Il micro-specchio dispositivo in questa ricerca è stato finanziato dalla sovvenzione della Fondazione Whitaker RG-02-0682 a N. Boustany. I lavori in corso è finanziato da concedere NSF-DBI-0852857 a N. Boustany. Pasternack RM è stato parzialmente sostenuto da una borsa di studio Rutgers presidenziale Graduate. Vorremmo anche ringraziare il Dott. E. White per le cellule iBMK utilizzati nei nostri studi e il Dr. Metaxas DN di discussione utile per quanto riguarda le strategie di filtraggio ottico.

<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>

Alcuni aspetti dei metodi sono stati inclusi nella divulgazione e invenzione, Rutgers University Docket # 09-049.

Il metodo sopra descritto produce mappe morfometriche dell&#39;oggetto che possono codificare la dimensione delle particelle o di orientamento per esempio. Queste informazioni strutturali possono essere utilizzati in diversi modi: <ol><li> Può essere utilizzato come una schermata iniziale per identificare le regioni tessuti o cellule che sono state modificate nel corso di un trattamento specifico e poi analizzare ulteriormente queste regioni, con specifiche analisi molecolari e biochimici. </li><li> Può essere uti...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<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>
</tr>
<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>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gemini Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Staurosporine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dymethylsulfoxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>Axiovert 200M</td>
<td>&nbsp;</td>
<tr>
<td>DMD</td>
<td>&nbsp;</td>
<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>
</tr>
<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

Si dimostra uno strumento microscopici in grado di misurare struttura subcellulare derivanti dalla morfologia degli organelli e organizzazione all&#39;interno delle cellule viventi senza macchia. Lo strumento proposto estende la sensibilità di etichetta senza microscopia ottica a cambiamenti nelle dimensioni degli organelli nanoscala e la forma e può essere utilizzato per accelerare lo studio della relazione struttura-funzione degli organelli dinamiche relative alla base dei processi biologici fondamentali, come la morte cellulare programmata o cellulare differenziazione. Il microscopio può essere facilmente implementata su piattaforme microscopia esistenti, e può quindi essere diffusi ai laboratori, dove gli scienziati possono implementare e utilizzare i metodi proposti con accesso illimitato. La tecnica proposta è in grado di caratterizzare la struttura subcellulare osservando la cellula attraverso filtri ottici bi-dimensionale Gabor. Questi filtri possono essere sintonizzati con senso su scala nanometrica (10 di nm) sensibilità, specifici attributi morfologici riguardanti le dimensioni e l&#39;orientamento di organuli subcellulari non sferiche. Mentre in base al contrasto generato dalla diffusione elastica, la tecnica non si basa su un modello dettagliato scattering inverso o sulla teoria di Mie per estrarre le misurazioni morfometriche. Questa tecnica è quindi applicabile alle obbligazioni extracontrattuali sferiche organelli per i quali una descrizione precisa dispersione teorica non è facilmente dato, e fornisce distintivo parametri morfometrici che possono essere ottenuti all&#39;interno di cellule viventi senza macchia per valutare la loro funzione. La tecnica è vantaggiosa in confronto con l&#39;elaborazione digitale delle immagini, nel senso che opera direttamente sul campo dell&#39;oggetto trasformare piuttosto che l&#39;intensità dell&#39;oggetto discretizzato è. Non si basa su alti tassi di campionamento delle immagini e può quindi essere usata per selezionare rapidamente l&#39;attività morfologica all&#39;interno di centinaia di cellule alla volta, facilitando in tal modo lo studio della struttura degli organelli organello di là di singoli segmentazione e ricostruzione di microscopia confocale di fluorescenza molto ingrandito le immagini digitali del campo di visuale limitato. In questa dimostrazione si mostrano i dati da una diatomea marina per illustrare la metodologia. Mostriamo anche i dati preliminari raccolti da cellule viventi per dare un&#39;idea di come il metodo può essere applicato in un contesto rilevante biologico.

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

Noi dimostrare un campo scuro metodo di microscopia in base a Gabor-come filtro per misurare la dinamica sub-cellulari all&#39;interno delle cellule viventi singolo. La tecnica è sensibile alle alterazioni nella struttura degli organelli, quali la frammentazione mitocondriale.

Scatter microscopia ottica Sulla base bidimensionale Filtri di Gabor

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 Views.  Rutgers University. Noi dimostrare un campo scuro metodo di microscopia in base a Gabor-come filtro per misurare la dinamica sub-cellulari all'interno delle cellule viventi singolo. La tecnica è sensibile alle alterazioni nella struttura degli organelli, quali la frammentazione mitocondriale.

Video: 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.

Manutenzione e pulizia adeguata del microscopio

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

Ricerca

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

Contrasto di fase e interferenza differenziale contrasto (DIC) Microscopia

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.

Valutazione bidimensionale Prove di cristallizzazione delle proteine ​​di membrana per piccoli studi di biologia strutturale di Cristallografia 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.

Lo xenotrapianto di cellule staminali umane in embrioni di pollo

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.

Risposta cellulare allo stimolo meccanico vivere Studiato da Integrated ottica e microscopia a forza atomica

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.

Visualizzazione dei raggi UV-indotta Intermedi replica in E. coli Utilizzando bidimensionale agarosio-gel Analisi

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

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

I filamenti intermedi e microtubuli con microscopia di ricostruzione ottico stocastico diretto 2-dimensionale di imaging

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.

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.

Saggio di screening basato su SA-Galactosidase per l'identificazione dei farmaci senoterapici

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 ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Imaging privo di etichette delle dinamiche di conservazione lipidica negli elegani di caenorhabditis utilizzando la microscopia a dispersione Raman stimolata

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

Divergenza del microbiota radicale in diversi habitat in base a reti di correlazione ponderate

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...