עבודה זו נתמכה על ידי תרומות של האמון Leverhulme, הנדסה המדעים הפיזיקליים מועצת המחקר [EP / H018301 / 1, EP / G037221 / 1]; אלצהיימר Research UK [ערוק-EG2012A-1]; קרן Wellcome [089,703 / Z / 09 / Z] המועצה למחקר רפואי [MR / K015850 / 1, MR / K02292X / 1]. אנו מודים E. Avezov ומ Lu עבור transfection של תאים LifeAct-GFP ו cytosolic-GFP בהתאמה, וו צ&#39;ן להכנת התרבות HEK293. אנו מודים גם ק O&#39;Holleran לסיוע עם העיצוב של המיקרוסקופ, ול &#39;שאו ור Heintzmann לדיונים והצעות שימושיים.

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The authors have nothing to disclose.

<html> שהותקנו מערכות TIRF-SIM כגון ההתקנה המפורטת בפרוטוקול זה מסוגלות הדמיה סופר-רזולוציה ססגונית במהירות גבוהה לעומת מיקרוסקופים זמינים מסחרי. היתרון הגלום של SIM כטכניקת סופר-רזולוציה הוא כי ההחלטה הזמנית אינה מוגבלת על ידי photophysics של fluorophore, בהשוואה לשיטות אחרות כגון מיקרוסקופיה לוקליזציה מולקולה בודדת (SMLM) או שיטות סריקת נקודה כגון מיקרוסקופיה דלדול פליטה מאולצת ( STED). שלא כמו טכניקות אחרות אלה, SIM אינו מחייב fluorophores photoswitchable או מתכלה כך ההדמיה ססגונית היא פשוטה. מערכות TIRF-SIM ללא, כגון אופטי חתך SIM ו- SIM מולטיפוקליות בדרך כלל ניתן להשיג שיפורים ברזולוציה של פי 1.7 או פחות בפועל בניגוד הגורם של שיפור 2 דיווחו כאן, ומערכות מסחריות הן גם בדרך כלל איטי יותר ופחות גמישה מאשר מערכת המובאים בפרוטוקול זה. &quot;&gt; שני הקשיים העיקריים ביישום הטכניקה הזו הם ראשית את הצורך עבור מיקום מדויק של שש קורה SIM בתוך אזור TIR של הצמצם האחורי של המטרה, מחייב הליך יישור אופטי מייגע זמן רב. שנית, לייצר בניגוד דפוס גבוה על מדגם, סיבוב הקיטוב חיוני. עבור מערכות NA 2D-SIM נמוך, סיבוב הקיטוב יכול להימנע על ידי בחירה זהירה של האוריינטציה קיטוב ליניארי, אבל זה הופך להיות בלתי אפשרי עבור TIRF-SIM 25. עבור הדמיה ססגוניות במהירות גבוהה, אלקטרו שליטת קיטוב אופטית יש צורך וזה מגדיל את מורכבות חשבון המערכת. מגבלות הטכניקה TIRF-SIM, כמו TIRF הקונבנציונלי, מוגבל באופן טבעי תצפית של מבנים ביולוגיים ותהליכים הממוקמים בקרום תאי הבסיס שיכול להיות מואר על ידי עומק חדירת 150-200 ננומטר של שדה החלוף. בזמןSIM מצוטט לעתים קרובות להיות פחות photodamaging לתאים מאשר כל אחד STED או SMLM, הכפלת רזולוציה לרוחב עדיין להגדיל את המספר הדרוש של פוטונים על ידי לפחות פי 4 5 לעומת מיקרוסקופיה TIRF קונבנציונאלי. עבור הדמית מסגרת חליפין גבוהים עם זמני חשיפות קצרים, עליית פוטון זה מחייבת שימוש בעוצמות תאורה מוגברת. בעוד כל fluorophore יכול לשמש הדמית SIM של דגימות מרגשות קבועות או איטיים, חלבוני ניאון בהירות גבוהה או צבע סינטטי הדור הבא עם photostability המשופרת מומלצי הדמית תא חי. למרות שיישום זה מסוגל הדמית צבע אחד מסגרת חליפי SIM העולה על 20 הרץ, הדמיה ססגונית במערכת הציגה מוגבלת על ידי זמן המעבר של גלגל מסנן פליטה הממונע. בשל גודלו של שבב המצלמה sCMOS, השימוש של אופטיקה פיצול תמונה מסנן ופליטה multiband יהיה אפשרי ולאפשר אני סימולטניmaging עם אורכי גל מרובים ללא עונש מהירות. אפשרות נוספת תהיה לסירוגין לייזרי העירור השונים ומשתמש מסנן חריץ multiband לדחות את אור העירור. השימוש SLM בינארי ferroelectric ביישום זה גם אינו אופטימלי. יעילות דיפרקציה של SLM כזה הוא נמוך מאוד, ולכן רוב לאור האירוע הוא בהשתקפות אפס הסדר, אשר סוננו על ידי מסכה מרחבית. עבור יישומים הדורשים שיעור גבוה מסגרת מאוד, מהירות הדמיה ולכן היא מוגבלת על ידי הספק של דיודות לייזר. SLM גם מציג כמה סְגַלגַלוּת בקיטוב עבור אורכי גל מן גל עיצוב 550 ננומטר שבו פיקסלים אינם פועלים כמו צלחות גל חצי אידיאליות. אמנם זה יכול להיות מתוגמל על ידי שימוש LCVR נוסף, הפתרון האידיאלי עשוי להיות השימוש בהתקן מייקרו-ראי דיגיטלי (DMD) כמחולל דפוס. שינויים אפשריים Prese ההתקנהnted כאן הוא גמיש ומשתנה בקלות רבה יותר מאשר מכשירים מסחריים כך שיטות הדמיה אחרות כגון 3D-SIM, SIM-2D מהר, SIM מולטיפוקליות (MSIM) ו- SIM שאינו ליניארי (NL-SIM) יכולות להיות מיושמות 21,34,35. 2D-SIM יכול להיות גם מתאים הדמיה שטוח יחסית, זז מהר מבנים כגון reticulum endoplasmic הפריפריה. חדר המיון הפריפריה טמון עמוק בתוך התא מאשר אפשר להאיר באמצעות שדה TIRF חלוף אך בשל המבנה השטוח ניתן הדמיה באמצעות-SIM 2D רגיל עם רקע זניח מחוץ לפוקוס. בנוסף, השימוש של אלגוריתמי שיקום חתך אופטיים משופרים לדכא out-of-פוקוס אור להרחיב את שימוש 2D-SIM כדי דגימות עבות אופטי, אם כי שם הכפלת רזולוציה הצירית אינה נדרשו 21. בשנת MSIM, המדגם הוא מואר על ידי שריג דליל של מוקדי עירור 36. טיפול זה יכול להיות מיושם על ידי הסרת המסכה מרחבית (SM) והחלפתו על ידי מקטב. SLM החברה פועלת כמו אפנן משרעת. בסורגי SIM בינארי מוצגים על SLM יכולים להיות מוחלף על ידי שריג 2D של כתמים, עם הגודל של הכתמים נבחרו להיות שווה לגודל של יכולת מיקוד עקיף במישור התמונה. באיור 7 א, סריג של 4 x 4 ריבועים פיקסל מוצג על SLM (ההבלעה) אשר כאשר demagnified על המדגם מייצר מוקדים עקיפים מוגבלים של 150 x 150 ננומטר, בהתחשב בגודל פיקסל SLM הפיזי של 13.62 מיקרומטר. מוקדי העירור אז יכולים להיות מתורגמים על ידי העברת דפוס הסריג על SLM וזה חוזר על עצמו מספר פעמים על מנת להאיר את השדה כולו מבט. תמונות נרכשות לכל תפקיד דפוס תורגם בערימה היא שלאחר עיבוד להניב תמונה משוחזרת עם רזולוציה משופרת של עד בפקטור של <img alt="משוואה" src="/files/ftp_upload/53988/53988eq19.jpg" /> והקטן מחוץ מוקד אור לעומת תמונת widefield המקבילה30. טיפול זה יכול להיות שימושי עבור הדמיה עבה, דגימות צפופות עבורו SIM סטנדרטי אינו מתאים עבור מבנים ניגודיות נמוכים למשל כגון תאי דם אדומים מוכתמים (איור 7C), אם כי פעם הרכישה גדלה בשל מספר הרב של מסגרות גלם נדרש לכל שדה הראיה (במקרה זה N = 168). לבסוף, ההתקנה יכולה להיות שונה כדי לאפשר או גבוה NA ליניארי TIRF-SIM או בדוגמת הפעלה שאינו ליניארי SIM (PA NL-SIM), כפי שהוצגה לאחרונה על ידי Li et al., על ידי שימוש אובייקטיבי ultrahigh 1.7 NA או תוספת של לייזר photoactivation 405 ננומטר ואופטימיזציה זהירה של דפוסי צורמת SLM 35. יישומים עתידיים SIM עדיין טכניקה המתפתחת במהירות ויישומים רבים במדעי החיים יופעלו בעתיד. מהירות, רזולוציה, ושיפורים הניגודיות של הטכניקה ואת יכולת השימוש מ fluorophores תקןEAN שעבור bioimaging, מוגדר SIM להחליף מערכות מיקרוסקופ רבות קונבנציונליות, כגון פלטפורמות שדה confocal ורחבות. מערכות SIM מסחריות זמינות כבר היום עם מפרט טכני יוצא מן הכלל, עם זאת, הם מעבר להישג ידם הכספי של מעבדות מחקר רבות, ואת, באופן מכריע, הם מאבדים את הגמישות להיות משונים פותח על מנת ליישם את התפתחויות המחקר העדכני ביותר בתחום. גם אין להם את היכולת החיונית &quot;להיות מותאמת עבור הניסוי בהישג היד&quot;, לעתים קרובות צוואר בקבוק קריטי חיתוך מחקר מדעי חי קצה. המערכת המתוארת כאן יהיה מתאים במיוחד גם ללמוד תהליכים דינמיים ליד פני התא, עבור במבחנה של מערכות bilayer מחדש, ללמוד כימיה של פני השטח של חומרים מדעי הטבע, למשל. חומרי 2D, ויישומים רבים אחרים.

<table border="0" cellpadding="0" cellspacing="0" width="1149">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">488 nm laser</td>
			<td style="width:143px;">Toptica</td>
			<td style="width:181px;">iBeam SMART</td>
			<td style="width:535px;">with digital modulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">561 nm laser</td>
			<td style="width:143px;">Coherent</td>
			<td style="width:181px;">OBIS LS</td>
			<td>with digital modulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">640 nm laser</td>
			<td style="width:143px;">Cobolt</td>
			<td style="width:181px;">MLD</td>
			<td>with digital modulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Long-pass dichroic mirrors&#160;</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;"></td>
			<td>for combining excitation beams</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Quad band dichroic mirror</td>
			<td style="width:143px;">Chroma</td>
			<td style="width:181px;">ZT405/488/561/640rpc</td>
			<td>3 mm thick, TIRF imaging flat, mounted in Olympus BX filter cube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Quad band dichroic mirror</td>
			<td style="width:143px;">Chroma</td>
			<td style="width:181px;">ZT405/488/561/640rpc</td>
			<td>From same batch as above, 25 x 25 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">1&#34; square kinematic mount</td>
			<td style="width:143px;">Edmund Optics</td>
			<td style="width:181px;">58-860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Glan-Taylor calcite polarizers</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">GT5-A</td>
			<td>For alignment of LCVR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Glan-Taylor mount</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">SM05PM5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Achromatic half wave plate</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">AHWP05M-600</td>
			<td>400 - 800 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rotation cage mount</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">CRM1/M</td>
			<td>For HWP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Liquid Crystal Variable Retarder</td>
			<td style="width:143px;">Meadowlark Optics</td>
			<td style="width:181px;">SWIFT</td>
			<td>Custom built to provide full wave retardance over the range 488 to 640 nm.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">LCVR controller</td>
			<td style="width:143px;">Meadowlark Optics</td>
			<td style="width:181px;">D3060HV</td>
			<td>Two channel high voltage controller for liquid crystal retarders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Achromatic quarter wave plate</td>
			<td style="width:143px;">Meadowlark Optics</td>
			<td style="width:181px;">AQM-100-0545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rotation cage mount</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">CRM1P/M</td>
			<td>For QWP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">10 mm achromatic doublet</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">AC080-010-A-ML</td>
			<td>For beam expander</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">200 mm achromatic doublet</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">AC254-200-A-ML</td>
			<td>For beam expander</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Cage XY Translators</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">CXY1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Ferroelectric spatial light modulator</td>
			<td style="width:143px;">Forth Dimension Displays</td>
			<td style="width:181px;">M0787-00249&#160;&#160;&#160;&#160;</td>
			<td>SXGA-3DM (IFF) Microdisplay Type M249, 1280 x 1024 pixels, with driver board</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">SLM mounting frame</td>
			<td style="width:143px;">Forth Dimension Displays</td>
			<td style="width:181px;">M0787-10014</td>
			<td>Fixed to custom built aluminium mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">&#216;50.8 mm Gimbal Mirror Mount</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">GM200/M</td>
			<td>For SLM mounting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Two-Axis Linear Translation Stage with Rotating Platform</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">XYR1/M</td>
			<td>For SLM mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rail carrier</td>
			<td style="width:143px;">Newport</td>
			<td style="width:181px;">M-PRC-3</td>
			<td>For SLM mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Precision Optical Rail</td>
			<td style="width:143px;">Newport</td>
			<td style="width:181px;">PRL-6</td>
			<td>For SLM mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">300 mm achromatic doublet lens</td>
			<td style="width:143px;">Qioptiq</td>
			<td style="width:181px;">G322 273 322&#160;</td>
			<td>f = 300 mm, 31.5 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">140 mm achromatic doublet lens</td>
			<td style="width:143px;">Qioptiq</td>
			<td style="width:181px;">G322 239 322</td>
			<td>f = 140 mm, 31.5 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Precision XY Translation Mounts</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">LM2XY</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Lens Mounting Adapters</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">SM2AD32</td>
			<td>For mounting 31.5 mm lenses in 2&#34; mounts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Translation stages</td>
			<td style="width:143px;">Comar</td>
			<td style="width:181px;">12XT65</td>
			<td>Dovetail, side drive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">XY Translator with Differential Drives</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">ST1XY-D/M</td>
			<td>for spatial filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rotation cage mount</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">CRM1/M</td>
			<td>for spatial filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">300 mm achromatic doublet</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">AC508-300-A-ML</td>
			<td>Excitation tube lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Automated XY stage with Z-piezo top plate</td>
			<td style="width:143px;">ASI</td>
			<td style="width:181px;">PZ-2150-XYFT-PZ-IX71&#160;</td>
			<td>with MS-2000 controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Inverted microscope frame</td>
			<td style="width:143px;">Olympus</td>
			<td style="width:181px;">IX-71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Objective lens</td>
			<td style="width:143px;">Olympus</td>
			<td style="width:181px;">UAPON100XOTIRF</td>
			<td>100X/1.49NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">High speed filter wheel</td>
			<td style="width:143px;">Prior Scientific</td>
			<td style="width:181px;">HF110A</td>
			<td>with Prior ProScan III controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Bandpass emission filters</td>
			<td style="width:143px;">Semrock</td>
			<td style="width:181px;">FF01-525/30, FF01-676/29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">sCMOS camera</td>
			<td style="width:143px;">Hamamatsu</td>
			<td style="width:181px;">ORCA Flash v4.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Stage top incubator</td>
			<td style="width:143px;">OKO Lab</td>
			<td style="width:181px;">H301-K-FRAME</td>
			<td>For live cell imaging, with Bold Line temperature and CO2 controllers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Stainless steel optical posts</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">TR series</td>
			<td>for mounting optical components</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Post holders</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">PH series</td>
			<td>for mounting optical components</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Kinematic mirror mounts</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">KM100</td>
			<td>for mounting 1&#34; mirrors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Shearing interferometer</td>
			<td style="width:143px;">Thorlabs</td>
			<td style="width:181px;">SI100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">100 nm fluorescent microspheres</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:181px;">T-7279</td>
			<td>Tetraspeck</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rhodamine 6G</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:181px;">83697-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">8 well glass bottom dishes</td>
			<td style="width:143px;">ibidi</td>
			<td style="width:181px;">80827</td>
			<td>with #1.5 coverglass</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Nunc Lab-Tek II Chambered Coverglass</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:181px;">155409</td>
			<td>with #1.5 coverglass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">0.01mm microscope reticle slide</td>
			<td style="width:143px;">EMS</td>
			<td style="width:181px;">68039-22</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">CellLight Tubulin-GFP, BacMam 2.0</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:181px;">C10613</td>
			<td></td>
		</tr>
	</tbody>
</table>

a guide to structured illumination tirf microscopy at high speed with multiple colors

הדמיה סופר-רזולוציה אופטית עם מיקרוסקופיה תאורה מובנה (SIM) היא טכנולוגיית מפתח עבור הדמיה של תהליכים ברמה המולקולרית בתחומי הכימיה ומדעי ביו. למרות שמערכות ה- SIM מסחריות זמינות, מערכות שמיועדות מותאם אישית במעבדה יכולה להכות מערכות מסחריות, האחרונות בדרך כלל תוכננו עבור קלות שימוש ויישומים למטרות כלליות, הן מבחינת נאמנות הדמיה ומהירות. מאמר זה מציג מדריך מעמיק בבניית מערכת ה- SIM המשתמשת השתקפות פנימית מוחלטת (TIR) ​​תאורה והוא מסוגל הדמיה במהירות של עד 10 הרץ בשלושה צבעים ברזולוציה לרמה של 100 ננומטר. בשל השילוב של ה- SIM TIRF, המערכת מספקת ניגודיות תמונה טובה יותר מאשר טכנולוגיות מתחרות. כדי להשיג מפרטים אלה, כמה אלמנטים אופטיים משמשים כדי לאפשר בקרה אוטומטית על מבנה מדינת הקיטוב המרחבי של אור התאורה לכל wav העירור הזמיןelengths. פרטים נוספים על יישום חומרה ובקרה ניתנים להשיג סנכרון בין דור דפוס אור העירור, אורך גל, מדינת קיטוב, ושליטת מצלמה תוך שימת דגש על השגת קצב פריימי רכישה מרבית. צעד אחר צעד פרוטוקול ליישור מערכת כיול מוצג והשיפור ברזולוצית השגת מקבלת תוקף על דגימות מבחן אידיאליות. יכולת הדמיה סופר-רזולוציה וידאו שער מודגם עם תאים חיים.

Watch this Scientific Journal Video about A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors at JoVE.com

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

A Guide to מיקרוסקופית TIRF תאורה מובנית ב- High Speed ​​עם צבעים מרובים

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

a-guide-to-structured-illumination-tirf-microscopy-at-high-speed-with

Research

JoVE Journal

Bioengineering

25.0K Views.  University of Cambridge. This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

Video: A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

זהב Nanostar סינתזה עם שיטת Seed כסף מתווכת צמיחה

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

קצב העברת נתונים גבוה תא יחיד ו מרובה תאים מיקרו אנקפסולציה

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Lensfree על שבב טומוגרפית מיקרוסקופית העסקת רב זווית תאורה פיקסל סופר רזולוציה

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.

The bottleneck for cellular 3D electron microscopy is feature extraction (segmentation) in highly complex 3D density maps. We have developed a set of criteria, which provides guidance regarding which segmentation approach (manual, semi-automated, or automated) is best suited for different data types, thus providing a starting point for effective segmentation.

מvoxels לידע: מדריך מעשי לפילוח אלקטרונים מורכבים מיקרוסקופית 3D-Data

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, ...

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 ...

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a non-contact measurement method that can be used to obtain direct readouts of viscoelastic material properties. It has been a useful tool in material characterization, structural monitoring and environmental sensing. In the past, Brillouin spectroscopy has usually employed scanning Fabry-Perot etalons to perform spectral analysis. This process requires high illumination power and long acquisition times, making the technique unsuitable for biomedical applications. A recently introduced novel spectrometer overcomes this challenge by employing two VIPAs in a cross-axis configuration. This innovation enables sub-Gigahertz (GHz) resolution spectral analysis with sub-second acquisition time and illumination power within the safety limits of biological tissue. The multiple new applications facilitated by this improvement are currently being explored in biological research and clinical application.

Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo.

ספקטרומטר תת-GHz מהירות גבוהה לניתוח פיזור רילואן

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

באמצעות Tomoauto: פרוטוקול לתפוקה גבוהה אוטומטית קריו אלקטרון טומוגרפיה

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

לכימות מיקרואורגניזמים בריכוזים נמוכים באמצעות מיקרוסקופ דיגיטלי הולוגרפי (סוג התושבות שלהם. מופיעים)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

ניצוח מספר מצבי הדמיה עם מיקרוסקופ זריחה אחת

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

יישום של מיקרוסקופ רפלקציה הפרעות עבור הדמיה ללא תווית, במהירות גבוהה של Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...

A Guide to מיקרוסקופית TIRF תאורה מובנית ב- High Speed ​​עם צבעים מרובים

A Guide to מיקרוסקופית TIRF תאורה מובנית ב- High Speed עם צבעים מרובים