超分辨熒光顯微鏡——顯納鏡

袁景和，方曉紅

（中國科學(xué)院化學(xué)研究所分子納米結(jié)構(gòu)與納米技術(shù)院重點(diǎn)實(shí)驗(yàn)室　北京 100190）

幾種超分辨率熒光顯微技術(shù)的原理和近期進(jìn)展

呂志堅(jiān)，陸敬澤，吳雅瓊，等

熱點(diǎn)追蹤

2014年諾貝爾化學(xué)獎(jiǎng)（超分辨率熒光顯微技術(shù)）

·編者按·

2014年10月8日，瑞典皇家科學(xué)院將2014年諾貝爾化學(xué)獎(jiǎng)授予美國霍華德·休斯醫(yī)學(xué)研究所的埃里克·白茲格（Eric Betzig）教授、美國斯坦福大學(xué)的威廉姆·莫納爾（William E Moerner）教授和德國馬普生物物理化學(xué)所的斯特凡·赫爾（Stefan W. Hell）教授，以表彰他們?cè)诔直媛薀晒怙@微技術(shù)領(lǐng)域取得的突出成就.他們的研究突破了光學(xué)顯微鏡分辨率不能高于0.2 μm的科學(xué)限制，將光學(xué)顯微鏡技術(shù)帶到了納米尺度，使科學(xué)家們可以觀察到蛋白質(zhì)的聚集，并可以在納米尺度上跟蹤細(xì)胞分裂.

所謂超分辨熒光顯微鏡是指超過衍射極限分辨率的熒光顯微鏡.17世紀(jì)，當(dāng)科學(xué)家首次在光學(xué)顯微鏡下研究血紅細(xì)胞、細(xì)菌、酵母細(xì)胞等生物活體時(shí)，微生物學(xué)由此誕生，光學(xué)顯微鏡成為生命科學(xué)研究中的重要工具之一.1873年，顯微鏡專家阿貝在其發(fā)表的論文中證明了阿貝方程，即顯微鏡的分辨率受到光的波長限制，人們無法利用光學(xué)顯微鏡觀察到比光波長的一半，即0.2 μm還小的物體.直至埃里克·白茲格、威廉姆·莫納爾和斯特凡·赫爾突破這一限制，利用熒光分子把光學(xué)顯微鏡帶到了一個(gè)新的天地.2000年，斯特凡·赫爾發(fā)明了受激發(fā)射損耗（STED）顯微技術(shù).該技術(shù)使用一束激光激發(fā)熒光分子使其發(fā)光，另一束激光則將一塊納米大小的區(qū)域之外的大部分熒光抵消.顯微鏡對(duì)樣本逐個(gè)進(jìn)行掃描，所生成圖像的分辨率高于阿貝分辨率的限制.埃里克·白茲格和威廉姆·莫納爾的研究成果為第2種方法——單分子顯微技術(shù)，這種方法的關(guān)鍵是能打開和關(guān)閉單個(gè)分子的熒光.科學(xué)家們對(duì)同一區(qū)域多次成像，每次只讓幾個(gè)零散的分子發(fā)出熒光，將得到的圖像疊加在一起，就得到了一幅分辨率在納米尺度的超稠密圖像.2006年，埃里克·白茲格首次將單分子顯微技術(shù)投入了實(shí)際運(yùn)用.

在生命化學(xué)研究領(lǐng)域，越來越多的化學(xué)家將生理狀態(tài)下的復(fù)雜生物體系，如活細(xì)胞/活體作為研究對(duì)象.如何在活細(xì)胞/活體中，對(duì)生物分子的結(jié)構(gòu)、分子間（內(nèi)）相互作用、生化反應(yīng)等，實(shí)現(xiàn)分子水平的實(shí)時(shí)動(dòng)態(tài)檢測是科研人員所面臨的首要問題.超分辨率熒光顯微鏡技術(shù)的發(fā)明，為人們突破生命化學(xué)研究的瓶頸，闡明生化反應(yīng)和生命活動(dòng)的分子機(jī)制，最終揭示生老病死的生命奧秘，帶來了新的手段和新的機(jī)遇.

自2006年以來，超分辨光學(xué)顯微技術(shù)發(fā)展迅速，各種新的超分辨成像方法不斷涌現(xiàn)，并展現(xiàn)了激動(dòng)人心的應(yīng)用前景.作為新興技術(shù)，超分辨光學(xué)顯微鏡的發(fā)展歷史不長，它們?cè)谏^程分子機(jī)制研究中的應(yīng)用，還處于起步階段，仍需要進(jìn)一步發(fā)展適合于活細(xì)胞/活體動(dòng)態(tài)變化研究的高時(shí)間分辨顯納鏡.但是，2014年諾貝爾化學(xué)獎(jiǎng)獲得者已經(jīng)為這項(xiàng)對(duì)人類最重要的技術(shù)的發(fā)展奠定了基礎(chǔ)，極大地促進(jìn)了分子水平上生化反應(yīng)過程及生物大分子構(gòu)效關(guān)系的研究，為生命化學(xué)的發(fā)展翻開了新的一頁.

本期專題得到了席鵬研究員（北京大學(xué)工學(xué)院生物醫(yī)學(xué)工程系）的大力支持.

·熱點(diǎn)數(shù)據(jù)排行·

截至2015年1月20日，根據(jù)中國知網(wǎng)（CNKI）和Web of Science數(shù)據(jù)報(bào)告顯示，有關(guān)超分辨率熒光顯微技術(shù)研究與應(yīng)用的期刊文獻(xiàn)分別為21與1652條，本刊將相關(guān)數(shù)據(jù)按照：研究機(jī)構(gòu)發(fā)文數(shù)、作者發(fā)文數(shù)、期刊發(fā)文數(shù)、被引用頻次進(jìn)行排行，結(jié)果如下.

研究機(jī)構(gòu)發(fā)文數(shù)量排名（CNKI）

研究機(jī)構(gòu)發(fā)文數(shù)量排名（WOS）

作者發(fā)文數(shù)量排名（CNKI）

作者發(fā)文數(shù)量排名（WOS）

期刊發(fā)文數(shù)量排名（CNKI）

期刊發(fā)文數(shù)量排名（WOS）

根據(jù)中國知網(wǎng)（CNKI）數(shù)據(jù)報(bào)告，篩選出有關(guān)超分辨率熒光顯微技術(shù)研究與應(yīng)用的高被引論文，排行結(jié)果如下.

國內(nèi)數(shù)據(jù)庫高被引論文排行

（數(shù)據(jù)來源：中國知網(wǎng)，檢索時(shí)間：2015.1.20）

根據(jù)Web of Science統(tǒng)計(jì)數(shù)據(jù)，有關(guān)超分辨率熒光顯微技術(shù)研究與應(yīng)用的高被引論文.*摘編自《化學(xué)通報(bào)》2014年77卷11期1029～1035頁

國外數(shù)據(jù)庫高被引論文排行

超分辨熒光顯微鏡——顯納鏡*

袁景和，方曉紅

（中國科學(xué)院化學(xué)研究所分子納米結(jié)構(gòu)與納米技術(shù)院重點(diǎn)實(shí)驗(yàn)室北京 100190）

化學(xué)是在分子水平研究物質(zhì)組成、結(jié)構(gòu)、性質(zhì)及變化的科學(xué).正如美國科學(xué)院院士、哈佛大學(xué)謝曉亮教授在《Science》評(píng)述中所指出的“活細(xì)胞已成為21世紀(jì)（科學(xué)研究中）的試管”，在生命化學(xué)研究領(lǐng)域，越來越多的化學(xué)家將生理狀態(tài)下的復(fù)雜生物體系如活細(xì)胞或活體作為研究對(duì)象.研究人員首先面臨的挑戰(zhàn)問題是，如何在活細(xì)胞/活體中實(shí)現(xiàn)分子水平上生物分子的結(jié)構(gòu)、分子間（內(nèi)）相互作用、生化反應(yīng)等的實(shí)時(shí)動(dòng)態(tài)檢測.2014年諾貝爾化學(xué)獎(jiǎng)獲得者Eric Betzig、Stefan W. Hell和William E. Moerner所發(fā)展的“超分辨率熒光顯微鏡”，為人們突破這一生命化學(xué)研究的瓶頸，闡明生化反應(yīng)和生命活動(dòng)的分子機(jī)制，最終揭示生老病死的生命奧秘，帶來了新的手段和新的機(jī)遇.所謂超分辨熒光顯微鏡是指超過衍射極限分辨的熒光顯微鏡.光學(xué)顯微鏡由于其非侵入、動(dòng)態(tài)表征等優(yōu)勢，廣泛應(yīng)用于生命科學(xué)研究.然而，物理學(xué)家Ernst. Abbe在1873年就指出，傳統(tǒng)光學(xué)顯微鏡的分辨率有一極限值.因?yàn)橐粋€(gè)無限小的點(diǎn)所發(fā)出的光，經(jīng)顯微光學(xué)系統(tǒng)成像后得到的是一個(gè)擴(kuò)展的焦斑，即艾里斑.對(duì)于常用可見光波長的顯微鏡，分辨率不超過200 nm，也就是說無法分辨距離小于200 nm的兩個(gè)點(diǎn).這個(gè)Abbe衍射極限一直被人們認(rèn)為是不可逾越的理論極限，阻礙了對(duì)生物體系的分子水平研究.

近年來隨著超分辨熒光顯微術(shù)的興起，研究人員研制了多種突破衍射極限的超分辨光學(xué)顯微鏡，使得光學(xué)顯微鏡進(jìn)入“顯納鏡”（Nanoscopy）時(shí)代.這些超分辨顯微鏡主要分為兩類：一類以Hell發(fā)明的受激輻射耗盡（Stimulated emission depletion，STED）顯微鏡為代表，通過調(diào)制光照明方式來實(shí)現(xiàn)超分辨，也可稱為結(jié)構(gòu)光照明超分辨顯微鏡；另一類是基于單分子定位的超分辨顯微鏡，典型代表是Betzig和Moerner等發(fā)展的光活化定位顯微鏡（Photoactivated localization microscopy，PALM），通過對(duì)具有光開關(guān)功能的熒光基團(tuán)進(jìn)行單分子成像和定位而實(shí)現(xiàn).下面將簡介這兩類顯微鏡的原理、方法、應(yīng)用進(jìn)展，以及三位諾獎(jiǎng)獲得者的貢獻(xiàn).

1STED熒光顯微鏡

1.1原理

早在1994年Hell就提出了通過受激輻射耗盡的方式實(shí)現(xiàn)超分辨成像的設(shè)想.其基本原理為，激發(fā)光將熒光分子從基態(tài)S0激發(fā)到激發(fā)態(tài)S1，經(jīng)過振動(dòng)弛豫，熒光分子躍遷到S1的最低振動(dòng)能級(jí)L2，當(dāng)熒光分子從該振動(dòng)能級(jí)躍遷回到基態(tài)S0時(shí)，能量以熒光的形式輻射出去，這就是自發(fā)熒光輻射過程，傳統(tǒng)的共聚焦顯微鏡等即是通過檢測這一自發(fā)輻射熒光信號(hào)來成像的.另一方面，處于激發(fā)態(tài)的熒光分子在外界輻射影響下，產(chǎn)生與外界輻射同頻率、同位相和同偏振的輻射，即受激熒光輻射過程，如果這一過程競爭超過自發(fā)熒光輻射，就導(dǎo)致激發(fā)態(tài)分子進(jìn)入非自發(fā)熒光輻射狀態(tài)，不發(fā)熒光，稱為受激輻射耗盡（STED）.諾貝爾化學(xué)獎(jiǎng)獲得者Hell創(chuàng)造性地同時(shí)使用兩束激光激發(fā)樣品，其中一束為中間空心的面包圈形狀STED光，兩束光疊加后使得只有激發(fā)光焦斑中心區(qū)域的熒光分子可以自發(fā)輻射熒光，而被STED光束照射的區(qū)域，發(fā)生STED效應(yīng)不再發(fā)熒光，這就使得有效的熒光激發(fā)半徑大大降低，從而突破了衍射極限的限制.

1.2方法與應(yīng)用

實(shí)現(xiàn)STED超分辨成像的關(guān)鍵在于獲得面包圈形STED光，并且要求STED光波長位于熒光發(fā)射波長的紅限，時(shí)間上比激發(fā)光有亞納秒量級(jí)的延遲，強(qiáng)度上遠(yuǎn)高于激發(fā)光.2000年，Hell領(lǐng)導(dǎo)其研究組，利用兩臺(tái)同步的鈦寶石飛秒脈沖激光器成功地構(gòu)建了超分辨STED顯微鏡，成像48 nm直徑熒光球，獲得104 nm橫向分辨率，第一次用實(shí)驗(yàn)實(shí)現(xiàn)了STED超分辨顯微成像.后來經(jīng)一系列設(shè)備改進(jìn)，分辨率提高到29 nm.隨后Hell等還發(fā)展了快速掃描STED熒光顯微鏡（80幀/s）、雙色STED成像、多壽命多色STED成像等一系列技術(shù).

早期的STED顯微鏡主要采用飛秒或者亞皮秒脈沖激光器激發(fā)，設(shè)備昂貴，調(diào)制難度大.后來Hell等發(fā)展了用連續(xù)激光激發(fā)的STED（CWSTED）顯微鏡，并實(shí)現(xiàn)了商品化.雖然成本降低，但為充分耗盡熒光分子，需使用比脈沖激光更高的激光功率，容易導(dǎo)致熒光樣品的光漂白和熱損傷.新的商品化超連續(xù)譜激光器，具有同時(shí)輸出激發(fā)光和STED光、方便STED波長選擇、避免同步失調(diào)等優(yōu)勢，將大大促進(jìn)STED顯微鏡的普及.

應(yīng)用方面，Hell實(shí)驗(yàn)室2006年在《Nature》上報(bào)道，STED顯微鏡分辨出直徑僅40 nm的神經(jīng)突觸囊泡，并發(fā)現(xiàn)突觸囊泡膜蛋白I（Synaptotagmin I）在突觸囊泡胞吐后仍保持獨(dú)立團(tuán)簇狀態(tài).隨后STED顯微鏡憑借其超高分辨能力很快在研究細(xì)胞膜微區(qū)結(jié)構(gòu)、細(xì)胞器、神經(jīng)突觸、病毒等方面取得不少新的重要發(fā)現(xiàn).如Hell及其合作者觀測140 nm大小的HIV-1病毒顆粒上包膜蛋白的分布，發(fā)現(xiàn)這些糖蛋白發(fā)生聚集是病毒高效?？克拗骷?xì)胞及隨后感染的必要條件.他們從對(duì)體外細(xì)胞成像進(jìn)一步實(shí)現(xiàn)了對(duì)活老鼠大腦神經(jīng)元的超高分辨率實(shí)時(shí)成像，展示了STED在活體成像中應(yīng)用前景.

Hell發(fā)明STED顯微鏡，其意義不僅是分析技術(shù)上的進(jìn)步，更重要的是觀念上的突破.Hell提出了STED顯微鏡的分辨率公式.公式指出，只要STED光強(qiáng)足夠大，顯微鏡的分辨率沒有理論上的極限.由于有衍射極限理論的思維禁錮，在STED提出后的很長一段時(shí)間，并未得到大多數(shù)學(xué)者的支持.直到2006年Hell研究組實(shí)現(xiàn)了神經(jīng)突觸囊泡的超分辨成像，展示了令人信服的生物體系表征結(jié)果，才得到廣泛認(rèn)可，并啟發(fā)和帶動(dòng)了一大批超分辨技術(shù)的迅速發(fā)展.值得一提的是，STED顯微鏡由于其技術(shù)難度，長期以來國際上只有少數(shù)實(shí)驗(yàn)室可實(shí)現(xiàn).在國內(nèi)，中科院化學(xué)所基于三態(tài)弛豫（T_Rex）概念研制成功了超連續(xù)脈沖激光激發(fā)SC-STED，獲得了好于50 nm的超分辨熒光成像，并在此基礎(chǔ)上研制了與AFM聯(lián)用的儀器，在國際上首次實(shí)現(xiàn)了熒光和力學(xué)信號(hào)的同時(shí)同步成像，可實(shí)現(xiàn)更高精度的光學(xué)引導(dǎo)AFM測量，以及光學(xué)和力學(xué)性質(zhì)的多參數(shù)測量.北京大學(xué)實(shí)現(xiàn)CWSTED顯微鏡，浙江大學(xué)使用方位角偏振光束，獲得了更銳的STED光.

2PALM——基于單分子熒光成像的超分辨顯微鏡

2.1成像原理

與STED顯微鏡需要復(fù)雜的光學(xué)系統(tǒng)不同，PALM等超分辨顯微鏡最初是在對(duì)傳統(tǒng)的全內(nèi)反射顯微鏡做簡單改造后實(shí)現(xiàn)的，通過對(duì)具有光開關(guān)性質(zhì)的熒光分子進(jìn)行單分子定位而重構(gòu)出超分辨顯微圖像.因?yàn)楸M管光學(xué)分辨極限為200 nm左右，如果這個(gè)衍射限范圍內(nèi)只有一個(gè)分子，那么通過對(duì)顯微物鏡點(diǎn)擴(kuò)展函數(shù)（PSF）的擬合，其定位的精度與波長無關(guān)，可達(dá)到1 nm，遠(yuǎn)小于200 nm的衍射極限.因此，可以對(duì)一個(gè)樣品采取多次成像的方法，每一次成像只激發(fā)少量的稀疏分布的單分子.利用一束激光使4個(gè)分子中的2個(gè)從非熒光態(tài)（暗態(tài)a）激活為熒光態(tài)（亮態(tài)b），對(duì)處于亮態(tài)的2個(gè)分子進(jìn)行擬合定位獲得納米級(jí)超分辨定位（c，定位于十字交叉中心點(diǎn)）；待這2個(gè)分子熒光猝滅后（d），再重復(fù)上述循環(huán)（e～f）可獲得新激活分子的納米級(jí)超分辨成像，這便是PALM的成像原理.由此可見，實(shí)現(xiàn)這類超分辨成像需要兩個(gè)關(guān)鍵技術(shù)：一是單分子成像，二是使用具有光調(diào)控暗態(tài)—亮態(tài)開關(guān)性質(zhì)的熒光分子.

2.2單分子成像

單分子熒光成像是PALM等超分辨成像的技術(shù)基礎(chǔ)，Moerner是單分子成像領(lǐng)域的先驅(qū).單個(gè)分子的成像檢測，即獲得1個(gè)分子的光學(xué)信號(hào)，對(duì)化學(xué)而言，可謂達(dá)到最高靈敏度，難度很大.1989年，Moerner等首次實(shí)現(xiàn)低溫下單個(gè)分子的光譜檢測，隨后人們相繼取得低溫單分子熒光光譜、室溫單分子熒光光譜、活細(xì)胞體系單分子熒光成像等一系列重要突破.1999年，《Science》以“單分子：化學(xué)研究的前沿”為題，出版了單分子研究的?？?，綜述了單分子研究進(jìn)展和未來發(fā)展方向，極大地推動(dòng)了單分子檢測技術(shù)的興起和發(fā)展.

需要指出的是，單分子檢測本身也是化學(xué)、生物、物理等交叉學(xué)科的前沿領(lǐng)域.單分子檢測對(duì)于生物分子的研究具有特別重要的意義.因?yàn)閭鹘y(tǒng)的生物體系研究往往都是對(duì)大量分子在一段時(shí)間內(nèi)的平均行為的描述，由于分子結(jié)構(gòu)（如構(gòu)象）的多樣性、生化反應(yīng)的非同步性和所處環(huán)境的（如細(xì)胞膜、質(zhì)、核等不同微區(qū)）非均相性，使得生物分子通常具有顯著的非均一性，在集群研究中單個(gè)分子的行為被平均化和掩蓋，阻礙了對(duì)其結(jié)構(gòu)和功能的深入認(rèn)識(shí).利用單分子成像，除了可實(shí)現(xiàn)超分辨單分子定位，還可研究生物單分子的物理化學(xué)性質(zhì)、動(dòng)態(tài)變化和生物學(xué)功能的分子機(jī)制，有望為生命科學(xué)和生命化學(xué)的研究帶來重大突破.最近，《Nature Methods》點(diǎn)評(píng)了在過去十年中對(duì)生物學(xué)研究影響最深的十大技術(shù)，其中單分子技術(shù)和超高分辨率顯微術(shù)共同上榜，今年的諾貝爾化學(xué)獎(jiǎng)也是對(duì)單分子技術(shù)的肯定Moerner長期從事單分子成像研究，為該領(lǐng)域的發(fā)展做出了許多重要貢獻(xiàn)，其中也包括一些超分辨成像新方法的發(fā)展，并一直推動(dòng)單分子技術(shù)在生命科學(xué)中的應(yīng)用.Moerner的獲獎(jiǎng)主要在于他對(duì)于PALM奠基技術(shù)－單分子成像的貢獻(xiàn).此外，他在1997年最早報(bào)道了具有光活化熒光開關(guān)效應(yīng)的熒光蛋白并實(shí)現(xiàn)了對(duì)該蛋白的單分子檢測.他發(fā)現(xiàn)綠色熒光蛋白（GFP）經(jīng)488 nm激光激發(fā)熒光后會(huì)進(jìn)入暗態(tài)，不再發(fā)熒光，但經(jīng)405 nm激光照射后，能返回到原來可發(fā)射熒光的狀態(tài).這種光活化特性的熒光蛋白正是實(shí)現(xiàn)PALM超分辨成像所需要的熒光基團(tuán).可惜的是，Moerner當(dāng)時(shí)并沒有想到將該熒光蛋白用于超分辨成像.

2.3PALM

2006年，Eric Betzig和Lippincott-Schwartz等提出了光激活定位顯微術(shù)（PALM）的概念，巧妙地利用一種GFP突變體作為光活化蛋白（PAFP）來標(biāo)記靶蛋白，并在細(xì)胞中表達(dá).用405 nm激光器低能量照射細(xì)胞表面，一次僅激活出稀疏分布的幾個(gè)熒光分子，然后用561 nm激光激發(fā)得到熒光圖像并精確定位這些熒光單分子.待長時(shí)間使用561 nm來漂白這些已經(jīng)定位正確的熒光分子之后，再分別用405 nm和561 nm激光來激活、激發(fā)和漂白其他的熒光分子，多次成像后，將這些分子的熒光圖像合成到一張圖上，得到了比傳統(tǒng)光學(xué)顯微鏡至少高10倍以上的分辨率.開發(fā)高性能的光活化蛋白是PALM技術(shù)發(fā)展的重要方向.一些常見的光活化蛋白存在光活化比率小、熒光信號(hào)弱等缺點(diǎn)，導(dǎo)致其成像質(zhì)量受限.研究人員從熒光蛋白的突變體中篩選pH和光穩(wěn)定性好、光活化速率快的各鐘新型的光活化蛋白如PAmCherry1，在固定細(xì)胞中獲得小于25 nm的空間分辨率.

2006年，Betzig等在《Science》發(fā)表首篇PALM論文，展示PALM顯微鏡可清楚分辨固定細(xì)胞粘著斑中的粘著斑蛋（Vinculin）、偽足上的肌動(dòng)蛋白、質(zhì)膜上逆轉(zhuǎn)錄蛋白Gag、線粒體等，隨后又發(fā)展了活細(xì)胞雙色共定位和單分子追蹤PALM技術(shù)等，觀察了多種亞細(xì)胞結(jié)構(gòu)變化，如活細(xì)胞中粘附復(fù)合物（adhesioncomplexes，ACs）的形成過程以及在形成過程中單個(gè)樁蛋白（paxillin）的捕獲和失去.PALM自提出以來受到了相關(guān)領(lǐng)域科研人員的廣泛重視，并得到了長足發(fā)展.例如，對(duì)神經(jīng)突觸后密集區(qū)結(jié)構(gòu)（PSD）高分辨成像，發(fā)現(xiàn)4種主要腳手架蛋白形成80 nm動(dòng)態(tài)變化的聚集體，有的能選擇性地富集AMPA受體而不是NMDA，提出PSD內(nèi)部結(jié)構(gòu)可能參與調(diào)節(jié)神經(jīng)傳遞的強(qiáng)度和可塑性.

Betzig獲得諾貝爾化學(xué)獎(jiǎng)的工作經(jīng)歷頗有些傳奇.他早年曾在建立近場光學(xué)顯微鏡SNOM方面做出過開創(chuàng)性的工作，1992年他在《Science》上發(fā)表利用SNOM實(shí)現(xiàn)單分子成像的成果，引起廣泛關(guān)注.SNOM通過納米光纖近場激發(fā)樣品，可實(shí)現(xiàn)納米尺度成像.Betzig意識(shí)到SNOM在生物體系研究的局限性，曾設(shè)想采用通過準(zhǔn)確定位不同波長的熒光分子以繞開衍射極限，并在1995年發(fā)表了可以說是PALM技術(shù)雛型的理論文章，但當(dāng)時(shí)無法在實(shí)驗(yàn)上實(shí)現(xiàn).他曾一度失望地離開了學(xué)術(shù)界，到父親的企業(yè)工作.不過他一直沒有放棄突破衍射極限的夢想.當(dāng)他了解到Lippincott-Schwartz實(shí)驗(yàn)室的光活化熒光蛋白后，重返實(shí)驗(yàn)室，終于夢想成真.

2.4隨機(jī)光學(xué)重構(gòu)顯微鏡（STORM）

與PALM同時(shí)誕生的，還有一種同樣是基于單分子點(diǎn)擴(kuò)展函數(shù)擬合定位實(shí)現(xiàn)超分辨的方法，即哈佛大學(xué)華裔女科學(xué)家Xiaowei Zhuang研究組開發(fā)的超分辨隨機(jī)光學(xué)重構(gòu)顯微（stochastic optical reconstruction microscopy，STORM）.STORM與PALM的基本原理一致，區(qū)別在于STORM使用的熒光開關(guān)基團(tuán)是有機(jī)染料而不是熒光蛋白.Zhuang等發(fā)現(xiàn)可使用一對(duì)染料分子Cy3和Cy5作為熒光標(biāo)記實(shí)現(xiàn)超分辨成像，因?yàn)椴煌ㄩL光可以控制Cy5在熒光發(fā)射態(tài)和暗態(tài)之間切換，而Cy3分子可以加速該開關(guān)過程.

Zhuang及合作者在STORM方法改進(jìn)和應(yīng)用上做了大量的工作，如發(fā)展可同時(shí)記錄2種甚至多種蛋白質(zhì)空間相對(duì)位置的方法、三維STORM等.應(yīng)用方面的成果包括：對(duì)神經(jīng)元樹突和軸突中肌動(dòng)蛋白等骨架蛋白成像，發(fā)現(xiàn)肌動(dòng)蛋白形成纏繞在神經(jīng)軸突外周的環(huán)狀結(jié)構(gòu)，軸突中的鈉離子通道呈現(xiàn)出與之關(guān)聯(lián)的周期性分布，表明軸突質(zhì)膜可能影響了軸突與其他細(xì)胞相互交流的方式；通過對(duì)端粒成像，發(fā)現(xiàn)了一種與端粒功能有關(guān)的t-loop構(gòu)象，并提出了調(diào)控t-loop構(gòu)象形成的新機(jī)制.

STORM和PALM一直是超分辨成像領(lǐng)域并駕齊驅(qū)、不分伯仲的兩種技術(shù).Zhuang在博士后期間就在單分子成像領(lǐng)域嶄露頭角，后來又以研究RNA構(gòu)象變化、病毒感染等出色工作成為單分子成像領(lǐng)域代表性人物之一.她的課題組在使用熒光共振能量轉(zhuǎn)移常用染料對(duì)進(jìn)行單分子成像時(shí)，觀察到其光開關(guān)效應(yīng)，很快便獨(dú)立地發(fā)展了超分辨成像新方法.STORM的工作與PALM于2006年同月發(fā)表，只是在投稿日期上晚了4個(gè)月.由于具有對(duì)細(xì)胞內(nèi)源性生物分子成像的優(yōu)勢，目前STORM在活細(xì)胞等生物體系的應(yīng)用似更領(lǐng)先一步，年僅40歲的Zhuang也因此當(dāng)選為美國科學(xué)院院士.此次遺憾地與諾獎(jiǎng)失之交臂，令人惋惜.不過，Zhuang及其發(fā)展的STORM對(duì)生物成像的重大貢獻(xiàn)是不會(huì)被人忘記的.

北京大學(xué)、中科院化學(xué)所、生物物理所、物理所等是國內(nèi)較早從事生物單分子成像和單分子研究的單位，在生物大分子相互作用、細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、蛋白質(zhì)折疊和分子馬達(dá)等領(lǐng)域的研究都取得過一些重要的突破.生物物理所和北京大學(xué)分別在發(fā)展新的PALM成像光活化蛋白以及雙分子熒光互補(bǔ)技術(shù)與PALM相結(jié)合等方面近期也有高水平的成果報(bào)道.不過總體來說，國內(nèi)在單分子研究這一前沿交叉領(lǐng)域的研究力量還較薄弱，在單分子檢測基礎(chǔ)上從事PALM和STORM的研究隊(duì)伍更小.可以說這一領(lǐng)域在國內(nèi)的發(fā)展不免受到傳統(tǒng)學(xué)科觀念、評(píng)價(jià)體系、經(jīng)費(fèi)支持力度和人才知識(shí)結(jié)構(gòu)等方面的影響.此次超分辨和單分子成像的工作獲得諾獎(jiǎng)，將對(duì)化學(xué)與生物前沿交叉領(lǐng)域的研究工作起到積極的推動(dòng)作用.

3小結(jié)與展望

自2006年以來，超分辨光學(xué)顯微鏡迅速發(fā)展，并且在生物體系的應(yīng)用中展現(xiàn)了激動(dòng)人心的前景，各種新的超分辨成像方法不斷涌現(xiàn).但是作為新興的技術(shù)，超分辨光學(xué)顯微鏡的發(fā)展歷史不長，STED PALM/STORM分別于2000年、2006年研制成功.它們應(yīng)用于生命過程分子機(jī)制的研究，還處于起步階段，其技術(shù)本身仍有待進(jìn)一步完善.例如，STED超分辨顯微鏡由于高強(qiáng)度STED光的使用，對(duì)熒光標(biāo)記分子光穩(wěn)定性的要求高，難以實(shí)現(xiàn)單分子水平檢測，還可能對(duì)生物樣品造成損傷.此外，STED顯微鏡的研制和維護(hù)通常需要具有一定光學(xué)專業(yè)技術(shù)知識(shí)的人員，影響了它的普及推廣.PALM和STORM雖然較容易實(shí)現(xiàn)，但需要進(jìn)行多次成像和擬合定位，時(shí)間分辨往往受到限制，對(duì)圖像數(shù)據(jù)處理要求高.因此仍需要進(jìn)一步發(fā)展適合于活細(xì)胞/活體動(dòng)態(tài)變化研究的高時(shí)間分辨顯納鏡.另一方面，超分辨光學(xué)顯微鏡與其他表征技術(shù)的聯(lián)用也是今后的發(fā)展方向，特別是實(shí)現(xiàn)在對(duì)分子位置進(jìn)行成像的同時(shí)，表征其化學(xué)組分和構(gòu)象.超分辨顯微技術(shù)的不斷進(jìn)步和廣泛應(yīng)用，將極大地促進(jìn)分子水平上生化反應(yīng)過程及生物大分子構(gòu)效關(guān)系的研究，為生命化學(xué)的發(fā)展翻開新的一頁.

·高被引論文摘要·

被引頻次：13

幾種超分辨率熒光顯微技術(shù)的原理和近期進(jìn)展

呂志堅(jiān)，陸敬澤，吳雅瓊，等

在生命科學(xué)領(lǐng)域，人們常常需要在細(xì)胞內(nèi)精確定位特定的蛋白質(zhì)以研究其位置與功能的關(guān)系.多年來，寬場/共聚焦熒光顯微鏡的分辨率受限于光的阿貝/瑞利極限，不能分辨出200 nm以下的結(jié)構(gòu).近年來，隨著新的熒光探針和成像理論的出現(xiàn)，研究者開發(fā)了多種實(shí)現(xiàn)超出普通共聚焦顯微鏡分辨率的三維超分辨率成像方法.主要介紹這些方法的原理、近期進(jìn)展和發(fā)展趨勢.介紹了光源的點(diǎn)擴(kuò)散函數(shù)（point spread function，PSF）的概念和傳統(tǒng)分辨率的定義，闡述了提高xy平面分辨率的方法.通過介紹單分子熒光成像技術(shù)，引入了單分子成像定位精度的概念，介紹了基于單分子成像的超分辨率顯微成像方法，包括光激活定位顯微技術(shù)（photoactivated localization microscopy，PALM）和隨機(jī)光學(xué)重構(gòu)顯微技術(shù)（stochastic optical reconstruction microscopy，STORM）.介紹了兩大類通過改造光源的點(diǎn)擴(kuò)散函數(shù)來提高成像分辨率的方法，分別是受激發(fā)射損耗顯微技術(shù)（stimulated emission depletion，STED）和飽和結(jié)構(gòu)照明顯微技術(shù)（saturated structure illumination microscopy，SSIM）.比較了不同的z軸提取信息的方法，并闡述了這些方法與xy平面上的超分辨率顯微成像技術(shù)相結(jié)合所得到的各種三維超分辨率顯微成像技術(shù)的優(yōu)劣.探討了目前超分辨率顯微成像的發(fā)展極限和方向.

超分辨率熒光顯微技術(shù)；點(diǎn)擴(kuò)散函數(shù)；PALM；STORM；STED；SSIM

來源出版物：生物化學(xué)與生物物理進(jìn)展, 2002, 36(12): 1626–1634聯(lián)系郵箱：陳良怡，chen_liangyi@yahoo.com

被引頻次：8

利用受激發(fā)射損耗(STED)顯微術(shù)突破遠(yuǎn)場衍射極限

陳文霞，肖繁榮，劉力，等

摘要：遠(yuǎn)場光學(xué)顯微鏡受衍射極限分辨率的限制，而近場光學(xué)顯微鏡南于缺乏層析能力，則無法實(shí)現(xiàn)超分辨的三維成像.研究了既可突破遠(yuǎn)場光學(xué)顯微術(shù)的衍射極限分辨率又可實(shí)現(xiàn)三維成像的成像技術(shù)——受激發(fā)射損耗（STED），綜述了STED的分辨率與STED光的光強(qiáng)、延遲時(shí)間、光斑空間分布等主要參數(shù)的關(guān)系，以及該技術(shù)的最新進(jìn)展和應(yīng)用前景.

關(guān)鍵詞：分辨率；衍射極限；受激發(fā)射損耗；時(shí)間特性；空間特性

來源出版物：激光與光電子學(xué)進(jìn)展, 2005, 42(10): 51–56

被引頻次：6

高分辨和超分辨光學(xué)成像技術(shù)在空間和生物中的應(yīng)用

姚保利，雷銘，薛彬，等

摘要：大到天文光學(xué)望遠(yuǎn)鏡觀察浩瀚的宇宙，小到光學(xué)顯微鏡探察細(xì)微的納米世界，光學(xué)成像技術(shù)在人類探索和發(fā)現(xiàn)未知世界奧秘的活動(dòng)中扮演著至關(guān)重要的角色.看得更遠(yuǎn)、看得更細(xì)、看得更清楚是人們不斷追求的目標(biāo)，傳統(tǒng)光學(xué)理論已證明所有經(jīng)典光學(xué)系統(tǒng)都是一個(gè)衍射受限系統(tǒng)，即光學(xué)系統(tǒng)空間分辨率的物理極限是由光的波長和系統(tǒng)的相對(duì)孔徑（或數(shù)值孔徑）決定的.能否突破這個(gè)極限？能否不斷提高光學(xué)系統(tǒng)的成分辨率？圍繞著這個(gè)問題，本文綜述了近年來開展的各種光學(xué)高分辨和超分辨成像技術(shù)，及其在空間探測和生物領(lǐng)域中的應(yīng)用.

關(guān)鍵詞：高分辨；超分辨；光學(xué)成像；空間光學(xué)遙感；顯微成像

來源出版物：光子學(xué)報(bào), 2010, 40(11): 1607–1618聯(lián)系郵箱：姚保利，yaobl@opt.ac.cn

被引頻次：5

HeLa細(xì)胞突起中微絲束的納米分辨熒光成像

陳丹妮，劉磊，于斌，等

摘要：在Matlab編程環(huán)境下模擬了單分子定位顯微的納米分辨成像，在傳統(tǒng)實(shí)驗(yàn)參數(shù)條件下，對(duì)不同間隔分子帶模型進(jìn)行了模擬成像.模擬結(jié)果表明，單分子定位顯微方法可以區(qū)分中心相隔20 nm的兩個(gè)分子帶.同時(shí)，也分析了不同像元大小對(duì)單分子定位精度的影響.此外，通過單分子定位顯微方法在IX71倒置熒光顯微鏡上實(shí)現(xiàn)了納米分辨，系統(tǒng)極限分辨率，即半高全寬為48 nm.在該系統(tǒng)上，獲得了HeLa細(xì)胞突起中微絲束結(jié)構(gòu)的納米分辨圖像.從重構(gòu)獲得的圖像中可以看到微絲束的直徑為75～200 nm.

關(guān)鍵詞：納米分辨；單分子定位；HeLa細(xì)胞；微絲束；細(xì)胞突起；

來源出版物：物理學(xué)報(bào), 2010, 59(10): 6948–6953聯(lián)系郵箱：牛憨笨，hhnin@5711edu. cn

被引頻次：5

生物超微弱發(fā)光的兩維探測

王蘇生，陳天明，俞信，等

摘要：本文介紹了自行研制的二套系統(tǒng)及其應(yīng)用.1.高靈敏熒光顯微鏡系統(tǒng)，該系統(tǒng)探測靈敏度達(dá)到10－6Ix量級(jí)，比普通CCD系統(tǒng)提高了104倍，系統(tǒng)用寬量程照度計(jì)對(duì)微弱光成像性能進(jìn)行了標(biāo)定，在給出細(xì)胞熒光圖像的同時(shí)，可以給出每一象元的發(fā)光強(qiáng)度，并可給出視覺更易分辨的光強(qiáng)的三維顯示和偽彩色圖像.在該系統(tǒng)上得到了分紅菌甲素在Hela細(xì)胞中的分布圖像，Hela細(xì)胞加入竹紅菌甲素后的光照損傷及抗氧化劑維生素E等對(duì)細(xì)胞的保護(hù)圖象.2.光子計(jì)數(shù)成像系統(tǒng)，該系統(tǒng)靈敏度達(dá)10-8Ix量級(jí)，可探測到單個(gè)光子及其分布，在其上得到了綠豆芽，樹葉，昆明鼠，人手及手指的超微弱發(fā)光的光子圖象，并用統(tǒng)計(jì)理論進(jìn)行了信號(hào)檢驗(yàn).

關(guān)鍵詞：納米分辨；單分子定位；HeLa細(xì)胞；微絲束；細(xì)胞突起；

來源出版物：激光生物學(xué), 1996, 5(1): 771–774

被引頻次：4

受激發(fā)射損耗熒光顯微鏡的模型設(shè)計(jì)及參量優(yōu)化

陳文霞，肖繁榮，劉力，等

摘要：受激發(fā)射損耗熒光顯微鏡利用熒光飽和激發(fā)態(tài)熒光受激損耗的非線性關(guān)系,通過限制損耗區(qū)域,可突破遠(yuǎn)場光學(xué)顯微術(shù)的衍射極限分辨力并實(shí)現(xiàn)三維成像.基于對(duì)粒子速率方程組的修正,建立了描述熒光團(tuán)各能級(jí)粒子數(shù)概率時(shí)間特性的模型,并定義了時(shí)間平均損耗效率判據(jù).采用高斯函數(shù)模擬兩束入射激光脈沖通過對(duì)模型的數(shù)值計(jì)算,模擬了激發(fā)脈沖的SIED激光脈沖的光強(qiáng)、脈沖寬度以及兩束光的延遲時(shí)間等參量與損耗效率之間的關(guān)系,并獲得了各參量的最佳值,優(yōu)化了損耗效率,為提高系統(tǒng)分辨力提供了有效的途徑.

關(guān)鍵詞：顯微；損耗效率；粒子數(shù)概率；光強(qiáng)；脈沖寬度；延遲時(shí)間；

來源出版物：光學(xué)學(xué)報(bào), 2006, 26(5): 720–725聯(lián)系郵箱：陳文霞，wenxia@siom.ac.cn

被引頻次：3

利用具有奇異非線性光學(xué)特性的納米粒子突破光學(xué)衍射極限

程亞，陳建芳

摘要：受激發(fā)射損耗熒光顯微鏡利用熒光飽和激發(fā)態(tài)熒光受激損耗的非線性關(guān)系,通過限制損耗區(qū)域,可突破遠(yuǎn)場光學(xué)顯微術(shù)的衍射極限分辨力并實(shí)現(xiàn)三維成像.基于對(duì)粒子速率方程組的修正,建立了描述熒光團(tuán)各能級(jí)粒子數(shù)概率時(shí)間特性的模型,并定義了時(shí)間平均損耗效率判據(jù).采用高斯函數(shù)模擬兩束入射激光脈沖通過對(duì)模型的數(shù)值計(jì)算,模擬了激發(fā)脈沖的SIED激光脈沖的光強(qiáng)、脈沖寬度以及兩束光的延遲時(shí)間等參量與損耗效率之間的關(guān)系,并獲得了各參量的最佳值,優(yōu)化了損耗效率,為提高系統(tǒng)分辨力提供了有效的途徑.

關(guān)鍵詞：遠(yuǎn)場光學(xué)成像；光學(xué)衍射極限；非線性光學(xué)

來源出版物：激光與光電子學(xué)進(jìn)展, 2009, (8): 35–40

被引頻次：3

超分辨成像中熒光分子定位算法性能比較

全廷偉，曾紹群，呂曉華

摘要：超分辨成像已成為活細(xì)胞結(jié)構(gòu)和功能成像的關(guān)鍵工具,熒光分子定位是超分辨成像過程中不可缺少的步驟，從超分辨成像角度研究各種熒光分子定位算法性能具有重要的意義.選擇5種典型的熒光分子定位算法：質(zhì)心法、廣義質(zhì)心法、高斯擬合、解線性方程組和極大似然法,以定位精度和定位時(shí)間來評(píng)價(jià)所選擇算法的性能.結(jié)果表明,1)高斯擬合、極大似然法和廣義質(zhì)心法能高精度對(duì)熒光分子定位,不受熒光分子所在子區(qū)域提取的影響；2)質(zhì)心法和解線性方程組法能應(yīng)用于圖像在線分析,但定位精度較低,受子區(qū)域提取影響較大；3)當(dāng)兩個(gè)熒光分子位于一個(gè)衍射斑時(shí),采用這5種算法的定位精度都會(huì)急劇下降.受激發(fā)射損耗熒光顯微鏡利用熒光飽和激發(fā)態(tài)熒光受激損耗的非線性關(guān)系,通過限制損耗區(qū)域,可突破遠(yuǎn)場光學(xué)顯微術(shù)的衍射極限分辨力并實(shí)現(xiàn)三維成像.基于對(duì)粒子速率方程組的修正,建立了描述熒光團(tuán)各能級(jí)粒子數(shù)概率時(shí)間特性的模型,并定義了時(shí)間平均損耗效率判據(jù).采用高斯函數(shù)模擬兩束入射激光脈沖通過對(duì)模型的數(shù)值計(jì)算,模擬了激發(fā)脈沖的SIED激光脈沖的光強(qiáng)、脈沖寬度以及兩束光的延遲時(shí)間等參量與損耗效率之間的關(guān)系,并獲得了各參量的最佳值,優(yōu)化了損耗效率,為提高系統(tǒng)分辨力提供了有效的途徑.

關(guān)鍵詞：遠(yuǎn)場光學(xué)成像；光學(xué)衍射極限；非線性光學(xué)

來源出版物：中國激光, 2010,37(11): 2714-2718

被引頻次：2

高分辨率光學(xué)顯微術(shù)在生命科學(xué)中的應(yīng)用

王娟，湯樂民

摘要：提高光學(xué)顯微鏡分辨率的研究主要集中在兩個(gè)方面進(jìn)行，一是利用經(jīng)典方法提高各種條件下的空間分辨率，如用于厚樣品研究的SPIM技術(shù)，用于快速測量的SHG技術(shù)以及用于活細(xì)胞研究的MPM技術(shù)等.二是將最新的非線性技術(shù)與高數(shù)值孔徑測量技術(shù)（如STED和SSIM技術(shù)）相結(jié)合.生物科學(xué)研究離不開超高分辨率顯微術(shù)的技術(shù)支撐，人們迫切需要更新顯微術(shù)來適應(yīng)時(shí)代發(fā)展的要求.近年來研究表明，光學(xué)顯微鏡的分辨率已經(jīng)成功突破200 nm橫向分辨率和400 nm軸向分辨率的衍射極限.高分辨率乃至超高分辨率光學(xué)顯微術(shù)的發(fā)展不僅在于技術(shù)本身的進(jìn)步，而且它將會(huì)極大促進(jìn)生物樣品的研究，為亞細(xì)胞級(jí)和分子水平的研究提供新的手段.

關(guān)鍵詞：光學(xué)顯微鏡；高分辨率；非線性技術(shù)；納米水平

來源出版物：南通大學(xué)學(xué)報(bào)（醫(yī)學(xué)版）, 2007, 27(1): 72–74

被引頻次：2

可逆飽和光轉(zhuǎn)移過程的熒光超分辨顯微術(shù)

郝翔，匡翠方，李旸暉，等

摘要：基于可逆飽和光轉(zhuǎn)移過程的熒光超分辨顯微技術(shù)，從原理上打破了原有的光學(xué)遠(yuǎn)場衍射極限對(duì)光學(xué)系統(tǒng)極限分辨率的限制，在生物、化學(xué)、醫(yī)學(xué)等多個(gè)學(xué)科擁有廣泛的應(yīng)用前景.回顧了近年來超分辨顯微研究的歷史，綜述了目前常見的幾種基于可逆飽和光轉(zhuǎn)移過程的熒光超分辨顯微方法，詳細(xì)描述了各自的技術(shù)特點(diǎn)并對(duì)比了其優(yōu)缺點(diǎn)，闡述了相關(guān)領(lǐng)域內(nèi)最新的研究工作進(jìn)展.

關(guān)鍵詞：顯微；超分辨；可逆飽和光轉(zhuǎn)移過程；受激發(fā)射損耗顯微鏡；基態(tài)損耗顯微鏡；飽和圖案激發(fā)顯微鏡；飽和結(jié)構(gòu)光照明顯微鏡

來源出版物：激光與光電子學(xué)進(jìn)展, 2012, 49(3): 030005聯(lián)系郵箱：匡翠方，cfkuang@zju. edu. cn

被引頻次：2

受激發(fā)射損耗顯微技術(shù)中0/π圓形相位板參數(shù)優(yōu)化

郝翔，匡翠方，王婷婷，等

摘要：采用光的矢量衍射理論，研究了0/π圓形相位板不同設(shè)計(jì)參數(shù)對(duì)于受激發(fā)射損耗（STED）顯微鏡中抑制光形成中空聚焦光斑聚焦質(zhì)量的影響，并進(jìn)行了數(shù)值模擬.相關(guān)計(jì)算結(jié)果表明，當(dāng)采用不同偏振態(tài)、不同光強(qiáng)分布的入射光作為STED光，0/π圓形相位板內(nèi)徑的合理取值范圍是不同的，而系統(tǒng)所用物鏡的數(shù)值孔徑對(duì)于內(nèi)徑的取值影響較小.在各種不同的參數(shù)組合下，使用線偏振光和圓偏振光作為入射光時(shí)，0/π圓形相位板內(nèi)徑取值范圍基本保持一致；當(dāng)入射光為徑向偏振光時(shí)，內(nèi)徑取值則相對(duì)較大；而當(dāng)使用切向偏振光時(shí)，任何取值均無法在系統(tǒng)焦平面附近得到滿意的中空聚焦光斑.

關(guān)鍵詞：受激發(fā)射損耗顯微技術(shù)；相位板；衍射理論；數(shù)值孔徑

來源出版物：光學(xué)學(xué)報(bào), 2011,31(3): 03180001聯(lián)系郵箱：匡翠方，cfkuang@zju. edu. cn

被引頻次: 2013

來源出版物: Science, 2006, 313(5793): 1642-1645.

聯(lián)系郵箱: Betzig E; betzige@janelia.hhmi.org

被引頻次: 1589

Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)

Rust, Michael J; Bates, Mark; Zhuang Xiaowei

Abstract: We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.

Keywords: Fluorescence Microscopy; Localization; resolution; superresolution

來源出版物: Nature Methods, 2006, 3(10): 793-795聯(lián)系郵箱: Zhuang Xiaowei; zhuang@chemistry.harvard.edu

被引頻次: 1216

Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy

Hell S W; Wichmann J.

Abstract: We propose a new type of scanning fluorescence microscope Capable of resolving 35 nm in the far field. We overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function. In contrast to near-field scanning optical microscopy, this method can produce three-dimensional images of translucent specimens.

來源出版物: Optics letters, 1994, 19(11): 780-782.

被引頻次: 693

Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution

Gustafsson M G L.

Abstract: Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.

Keywords: super resolution; moire; resolution extension; saturation

來源出版物: Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(37): 13081-13086.

聯(lián)系郵箱: Gustafsson M G L.; mats@msg.ucsf.edu

被引頻次: 647

Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy

Huang, Bo; Wang, Wenqin; Bates, Mark; et al.

Abstract: Recent advances in far- field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three- dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high- precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.

Keywords: Fluorescence nanoscopy; Axial resolution; localization; tracking; probes; endocytosis; precision; molecules; emitters; cells

來源出版物: Science, 2008, 319(5864): 810-813聯(lián)系郵箱: Zhuang Xiaowei; zhuang@chemistry.harvard.edu

被引頻次: 592

Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy

Gustafsson M G L

Abstract: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.

Keywords: actin; cytoskeleton; fluorescence microscopy; interference; lateral resolution; moire microscopy; optical transfer function; patterned excitation; resolution; structured illumination; super-resolution; wide-field microscopy

來源出版物: Journal of Microscopy, 2000, 198(2): 82-87.

被引頻次: 439

Multicolor super-resolution imaging with photo-switchable fluorescent probes

Bates, Mark; Huang, Bo; Dempsey, Graham T; et al.

Abstract: Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with a spatial resolution of 20 to 50 nanometers. Multicolor super-resolution imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photo-activation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, we demonstrate multicolor imaging of DNA model samples and mammalian cells with 20-to30-nanometer resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale.

Keywords: single molecules; energy-transfer; quantum dots; microscopy; localization; cells; resolution; nanoscopy

來源出版物: Science, 2007, 317(5845): 1749-1753聯(lián)系郵箱: Zhuang Xiaowei; zhuang@chemistry.harvard.edu

被引頻次: 401

STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis

Willig, KI; Rizzoli, SO; Westphal, V; et al.

Abstract: Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles and released by exocytosis upon activation. The vesicle membrane is then retrieved by endocytosis, and synaptic vesicles are regenerated and re-filled with neurotransmitter (1). Although many aspects of vesicle recycling are understood, the fate of the vesicles after fusion is still unclear. Do their components diffuse on the plasma membrane, or do they remain together? This question has been difficult to answer because synaptic vesicles are too small (similar to 40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. Here we use stimulated emission depletion (STED) (2) to reduce the focal spot area by about an order of magnitude below the diffraction limit, thereby resolving individual vesicles in the synapse. We show that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated. This suggests that at least some vesicle constituents remain together during recycling. Our study also demonstrates that questions involving cellular structureswith dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light.

Keywords: frog neuromuscular junction; hippocampal synapses; fluorescence microscopy; stimulated-emission; endocytosis; resolution; membrane; transmitter; release; domain

來源出版物: Science, 2006, 440(7086): 935-939聯(lián)系郵箱: Jahn, R; rjahn@gwdg.de

被引頻次: 383

Fluorescent probes for super-resolution imaging in living cells

Fernandez-Suarez, Marta; Ting, Alice Y.

Abstract: In 1873, Ernst Abbe discovered that features closer than similar to 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.

Keywords: hotoactivated localization microscopy; optical reconstruction microscopy; diffraction resolution barrier; small-molecule probes; live cells; stimulated-emission; quantum dots; in-vivo; sted microscopy; near-field

來源出版物: Nature Reviews Molecular Cell Biology, 2008, 9(12): 929-943聯(lián)系郵箱: Fernandez-Suarez, Marta; artafs@alum.mit.edu

被引頻次: 363

Super-Resolution Fluorescence Microscopy

Huang, Bo; Bates, Mark; Zhuang Xiaowei

Abstract: Achieving a spatial resolution that is not limited by the diffraction of light, recent developments of super-resolution fluorescence microscopy techniques allow the observation of many biological structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes.

Keywords: diffraction limit; fluorescence imaging; fluorescent protein; live-cell imaging; photoswitchable dye; single molecule

來源出版物: Annual Review of Biochemistry, 2009, 78: 993-1016聯(lián)系郵箱: Huang, Bo; bohuang@fas.harvard.edu

·推薦論文摘要·

Three dimensional multimodal sub-diffraction imaging with spinning-disk confocal microscopy using blinking/fluctuation probes

X. Chen; Z. Zeng; H. Wang; et al.

Abstract: Previous localization super-resolution techniques cannot offer three-dimensional imaging easily. Here we present a three-dimensional multimodal sub-diffraction imaging with spinning-disk (SD) confocal microscopy, 3D-MUSIC, which not only takes fully advantages of spinning-disk confocal microscopy, such as fast imaging speed, high signal-to-noise ratio, optical-sectioning capability, but also extends its spatial resolution limit along all three dimensions. Both axial and lateral resolution can be improved simultaneously by virtue of the blinking/fluctuation nature of the modified fluorescent probes, exemplified by the quantum dots (QDs). Further, dual-modality super-resolution image can be obtained, by super-resolution optical fluctuation imaging (SOFI), and bleaching/blinking assisted localization microscopy (BaLM). Therefore, fast super-resolution imaging can be achieved with SD-SOFI by only capturing 100 frames, yet a high-resolution imaging can be provided with SD-BaLM.

Keywords: multi-modality; super-resolution microscopy; three-dimensional; spinning-disk confocal

來源出版物：Nano Research, 2015, doi:10.1007/s12274–12015–10736–12278

聯(lián)系郵箱：XI Peng; xipeng@pku.edu.cn

Fast Super-Resolution Imaging with Ultra-High Labeling Density Achieved by Joint Tagging Super-Resolution Optical Fluctuation Imaging

Z. Zeng; X. Chen; H. Wang; et al.

Abstract: Previous stochastic localization-based super-resolution techniques are largely limited by the labeling density and the fidelity to the morphology of specimen. We report on an optical super-resolution imaging scheme implementing joint tagging using multiple fluorescent blinking dyes associated with super-resolution optical fluctuation imaging (JT-SOFI), achieving ultra-high labeling density super-resolution imaging. To demonstrate the feasibility of JT-SOFI, quantum dots with different emission spectra were jointly labeled to the tubulin in COS7 cells, creating ultra-high density labeling. After analyzing and combining the fluorescence intermittency images emanating from spectrally resolved quantum dots, the microtubule networks are capable of being investigated with high fidelity and remarkably enhanced contrast at sub-diffraction resolution. The spectral separation also significantly decreased the frame number required for SOFI, enabling fast super-resolution microscopy through simultaneous data acquisition. As the joint-tagging scheme can decrease the labeling density in each spectral channel, thereby bring it closer to single-molecule state, we can faithfully reconstruct the continuous microtubule structure with high resolution through collection of only 100 frames per channel. The improved continuity of the microtubule structure is quantitatively validated with image skeletonization, thus demonstrating the advantage of JT-SOFI over other localization-based super-resolution methods.

來源出版物：Scientific Reports, 2015, 5 Article number: 8359

聯(lián)系郵箱：XI Peng; xipeng@pku.edu.cn

Hundred-Thousand Light Holes Push Nanoscopy to go Parallel

Chen, Xuanze; Xi, Peng

Abstract: With the application of the elements of all major super-resolution techniques including stimulated emission depletion, structure illumination microscopy, and photo-activated localization microscopy, the incoherent crossed standing-wave microscopy achieves parallel super-resolution imaging.

Keywords: optical nanoscopy; STED; SIM; PALM

來源出版物: Microscopy Research and Technique, 2015, 78(1): 8–10

聯(lián)系郵箱: Xi, Peng ; xipeng@pku.edu.cn

Sub-diffraction imaging of nitrogen-vacancy centers in diamond by stimulated emission depletion and structured illumination

Yang, Xusan; Tzeng, Yan-Kai; Zhu, Zhouyang; et al.

Abstract: Stimulated emission depletion (STED) and structured illumination (SIM) are two commonly used techniques for super-resolution imaging. However, the performance of these two techniques has never been quantitatively compared side-by-side. Taking advantage of the non-photobleaching characteristic of NV centres in fluorescent nanodiamond (FND), we performed a comparative study for the resolution of these two methods with 35 nm FNDs at the single particle level, as well as with FND grown in bulk diamond material. Results show that STED provides more structural details, whereas SIM provides a larger field of view with a higher imaging speed. SIMmay induce deconvolution smooth and orientational artifacts during its post-processing.

Keywords: fluorescent nanodiamonds; unlimited resolution; state depletion; STED microscopy; color-centers; superresolution; limit

來源出版物: RSC Advances, 2014, 4(22): 11305–11310

聯(lián)系郵箱: Xi, Peng; xipeng@pku.edu.cn

Microscopy and its focal switch

Hell S W

Abstract: Until not very long ago, it was widely accepted that lens-based (far-field) optical microscopes cannot visualize details much finer than about half the wavelength of light. The advent of viable physical concepts for overcoming the limiting role of diffraction in the early 1990s set off a quest that has led to readily applicable and widely accessible fluorescence microscopes with nanoscale spatial resolution. Here I discuss the principles of these methods together with their differences in implementation and operation. Finally, I outline potential developments.

來源出版物: Nature Methods, 2009, 6(1): 24–32.

Nanoscopy with more than 100,000'doughnuts

Chmyrov A; Keller J, Grotjohann T; et al.

Abstract: We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 mm × 100 mm-sized fields of view in <1 s using 116000 such doughnuts.

Keywords: structured-illumination microscopy; fluorescence microscopy; optical resolution; limit; protein; breaking; GFP

來源出版物: Nature Methods, 2013, 10(8): 737–740

聯(lián)系郵箱: Hell S W; shell@gwdg.de

Toward fluorescence nanoscopy

Hell S W

Abstract: For more than a century, the resolution of focusing light microscopy has been limited by diffraction to 180 nm in the focal plane and to 500 nm along the optic axis. Recently, microscopes have been reported that provide three- to sevenfold improved axial resolution in live cells. Moreover, a family of concepts has emerged that overcomes the diffraction barrier altogether. Its first exponent, stimulated emission depletion microscopy, has so far displayed a resolution down to 28 nm. Relying on saturated optical transitions, these concepts are limited only by the attainable saturation level. As strong saturation should be feasible at low light intensities, nanoscale imaging with focused light may be closer than ever.

Keywords: emission depletion microscopy; diffraction resolution limit; stimulated-emission; axial resolution; light-microscopy; 4pi-confocal microscopy; 3-dimensional images; lateral resolution; opposing lenses; state-depletion

來源出版物: Nature Biotechnology, 2003, 21(11): 1347–1355.

聯(lián)系郵箱: Hell S W; hell@nanoscopy.de

Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution

Chen B C; Legant W R; Wang K; et al.

Abstract: Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

來源出版物: Science, 2014, 346(6208): 439

Nanoscopy in a living mouse brain

Berning S; Willig K I; Steffens H; et al.

Abstract: We demonstrated superresolution optical microscopy in a living higher animal. Stimulated emission depletion (STED) fluorescence nanoscopy reveals neurons in the cerebral cortex of a mouse with <70-nanometer resolution. Dendritic spines and their subtle changes can be observed at their relevant scales over extended periods of time.

Keywords: spines; GFP

來源出版物: Science, 2012, 335(6068): 551–551

聯(lián)系郵箱: Willig K I; kwillig@gwdg.de

Video-rate far-field optical nanoscopy dissects synaptic vesicle movement

Westphal V; Rizzoli S O; Lauterbach M A; et al.

Abstract: We present video-rate (28 frames per second) far-field optical imaging with a focal spot size of 62 nanometers in living cells. Fluorescently labeled synaptic vesicles inside the axons of cultured neurons were recorded with stimulated emission depletion (STED) microscopy in a 2.5-micrometer by 1.8-micrometer field of view. By reducing the cross-sectional area of the focal spot by about a factor of 18 below the diffraction limit (260 nanometers), STED allowed us to map and describe the vesicle mobility within the highly confined space of synaptic boutons. Although restricted within boutons, the vesicle movement was substantially faster in nonbouton areas, consistent with the observation that a sizable vesicle pool continuously transits through the axons. Our study demonstrates the emerging ability of optical microscopy to investigate intracellular physiological processes on the nanoscale in real time.

Keywords: fluorescence microscopy; nerve-terminals; STED microscopy; resolution; boutons; synapses; dynamics; recovery; mobility; pools

來源出版物: Science, 2008, 320(5873): 246–249

聯(lián)系郵箱: Hell S W; shell@gwdg.de

Imaging intracellular fluorescent proteins at nanometer resolution

Betzig E; Patterson G H; Sougrat R; et al.

We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to similar to 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method - termed photoactivated localization microscopy - to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

structured-illumination microscopy; diffraction limit; living cells; localization; spectroscopy; molecules