The financial support by DFG (German research foundation, project CZ 55/30-1) for parts of this work is gratefully acknowledged.

1. רכבת ההתקנה הניסיונית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנתי בצד הפרוקסימלי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים ולתקן את לייזר מתן קרן קרני אור מקבילות - או להשתמש בלייזר בשילוב סיבים עם אופטיקה collimation על היבט היציאה של סיבים. </li><li style=";text-align:right;direction:rtl"> שים ספליטר הקורה מקטב (PBS) לפצל את קרן הלייזר לתוך הפניה ו קרן האובייקט. סובבו את הכיוון של צלחות גל חצי (HWP) על ידי החלפה של HWP ברוטציה שלה הר עד כוח של קרן הייחוס קרן האובייקט (בצד הדיסטלית) הוא בערך אותו הדבר. בדוק זאת על ידי הצבת מסך לשני התייחסות קרן האובייקט. בחר את הכיוון של PBS כך הקיטוב של קרן ההתייחסות מתאים מאפנן אור מרחבי רגיש קיטוב (SLM). </li><li style=";text-align:right;direction:rtl"> שים מפצל קרן (BS) לתוך קרן ההתייחסות לפצל את קורה ההתייחסות לשתי קורות. מניחים את מאפננים אופטיים (OM) כגון ששני אלה קורות המגיעים BS1 יכול לעבור OM1 ו OM2, בהתאמה. &lt;/ Li&gt; <li style=";text-align:right;direction:rtl"> מערבבים את שתי אלומות עובר OM1 ו OM2 ב BS2 העסקת שתי מראות. התאם את מפצלי הקרן ומראה כך קורות שניהם מופרדים מרחבית. </li><li style=";text-align:right;direction:rtl"> בזהירות ליישר BS5 כדי להבטיח שהכיוון של שכיחות הן קורות הוא בניצב למישור פיקסל של SLM, התעלמות BS3 ו BS4 בהתחלה. בהתחלה, לא מוצג דבר על SLM, כלומר, היא פועלת כמו מראה עד סוף הכיול (בכל שלב 2). </li><li style=";text-align:right;direction:rtl"> להתאים את המיקום ואת המרחק בין שתי עדשות (L) המהווים טלסקופ קפלרי על מנת לקבל תמונה חדה של מטוס SLM על metal-oxide semiconductor משלימה המצלמה (CMOS). צפה הכיוון הנכון של L1 ו- L2 (צדדים שטוחים הם פונים זה לזה) כדי למזער סטיות. </li></ol></li><li style=";text-align:right;direction:rtl"> הכנתי בצד הדיסטלי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש BS7 לפצל את קרן האובייקט לשתי קורות ולשלב אותם BS8 העסיק שתי מראות. שוב, להתאים את מפצלי קרן ותערובותrrors כך קורות שניהם מופרדים מרחבית. </li><li style=";text-align:right;direction:rtl"> להסיט הוא קורה באמצעות BS9 לכוון אותם אל מטרת מיקרוסקופ (OBJ). פוקוס OBJ2 על הקצה הדיסטלי של סיבי multimode (MMF). בדוק את המיקוד על ידי התבוננות בהשתקפות בחזרה מן MMF העסקת L3 ומצלמה תשלום מצמידים מכשיר (CCD). </li></ol></li><li style=";text-align:right;direction:rtl"> הפרוקסימלי חיבור בצד הדיסטלי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Collimate האור מן הקורה אובייקט היציאה OBJ1 העסקת MMF. </li><li style=";text-align:right;direction:rtl"> פיצול קרן אובייקט באמצעות BS6, להתעלם מקטב ליניארי (LP) בהתחלה. שלב שני קורות האובייקט בשתי קורות ההתייחסות ב BS3 ו BS4 העסיק במראה. התאם את מפצלי הקרן ומראה כך שכל זוג של התייחסות חפיפת קרן עצם כלשהו על SLM, מצטלב עם זווית קטנה (פחות מ -1 °). </li><li style=";text-align:right;direction:rtl"> ודאו שאספקת המתח של ההפניה ואת קרן האובייקט הם שווה בערך ידי סיבוב האוריינטציה של HWP, בהתאם לשלב 1.1.2. </li><li style=";text-align:right;direction:rtl"> בדוק את ההפרעה pattern (מחוץ ציר הולוגרמה) אל מצלמת CMOS ולהתאים את זווית החיתוך בהתאם. הגדל את הזווית, עד ריווח שולי הפרעה בערך שווה לגודל של שני פיקסלים על מצלמת CMOS. </li><li style=";text-align:right;direction:rtl"> לשנות את הכיוון של LP כדי להתאים את הקיטוב של אובייקט קרן התייחסות על מנת לקבל ניגוד מרבי של תבנית ההתאבכות בתמונת מצלמת CMOS, כך שתמונת המצלמה מראה בשולים ברורים. </li></ol></li></ol> 2. כיול המערכת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כיול ביחס פיקסל בין SLM ו- CMOS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להאיר את כולו SLM להשתמש רק באחת קורה ההתייחסות ולחסום את ההתייחסות האחרת ואובייקטים קורים. </li><li style=";text-align:right;direction:rtl"> צילום תמונה של SLM עם מצלמת CMOS. </li><li style=";text-align:right;direction:rtl"> קבלת הקואורדינטות של בפינה השמאלית העליונה של SLM בתמונת מצלמת CMOS, למשל באמצעות תוכנת גרפיקת סמן העכבר על המחשב. השתמש קואורדינטות פיקסל אלה כנקודת המוצאלגבי SLM. </li><li style=";text-align:right;direction:rtl"> הסר את כל קוביות הקורה. </li></ol></li><li style=";text-align:right;direction:rtl"> כיול אות הנתיבים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חסום הן קרן התייחסות קרן 2 ואובייקט 2. </li><li style=";text-align:right;direction:rtl"> צילום תמונה של ההולוגרמה עם מצלמת CMOS. להעריך בשלב ב בהולוגרמה רשמה בשיטת ספקטרום זוויתי 19. לחשב את השלב הפוך באזור המקביל קורה 1. </li><li style=";text-align:right;direction:rtl"> הסר את קוביות קרן לשעבר ועכשיו לחסום את שתי קורות התייחסות קרן 1 ואובייקט 1. </li><li style=";text-align:right;direction:rtl"> צילום תמונה של ההולוגרמה עם מצלמת CMOS. מדוד את השלב ב בהולוגרמה רשמה בשיטת ספקטרום זוויתי שוב. לחשב את השלב הפוך על האזור המקביל קרן 2. </li><li style=";text-align:right;direction:rtl"> הסר את כל קוביות הקורה. </li></ol></li></ol> 3. שידור האותות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חסום את קרן האובייקט. </li><li style=";text-align:right;direction:rtl"> לתפור את התמונות בשלב הפוכות מחושבות לפי האזורים המקבילים של קרן 1 ו -2 יחד ולהציג אתהתמונה כולה על SLM, בדרך כלל באמצעות יציאת גרפיקה ממוחשבת. </li><li style=";text-align:right;direction:rtl"> הפעל את האפנון של אותות קלט 1 ו -2 על ידי הפעלת OM1 ו OM2. </li><li style=";text-align:right;direction:rtl"> שימו לב אותות הפלט 1 ו -2 על מצלמת CCD. </li></ol>

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The authors have nothing to disclose.

להרכבת הגדרת הניסוי (שלב 1 בפרוטוקול) דורשת התאמה יסודית של הרכיבים האופטיים עם כבוד זה לזה. ההיבט החשוב ביותר הוא השכיחות המלבנית של קורות הפניה על SLM כדי להבטיח גבוהה PBR. על מנת לשפר את ההתקנה יותר משני אותות משודרים, מפצלי קרן נוס...

העברת אותות אור מרובים באמצעות סיב multimode (MMF) בא לידי ביטוי קומוניקציות הנדסיות 1 ו Biophotonics 2. בהנדסת תקשורת, ריבוב שטח החלוק (SDM) הוא האמין להיות פתרון בר קיימא על מנת לשפר את יכולת ההולכה של סיבים אופטיים עבור יישומי העברת נתונים עתידיים מרוויחה ניצול גבוה של השטח המוגבל, לעומת סיבים במצב יחיד מרובים 3. בשנת Biophotonics, דגימות ביולוגיות הן מניפולציות על ידי שידור האור דרך אנדוסקופ MMF 4. לדוגמא, השליטה האופטית העצמאית של נוירונים בודדים באמצעות אנדוסקופים MMF היא עניין של optogenetics כדי ללמוד רשתות נוירונים במוח 5. עם זאת, האור מוקרן על היבט קלט MMF כפוף עיוות עקב ערבוב במצב ופיזור במהלך התפשטות אל outpuהיבט t של MMF. כתוצאה מכך, התקדמות האור משתנה, מה שהופך אות השידור מאתגר. Wavefront בעיצוב שיטות 6, 7 מיושמות פיזור מדיה באמצעות מאפנני אור מרחבים (SLM) ולאפשר פיצוי על העיוות בשל פיזור במהלך התקדמות אור 8. ישנן גישות איטרטיבי המביאות למרב את התפוקה באמצעות משוב אופטי 9. גישות אלה הן זמן רב למדי בגלל הצורך חזרות רבות ואת הרמה הגבוהה של חופש, מתאים למספר רב של אלמנטים אפננו. גישה נוספת היא לקבוע את העיוות לחלוטין בתוך MMF שתואר על ידי מטריקס התמסורת שלה 10. אם מספר מצבים להיות משודר הוא גדול, זה יהיה זמן רב גם כן. לעומת זאת, נטיית שלב דיגיטלית אופטי (dOPC) נחשבתמהיר יתרון כאן, מאז רק כמה מוקדי כתמים צריך להיווצר על היבט פלט של MMF. גישות נטיית שלב גם הוכחו למיקוד או הדמיה באמצעות רקמות ביולוגיות 12, 13, 14. עד כה, dOPC הועסק אות פעם אחת בלבד 15, 16, ויושם להעברת אור דרך MMF 17. גישת dOPC עבור אותות עצמאיים רבים לא הושגה. פתחנו שיטת dOPC משופרת לספק את השידור העצמאי של אותות אור מרובים באמצעות חזית גל פרט בעיצוב עבור כל אות העסקת יחיד שלב בלבד SLM 18. למטרה זו, את SLM הוא מקוטע לתוך אזורים, אחד לכל אות להיות משודר. הגדרת הניסוי המוצעת מתוארת באיור 1, שבו הכיול מתבצע) לפני השידור בפועל קורה ב). <img alt="איור 1" src="/files/ftp_upload/55407/55407fig1.jpg" /> איור 1: הגדרת ניסוי. BS = קרן splitter, CCD = מכשיר תשלום מצמיד, OM = אפנן אופטי, CMOS = metal-oxide semiconductor משלימה, HWP = חצי גל צלחת, L = עדשה, מקטב ליניארי LP =, סיבי multimode MMF =, מטרת OBJ = מיקרוסקופ, PBS = מקטב הקרן splitter, SLM = מאפנן אור מרחבים (שלב בלבד) - רק קורות רלוונטיים (א) הכיול (ב) ההעברה המתוארות <a href="http://ecsource.jove.com/files/ftp_upload/55407/55407fig1large.jpg" target="_blank">אנא לחצו כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>spatial light modulator</td>
			<td>Holoeye</td>
			<td>PLUTO&#8208;VIS&#8208;016</td>
			<td></td>
		</tr>
		<tr>
			<td>CMOS camera</td>
			<td>Mikrotron</td>
			<td>MC4082</td>
			<td></td>
		</tr>
		<tr>
			<td>diode&#8208;pumped solid state laser</td>
			<td>Laser Quantum</td>
			<td>torus 532</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>IDS</td>
			<td>U3&#8208;3482LE&#8208;M</td>
			<td>CMOS camera; suitable as well</td>
		</tr>
		<tr>
			<td>lens 1</td>
			<td>Qioptiq</td>
			<td>G063204000</td>
			<td></td>
		</tr>
		<tr>
			<td>lens 2</td>
			<td>Qioptiq</td>
			<td>G063203000</td>
			<td></td>
		</tr>
		<tr>
			<td>lens 3</td>
			<td>Thorlabs</td>
			<td>AC508&#8208;180&#8208;A&#8208;ML</td>
			<td></td>
		</tr>
		<tr>
			<td>multimode fiber</td>
			<td>Thorlabs</td>
			<td>M14L02</td>
			<td></td>
		</tr>
		<tr>
			<td>beam splitters</td>
			<td>Thorlabs</td>
			<td>BS013</td>
			<td>9x</td>
		</tr>
		<tr>
			<td>polarizing beam splitters</td>
			<td>Thorlabs</td>
			<td>PBS251</td>
			<td></td>
		</tr>
		<tr>
			<td>mirrors</td>
			<td>Thorlabs</td>
			<td>PF10&#8208;03&#8208;P01</td>
			<td>5x</td>
		</tr>
		<tr>
			<td>microscope objectives</td>
			<td>Thorlabs</td>
			<td>RMS20X</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>half wave plates</td>
			<td>Thorlabs</td>
			<td>WPH10M&#8208;532</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>linear polarizer</td>
			<td>Thorlabs</td>
			<td>LPVISB050&#8208;MP2</td>
			<td></td>
		</tr>
		<tr>
			<td>optical modulators</td>
			<td>Thorlabs</td>
			<td>MC2000B&#8208;EC</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>linear and rotation stage for CMOS camera</td>
			<td>Thorlabs</td>
			<td>XYR1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>fiber connector</td>
			<td>Thorlabs</td>
			<td>S120&#8208;SMA</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>reducing ring for microscope objectives</td>
			<td>Qioptiq</td>
			<td>G061621000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>xy adjustment for objective adapters</td>
			<td>Qioptiq</td>
			<td>G061025000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>z translation mount for fiber adapter</td>
			<td>Thorlabs</td>
			<td>SM1Z</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>rods for fiber alignment to objectives</td>
			<td>Qioptiq</td>
			<td>G061210000</td>
			<td>8x</td>
		</tr>
		<tr>
			<td>mounts for lenses 1 and 2 plus two phantom mounts</td>
			<td>Qioptiq</td>
			<td>G061047000</td>
			<td>4x</td>
		</tr>
		<tr>
			<td>rail carriers for objective and lens mounts</td>
			<td>Qioptiq</td>
			<td>G061372000</td>
			<td>6x</td>
		</tr>
		<tr>
			<td>rail for rail carriers</td>
			<td>Qioptiq</td>
			<td>G061359000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td colspan="2">adapter for CCD camera to 1 post</td>
			<td></td>
			<td>in-house</td>
		</tr>
		<tr>
			<td>adapter for laser to 4 posts</td>
			<td></td>
			<td></td>
			<td>in-house</td>
		</tr>
		<tr>
			<td>mount for lens 3</td>
			<td>Thorlabs</td>
			<td>LMR2/M</td>
			<td></td>
		</tr>
		<tr>
			<td>mounts for half wave plates</td>
			<td>Thorlabs</td>
			<td>RSP1D/M</td>
			<td>2</td>
		</tr>
		<tr>
			<td>mounts for mirrors</td>
			<td>Thorlabs</td>
			<td>KM100</td>
			<td>5x</td>
		</tr>
		<tr>
			<td>mount for linear polarizer</td>
			<td>Thorlabs</td>
			<td>RSP05/M</td>
			<td></td>
		</tr>
		<tr>
			<td>mounts for beam splitters and SLM</td>
			<td>Thorlabs</td>
			<td>KM100PM/M</td>
			<td>11x</td>
		</tr>
		<tr>
			<td>clamping arms for beam splitters and SLM</td>
			<td>Thorlabs</td>
			<td>PM4/M</td>
			<td>11x</td>
		</tr>
		<tr>
			<td>posts for mounts, rail carriers and adapters</td>
			<td>Thorlabs</td>
			<td>TR75/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>holders for posts</td>
			<td>Thorlabs</td>
			<td>PH50/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>pedestals for holders</td>
			<td>Thorlabs</td>
			<td>BE1/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>clamping forks for pedestals</td>
			<td>Thorlabs</td>
			<td>CF125</td>
			<td>29x</td>
		</tr>
	</tbody>
</table>

transmission of multiple signals through an optical fiber using wavefront shaping

העברת אותות אופטיים מרובים עצמאיים באמצעות סיב multimode מושגת באמצעות עיצוב חזית גל על ​​מנת לפצות על העיוות הקלה במהלך ההתפשטות בתוך הסיב. המתודולוגיה שלנו מבוססת על נטיית שלב דיגיטלית אופטי העסיקה רק מאפנן אור מרחבי יחיד, שבו חזית הגל האופטית מווסתת באופן פרטני על אזורים שונים של אפנן, באזור אחד לכל אות אור. גישות נטיית שלב אופטי דיגיטליות נחשבות מהר יותר מאשר גישות בעיצוב חזית גל שניות, שם (למשל) קביעה מלאה של התנהגות גל ההפצה של הסיבים מתבצעת. לעומת זאת, הגישה המוצגת היא זמן יעיל שכן הוא רק דורש כיול אחד לכל אות אור. השיטה המוצעת מתאימה פוטנציאלי עבור ריבוב חלוקת מרחבית בהנדסת תקשורת. שדות יישום נוספים הם משלוח האור אנדוסקופית ב Biophotonics, במיוחד optogenetics, שבו תאים בודדים ברקמות ביולוגיות צריכים להיות מואר באופן סלקטיבי עם רזולוציה גבוהה במרחב ובזמן.

<html> אותות פלט אופייניים בצד הדיסטלי של הסיבים הארוכים 2 מ &#39;מתוארים באיור 2. ראוי לציין, כי במקום מוקד הרצוי (שיא) מלווה דפוס רבב רצוי (ברקע), אשר בשל חוסר שלמות של dOPC כעניין של עיקרון. יחס שיא-רקע המקביל (PBR) מסתכם 53 (רק האות 1 הוא &#39;על&#39;), 36 (רק הא...

Watch this Scientific Journal Video about Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping at JoVE.com

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

העברת אותות מרובים דרך סיב אופטי באמצעות Wavefront לעיצוב

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

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9.7K Views.  TU Dresden. We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

Video: Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

תלת ממדית ברזולוציה אופטית photoacoustic מיקרוסקופית

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

הוספת בדיקות ביולוגיות Biosensors סיליקה אופטי באמצעות סוכני צימוד silane

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

אינדוקציה של אותות הדבקה תלויי שימוש בעוצמה נמוכה אולטראסאונד

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

בסינתזת מבחנה של ריווח ילידים, סיבי קולגן וארך ריווח המגזרי

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

ללא תווית מולקולה בודדת איתור באמצעות תהודה אופטית Microtoroid

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

ססגוניות קרינת איתור עבור מיקרופלואידיקה אגל באמצעות סיבים אופטיים

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

קביעת פוטנציאל זטה באמצעות Nanoparticle טרנסלוקציה מהירויות באמצעות nanopore מתכוונן: באמצעות חלקיקים שונה-DNA כמשל

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

באמצעות תמסורת אופטית יוצאת דופן לכמת סמנים לב בנסיוב אדם

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

פילוח סיבים עצביים ספינלי לטיפול גירוי מוחי עמוק באמצעות דגמים אינטראקטיביים, מטופל ספציפי

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

הקרנה וזיהוי של פפטידים קטנים פילוח הצמיחה פיברוהפיצוץ מקדם Receptor2 באמצעות ספריית Phage התצוגה פפטיד

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...