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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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry.">Flow cytometry.</a> Animal Cell Culture. , 543-558 (1990).</li><li>Eliceiri, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Imaging+Software+Tools.">Biological Imaging Software Tools.</a> Nat. Methods. 9 (7), 697-710 (2013).</li><li>Louis, K. S., Siegel, A. C., Stoddart, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+viability+analysis+using+Trypan+blue:+manual+and+automated+methods.">Cell viability analysis using Trypan blue: manual and automated methods.</a> Mammalian Cell Viability: Methods and Protocols. , 7-12 (2011).</li><li>Scott, W. N., Langdon, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+and+motility+assays.">Invasion and motility assays.</a> Cancer Cell Culture: Methods and Protocols. , 225-229 (2004).</li><li>Justus, C. R., Leffler, N., Ruiz-Echevarria, M., Yang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Cell+Migration+and+Invasion+Assays.">In vitro Cell Migration and Invasion Assays.</a> J. Vis. Exp. (88), e51046 (2014).</li><li>Luo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-378a-5p+promotes+trophoblast+cell+survival,+migration+and+invasion+by+targeting+Nodal.">MicroRNA-378a-5p promotes trophoblast cell survival, migration and invasion by targeting Nodal.</a> 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 border="0" cellpadding="0" cellspacing="0" width="1175">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">HyClone Classical Liquid Media: 
			RPMI 1640 - With L-Glutamine</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">SH3002702</td>
			<td style="width:692px;">Cell culturing media&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">Fetal bovian serum (FBS)</td>
			<td style="width:124px;">GIBCO BRL</td>
			<td style="width:119px;">P00015</td>
			<td>Media suppliment</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:107px;width:240px;">HTR8/SVneo trophoblast cell line</td>
			<td style="width:124px;">Cells were obtained from Dr. Charles Graham (Queen&#8217;s University, Kingston, Canada)</td>
			<td style="width:119px;"></td>
			<td>Software is designed to work with any cell line.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">Trypsin</td>
			<td style="width:124px;">GIBCO BRL</td>
			<td style="width:119px;">27250-018</td>
			<td>Prepared as 0.20% (w/v) in 10uM EDTA&#160; 1X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:240px;">Accutase</td>
			<td style="width:124px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT104</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">10 cm cell culture plates</td>
			<td style="width:124px;">SARSTEDT</td>
			<td style="width:119px;">833902</td>
			<td>Any tissue culture treated plates will be suitable</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">Transwell Polyester Membrane Inserts - 8.0&#181;m Pore size</td>
			<td style="width:124px;">Costar 3422 ordered from Fisher Scientific</td>
			<td style="width:119px;">7200150</td>
			<td>For 24-well plates; Pore size: 8.0&#181;m; 6.5mm diameter; 0.33cm2 growth area</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">HARLECO Hematology Stains and Reagents, EMD Millipore - Soluntions 1, 2 &#38; 3</td>
			<td style="width:124px;">EMD Millipore and ordered from VWR</td>
			<td style="width:119px;">65044A, B, &#38; C</td>
			<td>Hemacolor stain set consists of three 500mL (16.9oz.) poly bottles &#38; 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 height="60">
			<td height="60" style="height:60px;width:240px;">Cotton Tipped Applicator</td>
			<td style="width:124px;">Puritan Medical</td>
			<td style="width:119px;">806-WC</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Single-edge industrial razor blades</td>
			<td style="width:124px;">VWR</td>
			<td style="width:119px;">55411 - 055</td>
			<td>Thickness: 0.30 mm (0.012&#34;)</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">Microscope Slides - Precleaned/Plain</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">12550A3</td>
			<td>Dimentions: 25 X 75 X 1.0 mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">Fisherbrand Cover Glasses - Rectangles no. 1</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">12-545E</td>
			<td>Thickness: 0.13 to 0.17mm; Size: 50 x 22mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">Fisher Chemical Permount Mounting Medium</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">SP15-500</td>
			<td></td>
		</tr>
		<tr height="124">
			<td height="124" style="height:124px;width:240px;">Leica Stereo dissecting microscope</td>
			<td style="width:124px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td style="width:692px;">The microsope is equipped with Leica microscope camera Model MC170 HD &#38; camera software is Leica App. Suite (LAS E2) Version 3.1.1 [Build: 490]. Microscope parts:&#160; LED3000 Spot Light Illumination Model: MEB126, Leica M80 Optic Carrier Model M80, Objective achromat 1.0X, WD=90mm Model: MOB306 &#38; Objective achromat 0.32X, WD=303mm Model: MOB315, Video Objective 0.5X Model: MTU-293,&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">Hemacytometer</td>
			<td style="width:124px;">Assistant Germany&#160;</td>
			<td style="width:119px;"></td>
			<td>0.100 mm Depth - 0.0025 mm2</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">Olympus inverted light microscope</td>
			<td style="width:124px;">Olympus Corporation</td>
			<td style="width:119px;">CKX41SF</td>
			<td>The microsope is equipped with Lumenera Infinity 1-2&#160;&#160; 2.0 Megapixel CMOS Color Camera &#38; camera software is Infinity analyze Version 6.5.2</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">Laminar flow cabinet 1300 Series A2</td>
			<td style="width:124px;">Thermo Scientific&#160;</td>
			<td style="width:119px;">Model: 1375</td>
			<td>Any laminar flow cabinet for cell culture work will be suitable&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:240px;">Cell culture incubator</td>
			<td style="width:124px;">Thermo Scientific&#160;</td>
			<td style="width:119px;">Model: 370</td>
			<td>Any cell culture incubator will be suitable - Cells were cultured under humidefied environment, 5% CO2, 37 &#176;C&#160;</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;width:240px;">ImageJ</td>
			<td style="width:124px;">NIH</td>
			<td>Version 1.50e</td>
			<td>Minimum version required</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:240px;">Java Runtime Environment</td>
			<td style="width:124px;">Oracle</td>
			<td style="width:119px;">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 ...

automated-quantification-analysis-cell-counting-procedures-using

Research

JoVE Journal

Biology

46.6K 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

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

ספירת ועל קביעת הכדאיות של תאים בתרבית

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

חצי אוטומטיות Heartbeat ניתוח אופטי של לבבות קטנים

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

פיתוח הדמיה ניתוח אוטומטיות עבור מסכי דג הזברה כימיים.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

קביעת ספירת תאים יונקים, גודל התא ובריאות נייד באמצעות ת Moxi מיני מונה אוטומטי Cell

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

ניתוח ספציפי של תא<em&gt; ארבידופסיס</em&gt; עלים באמצעות מיון תא הופעל פלואורסצנטי

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools5,6. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS)7. Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices8. To date, leaf shape programs (e.g. SHAPE9, LAMINA10, LeafAnalyzer11, LEAFPROCESSOR12) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size13. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping.

Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.

LeafJ: תוסף ImageJ למדידת צורת עלה חצי אוטומטית

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

שעון סריקה פרוטוקול ניתוח תמונה: Plugins ImageJ

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

מדידת המפגש של iPSCs באמצעות מערכת הדמיה אוטומטית.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

הכנת רשת מיקרוסקופית קריו-אלקטרון למחקרים שנפתרו בזמן באמצעות מערכת רובוטית חדשנית, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

זיהוי וניתוח אוטומטיים של אקסוציאטוזיס

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...