnóstico por imagem , Pessoa de Meia-Idade , Holografia/métodos , Artéria Pulmonar/diagnóstico por imagem , Artéria Pulmonar/anatomia & histologia , Imageamento Tridimensional , Padrões de Referência , Pulmão/diagnóstico por imagem , Pulmão/irrigação sanguínea , Pulmão/cirurgia
2.
Cells ; 13(8)2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38667312

RESUMO

The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC50) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.


Assuntos
Nanopartículas , Animais , Camundongos , Células NIH 3T3 , Nanopartículas/toxicidade , Nanopartículas/química , Células RAW 264.7 , Sobrevivência Celular/efeitos dos fármacos , Holografia/métodos , Imageamento Quantitativo de Fase
3.
Appl Opt ; 61(5): B77-B82, 2022 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-35201128

RESUMO

A digital lensless holographic microscope (DLHM) sensitive to the linear diattenuation produced by biological samples is reported. The insertion of a linear polarization-states generator and a linear polarization-states analyzer in a typical DLHM setup allows the proper linear diattenuation imaging of microscopic samples. The proposal has been validated for simulated and experimental biological samples containing calcium oxalate crystals extracted from agave leaves and potato starch grains. The performance of the proposed method is similar to that of a traditional polarimetric microscope to obtain linear diattenuation images of microscopic samples but with the advantages of DLHM, such as numerical refocusing, cost effectiveness, and the possibility of field-portable implementation.


Assuntos
Holografia , Microscopia , Análise Custo-Benefício , Holografia/métodos , Microscopia/métodos
4.
Sensors (Basel) ; 22(2)2022 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-35062512

RESUMO

Lensless holographic microscope (LHM) is an emerging very promising technology that provides high-quality imaging and analysis of biological samples without utilizing any lens for imaging. Due to its small size and reduced price, LHM can be a very useful tool for the point-of-care diagnosis of diseases, sperm assessment, or microfluidics, among others, not only employed in advanced laboratories but also in poor and/or remote areas. Recently, several LHMs have been reported in the literature. However, complete characterization of their optical parameters remains not much presented yet. Hence, we present a complete analysis of the performance of a compact, reduced cost, and high-resolution LHM. In particular, optical parameters such as lateral and axial resolutions, lateral magnification, and field of view are discussed into detail, comparing the experimental results with the expected theoretical values for different layout configurations. We use high-resolution amplitude and phase test targets and several microbeads to characterize the proposed microscope. This characterization is used to define a balanced and matched setup showing a good compromise between the involved parameters. Finally, such a microscope is utilized for visualization of static, as well as dynamic biosamples.


Assuntos
Holografia , Lentes , Calibragem , Análise Custo-Benefício , Microscopia
5.
IEEE J Biomed Health Inform ; 26(3): 1318-1328, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34388103

RESUMO

This study presents a novel approach to automatically perform instant phenotypic assessment of red blood cell (RBC) storage lesion in phase images obtained by digital holographic microscopy. The proposed model combines a generative adversarial network (GAN) with marker-controlled watershed segmentation scheme. The GAN model performed RBC segmentations and classifications to develop ageing markers, and the watershed segmentation was used to completely separate overlapping RBCs. Our approach achieved good segmentation and classification accuracy with a Dice's coefficient of 0.94 at a high throughput rate of about 152 cells per second. These results were compared with other deep neural network architectures. Moreover, our image-based deep learning models recognized the morphological changes that occur in RBCs during storage. Our deep learning-based classification results were in good agreement with previous findings on the changes in RBC markers (dominant shapes) affected by storage duration. We believe that our image-based deep learning models can be useful for automated assessment of RBC quality, storage lesions for safe transfusions, and diagnosis of RBC-related diseases.


Assuntos
Aprendizado Profundo , Holografia , Envelhecimento , Eritrócitos , Holografia/métodos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Redes Neurais de Computação
6.
Appl Opt ; 60(4): A205-A214, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33690371

RESUMO

In this work, the design, construction, and testing of the most cost-effective digital lensless holographic microscope to date are presented. The architecture of digital lensless holographic microscopy (DLHM) is built by means of a 3D-printed setup and utilizing off-the-shelf materials to produce a DLHM microscope costing US$52.82. For the processing of the recorded in-line holograms, an open-source software specifically developed to process this type of recordings is utilized. The presented DLHM setup has all the degrees of freedom needed to achieve different fields of view, levels of spatial resolution, and 2D scanning of the sample. The feasibility of the presented platform is tested by imaging non-bio and bio samples; the resolution test targets, a section of the head of a Drosophila melanogaster fly, red blood cells, and cheek cells are imaged on the built microscope.


Assuntos
Holografia/instrumentação , Microscopia/instrumentação , Animais , Bochecha/diagnóstico por imagem , Análise Custo-Benefício , Drosophila melanogaster/metabolismo , Eritrócitos/metabolismo , Cabeça/diagnóstico por imagem , Holografia/métodos , Microscopia/métodos , Impressão Tridimensional
7.
Appl Opt ; 59(10): 2969-2975, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32400572

RESUMO

In this work, the development and application of a cost-effective and robust digital lensless holographic microscopy (DLHM) system is presented. In the simple architecture of DLHM based on a point source and a digital camera, the production of the former is introduced by means of an engineered step-index optical fiber with a cone-shaped end tip. The conventional and regularly expensive point source in DLHM is produced by means of a high-numerical-aperture microscope objective and a metallic wavelength-sized pinhole. The proposed replacement renders to DLHM additional simplicity of building, in addition to mechanical stability and robustness, and further reduces the cost of the microscope. The simplified cost-effective DLHM architecture is utilized for imaging resolution test targets and samples of human blood and pond water, revealing competitive mechanical stability and trustable phase images of the imaged specimens.


Assuntos
Eritrócitos/classificação , Holografia/instrumentação , Microscopia/instrumentação , Fibras Ópticas , Água/química , Sangue , Análise Custo-Benefício , Desenho de Equipamento , Holografia/economia , Humanos , Processamento de Imagem Assistida por Computador , Microscopia/economia
8.
Surg Endosc ; 34(4): 1678-1687, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31286252

RESUMO

BACKGROUND: Suturing is a fundamental skill in undergraduate medical education. It can be taught by faculty-led, peer tutor-led, and holography-augmented methods; however, the most educationally effective and cost-efficient method for proficiency-based teaching of suturing is yet to be determined. METHODS: We conducted a randomized controlled trial comparing faculty-led, peer tutor-led, and holography-augmented proficiency-based suturing training in pre-clerkship medical students. Holography-augmented training provided holographic, voice-controlled instructional material. Technical skill was assessed using hand motion analysis every ten sutures and used to construct learning curves. Proficiency was defined by one standard deviation within average faculty surgeon performance. Intervention arms were compared using one-way ANOVA of the number of sutures placed, full-length sutures used, time to proficiency, and incremental costs incurred. Surveys were used to evaluate participant preferences. RESULTS: Forty-four students were randomized to the faculty-led (n = 16), peer tutor-led (n = 14), and holography-augmented (n = 14) intervention arms. At proficiency, there were no differences between groups in the number of sutures placed, full-length sutures used, and time to achieve proficiency. The incremental costs of the holography-augmented method were greater than faculty-led and peer tutor-led instruction ($247.00 ± $12.05, p < 0.001) due to the high cost of the equipment. Faculty-led teaching was the most preferred method (78.0%), while holography-augmented was the least preferred (0%). 90.6% of students reported high confidence in performing simple interrupted sutures, which did not differ between intervention arms (faculty-led 100.0%, peer tutor-led 90.0%, holography-augmented 83.3%, p = 0.409). 93.8% of students felt the program should be offered in the future. CONCLUSION: Faculty-led and peer tutor-led instructional methods of proficiency-based suturing teaching were superior to holography-augmented method with respect to costs and participants' preferences despite being educationally equivalent.


Assuntos
Competência Clínica , Educação de Graduação em Medicina/economia , Holografia/economia , Aprendizagem Baseada em Problemas/economia , Técnicas de Sutura/educação , Adulto , Análise Custo-Benefício , Educação de Graduação em Medicina/métodos , Feminino , Holografia/métodos , Humanos , Curva de Aprendizado , Masculino , Aprendizagem Baseada em Problemas/métodos , Estudantes de Medicina/estatística & dados numéricos
9.
JAMA Netw Open ; 2(4): e192633, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-31002326

RESUMO

Importance: Nephron-sparing surgery can be considered in well-defined cases of unilateral and bilateral Wilms tumors, but the surgical procedure can be very challenging for the pediatric surgeon to perform. Objective: To assess the added value of personalized 3-dimensional (3-D) kidney models derived from conventional imaging data to enhance preoperative surgical planning. Design, Setting, and Participants: In a survey study, the conventional imaging data of 10 Dutch children with Wilms tumors were converted to 3-D prints and augmented reality (AR) holograms and a panel of pediatric oncology surgeons (n = 7) assessed the quality of the different imaging methods during preoperative evaluation. Kidney models were created with 3-D printing and AR using a mixed reality headset for visualization. Main Outcomes and Measures: Differences in the assessment of 4 anatomical structures (tumor, arteries, veins, and urinary collecting structures) using questionnaires. A Likert scale measured differences between the imaging methods, with scores ranging from 1 (completely disagree) to 5 (completely agree). Results: Of the 10 patients, 7 were girls, and the mean (SD) age was 3.7 (1.7) years. Compared with conventional imaging, the 3-D print and the AR hologram models were evaluated by the surgeons to be superior for all anatomical structures: tumor (median scores for conventional imaging, 4.07; interquartile range [IQR], 3.62-4.15 vs 3-D print, 4.67; IQR, 4.14-4.71; P = .008 and AR hologram, 4.71; IQR, 4.26-4.75; P = .002); arteries (conventional imaging, 3.62; IQR, 3.43-3.93 vs 3-D print, 4.54; IQR, 4.32-4.71; P = .002 and AR hologram, 4.83; IQR, 4.64-4.86; P < .001), veins (conventional imaging, 3.46; IQR 3.39-3.62 vs 3-D print, 4.50; IQR, 4.39-4.68; P < .001 and AR hologram, 4.83; IQR, 4.71-4.86; P < .001), and urinary collecting structures (conventional imaging, 2.76; IQR, 2.42-3.00 vs 3-D print, 3.86; IQR, 3.64-4.39; P < .001 and AR hologram, 4.00; IQR, 3.93-4.58; P < .001). There were no differences in anatomical assessment between the two 3-D techniques (the 3-D print and AR hologram). Conclusions and Relevance: In this study, the 3-D kidney models were associated with improved anatomical understanding among the surgeons and can be helpful in future preoperative planning of nephron-sparing surgery for Wilms tumors. These models may be considered as a supplementary visualization in clinical care.


Assuntos
Realidade Aumentada , Holografia/métodos , Neoplasias Renais/diagnóstico por imagem , Modelos Anatômicos , Impressão Tridimensional , Tumor de Wilms/diagnóstico por imagem , Pré-Escolar , Feminino , Humanos , Masculino , Período Pré-Operatório
10.
Sci Rep ; 9(1): 4823, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30886325

RESUMO

Raman microspectroscopy (RM) and polarization sensitive digital holographic imaging (PSDHI) are valuable analytical tools in biological and medical research, allowing the detection of both biochemical and morphological variations of the sample without labels or long sample preparation. Here, using this multi-modal approach we analyze in vitro human sperm capacitation and the acrosome reaction induced by heparin. The multimodal microscopy provides morphofunctional information that can assess the sperms ability to respond to capacitation stimuli (sperm function). More precisely, the birefringence analysis in sperm cells can be used as an indicator of its structural normality. Indeed, digital holography applied for polarization imaging allows for revelation of the polarization state of the sample, showing a total birefringence of the sperm head in non-reacted spermatozoa, and a birefringence localized in the post-acrosomal region in reacted spermatozoa. Additionally, RM allows the detection and spectroscopic characterization of protein/lipid delocalization in the plasma and acrosomal membranes that can be used as valuable Raman biomarkers of sperm function. Interestingly, these spectral variations can be correlated with different time phases of the cell capacitation response. Although further experimentation is required, the proposed multimodal approach could represent a potential label-free diagnostic tool for use in reproductive medicine and the diagnosis of infertility.


Assuntos
Holografia/métodos , Infertilidade/diagnóstico , Análise do Sêmen/métodos , Análise Espectral Raman/métodos , Espermatozoides/fisiologia , Reação Acrossômica/fisiologia , Voluntários Saudáveis , Humanos , Infertilidade/fisiopatologia , Masculino , Microscopia de Fluorescência/métodos , Microscopia de Polarização/métodos , Capacitação Espermática/fisiologia
11.
J Microsc ; 273(3): 199-209, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30561003

RESUMO

Commercial high-resolution optical microscopes are essential for microscopy imaging; however, they are expensive and bulky, which limits their use in point-of-care devices, resource-limited areas, and real-time imaging of a sample in a large apparatus. In this study, we report a novel compact (10 cm × 5 cm × 5 cm, without the light source) lightweight (∼0.5 kg) submicron-resolution inverted optical microscope at low cost (∼$ 300). Our technique utilises the proximity of the image sensor to a commercial microscope objective lens for compactness of the microscope. The use of an image sensor with a small pixel size helps to reduce the information loss, which provides high-resolution images. Moreover, our technique offers a freedom to tailor the design of microscope according to the required resolution, cost, and portability for specific applications, which makes it a suitable candidate for affordable point-of-care devices. Images of several micron-to-submicron scale patterns and spherical beads are acquired to observe the resolution and quality of the images obtained using our microscope. In addition, we demonstrate the applications of our microscope in various fields such as recording of high-speed water microdroplet formation inside a microfluidic device, high-resolution live cell imaging inside an incubator, and real-time imaging of crack propagation in a sample under stretching by a material testing system (MTS). Therefore, this portable and inexpensive microscope provides the essential functionalities of a bulky expensive high-performance microscope at a lower cost. LAY DESCRIPTION: Microscope is an essential tool in research allowing for observation of microsized objects and life forms. Contemporary commercial high-resolution microscopes have long optical paths involving series of lenses and filters. Although this configuration precisely corrects for optical distortions and produces clear images, it makes modern microscopes very costly and bulky, restricting their usage to low-funded research laboratories and at remote places. We have developed a simple digital microscope with high-resolution but with much smaller size and lighter in weight at low cost by removing the long optical terrain. Our microscope consists of a commercial microscope objective lens for magnification and semiconductor image sensor with small pixels placed right after the lens, both of which are affordable and easily available. The small pixel size helps to translate the magnified analogue sample image to high-resolution digital image. In our paper, we show that our microscope can view micro and submicron-sized patterns and beads. Moreover, our fist-sized microscope can be placed inside an incubator for real-time imaging of cells or rotated sideways for recording submicron-sized crack generation due stretching of novel materials, both of which could not be accomplished with the 2 feet tall laboratory microscopes.


Assuntos
Desenho de Equipamento , Dispositivos Lab-On-A-Chip , Microscopia de Polarização/instrumentação , Animais , Escherichia coli/ultraestrutura , Fibroblastos/ultraestrutura , Holografia , Lentes , Camundongos , Microscopia de Polarização/métodos , Células NIH 3T3 , Oócitos/ultraestrutura , Peixe-Zebra
12.
Cytometry A ; 93(3): 334-345, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29283496

RESUMO

The noninvasive, fast acquisition of quantitative phase maps using digital holographic microscopy (DHM) allows tracking of rapid cellular motility on transparent substrates. On two-dimensional surfaces in vitro, MDA-MB-231 cancer cells assume several morphologies related to the mode of migration and substrate stiffness, relevant to mechanisms of cancer invasiveness in vivo. The quantitative phase information from DHM may accurately classify adhesive cancer cell subpopulations with clinical relevance. To test this, cells from the invasive breast cancer MDA-MB-231 cell line were cultured on glass, tissue-culture treated polystyrene, and collagen hydrogels, and imaged with DHM followed by epifluorescence microscopy after staining F-actin and nuclei. Trends in cell phase parameters were tracked on the different substrates, during cell division, and during matrix adhesion, relating them to F-actin features. Support vector machine learning algorithms were trained and tested using parameters from holographic phase reconstructions and cell geometric features from conventional phase images, and used to distinguish between elongated and rounded cell morphologies. DHM was able to distinguish between elongated and rounded morphologies of MDA-MB-231 cells with 94% accuracy, compared to 83% accuracy using cell geometric features from conventional brightfield microscopy. This finding indicates the potential of DHM to detect and monitor cancer cell morphologies relevant to cell cycle phase status, substrate adhesion, and motility. © 2017 International Society for Advancement of Cytometry.


Assuntos
Neoplasias da Mama/patologia , Movimento Celular/fisiologia , Holografia/métodos , Aprendizado de Máquina , Microscopia de Fluorescência/métodos , Actinas/análise , Adesão Celular/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/fisiologia , Humanos , Invasividade Neoplásica/patologia
13.
Plast Reconstr Surg ; 140(5): 1066-1070, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29068946

RESUMO

Virtual reality and augmented reality devices have recently been described in the surgical literature. The authors have previously explored various iterations of these devices, and although they show promise, it has become clear that virtual reality and/or augmented reality devices alone do not adequately meet the demands of surgeons. The solution may lie in a hybrid technology known as mixed reality, which merges many virtual reality and augmented realty features. Microsoft's HoloLens, the first commercially available mixed reality device, provides surgeons intraoperative hands-free access to complex data, the real environment, and bidirectional communication. This report describes the use of HoloLens in the operating room to improve decision-making and surgical workflow. The pace of mixed reality-related technological development will undoubtedly be rapid in the coming years, and plastic surgeons are ideally suited to both lead and benefit from this advance.


Assuntos
Holografia/instrumentação , Procedimentos de Cirurgia Plástica/instrumentação , Cirurgia Assistida por Computador/instrumentação , Interface Usuário-Computador , Tomada de Decisão Clínica , Humanos , Procedimentos de Cirurgia Plástica/métodos , Cirurgia Assistida por Computador/métodos , Fluxo de Trabalho
14.
Sci Rep ; 7(1): 4856, 2017 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-28687769

RESUMO

Caused by the herpes simplex virus (HSV), herpes is a viral infection that is one of the most widespread diseases worldwide. Here we present a computational sensing technique for specific detection of HSV using both viral immuno-specificity and the physical size range of the viruses. This label-free approach involves a compact and cost-effective holographic on-chip microscope and a surface-functionalized glass substrate prepared to specifically capture the target viruses. To enhance the optical signatures of individual viruses and increase their signal-to-noise ratio, self-assembled polyethylene glycol based nanolenses are rapidly formed around each virus particle captured on the substrate using a portable interface. Holographic shadows of specifically captured viruses that are surrounded by these self-assembled nanolenses are then reconstructed, and the phase image is used for automated quantification of the size of each particle within our large field-of-view, ~30 mm2. The combination of viral immuno-specificity due to surface functionalization and the physical size measurements enabled by holographic imaging is used to sensitively detect and enumerate HSV particles using our compact and cost-effective platform. This computational sensing technique can find numerous uses in global health related applications in resource-limited environments.


Assuntos
Herpes Simples/diagnóstico , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Simplexvirus/isolamento & purificação , Análise Custo-Benefício , Holografia/métodos , Processamento de Imagem Assistida por Computador/economia , Microscopia/economia , Sensibilidade e Especificidade
15.
Cytometry A ; 91(5): 460-469, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28437571

RESUMO

Cell proliferation assays are widely applied in biological sciences to understand the effect of drugs over time. However, current methods often assess cell population growth indirectly, that is, the cells are not actually counted. Instead other parameters, for example, the amount of protein, are determined. These methods often also demand phototoxic labels, have low temporal resolution, or employ end-point assays, and frequently are labor intensive. We have developed a robust and label-free kinetic cell proliferation assay with high temporal resolution for adherent cells using digital holographic microscopy (DHM), one of many quantitative phase microscopy techniques. As no labels or stains are required, and only very low intensity illumination is necessary, the technique allows for noninvasive continuous cell counting. Only two image processing settings were adjusted between cell lines, making the assay practical, user friendly, and free of user bias. The developed direct assay was validated by analyzing cell cultures treated with various concentrations of the anti-cancer drug etoposide, a well-established topoisomerase inhibitor that causes DNA damage and leads to programmed cell death. After treatment, the unstained adherent cells were nondestructively imaged every 30 min for 36 h inside a cell incubator. In the recorded time-lapse image sequences, individual cells were automatically identified to provide detailed growth curves and growth rate data of cell number, confluence, and average cell volume. Our results demonstrate how these parameters facilitate a deeper understanding of cell processes than what is achievable with current single-parameter and end-point methods. © 2017 International Society for Advancement of Cytometry.


Assuntos
Contagem de Células/métodos , Holografia/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Linhagem Celular , Proliferação de Células/genética , Humanos
16.
Lab Chip ; 16(22): 4350-4358, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27713987

RESUMO

Monitoring yeast cell viability and concentration is important in brewing, baking and biofuel production. However, existing methods of measuring viability and concentration are relatively bulky, tedious and expensive. Here we demonstrate a compact and cost-effective automatic yeast analysis platform (AYAP), which can rapidly measure cell concentration and viability. AYAP is based on digital in-line holography and on-chip microscopy and rapidly images a large field-of-view of 22.5 mm2. This lens-free microscope weighs 70 g and utilizes a partially-coherent illumination source and an opto-electronic image sensor chip. A touch-screen user interface based on a tablet-PC is developed to reconstruct the holographic shadows captured by the image sensor chip and use a support vector machine (SVM) model to automatically classify live and dead cells in a yeast sample stained with methylene blue. In order to quantify its accuracy, we varied the viability and concentration of the cells and compared AYAP's performance with a fluorescence exclusion staining based gold-standard using regression analysis. The results agree very well with this gold-standard method and no significant difference was observed between the two methods within a concentration range of 1.4 × 105 to 1.4 × 106 cells per mL, providing a dynamic range suitable for various applications. This lensfree computational imaging technology that is coupled with machine learning algorithms would be useful for cost-effective and rapid quantification of cell viability and density even in field and resource-poor settings.


Assuntos
Aprendizado de Máquina , Microscopia/economia , Microscopia/instrumentação , Saccharomyces cerevisiae/citologia , Análise Custo-Benefício , Holografia , Fatores de Tempo
17.
Int J Radiat Biol ; 92(12): 796-805, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27586164

RESUMO

PURPOSE: To assure the quality of cells to be used in cell therapy, we examined the applicability of digital holographic microscopy (DHM) for non-invasive, quantitative assessment of changes in cell morphology. MATERIALS AND METHODS: Mesenchymal stem cells derived from adipose tissue (MSC-AT) and bone marrow (MSC-BM), in addition to human alveolar periosteal cells (PC) as a reference, were γ-ray irradiated (1 and 4 Gy), and their morphological changes were quantified without fixation using holographic microscopy. After detachment and fixation with ethanol, cell number and surface antigen expression were determined using an automated cell counter kit and flow-cytometry, respectively. RESULTS: Among various indexes, only indexes related to cell size were significantly changed after γ-irradiation. Both BMC-AT and BMC-BM were enlarged and more sensitive to a low dose of γ-irradiation than PC. In contrast to PC, proteins related to DNA damage repair (γ-H2AX, p21waf1, p53 and Rb) were not substantially upregulated or sustained for a week in either MSC-AT or MSC-BM. CONCLUSION: Instead of DNA damage markers, we suggest that cell morphological parameters (e.g. cell volume) that are monitored by DHM could be a useful and more stable marker of MSC quality.


Assuntos
Holografia/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos da radiação , Microscopia/métodos , Periósteo/citologia , Periósteo/efeitos da radiação , Tamanho Celular/efeitos da radiação , Rastreamento de Células/métodos , Células Cultivadas , Relação Dose-Resposta à Radiação , Raios gama , Humanos , Imageamento Tridimensional/métodos , Doses de Radiação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Processamento de Sinais Assistido por Computador
18.
Theranostics ; 6(10): 1603-10, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27446494

RESUMO

Low-cost, rapid and accurate detection technologies are key requisites to cope with the growing global cancer challenges. The need is particularly pronounced in resource-limited settings where treatment opportunities are often missed due to the absence of timely diagnoses. We herein describe a Holographic Assessment of Lymphoma Tissue (HALT) system that adopts a smartphone as the basis for molecular cancer diagnostics. The system detects malignant lymphoma cells labeled with marker-specific microbeads that produce unique holographic signatures. Importantly, we optimized HALT to detect lymphomas in fine-needle aspirates from superficial lymph nodes, procedures that align with the minimally invasive biopsy needs of resource-constrained regions. We equipped the platform to directly address the practical needs of employing novel technologies for "real world" use. The HALT assay generated readouts in <1.5 h and demonstrated good agreement with standard cytology and surgical pathology.


Assuntos
Holografia/métodos , Linfoma/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Imagem Óptica/métodos , Smartphone , Coloração e Rotulagem/métodos , Biópsia por Agulha Fina , Linfonodos/patologia , Linfoma/patologia , Microesferas , Fatores de Tempo
19.
Appl Opt ; 54(10): 2857-65, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25967200

RESUMO

The use of a low-power laser beam to characterize self-phase modulation (SPM) and bubble formation during thermal blooming (TB), as well as manipulation of the bubbles, is reported. First, a low-power 633 nm laser beam is used to characterize the induced refractive index profile during SPM of a focused 514 nm pump beam in absorbing liquid media, e.g., a solution of red dye in isopropyl alcohol. The induced phase change is also characterized using digital holography via the 633 nm source as the probe and reference. During TB at higher pump powers, bubble formation occurs in the liquid. Using a modified setup, which minimizes the effects of gravity, buoyancy, and convection, stable bubbles are generated. These are characterized using in-line digital holography with the 633 nm probe beam. It is shown that the bubble size depends on exposure time of the pump and that the bubble can be steered by moving a focused low-power laser beam. Finally, possible applications of these thermally generated bubbles are discussed.


Assuntos
Holografia/instrumentação , Lasers , 2-Propanol/química , Algoritmos , Corantes/química , Simulação por Computador , Sistemas de Liberação de Medicamentos , Desenho de Equipamento , Holografia/métodos , Processamento de Imagem Assistida por Computador/métodos , Interferometria , Íons , Distribuição Normal , Refratometria , Temperatura
20.
ACS Nano ; 9(3): 3265-73, 2015 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-25688665

RESUMO

Sizing individual nanoparticles and dispersions of nanoparticles provides invaluable information in applications such as nanomaterial synthesis, air and water quality monitoring, virology, and medical diagnostics. Several conventional nanoparticle sizing approaches exist; however, there remains a lack of high-throughput approaches that are suitable for low-resource and field settings, i.e., methods that are cost-effective, portable, and can measure widely varying particle sizes and concentrations. Here we fill this gap using an unconventional approach that combines holographic on-chip microscopy with vapor-condensed nanolens self-assembly inside a cost-effective hand-held device. By using this approach and capturing time-resolved in situ images of the particles, we optimize the nanolens formation process, resulting in significant signal enhancement for the label-free detection and sizing of individual deeply subwavelength particles (smaller than λ/10) over a 30 mm(2) sample field-of-view, with an accuracy of ±11 nm. These time-resolved measurements are significantly more reliable than a single measurement at a given time, which was previously used only for nanoparticle detection without sizing. We experimentally demonstrate the sizing of individual nanoparticles as well as viruses, monodisperse samples, and complex polydisperse mixtures, where the sample concentrations can span ∼5 orders-of-magnitude and particle sizes can range from 40 nm to millimeter-scale. We believe that this high-throughput and label-free nanoparticle sizing platform, together with its cost-effective and hand-held interface, will make highly advanced nanoscopic measurements readily accessible to researchers in developing countries and even to citizen-scientists, and might especially be valuable for environmental and biomedical applications as well as for higher education and training programs.


Assuntos
Microscopia/métodos , Nanopartículas/química , Tamanho da Partícula , Análise Custo-Benefício , Holografia , Microscopia/economia , Microscopia/instrumentação , Poliestirenos/química , Volatilização
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