This work was supported by the Canadian Institute of Health Research to CP (OR 142730 and OR 89931). We would like to thank Jelena Brkic for her initial idea of binary particle analysis in ImageJ.

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	<li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+and+Determining+the+Viability+of+Cultured+Cells.">Counting and Determining the Viability of Cultured Cells.</a> J. Vis. Exp. (16), e752 (2008).</li><li>Using a Hemacytometer to Count Cells. Basic Methods in Cellular and Molecular Biology Available from: <a target="_blank" href="https://www.jove.com/science-education/5048/using-a-hemacytometer-to-count-cells">https://www.jove.com/science-education/5048/using-a-hemacytometer-to-count-cells</a> (2016)</li><li>Graham, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coulter+principle:+foundation+of+an+industry.">The coulter principle: foundation of an industry.</a> J. Lab. Autom. 8 (6), 72-81 (2003).</li><li>Ormerod, M. G., Imrie, P. R., Walker, J. M., Pollard, J. 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The authors declare that they have no competing financial interests.

שלבים קריטיים, פתרון בעיות, ומגבלות עצם טבעו של שיטות חישוביות אוטומטיות, במיוחד אלה של ניתוח חלקיקים, מחייבת היכולת המתמטית להגדיר חלקיקים אלה. כתוצאה מכך, את הדיוק של שני מחשבון נייד ריכוזיות דלפק assay ההגירה תלוי majorly על נאמנות תמ?...

בשנת ספירת תאים במבחנה היא טכניקה בסיסית חשובה במגוון רחב של ניסויים בתרבית רקמה. קביעת מספר התאים במדויק בתרבות חיונית שחזור ניסיוני 1,2 סטנדרטיזציה. ספירת תאים יכול להתבצע באופן ידני באמצעות hemocytometer וכן באמצעות מגוון של שיטות אוטומטיות, כל אחד עם יתרונות וחסרונות משלהם 3,4,5. רוב השיטות האוטומטיות ספירת תאים שייך לאחת משתי כיתות, אלה העושים שימוש עיקרון קולטר או cytometry זרימה. מונה קולטר לנצל התנגדות חשמלית תאים כדי לקבוע את מספר נייד וגודל. הם מהירים, מדויקים יותר זולים מאשר cytometers זרימה. עם זאת, הם משמשים לעתים נדירות עבור ספירת תאים רק בשל הניכר בעלויות שלהם לעומת ספירה ידנית 3. זרימת cytometers, מצד השני, הם יקרים, אבל יש להם יישומים רבים כגון ספירת תאים, ניתוח של צורת התאים, structure ותא פנימי למדידת סמני 4,5. מכונות המשתמשות אחד משני עקרונות אלה זמינות מיצרנים רבים. ספירה ידנית היא זולה אך זמן רב ובכפוף ההטיה בעוד בשיטות האוטומטיות לבוא עם חלק מן הזמן הדרוש הספירה הידנית אלא באמצעות 6 מכונות יקרות. הליכים נפוצים תרבית תאים אחרים נמצאים מבחני תנועתיות תא במבחנה, כלומר, נדידת תאים ופלישה 7. מבחני הגירה ופלישה משמשים בדרך כלל כדי לחקור תנועתיות התא הפולשנות בתגובה לתגובה chemotactic. בנוסף, הם נמצאים בשימוש נרחב ללמוד התפתחות עוברית, בידול, תגובה דלקתית, וגרורות של סוגי תאים מרובים 7-11. תאים שתהליך ההתפתחות שלהם היגרו או פלשו דרך הממברנה נקבובי של assay הגירה ניתן לכמת בשתי דרכים שונות. ראשית, על ידי צביעה את התאים עם צבע פלואורסצנטי, דיסוציאציה הלוך ושובמ הממברנה, וכימות באמצעות קורא פלורסנט 12. הגבלה של שיטה זו של כימות היא שאף שיא ניתן להיעזר של ממברנות ואין אפשרות לניתוח נוסף 13. שיטת הכימות השנייה עבור היגרו / פלש תא להיות קבוע מוכתם צבע פלואורסצנטי או יותר נפוץ, עם צבעי ציטולוגיה כגון סגול קריסטל, צבע כחול toluidine או hematoxylin; אז תאים לכמת באמצעות תמונות מיקרוסקופיות הפוכות ידני של הקרומים האלה אשר הוא משימה מאוד זמן רב 12,13. כדי להתגבר על החסרונות של ספירת תאים ידנית, שני מוני תא אוטומטיים אמינים ומדויקים עבור ריכוז תא עבור assay הגירה פותחו. אלגוריתמים נגד תא אוטומטי אלו פותחו עבור ImageJ בתור תוסף באמצעות שפת מחשב Java של אורקל. ImageJ הוא כלי עיבוד תמונה ציבורי הנפוצה ביותר לשימוש שפותח על ידי המכון הלאומי של חימוםLTH (NIH) 14,15; וכך, כותב תוספים אלה עבור ImageJ מקל אינטגרציה קלה לתוך הקהילה הביולוגית. אוטומציה של ספירת תאים מבטיחה תפוקת שחזור גבוהות לעומת ספירה ידנית. למרות תוכנות ותוספות אחרות זמינות שניתן להשתמש בם כדי לחשב ריכוז תא באמצעות ניתוח תמונת 5,16,17, תוסף מחשבון ריכוז תא הוא מהיר והוא יכול גם להתמודד עם דילולים של תאים וטיפולים. יתר על כן, כל התוצאות והחישובים משני מונים אלה ניתן לשמור וייצא. שני תוספים המתואר במאמר זה מותאמים לשימוש מיקרוסקופ לעומת שלב הדמיה תא חי ושדה גדול של תצוגה (לכידת הממברנה כולה) הדמיה עבור ממברנות assay הגירה באמצעות היקף הניתוחים. את התוספים זמינים באופן חופשי להורדה עם הוראות התקנה מ: http://peng.lab.yorku.ca/imagej-plugins.

<table><tbody><tr><td>HyClone Classical Liquid Media: RPMI 1640 - With L-Glutamine</td><td>Fisher Scientific</td><td>SH3002702</td><td>Cell culturing media&amp;nbsp;</td></tr><tr><td>Fetal bovian serum (FBS)</td><td>GIBCO BRL</td><td>P00015</td><td>Media suppliment</td></tr><tr><td>HTR8/SVneo trophoblast cell line</td><td>Cells were obtained from Dr. Charles Graham (Queen&amp;rsquo;s University, Kingston, Canada)</td><td></td><td>Software is designed to work with any cell line.</td></tr><tr><td>Trypsin</td><td>GIBCO BRL</td><td>27250-018</td><td>Prepared as 0.20% (w/v) in 10 &amp;micro;M EDTA 1x PBS</td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT104</td><td></td></tr><tr><td>10 cm cell culture plates</td><td>SARSTEDT</td><td>833902</td><td>Any tissue culture treated plates will be suitable</td></tr><tr><td>Transwell Polyester Membrane Inserts - 8.0&amp;nbsp;&amp;micro;m Pore size</td><td>Costar 3422 ordered from Fisher Scientific</td><td>7200150</td><td>For 24-well plates; Pore size: 8.0 &amp;micro;m; 6.5 mm diameter; 0.33 cm&lt;sup&gt;2&lt;/sup&gt; growth area</td></tr><tr><td>HARLECO Hematology Stains and Reagents, EMD Millipore - Soluntions 1, 2 &amp;amp; 3</td><td>EMD Millipore and ordered from VWR</td><td>65044A, B, &amp;amp; C</td><td>Hemacolor stain set consists of three 500 ml (16.9 oz.) poly bottles &amp;amp; includes a methanol fixative (Solution 1), an eosin or acid stain (Solution 2), and a methylene blue or basic stain (Solution 3)</td></tr><tr><td>Cotton Tipped Applicator</td><td>Puritan Medical</td><td>806-WC</td><td></td></tr><tr><td>Single-edge industrial razor blades</td><td>VWR</td><td>55411 - 055</td><td>Thickness: 0.30 mm (0.012&quot;)</td></tr><tr><td>Microscope Slides - Precleaned/Plain</td><td>Fisher Scientific</td><td>12550A3</td><td>Dimentions: 25 mm x 75 mm x 1.0 mm</td></tr><tr><td>Fisherbrand Cover Glasses - Rectangles no. 1</td><td>Fisher Scientific</td><td>12-545E</td><td>Thickness: 0.13 to 0.17 mm; Size: 50 mm x 22 mm</td></tr><tr><td>Fisher Chemical Permount Mounting Medium</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>Leica Stereo dissecting microscope</td><td>Leica Microsystems</td><td></td><td>The microsope is equipped with Leica microscope camera Model MC170 HD &amp;amp; camera software is Leica App. Suite (LAS E2) Version 3.1.1 [Build: 490]. Microscope parts:&amp;nbsp; LED3000 Spot Light Illumination Model: MEB126, Leica M80 Optic Carrier Model M80, Objective achromat 1.0X, WD=90&amp;nbsp;mm Model: MOB306 &amp;amp; Objective achromat 0.32X, WD=303&amp;nbsp;mm Model: MOB315, Video Objective 0.5X Model: MTU-293</td></tr><tr><td>Hemacytometer</td><td>Assistant Germany&amp;nbsp;</td><td></td><td>0.100 mm Depth - 0.0025 mm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Olympus inverted light microscope</td><td>Olympus Corporation</td><td>CKX41SF</td><td>The microsope is equipped with Lumenera Infinity 1-2&lt;br /&gt;2.0 Megapixel CMOS Color Camera &amp;amp; camera software is Infinity analyze Version 6.5.2</td></tr><tr><td>Laminar flow cabinet 1300 Series A2</td><td>Thermo Scientific&amp;nbsp;</td><td>Model: 1375</td><td>Any laminar flow cabinet for cell culture work will be suitable&amp;nbsp;</td></tr><tr><td>Cell culture incubator</td><td>Thermo Scientific&amp;nbsp;</td><td>Model: 370</td><td>Any cell culture incubator will be suitable - Cells were cultured under humidefied environment, 5% CO&lt;sub&gt;2&lt;/sub&gt;, 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>ImageJ</td><td>NIH</td><td>Version 1.50e</td><td>Minimum version required</td></tr><tr><td>Java Runtime Environment</td><td>Oracle</td><td>Version 1.8.0_66</td><td>Minimum version required</td></tr></tbody></table>

automated quantification and analysis of cell counting procedures using imagej plugins

המכון הלאומי ImageJ של בריאות היא חבילת תוכנת עיבוד תמונה רבת עוצמה, זמין באופן חופשי. ImageJ יש אלגוריתמי ניתוח חלקיקים מקיפים אשר ניתן להשתמש בם ביעילות למנות חלקיקים ביולוגיים שונים. כאשר כותבים מספרים גדולות של דגימות תאים, hemocytometer מציג צוואר בקבוק בכל הנוגע לזמן. כמו כן, ספירת ממברנות מבחני הגירה / פלישה עם דלפק תא תוסף ImageJ, למרות מדויק, הוא עבודה באופן יוצא מן הכלל אינטנסיבי, סובייקטיבי, לשמצה בשל גרימת כאב בפרקי יד. כדי לתת מענה לצורך זה, פיתחנו שני תוספים בתוך ImageJ למשימה הבלעדית של hemocytometer אוטומטיות (או נפח ידוע) ועוד היד נטויה תא הגירה / הפלישה. שני תוספים להסתמך על היכולת לרכוש micrographs באיכות גבוהה עם רקע מינימלי. הם קלים לשימוש אופטימיזציה עבור ספירה וניתוח מהיר של מדגמים גדולים עם כלי ניתוח מובנים כדי לעזור כיול של ספירות. על ידי שילוב של עיקרון הליבהים של מונה סלולארי עם ניתוח אלגוריתם ספירה אוטומטי שלאחר ספירה, זה מגדיל מאוד את הקלות שבה מבחני הגירה יכולים להיות מעובד ללא כל אובדן של דיוק.

מחשבון ריכוז התא איור 1 מציג את התהליך הכולל של כיול CCC ורכישת תמונת ספיר. איור 1 א ו -1 B לתאר את תמונת כיול מרובע P וחישוב אורך P-מרובע בפיקסלים. CCC קובע ריכוז התאים בנפח נת...

Watch this Scientific Journal Video about Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins at JoVE.com

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

כימותים אוטומטיים וניתוח הליכי ספירה הניידת באמצעות תוספי ImageJ

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

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Research

JoVE Journal

Biology

46.7K Views.  York University. This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

Video: Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

A Method of Targeted Cell Isolation via Glass Surface Functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

שיטה של ​​בידוד תא ממוקד באמצעות Functionalization משטח הזכוכית

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

Cancer Research

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

ויזואליזציה שיפור ניתוח כמותי של השפעות התרופה באמצעות תאים micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Bioengineering

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

אוטומטי כימות של תא המטופואטיות - אינטראקציות תא סטרומה בתמונות היסטולוגית של עצם Undecalcified

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Developmental Biology

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

Immunofluorescence כמותי מודדים הגלובלי לשפות אחרות תרגום

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemistry

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

ניטור הצטברות חלבונים קינטיקה ב Vivo באמצעות ספירת הכללה אוטומטית ב Elegans Caenorhabditis

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

מדידת כמותית של Proteolysis מטריקס invadopodia בתיווך בקשרים יחידים ורבים תאיים

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Area-based Image Analysis Algorithm for Quantification of Macrophage-fibroblast Cocultures

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either fluorescence detection approaches, such as immunofluorescent staining or transfection with fluorescent proteins or edge detection techniques, which are often error-prone due to noise and other non-idealities in the image background. 
We designed a new algorithm that could accurately count and distinguish macrophages and fibroblasts, cells of different phenotypes that often colocalize during tissue regeneration. MATLAB was used to implement the algorithm, which differentiated distinct cell types based on differences in height from the background. A primary algorithm was developed using an area-based method to account for variations in cell size/structure and high-density seeding conditions. 
Non-idealities in cell structures were accounted for with a secondary, iterative algorithm utilizing internal parameters such as cell coverage computed using experimental data for a given cell type. Finally, an analysis of coculture environments was carried out using an isolation algorithm in which various cell types were selectively excluded based on the evaluation of relative height differences within the image. This approach was found to accurately count cells within a 5% error margin for monocultured cells and within a 10% error margin for cocultured cells.

We present a method, which utilizes a generalizable area-based image analysis approach to identify cell counts. Analysis of different cell populations exploited the significant cell height and structure differences between distinct cell types within an adaptive algorithm.

אלגוריתם ניתוח תמונה מבוסס אזור לכימות של קוקולטורות מקרופאגים-פיברובלסטים

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

לימוד חלבון מערכת בקרת איכות של<em&gt; D. discoideum</em&gt; שימוש הדמית תא חי בקרת טמפרטורה

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

קוונפיקציה של אזור הדבקה בתשתית התא והפצות צורות התא ב-MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

הדמיה וניתוח אוטומטיים לכימות של מקרופינומיסומים המסומנים בפלואורסצנטות

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

כימותים אוטומטיים וניתוח הליכי ספירה הניידת באמצעות תוספי ImageJ

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שיטה של בידוד תא ממוקד באמצעות Functionalization משטח הזכוכית

ויזואליזציה שיפור ניתוח כמותי של השפעות התרופה באמצעות תאים micropatterned

אוטומטי כימות של תא המטופואטיות - אינטראקציות תא סטרומה בתמונות היסטולוגית של עצם Undecalcified

Immunofluorescence כמותי מודדים הגלובלי לשפות אחרות תרגום

ניטור הצטברות חלבונים קינטיקה ב Vivo באמצעות ספירת הכללה אוטומטית ב Elegans Caenorhabditis

מדידת כמותית של Proteolysis מטריקס invadopodia בתיווך בקשרים יחידים ורבים תאיים

אלגוריתם ניתוח תמונה מבוסס אזור לכימות של קוקולטורות מקרופאגים-פיברובלסטים

לימוד חלבון מערכת בקרת איכות של

קוונפיקציה של אזור הדבקה בתשתית התא והפצות צורות התא ב-MCF7 Cell Monolayers

הדמיה וניתוח אוטומטיים לכימות של מקרופינומיסומים המסומנים בפלואורסצנטות

כימותים אוטומטיים וניתוח הליכי ספירה הניידת באמצעות תוספי ImageJ

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Chapters in this video

שיטה של ​​בידוד תא ממוקד באמצעות Functionalization משטח הזכוכית

ויזואליזציה שיפור ניתוח כמותי של השפעות התרופה באמצעות תאים micropatterned

אוטומטי כימות של תא המטופואטיות - אינטראקציות תא סטרומה בתמונות היסטולוגית של עצם Undecalcified

Immunofluorescence כמותי מודדים הגלובלי לשפות אחרות תרגום

ניטור הצטברות חלבונים קינטיקה ב Vivo באמצעות ספירת הכללה אוטומטית ב Elegans Caenorhabditis

מדידת כמותית של Proteolysis מטריקס invadopodia בתיווך בקשרים יחידים ורבים תאיים

אלגוריתם ניתוח תמונה מבוסס אזור לכימות של קוקולטורות מקרופאגים-פיברובלסטים

לימוד חלבון מערכת בקרת איכות של

קוונפיקציה של אזור הדבקה בתשתית התא והפצות צורות התא ב-MCF7 Cell Monolayers

הדמיה וניתוח אוטומטיים לכימות של מקרופינומיסומים המסומנים בפלואורסצנטות

שיטה של בידוד תא ממוקד באמצעות Functionalization משטח הזכוכית