Name: fluorescence imaging with one-nanometer accuracy (fiona)
Uploaded: 2014-09-26T15:00:00
Description: Watch this Scientific Journal Video about Fluorescence Imaging with One-nanometer Accuracy FIONA at JoVE.com

Работа выполнена при поддержке грантов НИЗ 068625, NSF грантов 1063188 и центра физике живых клеток 0822613. Особая благодарность доктору Марина Марьянович в Beckman Института передовой науки и технологии за дар глаз кролика.

О себе Этика: ткани роговицы от кроликов собирали в соответствии с Университетом Иллинойса Уходу за животными и использованию принципов. 1 TIRFM Setup ПРИМЕЧАНИЕ: носить очки лазерной безопасности все время. <ol><li> Убедитесь, что все необходимые оптические ком...

<ol>
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T., 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=Multicolor+Super-Resolution+Imaging+with+Photo-Switchable+Fluorescent+Probes.">Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes.</a> Science. 317 (5845), 1749-1753 (2007).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Intracellular+Fluorescent+Proteins+at+Nanometer+Resolution.">Imaging Intracellular Fluorescent Proteins at Nanometer Resolution.</a> Science. 313 (5793), 1642-1645 (2006).</li><li>Abramoff, M. D., Magalh&#227;es, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics international. 11 (7), 36-42 (2004).</li><li>Enderlein, J., Toprak, E., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+effect+on+position+accuracy+of+fluorophore+localization.">Polarization effect on position accuracy of fluorophore localization.</a> Optics Express. 14 (18), 8111-8120 (2006).</li><li>Cheezum, M. K., Walker, W. F., Guilford, W. 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ФИОНА это техника локализовать положение флуоресцентного излучателя (органического флуорофором или квантовой точки) с нанометровой точностью и временным разрешением до 1 мс 4 8. Когда достаточное количество фотонов собирают, этот метод позволяет определить положение флуоресцен...

Вокруг 1882, Эрнст Аббе установлено, что разрешение на видном светового микроскопа ~ λ / 2NA, или ~ 200 нм (где λ длина волны и Н.А. является числовая апертура) 1,2. Поэтому любой объект меньше, чем этого измерения будет отображаться как дифракционной месте в оптическом микроскопе. Тем не менее, можно определить центр на месте, то есть местоположение объекта, с гораздо более высокой точностью 3. Флуоресценции изображений с точностью один-нанометровом (Fiona) является простой, но полезный метод для локализации одиночных флуорофоры с нанометровой точностью в плоскости 4. Точность локализации, σ μ (то есть, стандартная ошибка среднего), зависит от общего количества собранных фотонов, <img alt="Уравнение 1" fo:content-width="1in" src="/files/ftp_upload/51774/intro_eqn.jpg" width="100" /> , Где N является количество фотонов, с стандартное отклонение флуоресцентного месте, эторазмер пикселя детектора изображения, и б является стандартное отклонение фоне 3,4. Для флуорофором излучающей ~ 10000 фотоны, ФИОНА может достичь ~ 1 нм точность 4. ФИОНА может быть использован для точного определения положения стационарного излучателя или движущегося один (при условии, изображения могут быть приняты достаточно быстро). ФИОНА может быть применен последовательно в рамках фильма и, таким образом отслеживать движение одной молекулы 4- 8. Фото-защитная реагенты могут быть необходимы для того, чтобы образец не этого разрушению. Кроме того, сама по себе флуоресцентный объект может быть любого размера, меньше или больше, чем дифракционные ограничения, например, он может состоять из органелл (~ 1 мкм) с множеством флуоресцентных белков, диспергированных на ее мембране. Использование ФИОНА еще может дать очень точную (нанометровой) среднем его средней центра масс. Значительное улучшение в точности локализации Фионой позволяет определить nanomeтер Массовое перемещение во времени. Это подтолкнуло микроскопии в молекулярном масштабе длины 4 8. С момента своего изобретения, варианты ФИОНЫ были разработаны. Например, ярко-поле изображения с точностью один-нанометровом (bFIONA) 9, небольшой варианта Фиона, изображений и локализует плотные объекты, такие как Меланосомы естественных условиях (темных объектов, содержащих пигмент меланин) в с проходящем свете. Кроме того, ФИОНА был принят на работу, чтобы решить несколько красителей. Например, одной молекулы с высоким разрешением изображения с фотообесцвечивания (креветки) 10,11 или одной молекулы с высоким разрешением колокализация (SHREC) 12 были разработаны для решения двух красителей в течение примерно 10 нм. (Заметим, что это разрешение, то есть насколько точно можно сказать, одинаковые красители друг от друга.) Совсем недавно, анализ ФИОНА внесла свой ​​вклад в процесс локализации определенного сверхвысокого разрешения микроскопии, такие как стохастический оптического RECOnstruction микроскопии (ШТОРМОВАЯ) 13-15 и фото-активированный локализации микроскопии (Palm) 16, в котором временные темные флуорофоров возбуждены, а затем флуоресценции локализован. При повторном захватывающей довольно низкой плотности красителей (менее одного дифракционного предела спот), и затем сбора флуоресценции, анализируя каждый из них по Фиона, можно построить в высоком разрешении карту. Резолюция затем просто ограничивается числом фотонов друг краситель звук производится, а также вещей, как хранение образца в неподвижном (в том числе, например, микроскоп) при приобретении. В этой статье, краткое изложение методики FIONA и кратко описать примеры исследований, которые были выполнены с помощью ФИОНА сообщается. Во-первых, как настроить необходимое оборудование для Фиона экспериментов, то есть полное внутреннее отражение флуоресценции микроскопии (TIRFM), с подробной информацией о совместив оптику, описывается. Тогда, какпровести простой эксперимент Фиона по локализации иммобилизованных Cy3-ДНК одиночных молекул с использованием соответствующих протоколов, иллюстрируется. После этого, использование ФИОНЫ для измерения 36 нм размер шага одной усеченной миозина Va двигателя с надписью с квантовой точки представлена. Миозин Va является важным processive двигателя белок, который осуществляет сотовой груз во время транслокации вдоль нитей актина. Здесь миозина Va построить усеченный используется для удаления доменов не имеющие отношения к размером шага, и с флагом тег добавлен в С-конца, чтобы позволить легкость маркировки с квантовыми точками функционализированными Anti-Flag антител. Этот эксперимент проводится при низкой АТФ замедлить миозин и позволяют использовать достаточно длинных выдержках, чтобы получить хороший счета фотонов в каждом кадре. Любая достаточно ярким флуоресцентную метку можно заменить следующим протоколом. Наконец, сообщается последнее усилие расширения сферы применения ФИОНЫ к толстых образцов. Как доказательство-о-принципе, квантовые точки были пропитаныв Соль-гелей и кролик глазных роговиц, а затем в образ и локализованы с помощью Фиона. Для визуализации, погружения цель 60X вода с NA = 1.2 был использован, потому что эта цель имеет больше рабочее расстояние, чем ранее использовавшийся 100X иммерсионным объективом. Чтобы компенсировать потери в увеличении в цели, экстра-увеличение объектива (3.3x или 4.0X) был вставлен в пути излучения. Кроме того, эпи-флуоресцентный (не МДП) микроскопии должен быть использован для доступа глубокие участки в толстых образцов. Показано, что квантовые точки, смоченные в глубине золь-гелей и в глаз кролика роговицы (Z&gt; 200 мкм) можно локализовать с 2-3 точностью нм.

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			<td height="21" style="height:21px;width:220px;">Name of Material/ Equipment</td>
			<td style="width:211px;">Company</td>
			<td style="width:211px;">Catalog Number</td>
			<td style="width:443px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Double-sided tape</td>
			<td style="width:211px;">3M</td>
			<td style="width:211px;">--</td>
			<td>~ 75 um thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">EMCCD camera</td>
			<td style="width:211px;">Andor Technology</td>
			<td style="width:211px;">DU-897E-CS0-#BV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Ultrasonic cleaner</td>
			<td style="width:211px;">Branson</td>
			<td style="width:211px;">2510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Fluorescence filter set</td>
			<td style="width:211px;">Chroma</td>
			<td style="width:211px;">49016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Actin polymerization buffer&#160;</td>
			<td style="width:211px;">Cytoskeleton&#160;</td>
			<td style="width:211px;">BSA02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Biotin G-actin</td>
			<td style="width:211px;">Cytoskeleton&#160;</td>
			<td style="width:211px;">AB07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">G-actin&#160;</td>
			<td style="width:211px;">Cytoskeleton&#160;</td>
			<td style="width:211px;">AKL95</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">General actin buffer&#160;</td>
			<td style="width:211px;">Cytoskeleton&#160;</td>
			<td style="width:211px;">BSA01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Laser shutter (with driver)</td>
			<td style="width:211px;">Electro-Optical Products Corp.&#160;</td>
			<td style="width:211px;">SH-10-MP</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">IDL</td>
			<td style="width:211px;">Exelis Visual Information Solutions</td>
			<td style="width:211px;">--</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Neutravidin&#160;</td>
			<td style="width:211px;">Fisher Scientific</td>
			<td style="width:211px;">PI-31000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Coverslip</td>
			<td style="width:211px;">Fisherbrand&#160;</td>
			<td style="width:211px;">22X30-1.5</td>
			<td>0.16-0.19 mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Microscope slide</td>
			<td style="width:211px;">Gold Seal Microslides&#160;</td>
			<td style="width:211px;">30103X1</td>
			<td>0.93-1.05 mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plasma cleaner</td>
			<td style="width:211px;">Harrick Plasma</td>
			<td style="width:211px;">PDC-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Glass bottom dish&#160;</td>
			<td style="width:211px;">In Vitro Scientific</td>
			<td style="width:211px;">D35-20-1.5-N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Cy3-DNA oligos</td>
			<td style="width:211px;">Integrated DNA Technologies</td>
			<td style="width:211px;">--</td>
			<td>5&#39;-Cy3/GCCTCGCTGCCGTCGCCA-3&#39;Bio</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Fluorescent beads&#160;</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">T-7280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Qdot 605-streptavidin&#160;</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q10101MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Qdot605&#160;</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q21301MP</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Qdot705</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q22021MP&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Qdot705 Antibody Conjugation Kit</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q22061MP</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Matlab</td>
			<td style="width:211px;">MathWorks</td>
			<td style="width:211px;">--</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Optical table</td>
			<td style="width:211px;">Newport Corp</td>
			<td style="width:211px;">--</td>
			<td>RS4000 Series</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">60X Objective</td>
			<td style="width:211px;">Nikon</td>
			<td style="width:211px;">Plan Apo VC 60x WI</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">100X Objective</td>
			<td style="width:211px;">Olympus</td>
			<td style="width:211px;">PlanApo 100X/1.45 Oil &#8734;/0.17</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">60X Objective</td>
			<td style="width:211px;">Olympus</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Inverted microscope</td>
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			<td height="21" style="height:21px;width:220px;">Origin</td>
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			<td height="21" style="height:21px;width:220px;">Anti-FLAG antibody</td>
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			<td style="width:211px;">63689-25ML-F</td>
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			<td>Creatine Phosphokinase from rabbit muscle</td>
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			<td>Phosphocreatine di(tris) salt</td>
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			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
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			<td height="21" style="height:21px;width:220px;">NaCl</td>
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			<td style="width:211px;">03930590</td>
			<td>Protocatechuic acid&#160;</td>
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			<td height="21" style="height:21px;width:220px;">PCD</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">P8279</td>
			<td>Protocatechuate-3,4-dioxygenase&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">TMOS</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">341436-25G</td>
			<td>Tetramethyl orthosilicate</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Tris-HCl&#160;</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">93363</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Trolox</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">238813</td>
			<td>6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">1&#8221; diameter broadband dielectric mirrors with mounts</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BB1-E02, KM100</td>
			<td>Quantity: 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">&#189;&#8221; diameter posts</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">TR4</td>
			<td>Quantity &#8805; 6</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">10x beam expander</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BE10M-A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">2&#8221; diameter broadband dielectric mirrors with mounts</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BB2-E02, KM200</td>
			<td>Quantity: 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">2&#8221; diameter f = 300 mm lens with mount</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">LA1256-A, LMR2</td>
			<td>TIR lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Fluorescent alignment target&#160;</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">VRC2SM1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Laser safety goggles</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">LG3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">ND filter(s)</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">FW1AND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Optical beam profiler</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BP209-VIS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Post-mounted iris diaphragm</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">ID25</td>
			<td>Quantity: 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Shearing interferometer</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">SI100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">XYZ translation stage, &#189;&#8221; travel&#160;</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">T12XYZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Laser</td>
			<td style="width:211px;">World Star Technologies&#160;</td>
			<td style="width:211px;">TECGL-30</td>
			<td>532 nm, 30 mW</td>
		</tr>
	</tbody>
</table>

fluorescence imaging with one-nanometer accuracy (fiona)

Флуоресценции изображений с точностью один-нанометровом (Fiona) является простой, но полезный метод для локализации одиночных флуорофоры с нанометровой точностью в плоскости ху. Вот краткое изложение методики FIONA Сообщается и примеры исследований, которые были проведены с использованием Фиона кратко описаны. Во-первых, как настроить необходимое оборудование для Фиона экспериментов, то есть полное внутреннее отражение флуоресценции микроскопии (TIRFM), с подробной информацией о совместив оптику, описывается. Тогда, как провести простой эксперимент Фиона по локализации иммобилизованных Cy3-ДНК одиночных молекул с использованием соответствующих протоколов, а затем с помощью ФИОНЫ для измерения 36 нм размер шага одной усеченной миозина Va двигателя с надписью с квантовой точки, иллюстрируется. Наконец, сообщается недавняя попытка распространить применение Фионы в толстых образцах. Показано, что, используя погружением в воду цели и квантовые точки замачивают в глубине золь-гелей и кролик глазных роговиц (&gt;200 мкм), точность локализации 2-3 нм может быть достигнуто.

Типичная задача типа установки TIRFM показано на рисунке 3. Сначала поверхность с иммобилизованным образец Cy3-ДНК образ. Типичный изображение показано на рисунке 4а. Изображение было получено с воздействием 0,5 сек, с Е.М. усиления = 50 и чувствительности ПЗС = 12,13 для камеры. ...

Watch this Scientific Journal Video about Fluorescence Imaging with One-nanometer Accuracy FIONA at JoVE.com

Fluorescence Imaging with One-nanometer Accuracy FIONA

Холост флуорофоры могут быть локализованы с нанометровой точностью, используя Фиона. Вот краткое изложение методики FIONA Сообщается, и как проводить Фиона эксперименты описывается.

Флуоресценции изображений с Один-нм Точность (Fiona)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

fluorescence-imaging-with-one-nanometer-accuracy-fiona

Research

JoVE Journal

Biology

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

Родился Нормализация для флуоресцентной оптической томографии для проекции изображений Всего Сердца

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Мезоскопические флуоресцентная томография для В естественных условиях Изображений развивающихся Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Визуализация одиночных молекул ДНК репликации с помощью флуоресцентной микроскопии

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Покадровый Визуализация Митоз После трансфекции миРНК

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Сотовые Электромуфтовая Визуализированные с флуоресцентной микроскопии

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Одновременное Многоцветный изображений биологических структур с флуоресцентной фотоактивации локализации микроскопии

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Экспресс-анализ и разведка флуоресцентной микроскопии Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Визуальное стромул Частота с флуоресцентной микроскопией

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Смешанные первичные культуры мышиных тонкой кишки, предназначенные для изучения Gut Гормон Секреции и живых изображений клеток из клеток энтероэндокринных

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

Флуоресценции восстановления после Фотообесцвечивание Жёлтый флуоресцентный белок, помеченный p62 в Aggresome-индуцированной структуры

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...