Name: a protocol for real-time 3d single particle tracking
Uploaded: 2018-01-03T13:00:00
Description: Watch this Scientific Journal Video about A Protocol for Real-time 3D Single Particle Tracking at JoVE.com

यह काम राष्ट्रीय स्वास्थ्य संस्थान के जनरल मेडिकल साइंसेज के पुरस्कार संख्या R35GM124868 के तहत और ड्यूक विश्वविद्यालय द्वारा समर्थित किया गया था ।

1. सेटअप लेआउट और संरेखण
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	<li>ट्रैकिंग उत्तेजना लेज़र की स्थापना और collimation<ol>
		<li>एक घर का उपयोग कर ऑप्टिकल मेज पर लेजर प्रत्यय माउंट बनाया । माउंट लेजर सिर और ऑप्टिकल टेबल के लिए बढ़ते छेद के साथ एक सरल एल्यू?...

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	<li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat. Methods. 3 (10), 793-796 (2006).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Super-Resolution+Imaging+by+Stochastic+Optical+Reconstruction+Microscopy.">Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy.</a> Science. 319 (5864), 810-813 (2008).</li><li>Jones, S. A., Shim, S. H., He, J., Fast Zhuang, X. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+Nanometer+Localization+Analysis+for+Individual+Fluorescent+Probes.">Precise Nanometer Localization Analysis for Individual Fluorescent Probes.</a> Biophys. J. 82 (5), 2775-2783 (2002).</li></ol>

हालांकि 3 डी एकल कण ट्रैकिंग तरीकों की कई किस्मों हाल के वर्षों में उभरा है, मजबूत वास्तविक समय उच्च गति 3 डी प्रसार के एक साधारण सेटअप के साथ कम फोटॉन गिनती दरों पर ट्रैकिंग अभी भी एक चुनौती है, जो महत्वपू...

उंनत इमेजिंग तकनीक के उद्भव के लिए एक खिड़की खोल दिया है कभी सेलुलर घटनाएं, सभी तरह के आणविक स्तर पर नीचे की संरचना अधिक विस्तृत देखें । stochastic ऑप्टिकल पुनर्निर्माण माइक्रोस्कोपी (तूफान) के रूप में तरीके1,2,3, फोटो-सक्रिय स्थानीयकरण माइक्रोस्कोपी (पाम)4,5,6,7 , संरचित दीप्ति माइक्रोस्कोपी (सिम)8,9,10,11, और प्रेरित उत्सर्जन घट माइक्रोस्कोपी (STED)12,13, 14 दूर विवर्तन सीमा से परे चले गए है संरचना और जीने की कोशिकाओं के समारोह में अभूतपूर्व विस्तार से उद्धार । हालांकि, कैसे इन प्रणालियों व्यवहार की पूरी समझ में गतिशील जानकारी की आवश्यकता है और साथ ही संरचनात्मक जानकारी । ऊपर सूचीबद्ध सुपर संकल्प तरीकों स्थानिक संकल्प और लौकिक संकल्प के बीच एक व्यापार बंद शामिल है, जिसके साथ गतिशील प्रक्रियाओं जांच की जा सकती है लौकिक शुद्धता सीमित । एक विधि जो दोनों उच्च स्थानिक परिशुद्धता और लौकिक संकल्प प्रदान करता है RT-3d-SPT15,16,17,18,19,20, 21,22,23,24,25,26,27,28,29. यहां, हम पारंपरिक 3d-SPT30 और RT-3d-SPT के बीच एक अंतर आकर्षित करते हैं । पारंपरिक 3d-SPT बस तीन आयामी छवि डेटा (जो या तो एक फोकल माइक्रोस्कोप या एक epifluorescence माइक्रोस्कोप का उपयोग कर प्राप्त किया जा सकता है की एक समय श्रृंखला की आवश्यकता है सही विंयास दिया) । पारंपरिक 3d-SPT में, कण के निर्देशांक प्रत्येक छवि स्टैक में कण का पता लगाने और एक पथ बनाने के लिए क्रमिक वॉल्यूंस में स्थानों को श्रेणीबद्ध करके डेटा संग्रह के बाद निर्धारित किए जाते हैं । इन विधियों के लिए, अंतिम लौकिक रिज़ॉल्यूशन volumetric इमेजिंग दर द्वारा निर्धारित होता है । फोकल माइक्रोस्कोप के लिए, यह सेकंड के दसियों सेकंड के पैमाने पर आसानी से होता है । epifluorescence तरीकों के लिए, जिसमें ऑप्टिकल पथ इतनी हेर है कि अक्षीय स्थान जानकारी निकाला जा सकता है, लौकिक संकल्प कैमरा जोखिम या readout समय तक सीमित है । इन epifluorescent तरीकों सीमा से अधिक है जो अक्षीय जानकारी एकत्र किया जा सकता है में सीमित हैं, हालांकि हाल ही में प्रगति का रूपान्तर विमान चरण मास्क डिजाइन और अनुकूली प्रकाशिकी इन पर्वतमाला का विस्तार है 10 µm या अधिक31,३२ , ३३ , ३४.
इसके विपरीत, RT-3d-SPT एक 3d छवि स्टैक प्राप्त करने और इस तथ्य के बाद कणों को ढूँढने पर निर्भर नहीं करता है. इसके बजाय, वास्तविक समय स्थान की जानकारी एकल बिंदु डिटेक्टरों के माध्यम से निकाला जाता है और प्रतिक्रिया प्रभावी ढंग से "बंद" एक highspeed piezoelectric मंच के उपयोग के माध्यम से उद्देश्य लेंस के फोकल मात्रा में कण को लागू किया जाता है । यह केवल कितने फोटॉनों एकत्र किया जा सकता द्वारा सीमित कण की स्थिति के सतत माप की अनुमति देता है । इसके अलावा, इस विधि के कण के वर्णक्रम पूछताछ सक्षम बनाता है के रूप में यह लंबी पर्वतमाला पर चलता है । RT-3 डी-प्रभाव में SPT नेनो वस्तुओं के लिए एक बल मुक्त ऑप्टिकल जाल के सदृश काम करता है, जिसमें कण लगातार जांच की है और बड़े लेजर शक्तियों या ऑप्टिकल बलों के लिए आवश्यकता के बिना वास्तविक समय में मापा जाता है । यह देखते हुए कि RT-3d-SPT तेजी से प्रसार करने वाली वस्तुओं की सतत पूछताछ के लिए एक साधन प्रदान करता है (अप करने के लिए 20 µm2/25,29 पर तीन आयामों में कम फोटॉन गणना दर20,29, ३५, यह intracellular कार्गो परिवहन, ligand रिसेप्टर बाइंडिंग, और एकल virions की extracellular गतिशीलता के रूप में तेजी से या परिवर्तनीय जैविक प्रक्रियाओं में एक खिड़की प्रदान करना चाहिए । हालांकि, इस बिंदु के लिए, RT-3d-SPT के आवेदन इस प्रौद्योगिकी अग्रिम करने के लिए काम कर रहे समूहों की मुट्ठी भर तक ही सीमित किया गया है.
एक बाधा ऑप्टिकल आरटी-3d-SPT तरीकों, जो विविध है द्वारा आवश्यक लेआउट की जटिलता है । अधिकांश विधियों के लिए, ऑप्टिकल प्रतिक्रिया एक piezoelectric चरण द्वारा प्रदान की गई है । कण के रूप में एक्स, वाई, या जेड में छोटे आंदोलनों बनाता है, एकल बिंदु डिटेक्टरों से readouts त्रुटि कार्यों में परिवर्तित कर रहे है और उच्च गति पर खिलाया एक piezoelectric nanopositioner है, जो बारी में नमूना है कण गति प्रतिक्रिया, प्रभावी ढंग से ले जाता है यह उद्देश्य लेंस के सापेक्ष जगह में ताला लगा । एक्स, वाई, और जेड में छोटे स्थिति आंदोलनों को मापने के लिए, या तो एकाधिक डिटेक्टरों (4 या 5 कार्यांवयन के आधार पर)15,18,21 या एकाधिक उत्तेजना स्पॉट (2-4, जिनमें से कम लागू किया जा सकता है अगर एक ताला एम्पलीफायर में एक्स और वाई स्थिति एक घूर्णन लेजर स्थान का उपयोग कर निकालने के लिए प्रयोग किया जाता है)25,28 लागू होते हैं. इन एकाधिक डिटेक्शन और उत्सर्जन धब्बों के ओवरलैप सिस्टम को संरेखित और बनाए रखना कठिन बनाते हैं ।
इस के साथ साथ, हम एक उच्च गति लक्ष्य-3 डी-DyPLoT29कहा जाता है एक सरलीकृत ऑप्टिकल डिजाइन के साथ 3d-SPT विधि बंद प्रस्तुत करते हैं । 3 डी-DyPLoT एक 2d-ईओडी और एक टैग लेंस का उपयोग करता है३६,३७,३८ गतिशील एक उच्च दर (५० khz XY, ७० khz जेड) पर उद्देश्य फोकल मात्रा के माध्यम से एक केंद्रित लेजर स्थान ले जाने के लिए । लेजर फोकस स्थिति और फोटॉन आगमन के समय के संयोजन है कण 3 डी की स्थिति को तेजी से भी कम फोटॉन गिनती दरों पर प्राप्त करने में सक्षम बनाता है । 2d-ईओडी एक नाइट टूर पैटर्न में लेजर फोकस ड्राइव३९ x-Y विमान और टैग लेंस में 1 x 1 µm के एक वर्ग आकार के साथ 2-4 µm की एक सीमा के साथ अक्षीय दिशा में लेजर ध्यान चलता रहता है । 3 डी कण स्थिति 3 डी में एक अनुकूलित स्थिति अनुमान एल्गोरिथ्म29,४० के साथ प्राप्त की है । 3 डी का नियंत्रण गतिशील चलती लेजर स्पॉट, फोटॉन हिमस्खलन photodiode से गिनती (APD), वास्तविक समय कण स्थिति गणना, piezoelectric मंच प्रतिक्रिया, और डेटा रिकॉर्डिंग एक क्षेत्र प्रोग्राम गेट सरणी (FPGA) पर प्रदर्शन कर रहे हैं ।इस प्रोटोकॉल में, हम का वर्णन कैसे एक 3d-DyPLoT माइक्रोस्कोप का निर्माण करने के लिए कदम दर कदम, सहित ऑप्टिकल संरेखण, तय कणों के साथ अंशांकन, और अंत में मुक्त कण ट्रैकिंग. एक प्रदर्शन के रूप में, ११० एनएम फ्लोरोसेंट मोती एक समय में मिनट के लिए पानी में लगातार ट्रैक किए गए ।
यहां वर्णित विधि किसी भी आवेदन के लिए एक आदर्श विकल्प है जहां यह लगातार कम प्रकाश स्तर पर एक तेजी से बढ़ फ्लोरोसेंट जांच की निगरानी करना वांछित है, वायरस, नैनोकणों सहित, और बुलबुले जैसे endosomes । पिछले तरीकों के विपरीत, वहां केवल एक एकल उत्तेजना और एकल पहचान मार्ग, बनाने संरेखण और रखरखाव सीधा है । इसके अलावा, बड़े क्षेत्र का पता लगाने के इस माइक्रोस्कोप आसानी से जल्दी से फैलाना कणों लेने के लिए सक्षम बनाता है, जबकि कम संकेत स्तर पर ट्रैक करने की क्षमता (10 kHz करने के लिए नीचे) कम प्रकाश अनुप्रयोगों के लिए इस विधि आदर्श बनाता है29.

<table border="0" cellpadding="0" cellspacing="0" width="1075">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">2D Electro-optic Deflector&#160;</td>
			<td style="width:145px;">ConOptics</td>
			<td style="width:160px;">M310A</td>
			<td style="width:541px;">2 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Power supply for EOD</td>
			<td style="width:145px;">ConOptics</td>
			<td style="width:160px;">412</td>
			<td>Converts FPGA ouput to high voltage for EOD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">TAG&#160; Lens</td>
			<td style="width:145px;">TAG Optics</td>
			<td style="width:160px;">TAG 2.0</td>
			<td>Used to deflect laser along axial direction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">XY piezoelectric nanopositioner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">Nano-PDQ275HS</td>
			<td>Used for moving the sample to lock the particle in the objective focal volume in&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Z piezoelectric nanoposiitoner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">Nano-OP65HS</td>
			<td>Used to move the objective lens to follow the diffusing particle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Micropositioner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">MicroDrive</td>
			<td>Used to coarsely position sample and evaluate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Objective Lens</td>
			<td style="width:145px;">Zeiss</td>
			<td style="width:160px;">PlanApo</td>
			<td>High numerical aperture required for best sensitivity. 100X, 1.49 NA, M27, Zeiss</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">sCMOS camera</td>
			<td style="width:145px;">PCO</td>
			<td style="width:160px;">pco.edge 4.2</td>
			<td>Used to monitor the particle&#39;s position</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">APD</td>
			<td style="width:145px;">Excelitas</td>
			<td style="width:160px;">SPCM-ARQH-15</td>
			<td>Lower dark counts beneficial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Field programmable gate array</td>
			<td style="width:145px;">National Instruments</td>
			<td style="width:160px;">NI-7852r</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Software</td>
			<td style="width:145px;">National Instruments</td>
			<td style="width:160px;">LabVIEW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Tracking excitation laser</td>
			<td style="width:145px;">JDSU</td>
			<td style="width:160px;">FCD488-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-150-A-ML</td>
			<td>L1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Pinhole</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">P75S</td>
			<td>PH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Glan-Thompson Polarizer</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">GTH5-A</td>
			<td>GT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Half-wave plate</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">WPH05M-488</td>
			<td>WP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-75-A-ML</td>
			<td>L3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-250-A-ML</td>
			<td>L4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Dichroic Mirror</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">ZT405/488/561/640rpc</td>
			<td>DC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Fluorescence Emission Filter</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">D535/40m</td>
			<td>F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">10/90 beamsplitter</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">21012</td>
			<td>BS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">190 nm fluorescent nanoparticles</td>
			<td>Bangs laboratories</td>
			<td>FC02F/9942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">110 nm fluorescent nanoparticles</td>
			<td>Bangs laboratories</td>
			<td>FC02F/10617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-545A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Powermeter</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">PM100D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">CMOS</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">DCC1545M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Iris</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">SM1D12D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:145px;">Mad City Labs</td>
			<td style="width:160px;">RM21</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for real-time 3d single particle tracking

रीयल-टाइम तीन-आयामी एकल कण ट्रैकिंग (RT-3d-SPT) में तेज, सेलुलर सिस्टम में 3d प्रक्रियाओं पर प्रकाश डाला जाने की क्षमता है. हालांकि विभिन्न आरटी-3d-SPT तरीकों को हाल के वर्षों में आगे रखा गया है, कम फोटॉन गणना दरों पर उच्च गति वाले 3d फैलाना कणों पर नज़र रखना एक चुनौती बनी हुई है । इसके अलावा, आरटी-3d-SPT setups आम तौर पर जटिल है और लागू करने के लिए मुश्किल है, जैविक समस्याओं के लिए अपने व्यापक आवेदन सीमित । इस प्रोटोकॉल 3 डी गतिशील फोटॉन स्थानीयकरण ट्रैकिंग (3 डी-DyPLoT), जो उच्च प्रसार गति (अप करने के लिए 20 µm2/कम फोटॉन गिनती दर पर (10 kHz करने के लिए नीचे) के साथ कणों को ट्रैक कर सकते है नामक एक RT-3d-SPT प्रणाली प्रस्तुत करता है । 3 डी-DyPLoT एक 2d इलेक्ट्रो ऑप्टिक झुकानेवाला (2d-ईओडी) और एक स्वरित्र ध्वनिक ढाल (टैग) लेंस एक एकल केंद्रित लेजर स्थान गतिशील रूप से 3 डी में ड्राइव को रोजगार । एक अनुकूलित स्थिति अनुमान एल्गोरिथ्म के साथ संयुक्त, 3 डी-DyPLoT उच्च ट्रैकिंग गति और उच्च स्थानीयकरण परिशुद्धता के साथ एकल कणों पर ताला कर सकते हैं । एकल उत्तेजना और एकल पहचान पथ लेआउट के कारण, 3d-DyPLoT मजबूत और स्थापित करने के लिए आसान है । यह प्रोटोकॉल चर्चा करता है कि 3d-DyPLoT चरण द्वारा चरण कैसे बनाएं । सबसे पहले, ऑप्टिकल लेआउट वर्णन किया गया है । अगले, प्रणाली और तुले है रैस्टर द्वारा अनुकूलित piezoelectric nanopositioner के साथ एक १९० एनएम फ्लोरोसेंट मनका स्कैनिंग । अंत में, वास्तविक समय 3 डी ट्रैकिंग क्षमता का प्रदर्शन करने के लिए, ११० एनएम फ्लोरोसेंट मोती पानी में नज़र रखी हैं ।

फिक्स्ड कण स्कैनिंग (चित्रा 4) और आज़ादी ११० एनएम फ्लोरोसेंट कण ट्रैकिंग (चित्रा 5) के ऊपर प्रोटोकॉल के बाद प्रदर्शन किया गया । कण स्कैनिंग piezoelectric nanopositioner और बिन फोटॉनों चल?...

Watch this Scientific Journal Video about A Protocol for Real-time 3D Single Particle Tracking at JoVE.com

A Protocol for Real-time 3D Single Particle Tracking

इस प्रोटोकॉल के निर्माण और एक वास्तविक समय 3 डी एकल कण ट्रैकिंग माइक्रोस्कोप उच्च प्रसार गति और कम फोटॉन गिनती दरों पर नेनो फ्लोरोसेंट जांच पर नज़र रखने में सक्षम का विवरण ।

रीयल-टाइम 3d एकल कण ट्रैकिंग के लिए एक प्रोटोकॉल

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

The overall goal of this protocol is to implement a real-time, 3D, single particle tracking microscope. The main advantage of this technique is that a single diffusing nano scale object can be monitored continuously with photon limited temporal resolution. This technique can help answer key questions in the field of virology, such as following a single virion through the entire infection process.Prepare the apparatus on an optical table. This setup is ready for calibration. Key components include a 488 nanometer solid state laser, two electro-optic deflectors that are along the beam's path, a tunable acoustic gradient lens, and a sample stage with micro and nano positioners.This schematic of the setup provides additional details. Here is the laser beam path to the sample stage. The electro-optic deflectors and the tunable acoustic lens are along the beam's path.These devices dynamically drive the laser spot at the sample stage. Photons from the sample are counted by an avalanche photo diode. A camera monitors the position of the particle.The position of the sample is set using micro and nano controllers. A field programmable gate array controls the laser spot, real-time particle position calculation, stage feedback, and photon counting. Two samples have to be prepared.The first, requires 190 nanometer fluorescent beads diluted in phosphate buffered saline. Use a cover slip to prepare a fixed particle sample. Place 400 microliters of the bead solution onto the cover slip.Now, produce the free moving particles sample. It requires 110 nanometer fluorescent beads diluted in water. Create a free moving particle sample by placing 400 microliters of the solution onto a cover slip.Take the fixed particle sample to the microscope to mount. The sample is on the XY nano positioner. Turn on the laser, the photo detector, the controllers for the beam line elements, and for the positioners.Run the Piezo nano positioner in a closed loop. Next, remove the fluorescence emission filter from in front of the photo detector. Then, vary the sample's z-position with the micro positioner.As the position varies, use the photo detector to monitor the intensity of the laser's reflection. Maximize the intensity to identify the focal plane of the objective lens. With the sample in the focal plane, replace the fluorescence emission filter.Now, work with the computer control software. Use custom software to raster scan the sample. First, set a large scanning range, the difference between start and finish.Here, the range is 10 micrometers in Y and 10 micrometers in X.Also, set a large step size in each direction. Here, 200 nanometers. Set both Z fields to the previously identified position of the focal plane.Pushing scan will start the Raster scan. Start the scan. The software drives the electro-optic deflectors in a night's tour, collects counts from the photo detector, drives the nano positioner, and performs position calculations.The scan will find a particle that is identifiable in the software through a plata fluorescence. For each site in the scan, the software will also determine estimates of the particle position in X and Y, relative to the center of the electro-object deflector scan. After finding the particle, shrink the scanning range around it to two micrometers in X and Y.Also, decrease the step size for both to 100 nanometers.Add a scan range in the Z direction of two micrometers to bracket the fluorescent focus. Set the Z step size to 200 nanometers. Start the 3D raster scan to find the fluorescent focus.The data produced from the scan show the focal plane before the tunable acoustic gradient lens is powered on. Move on to work with the tunable acoustic lens setting within its control software. First, click connect, then, power on.Go to the pulse and gating control section, change the output trigger mode from RGB to multi-plane. Then, change it back to RGB to allow changing the output phase. Use the sliders to set the output phase between zero degrees, 90 degrees, and 270 degrees.Next, go to the frequency control section. There, select a resonance frequency of 68, 500 hertz. Move to the top of the screen and click advanced.Then, choose the settings tab. On the new screen, find the maximum frequency of the zero column. Adjust it to change the frequency search range.Click, save calibration, then, exit calibration. Return to frequency controls and change the resonance frequency. Immediately change it back to 68, 500 hertz to have the calibration take effect.In the amplitude control section, use the slider to slowly raise the amplitude to 35%After this, click lock resonance. When the frequency is locked, click unlock resonance. Return to the raster scan software.Input two micrometer scanning ranges centered on the particle in X and Y.Use a step size of 100 nanometers. Center the particle in a four micrometer range in Z, with a step size of 200 nanometers. In the scanning image that results the region of illumination representing the particle is about one micrometer wide.Based on this, adjust the XY scale parameter so the estimated positions in X and Y range from negative 0.5 to 0.5 micrometers. After a similar procedure with the Z scale set the nano positioner to the zero position. Turn off the power to the nano positioner before continuing.Replace the fixed particle sample with the freely moving particle sample. Then, make sure all of the equipment is on. Run the Piezo nano positioner in open loop.At the computer, set the tunable acoustic gradient lens software as before. Remove the fluorescence emission filter from the path to the photo detector. Find the focal plane by varying the sample Z position and maximizing the intensity of laser reflection, from the cover slip.After finding the focal plane, increase the focus position 15 micrometers into the solution. Replace the fluorescence emission filter. Open the custom tracking software.Set the position estimation parameters to the values found during optimization. Now, set the integral control constants, which dictate the response speed of the nano positioner. Start with the X constant.Enter a low value and increase it slowly. When oscillations begin in the particle position read-out, note the value of the control constant. Reduce the integral control constant to about 70%of its value at the onset of oscillation.Repeat the steps with the Y and Z control constants. Set he trigger to start tracking the track threshold to three kilohertz. Set track minimum, the trigger to end tracking, at 1.5 kilohertz.Start the experiment by clicking search and then clicking auto track. This movie shows the result of using the three dimensional, dynamic photon localization tracking microscope to track the diffusive motion of a 110 nanometer fluorescent nano particle in water. The particle's position, measured using the position read-out of the nano positioner is shown on the 3D axes.The inset is the read-out of the scientific sea moss camera. It demonstrates that the particle is held, locked in the focal volume, by the tracking system. The particle was tracked and its position recorded for more than two minutes.Once mastered, this technique can be done in under 50 minutes, if the system is properly aligned, and the calibration parameters set. After its development, this technique will help researchers in the field of drug delivery explore the pathways of internalization and transport of cellular cargo. After watching this video, you should have a good understanding of how to operate a zero-time, 3D single particle tracking microscope.

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Research

JoVE Journal

Bioengineering

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

3D spheroids की पीढ़ी के लिए एक सरल हैंगिंग ड्रॉप सेल संस्कृति प्रोटोकॉल

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

कण ट्रैकिंग Microrheology 3 डी का उपयोग ट्यूमर मॉडल में बाह्य मैट्रिक्स कठोरता के अनुदैर्ध्य मापन

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

एक संशोधित दो photon माइक्रोस्कोप में 3 डी कक्षीय ट्रैकिंग: Intracellular vesicles की ट्रैकिंग के लिए एक आवेदन

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

एकल कक्षों और 3 डी कारावास में एकल भ्रूण आदेश: उच्च सामग्री स्क्रीनिंग के लिए एक नई डिवाइस

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

कण-ट्रैकिंग Microrheology द्वारा biofilms के यांत्रिक गुणों के सीटू मानचित्रण में

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

एक आनुवंशिक रूप से इनकोडिंग झल्लाहट आधारित पित्त अम्ल सेंसर का उपयोग Intracellular पित्त अम्ल गतिशीलता की वास्तविक समय की निगरानी

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

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.

सीटू में बायोप्रोसेस में सिंगल-सेल मॉर्फोलॉजी के वास्तविक समय निर्धारण के लिए माइक्रोस्कोपी

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

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

एक Nafion-लेपित, कम ग्राफीन ऑक्साइड/Polyaniline Chemiresistive संवेदक के निर्माण के लिए एक माइक्रोबियल किण्वन के दौरान वास्तविक समय में पीएच की निगरानी

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

मानव Mesenchymal स्टेम सेल द्वारा Falcarindiol युक्त नए लिपिड लेपित नैनोकणों के ऊपर ले

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

वास्तविक समय अवलोकन के लिए एक Microfluidic विकास त्वरक पर कैंसर आबादी के पार Heterogeneous दवा gradients की पीढ़ी

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...