מחקר זה נתמך על ידי מענק מNIH: R21AG042066. ברצוננו להודות לפיליפ שולץ על תרומתו ביצירת קטעי וידאו לכידת מסך.

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The authors have nothing to disclose.

Protein variants have been shown to be involved in the progression of many neurodegenerative diseases such as Alzheimer&#8217;s, Parkinson&#8217;s, ALS and FTD1,2,3,4,5,6,7,8,9,10,11. Isolation of antibodies that can selectively recognize these different protein variant targets can be effective reagents to study, diagnose and potentially treat such ailments. To generate such variant specific antibodies we have developed a novel biopanning process that utilizes atomic force microscopy to monitor the progress ...

הנוכחות של גרסאות חלבון הייתה מעורבת כגורם בהתפתחות של מחלות רבות, כולל מחלות ניווניות כגון אלצהיימר, פרקינסון, ALS ופרונטוטמפורל דמנציה (FTD) 1,2,3,4,5,6,7,8,9 , 10,11. צורות Oligomeric של בטא-עמילואיד החלבונים ואלפא-סינוקלאין נחשבות למינים הרעילים האחראים לאלצהיימר ופרקינסון, בהתאמה 2,3,4,5. מצרפים של חלבון (43 TDP-43) TAR DNA מחייבים נקשרו לALS וFTD 12,13,14. לכן ריאגנטים כמו נוגדנים שיכולים באופן סלקטיבי למקד את גרסאות חלבון השונות יכולות להיות כלי רב עוצמה כדי לשמש כסמנים לאבחון וכתרופות פוטנציאליות. במחקר זה, אנו מתמקדים בפיתוח חומרים כימיים שסלקטיבי לאגד גרסאות של חלבון TDP-43 המעורבים בALS, אך הטכניקה שתוארה במאמר זה צריכה להיות ישימה לבידוד של חומרים כימיים נגד מגוון רחב של protein גרסאות. צבירת cytoplasmic של TDP-43 זוהתה כתכונה פתולוגית בALS 15,16,17,18,19. בדרך כלל TDP-43 נמצאים בגרעין של כל תאי מאדם נורמלי, למרות שהיא נוטה לעבור בין cytosol והגרעין 15,17. צורות עם זאת, בALS מצטברים של TDP-43 מזוהות בציטופלסמה של תאי עצב בחר וגליה עם ריכוזים נמוכים יותר נמצאו בגרעין טוען התנועה של TDP-43 מהגרעין אל הציטופלסמה במהלך התקדמות מחלה 16,20. בעוד צבירה של TDP-43 נמצאת ברוב המכריע של המקרים ALS, זה אינו לוקח בחשבון את כל המקרים מאז 1% -2% מהמקרים כולל ALS (או 15% -20% מהמקרים ALS משפחתיים) צמודים למוטציות ב סופראוקסיד דיסמוטאז 1 15,17 גן (SOD1). בגלל התפקיד החשוב של TDP-43 ברוב המכריע של המקרים ALS, כאן אנו מתמקדים בפיתוח חומרים כימיים נוגדנים מבוססים שיכול באופן סלקטיבי להיקשר לTDP-43 וריאנטים שקיים ברקמת המוח אנושית ALS תוך שימוש בטכניקות biopanning מבוסס רומן AFM שלנו. בתחילה אנו זקוקים לרפרטואר מגוון של תחומים נוגדן מחייב. אנחנו שילבנו שלוש ספריות בר שונה תצוגת הפאג שרשרת אחת תחום משתנה נוגדן (scFvs), (טומלינסון אני וJ וספריות גיליונות 21). תהליך צילום פנורמי מחולק לשלבי צילום פנורמי שליליים וחיוביים. פאגים מהספריות כפופים ראשון לתהליך צילום פנורמי השלילי שבמהלכו פאגים תגובה לאנטיגנים מחוץ יעד מרובים אינם נכללים. לאחר השלמת כל סיבוב של צילום פנורמי שלילי נגד כל אנטיגן מחוץ היעד, התהליך מנוטר על ידי ההדמיה AFM כדי להבטיח שכל הפאג מחייב אנטיגנים מחוץ היעד הוסר. רק אחרי שאימתי על ידי ההדמיה AFM שכל פאגים התגובה יוסרו אנחנו להמשיך ליעד הבא. כדי לבודד ריאגנטים נגד TDP-43 גרסאות מעורבות בALS אנו מנוצלים אנטיגנים פנורמי השליליים הבאים:<html> 1) BSA כדי להסיר הפאג אשר נקלט חלש או שאינם ספציפי לחלבונים; 2) אלפא-סינוקלאין מצטבר להסיר הפאג אשר נקלט על ידי אלמנטים מבניים הגנרית של חלבונים מצטברים; 3) homogenates רקמת המוח האנושי כדי להסיר הפאג אשר נקלט על ידי כל חלבונים או רכיבים אחרים הנמצאים בדגימות שלאחר המוות של רקמת מוח אדם בריאה; 4) immunoprecipitated TDP-43 ממוח האנושי בריא כדי להסיר הפאג אשר נקלט על ידי כל TDP-43 טפסים הקשורים למוח אנושי בריא; ו -5) immunoprecipitated TDP-43 מבודד מhomogenates מוח FTD להסיר הפאג אשר נקלט TDP-43 וריאנטים הקשורים לפתולוגיה שאינו ALS. לאחר הסרת כל הפאג התגובה לכל אנטיגנים מחוץ היעד, אנחנו לאחר מכן המשכנו את שלב צילום פנורמי החיובי שבמהלכו שברי נוגדנים שקושרים את האנטיגן של עניין מבודדים, במקרה זה TDP-43 immunoprecipitated מרקמת המוח אנושי ALS. נוגדנים מבודדים אלה עשויים להיות תגובתי לצורות מצטברות או שונה של TDP-43. &quot;&gt; Biopanning הפאג הקונבנציונלי מתמקד בעיקר בשלב צילום פנורמי החיובי 22,23. בדרך כלל המטרה של עניין היא משותקת, ספריית הפאג הוסיפה ופאגים כבול eluted. אז פאגים הם מוגברים והוסיפו ליעד שוב. תהליך ההגברה והדגירה בדרך כלל חוזרים על עצם מספר פעמים כדי להגדיל את האחוז של הפאג מחייב חיובי. בעוד וריאציות של תהליך זה נעשו שימוש נרחב לבודד את ריאגנטים נוגדנים נגד מגוון רחב של אנטיגנים יעד, הם בדרך כלל דורש כמויות גדולות של אנטיגן היעד המטוהר 24,25,26, 27, ואילו התהליך שלנו דורש רק כמויות זעירות של האנטיגן היעד. הפרוטוקול המתואר כאן יכול לשמש כדי לבודד ריאגנטים שאנטיגנים באופן סלקטיבי לאגד יעד שנמצאים בריכוזים נמוכים מאוד, ללא הצורך בטיהור והצילום פנורמי יכולים להתבצע ישירות נגד הווה אנטיגן בדגימות רקמה מורכבות. השימוש בפרוטוקולי צילום פנורמי שליליים ממצים כפי שאומתעל ידי AFM מבטיח כי שיבוטים המבודדים נגד אנטיגן החיובי צריכים סלקטיבי יחייבו את היעד גם כאשר לא מטוהרים או מועשר. Kasturirangan ועמיתיו (2003) ביצעו תהליך biopanning שלילי וחיובי דומה לבודד נוגדנים מגיבים לביתא-עמילואיד oligomeric באמצעות ריכוז ננוגרם של היעד 5. כאן אנו להרחיב על תהליך זה כדי לאפשר את הדור של חומרים כימיים שסלקטיבי לקשור חלבון ספציפי למחלה גרסאות ישירות מדגימות רקמה אנושיות. במחקרים עתידיים בכוונתנו להמשיך לחקור לא רק את הערך האבחנתי של ריאגנטים המבודדים כאן, אלא גם להעריך את הרלוונטיות הטיפוליות שלהם לטיפול ב- ALS. בסך הכל, הטכנולוגיה הייחודית שלנו לAFM מבוססת biopanning צריכה להיות ישימה לבידודה של כל חלופת חלבון ספציפית למחלה בכל חומר ביולוגי ללא הצורך בטיהור חלבון או שינוי, גם כאשר concentratio אנטיגן היעדns הם נמוכים מאוד.

<table border="0" cellpadding="0" cellspacing="0" width="940">
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			<td height="21" style="height:21px;width:323px;">Name of Reagent/ Equipment</td>
			<td style="width:287px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:212px;">Comments/Description</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:323px;">Tomlinson I and J Libraries</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sheets Library</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2xYT</td>
			<td style="width:287px;">BD Sciences</td>
			<td style="width:119px;">244020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Glucose</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0188-2.5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Ampicillin</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0339-25G</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">KM13 Helper Phage</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Kanamycin</td>
			<td style="width:287px;">OmniPur</td>
			<td style="width:119px;">5880</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Polyethylene Glycol 8000</td>
			<td style="width:287px;">OmniPur</td>
			<td style="width:119px;">6510</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium Chloride</td>
			<td style="width:287px;">Macron</td>
			<td style="width:119px;">7647-14-5</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium Phosphate Dibasic</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0404-1KG</td>
			<td>Irritant</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Potassium Chloride</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;">PX1405-1</td>
			<td>Irritant</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Potassium Phosphate Monobasic</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0781-500G</td>
			<td>Irritant</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">TG1 Cells</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Luria-Bertani Agar</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;">1.10283.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Bovine Serum Albumin</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0332-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">STEN buffer</td>
			<td style="width:287px;">Crystalgen Inc.</td>
			<td style="width:119px;">33429775</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Immunotubes</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">470319</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Mica</td>
			<td style="width:287px;">Spruce Pine Mica</td>
			<td style="width:119px;">24365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Tween 20</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Trypsin</td>
			<td style="width:287px;">Sigma</td>
			<td style="width:119px;">T-0303</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethylamine</td>
			<td style="width:287px;">Sigma</td>
			<td style="width:119px;">T-0886</td>
			<td>Flammable</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Glycerol</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0854-1L</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DNA Plasmid Prep Kit</td>
			<td style="width:287px;">qiagen</td>
			<td style="width:119px;">27106</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Non-Fat Milk Powder</td>
			<td style="width:287px;">Carnation</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">96-Well High Binding ELISA Plate</td>
			<td style="width:287px;">Costar</td>
			<td style="width:119px;">3590</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-M13 HRP</td>
			<td style="width:287px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">27-9421-01</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:323px;">ELISA Femto Chemiluminescence Substrate Kit</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">37074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-TDP 43 Polyclonal Antibody</td>
			<td style="width:287px;">ProteinTech</td>
			<td style="width:119px;">10782-2-AP</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">A/G Agarose Beads</td>
			<td style="width:287px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-2003</td>
			<td></td>
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			<td height="21" style="height:21px;width:323px;">HB 2151 Cells</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Isopropylthiogalactoside</td>
			<td style="width:287px;">Teknova</td>
			<td style="width:119px;">13325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">9e10 HRP</td>
			<td style="width:287px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Nitrocellulose Membrane</td>
			<td style="width:287px;">Biorad</td>
			<td style="width:119px;">162-0115</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Centrifuge</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">Sorvall RC 6+</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanoscope IIIa Atomic Force Microscope</td>
			<td style="width:287px;">Veeco</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AFM Probes</td>
			<td style="width:287px;">VistaProbes</td>
			<td style="width:119px;">T300R-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

novel atomic force microscopy based biopanning for isolation of morphology specific reagents against tdp-43 variants in amyotrophic lateral sclerosis

בגלל גרסאות חלבון תפקיד קריטי במחלות רבות, כולל TDP-43 בטרשת לרוחב amyotrophic (ALS), אלפא-סינוקלאין במחלה וביתא-עמילואיד וטאו במחלת אלצהיימר, זה חשוב באופן קריטי לפיתוח חומרים כימיים מסוימים מורפולוגיה שיכולה באופן סלקטיבי יעד פרקינסון חלבון ספציפי למחלה אלה גרסאות כדי ללמוד את התפקיד של גרסאות אלה בפתולוגיה מחלה ועבור יישומים אבחוניים וטיפוליים פוטנציאליים. פיתחנו טכניקות חדשניות במיקרוסקופ כוח אטומי (AFM) המבוססות biopanning המאפשרות בידוד של חומרים כימיים שסלקטיבי להכיר גרסאות חלבון ספציפית למחלה. ישנם שני שלבים עיקריים מעורבים בתהליך, שלבי צילום פנורמי השליליים וחיוביים. בשלב צילום פנורמי השלילי, פאגים שמגיבים לאנטיגנים מחוץ היעד בוטלו באמצעות סבבים רבים של צילום פנורמי תוסף ניצול סדרה נבחרו בקפידה אנטיגנים מחוץ היעד. תכונה מרכזית בשלילהשלב פנורמי הוא ניצול ההדמיה AFM כדי לפקח על התהליך ולוודא שכל חלקיקי phage לא רצויים יוסרו. בשלב צילום פנורמי החיובי, אנטיגן היעד של ריבית קבועה על משטח נציץ ופאגים מחויבים הם eluted והוקרנו לזהות פאגים שסלקטיבי לקשור את האנטיגן היעד. גרסת חלבון המטרה לא צריכה להיות מטוהרת מספקת בקרות צילום פנורמי השליליות המתאימות כבר בשימוש. אף למקד את גרסאות חלבון שנמצאים רק בריכוזים נמוכים מאוד בחומר ביולוגי מורכב יכול להיות מנוצל בצעד החיובי צילום פנורמי. באמצעות יישום של טכנולוגיה זו, שרכשנו את הנוגדנים לחלבון גרסאות של TDP-43 הנמצאות באופן סלקטיבי ברקמת המוח אנושית ALS. אנו צופים כי פרוטוקול זה צריך להיות ישים ליצירת חומרים כימיים שסלקטיבי לאגד גרסאות חלבון קיימים במגוון רחב של תהליכים ביולוגיים שונים ומחלות.

באיור 1, סכמטי מדגים את תהליך צילום פנורמי השלילי שבו אנו הוסרו הפאג מחייבים אנטיגנים מחוץ יעד מהספרייה שלנו באמצעות immunotubes. אנחנו בתחילה התחלנו עם BSA שכן מדובר בסוכן חסימה נפוץ וכל הפאג שיגיב nonspecifically עם יעד זה יהיה בעייתי בimmunoassays העתיד. בשלב הבא, הסרנו קלסרי?...

Watch this Scientific Journal Video about Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis at JoVE.

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

רומן הכוח האטומי מיקרוסקופית מבוסס Biopanning לבידוד של חומרים כימיים מסוימים מורפולוגיה נגד גרסאות TDP-43 בטרשת לרוחב amyotrophic

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

novel-atomic-force-microscopy-based-biopanning-for-isolation

Research

JoVE Journal

Bioengineering

8.7K Views.  Arizona State University. Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

Video: Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Immobilization חיידקית עבור הדמיה על ידי מיקרוסקופית כוח אטומי

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

ויזואליזציה של דנ&quot;א רקומביננטי ו קומפלקסים חלבונים באמצעות מיקרוסקופ כוח אטומי

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

כימות תכונות המכאניות של Glycocalyx האנדותל עם מיקרוסקופי כוח אטומי

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

מדידת התכונות המכאניות של תאי חיים באמצעות מיקרוסקופ כוח אטומי

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

שיטה חדשה ללוקליזציה חרוזי ניאון כתב סמוך לפני שטח תרבית תאים לגרירה מיקרוסקופית כוח

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

כוח האטומי מיקרוסקופית הדמיה וחיל ספקטרוסקופיה של bilayers שומנים הנתמכים

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

באתרו מיקרוסקופיה לקביעת בזמן אמת של מורפולוגיה תא חד בBioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

יישום של מיקרוסקופ כוח אטומי כדי לזהות אוסטיאוארתריטיס מוקדם

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

אוטומציה של מדידות מיקרוסקופ כוח ביו-אטומי על מאות תאי C. אלביקנס

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

ליד סריקת לייזר בו זמנית מיקרוסקופיה קונפוקל וכוח אטומי (Conpokal) על תאים חיים

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...