O dispositivo de micro-espelho nesta pesquisa foi financiada pela Fundação Whitaker conceder RG-02-0682 a N. Boustany. Trabalhos em curso é financiado pela concessão do NSF-DBI-0852857 para N. Boustany. Pasternack RM foi parcialmente financiado por uma Bolsa de Pós-Graduação Rutgers Presidencial. Gostaríamos também de agradecer ao Dr. E. White para as células iBMK utilizado em nossos estudos e Metaxas Dr. DN para discussão útil sobre óptica estratégias de filtragem.

<ol>
	<li>Pasternack, R. M., Qian, Z., Zheng, J. -. Y., Metaxas, D. N., White, E., Boustany, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Subcellular+Texture+by+Optical+Gabor-Like+Filtering+with+a+Digitial+Micromirror+Device.">Measurement of Subcellular Texture by Optical Gabor-Like Filtering with a Digitial Micromirror Device.</a> Optics Letters. 33 (19), 2209-2211 (2008).</li><li>Pasternack, R. M., Qian, Z., Zheng, J. -. Y., Metaxas, D. N., Boustany, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+size+discrimination+of+submicron+objects+using+optical+Fourier+filtering+based+on+two-dimensional+Gabor+filters.">Highly sensitive size discrimination of submicron objects using optical Fourier filtering based on two-dimensional Gabor filters.</a> Optics Express. 17 (14), 12001-12012 (2009).</li><li>Zheng, J. -. Y., Pasternack, R. M., Boustany, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+scatter+imaging+with+a+digital+micromirror+device.">Optical scatter imaging with a digital micromirror device.</a> Optics Express. 17 (22), 20401-20414 (2009).</li></ol>

Alguns aspectos dos métodos foram incluídos na invenção e divulgação, Rutgers University Docket # 09-049.

O método descrito acima produz mapas morfométricos do objeto que pode codificar tamanho de partícula ou orientação, por exemplo. Esta informação estrutural pode ser usado de várias maneiras: <ol><li> Ele pode ser usado como uma tela inicial para identificar as regiões de tecidos ou células que são alteradas durante um tratamento específico e, em seguida, aprofundar a análise destas regiões com os ensaios moleculares e bioquímicas. </li><li> Pode ser usado em conjunto com fluorescência para produzir u...

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>Low glucose DMEM</td>
</tr>
<tr>
<td>Liebowitz L15 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>Without phenol red</td>
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<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mitotracker Green</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Brain Extract</td>
<td>&nbsp;</td>
<td>Clonetics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gemini Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
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<td>Staurosporine</td>
<td>&nbsp;</td>
<td>Sigma</td>
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<td>Dymethylsulfoxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
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<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>Axiovert 200M</td>
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<td>DMD</td>
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<td>Texas Instruments</td>
<td>TI 0.7 XGA DMD 1100</td>
<td>&nbsp;</td>
<tr>
<td>CCD</td>
<td>&nbsp;</td>
<td>Roper Scientific</td>
<td>Cascase 512B</td>
<td>High (16 bit) dynamic range CCD</td>
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<tr>
<td>CCD</td>
<td>&nbsp;</td>
<td>Roped Scientific </td>
<td>Coolsnap cf</td>
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optical scatter microscopy based on two-dimensional gabor filters

Demonstramos um instrumento microscópicas que podem medir textura subcelulares decorrentes da organela morfologia e organização dentro de células vivas sem mácula. O instrumento proposto se estende a sensibilidade da microscopia óptica rótulo sem a mudanças em nanoescala em organela tamanho e forma e podem ser usados ​​para acelerar o estudo da relação estrutura-função pertencentes a organela dinâmica subjacente processos biológicos fundamentais, como a morte celular programada ou celular diferenciação. O microscópio pode ser facilmente implementado em plataformas de microscopia existentes, e pode, portanto, ser divulgada a laboratórios específicos, onde os cientistas podem implementar e utilizar os métodos propostos com acesso irrestrito. A técnica proposta é capaz de caracterizar a estrutura subcelular, observando a célula através de duas dimensões filtros ópticos Gabor. Esses filtros podem ser ajustados para sentir com a sensibilidade (10 de nm) em nanoescala, atributos específicos morfológicas relativas ao tamanho e orientação de não-esférica organelas subcelulares. Embora baseado em contraste gerado por espalhamento elástico, a técnica não se baseia em um modelo de espalhamento inverso ou detalhada sobre a teoria Mie para extrair medidas morfométricas. Esta técnica é, portanto, aplicável a não-esférica organelas para a qual uma descrição precisa dispersão teórica não é facilmente determinada, e fornece parâmetros morfométricos distintivo que pode ser obtido dentro de células vivas sem mácula para avaliar a sua função. A técnica é vantajosa em comparação com processamento digital de imagens em que ele atua diretamente no campo do objeto, em vez de transformar a intensidade do objeto é discretizada. Ele não depende de altas taxas de amostragem de imagem e, portanto, pode ser usada para rapidamente tela de atividades morfológicas dentro de centenas de células de cada vez, portanto, facilitando bastante o estudo da estrutura organela organela além da segmentação individual e reconstrução por microscopia de fluorescência confocal de imagens digitais altamente ampliada de campos de vista limitado. Nesta demonstração vamos mostrar dados de uma diatomácea marinha para ilustrar a metodologia. Nós também mostram os dados preliminares coletados em células vivas para dar uma idéia de como o método pode ser aplicado em um contexto biológicas relevantes.

Watch this Scientific Journal Video about Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters at JoVE.com

Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

Demonstramos um método de microscopia de campo escuro com base em Gabor-como filtragem para medir a dinâmica subcelulares dentro de células vivas único. A técnica é sensível a alterações na estrutura das organelas, tais como a fragmentação mitocondrial.

Microscopia óptica de dispersão com base em filtros Gabor Two-Dimensional

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living ...

optical-scatter-microscopy-based-on-two-dimensional-gabor-filters

Research

JoVE Journal

Biology

9.5K visualizações.  Rutgers University. Demonstramos um método de microscopia de campo escuro com base em Gabor-como filtragem para medir a dinâmica subcelulares dentro de células vivas único. A técnica é sensível a alterações na estrutura das organelas, tais como a fragmentação mitocondrial.

Vídeo: Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Cuidados e limpeza do microscópio

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Biologia

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Contraste de fase e de interferência diferencial Contrast Microscopy (DIC)

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Avaliar Trials bidimensional cristalização de proteínas da membrana Pequenas de Estudos Biologia Estrutural por Cristalografia Electron

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

Xenotransplante de células-tronco humanas no embrião de galinha

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

Resposta celular ao vivo para Estimulação mecânicas estudadas por Microscopia de Força Atômica e Óptica Integrada

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Visualização dos Intermediários UV-induzida replicação em E. coli Usando bidimensional Análise Agarose-gel

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Preparação e imagem longitudinal do coração Função In Vivo Usando Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Caracterização de calcificação eventos usando o Live Óptica e Microscopia Eletrônica de técnicas em um tubeworm Marinha

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

Filamentos intermediários e microtúbulos com microscopia 2-dimensional direta estocástico reconstrução óptico de imagem

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Ensaio de triagem à base de SA-β-galactosidase para a identificação de medicamentos Senoterapêuticos

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Methods Article

Microscopia óptica de dispersão com base em filtros Gabor Two-Dimensional