答老婆的疑惑

爱尔美差再次说：大师，那么关于婚姻又是如何

他回答道：
你们一同出生，而且永远相伴。
当死亡白色的羽翼掠过你们的生命时，你们将会合一。
是的，甚至在神静默的记忆中，你们也在一起。
但要在你们的依偎里留有余地，
让天堂的风儿在你们中间舞蹈。

彼此相爱，却不要让爱成为束缚：
让它成为涌动在你们魂灵岸间的大海。
要斟满每个人的杯盏却不是从一只中啜饮。
将你的面包送给另外一个，而不是从同一片上分食。
一起快乐的唱歌跳舞，但要让你们中的每个人都能独立。
即使是竖琴上的琴弦也是独立的，尽管它们在同一首乐曲中震颤。

给出你的心灵，但不是要交给对方保留。
因为只有生活之手才能容纳你们的心灵。
要站在一起却不能靠得太近：
因为庙堂里的廊柱是分开而立，
而橡树和松柏也不能在彼此的树荫里生长。

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很多年前看到纪伯伦这样写，我真的很不明白，但是现在我开始觉得他的睿智了。

老婆穿新衣服的可爱姿态

又地震了一次

刚刚又地震了一次，连我这样迟钝的人也感觉到了！唉！多事之秋！

地震了

地震了，在床上失眠，感觉到床在抖，门在晃！第一反应是给刘莎打个电话，一看表，两点多了，赶快穿好衣服去实验室，很多人已经到园区里了，给刘莎发了短信，如果有什么不测，祝福她幸福快乐健康！

倒板子的小男孩(转载)

在一个下着雪的圣诞节前夜，一个饥寒交迫的小男孩，手里拿着一个破旧的三角瓶，在
大街上走着。
他敲开一家大门：“先生，你有板子要倒么？”
结果给踹了出去：“没有！扫兴。”
再一次：“女士，你有不愿做的板子要我帮忙么？一美分2板。”
“谁还倒板子啊，都在做液体培养基。”
可怜的小男孩就这样不断的被拒绝。
他实在走不动了，疲乏地缩在一个墙角里。他不敢回家，因为他没有倒掉一个
板子。家里而且也很冷，风可以从许多地方刮进屋子里来。
他冻得发抖，需要温暖。哪怕一个板子的温暖也好。
他的一双手 几乎冻僵了。太冷了。他决定倒一个板子。
“哧！”板子倒出来了，象一瓶温暖、光明的LB，小男孩觉得象坐在火炉旁一样
。火烧得那么欢，那么暖，那么美！这是怎么回事呢？
火焰突然熄灭了，LB也不见了。他坐在那儿，手中只有做出的板子。
他又倒了一次板子，变成一朵粉红色的光焰。
他发现自己坐在一根美丽的亲和柱下，比中午见到的那根亲和柱还要大，还要
美丽。
它的管壁里有几千万个beads。小男孩把双手伸过去，板子又熄灭了。几千万个
beads都变成了明亮的星星。这些星星中有一颗落下来，在天空中划出一条长长的亮光
。 他又倒了一次板子。
啊，火光中出现了他日日夜夜思念的老沃森，他扑进老沃森的怀抱。
“沃森！”小男孩叫起来。“请把我带走吧！带到那没有寒冷（冷泉港到底冷不
冷呢），没有饥饿的地方。我知道，这块板子一熄灭，你就会不见了。就象那温暖的LB
，那美丽的confocal，那幸福的亲和柱一样，我什么也看不见了。”
于是，小男孩把所有的板子全倒了，因为他非常想把沃森留住。
板子发出更加强烈的光芒，照得周围比白天还要明亮，沃森是那样慈祥，他把
小男孩抱起来了，他们在光明和幸福中飞走了。越飞越高，真的到了没有寒冷，没有饥
饿的地方。
新年的早晨，人们看到小男孩仍坐在墙角里，他双颊通红，脸上带着幸福的微
笑。可是，他已经死了，冻死在圣诞节的夜晚，他手里仍握着一打倒好的板子。

《粮食危机》读后感

粮食危机

毫无疑问，全球的粮食供求是不平衡的，否则 粮食危机就不能成立，仔细考虑下，我国吃饱肚子的日子没有多久，刚刚摆脱了饥荒的中国目却要面临的经济危机和粮食涨价。我想这大概就是国家大力扶持农业的 原因，我们的粮食需求在增加，无论是对粮食的量和质的需求都远远没有饱和，但是我们可供耕种的土地却在减少，农业人口数目也在下降。家庭联产承包责任制的 不利于土地的集中，因此导致了中国农业的发展速度缓慢，但是如果允许土地集中，国家则一时无法安置如此多的无生产资料的农民。而现代化农业本身就是劳动力 的解放和机器代替人的过程，因此，从这点出发，我认为，我国粮食的产量在最近年内可能不会有质的变化。分析世界粮食的供需非常困难，因为粮食已经被作为一 种战略资源和武器，在国家与国家的博弈中扮演重要的一部分。但最明显的势态是目前发达国家都是粮食输出国，贫穷国家主要是粮食短缺国。粮食的产能其实是一 个国家经济、技术、权利的综合体现。既然粮食成为了战略资源，因此其供求就不会完全按照市场经济规律运行，美国宁愿用一个州的玉米去做生物酒精也不会便宜 卖给非洲。从这点看待，我国的粮食问题外交是一致的，只有靠自己。更可怕的问题是，我国从封建农业大国到现在的要建设成为的现代化工业大国之间的跃进过于 激进，使得一方面国家喊着农业很重要，一方面对农业的投资有滞后于对工业的投资，普通民众对农业的认识落后，种地被认为是一种低级工种。国家的土地政策对 耕地的保障也是有法无力，有名无实。因此，我国不但面临粮食危机，而且将会长期面临这种危机，我们粮食储备如果无法维持一个粮食丰收周期的话，就不能忽视 粮食问题。

种子从哪里来

袁隆平院士的杂交水稻使得中国粮食产量有了质的飞跃，但是杂交育种周期非常长而且受到天然遗传资源的限制，我想这个领域已经几乎无法再发生质的飞跃，甚至是要的到稍微显著的量的提升也很难。美国至少在40年前就开始研究转基因作物，包括美国农业部和以洛克菲勒基金会为首的私人基金在转基因作物方面投入了大量的经费。全球四大综合农业公司有三家在美国（孟山都、陶氏化学、先锋良种），而且洛克菲勒基金资助的菲律宾水稻研究所拥有全球最大的水稻种质资源库。早在上世纪90年， 美国的很多生物技术公司和农业公司就在全球处心积虑搜集良种，进行改造。而从老布什到小布什的美国历届总统对转基因食品在法律和政策上敞开大门。美国法律 规定“基因改造作物和天然作物实质相同”，并且允许转基因作物可以不用特别标识就出售，这助长了各生物技术公司对转基因种子的开发。孟山都、陶氏化学、先 锋良种有非常强大的政治背景，无一不和五角大楼有密切的联系，孟山都发明了越战时期广泛使用的橘剂、陶氏化学发明了固体汽油燃烧弹、杜邦则是石油化工和塑 料纤维的巨头。这三家公司在美国政府的帮助下，将其种子迅速散布世界。其中以大豆为例，孟山都在巴西和阿根廷推广其转基因大豆的时候，采取了微软进入中国 的策略。首先允许走私转基因种子或者馈赠，然后在通过国家施压，允许该国解禁销售和出口转基因粮食，其次再通过国家和WTO以 知识产权为借口收取高额专利费用。孟山都的转基因大豆是导入了抗药性的基因，再通过喷洒其专利的农药抑制杂草。这种转基因大豆的推广，导致种植者在种子和 农药方面对孟山都的依赖，由于该农药无法迅速分解，使用过的该农药的土地无法种植传统的大豆。而且杂草对该药物抗性也在逐渐增强，需要的农药剂量也在加 大，种植成本不断攀升。孟山都的大豆，如鸦片般控制了巴西和阿根廷的农业。当然，这个只是比较极端的例子，从这点我们可以看到，种子的开发将是未来农业科 学研究的重点。然而，种子的开发需要丰富的种质资源，我不清楚我国对种质资源的保护和重视是力度足够。但从云南在中国植物资源的地位来看，我们绝对具有得 天独厚的优势。因此，我想我们要开发良种，因该走这样的一条路：收集种质资源（通过合作可以实现）——鉴定有价值基因（目前正在做）——开发高效的转基因 技术特别是多基因转入系统——开发高效筛选系统——保护机制（不稳定遗传，让转基因种子无法再次种植）。

机遇和困境

对于我国研究人员，转基因作物最大的机遇就 是庞大的市场和相对较小的国内竞争环境。首先我国需要粮食供养的人口巨大，任何一个小有成效的转基因作物一旦推广，将带动巨大的经济利益。相对于欧美国 家，特别是美国和瑞士，我国正真有能力从事转基因植物研究的企业和科研机构依然不多，特别是在成果转化方面的力度远远不及欧美，这为我们提供了很大的生存 空间，只要快速切入，迅速建立技术和品台。从国家的政策和法规来看，对转基因作物研究比较扶持，而且市场上没有明确规定转基因作物的性质，因此转基因作物 比较容易由实验走向生产。此外，我国可利用的种质资源比较丰富，杂交水稻研究非常成熟，转基因棉花已经有很大的种植面积，也就说说上游产业和下游产业都有 一定的基础。目前的困境是转基因作物的安全问题，这点无论是国家和民众都会存在很大的顾虑，也是目前阻碍转基因作物推广的最大障碍。如果要推出自己的转基 因种子，我想因该配套的对其生物安全性做一个评估，一方面减少非议，一方面提升竞争力。民众对转基因作物的接受程度远远不像国家那样热情，但是这只是时间 问题，国家可以通过提高粮价将粮食危机的气氛烘托的更浓，这样农民就有可能和有能力接受良种。普通消费者对转基因作物依然持观望态度，科学界在神话转基因 作物性能的同时，民间特别是是欧美国家的民间组织却不断丑化转基因。如何真确引导农民、消费者、媒体等接受转基因作物将面临很大的挑战。其次的壁垒就是如 何与国际农业巨头的竞争。这些公司都与政府关系暧昧，渗透力极强，如果我们的研究与其构成竞争，他们会用知识产权作为借口前方百计打压，一旦形成这种局 面，我们唯一能做的就是找棵“大树”，然后等待羽翼丰满时再与其正面交锋。

生物技术与农业

农业为通过培育动植物生产食品及工业原料的产业。因此放开了思考农业，我觉得转基因粮食只是很小的一部分。如果政府在转基因作物上无法放开的话，我们还有其他的路可以走：1、基因改造的牧草。既然国家对人吃的还犹豫不决，我们可以去考虑提供动物饲料。由于土地和资源的限制，国内畜牧业只能通过集约化养殖，这样的话动物饲料的需求就很大，如果在这方面有所突破，我相信可以夹缝中求生存。2、转基因家蚕。这个不会涉及食品安全的问题，因此门槛较低，而且我们有比较好的果蝇遗传基础，做鳞翅目昆虫的基因操作应该容易上手。3、抗菌肽应用。目前全球70%的抗生素都应有于动物。但是这样会使得畜牧业成本过高和品质下降。例如在奶牛的乳腺表达抗菌肽，这样就不需要在牛奶中添加高计量的抗生素。

QQ视频收集的表情

老板给我的回信

前几天和老板发生了分歧，受到批评，老板回信如下：

人 总是要前进的。不经历一些事情，就不能好好前进。对外交往说话也需要试错。最后学会根据不同场合拿捏分寸，在实事求是和忽悠之间，在豪气和可行性之间，在 夸大一点和不带来负面影响之间要根据所对的人、所对的事取得好平衡。一般而言，要对所有涉及的各方都是正面积极的方面说就比较好。比如这次这个，只是惯例 的知情模式，不涉及多大的困难和面子，你只要告诉他们应该没问题，但跟老板说一下就好了，比如我们给饶毅lab要东西，肯定我也得跟饶毅先打个招呼，此乃惯例，不涉及你说No的问题。

你尽力而为是好的，人只要尽力而为，做不到说No也 没有丝毫内疚。总之，人要在试错中前进，不完全尽力，很多时候呢又会丧失机会。很多人其实就是事不关己的态度丧失很多机会的。所以你勇往直前做事的方式是 非常重要的优点。我也经常跟他们说，没有气势是做不了大事的，仅就试验而言也会缓慢。看文献和努力做试验都很重要，但付诸行动比空谈更有效。即便做试验也 需要看文献不是？实验也是学习进步的一种，不是什么技工之类的，事实上只是技工是不能trouble shooting的。你能把这些挑战性和创新性的实验做好，我也不太担忧你在科学上能否做好。只是男子汉，要不断提高自己，全方位地前进。

皇后的新装

委屈了好多年，终于爆发了，刘莎有了很多新衣服，特此发图庆祝！还有双PUMA的七夕特别款！

Slow shipping hobbles Chinese science

Shanghai
Earlier this year, a researcher in Shanghai requested antibodies from a collaborator at Harvard University. Working with customs and shippers to arrange the transfer, including extra dry ice to keep the materials cool, cost US$1,500 and took well over a month. "It was very tedious and very expensive," says the scientist, who did not want to be identified.

Yet this experience is speedy for China, where reagents, biological materials and other laboratory materials can take weeks or months longer to obtain than in Europe or the United States. With few local suppliers, researchers in China rely on deliveries from overseas. But the shipments regularly get hung up in customs and in a network of distributors, and product quality is sometimes compromised by the tortuous route.

The reagents problem could crimp China's ambitious plans to become one of the top five biotechnology powers by 2020. The Chinese can build a five-star hotel, a highway or a research institute before they can get a mouse from Boston to Beijing. "If a researcher from the United States and I have the same idea at the same time, I won't even try," says Duanqing Pei, director of the Guangzhou Institute of Biomedicine and Health. "I'd never be able to keep up."

Chinese scientists like to say that a 10-million renminbi (US$1.5-million) grant in China will go as far as a $10-million grant in the United States, because labour and overheads are cheaper. But reagents remain some of the biggest expenses — and hassles.

This year's Beijing Olympics made a tough situation even worse. Because of tightened security restrictions, the China branches of Sigma Aldrich, one of the world's biggest suppliers of laboratory reagents, stopped importing from US plants between early August and mid-September. As a result, its China sector was the only one that failed to meet target goals for the company for the third quarter of 2008. Employees at Stealth Peptides International had to drive for two days to bring peptides from its Shanghai headquarters to a contract laboratory in Shenzen, says chief executive Dajun Yang. In Beijing, neuroscientist Minmin Luo of the National Institute of Biological Sciences couldn't get oxygen in compressed tanks, or the radioactive probes to do Southern blots. "It was very bizarre," he says.

Now things are mostly back to normal — that is, normal for China. Researchers share stories of waiting a year for a shipment of basic materials. Yuqiang Ding of the Institute of Neuroscience in Shanghai says that part of the problem is that distributors pile up orders and send them all together to reduce shipment fees. Animals, cell lines or reagents with bovine serum are particularly slow to arrive because of strict regulations and confusion at customs. Knockout mice take six months to arrive from most places, says Luo. Shipping from the United States is often a little faster — about five months — because the greater number of orders has made the paperwork more routine.

The delays mean that interesting leads turned up during experiments frequently have to wait. "Sometimes you just can't plan ahead," says Luo. He often ends up saving the exciting parts of the experiment for later and doing controls — experiments that might not even be needed if the more critical parts don't turn out — until he can get what he needs.

Some researchers have to make substitutions using less-than-ideal alternatives. Luo has applied several times for the right to obtain ketamine from Beijing manufacturers, to no avail. The drug, which can be used recreationally, is tightly controlled in China. Instead he uses phenobarbiturate, which is more difficult to dose than ketamine. "We end up killing some mice," he says. "The experiments go much slower."

Multinational pharmaceutical companies and contract research organizations, which have poured hundreds of millions of dollars into research units in China over the past few years (see Nature 455, 1168–1170; 2008), are not immune to the shortages. Sometimes they innovate around the problem. In one instance, Shanghai-based researchers at GlaxoSmithKline took just a week to express and purify more than 10 milligrams of a protein for assay. It would have cost more than $200,000 and taken at least a month if ordered from a US vendor, says Jingwu Zang, head of the research centre.

But that only works for some reagents. GlaxoSmithKline wants to use its influence and size to sort out the rest. The company intends to increase its staff in China from 200 to 1,000 over the next decade and is pushing local vendors to stock reagents nearby, and others to expedite shipping. It has paid off: some shipments are down to seven days from the United States and one day within China. Zang says he also plans to collaborate with other pharmaceutical companies, many of which are now represented in Shanghai, to establish a centralized local stock facility for all users.

In Guangzhou, Anlong Xu, the vice-president for research at Sun Yat-Sen University, is working to build expertise at the custom houses and exchange agencies that must test and approve imported reagents. "They often don't know how to test the materials, and so they leave them overnight", by which time the dry ice has gone, Xu says. "We can help them." But pulling together the regulatory authorities is a headache; Xu has dedicated one assistant entirely to the task. Luo suggests a quicker alternative: a fast-track system that would accelerate shipments for accredited research institutions.

None of these innovations can arrive too soon for researchers. "Sometimes I feel so frustrated I wonder if I can still do research here," says Luo, who spent nine years in the United States. "It just goes so much faster there."

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终于说了我想说的了！

老哥俩的巨作！

Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

Eric Betzig,^1,2* George H. Patterson,³ Rachid Sougrat,³ O. Wolf Lindwasser,³ Scott Olenych,⁴ Juan S. Bonifacino,³ Michael W. Davidson,⁴ Jennifer Lippincott-Schwartz,³ Harald F. Hess^5*

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 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.

¹ Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147, USA.
² New Millennium Research, LLC, Okemos, MI 48864, USA.
³ Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development (NICHD), Bethesda, MD 20892, USA.
⁴ National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310, USA.
⁵ NuQuest Research, LLC, La Jolla, CA 92037, USA.

Transfected cells expressing fluorescent proteins (1) contain information that is accurate at the molecular level about the spatial organization of the target proteins to which they are bound. However, the best resolution that can be obtained by diffraction-limited conventional optical techniques is coarser than the molecular level by two orders of magnitude. Great progress has been made with superresolution methods that penetrate beyond this limit, such as near field (2), stimulated emission depletion (3), structured illumination (4, 5), and reversible saturable optical fluorescence transitions microscopy (6), but the goal remains a fluorescence technique capable of achieving resolution closer to the molecular scale.

Early results (7) in single-molecule microscopy (8) and the spatiospectral isolation of individual exciton recombination sites in a semiconductor quantum well (9) led to a proposal for a means of molecular resolution fluorescence microscopy a decade ago (10). In brief, individual molecules densely packed within the resolution limit of a given instrument [as defined by its point-spread function (PSF)] are first isolated from one another on the basis of one or more distinguishing optical characteristics. Each molecule is then localized to much higher precision by determining its center of fluorescence emission through a statistical fit of the ideal PSF to its measured photon distribution (Fig. 1). When the background noise is negligible compared with the molecular signal, the error in the fitted position is _{x, y} s/(N), where s is the standard deviation of a Gaussian approximating the true PSF (200 nm for light of wavelength = 500 nm) and N is the total number of detected photons (11, 12). Given that it is possible to detect many more than 10⁴ photons from a single fluorophore before it bleaches, single-molecule localization to nearly 1-nm precision has already been demonstrated (13–15) and applied to studies of molecular motor dynamics (13).

Fig. 1. The principle behind PALM. A sparse subset of PA-FP molecules that are attached to proteins of interest and then fixed within a cell are activated (A and B) with a brief laser pulse at act = 405 mm andthenimagedat exc = 561 mm until most are bleached (C). This process is repeated many times (C and D) until the population of inactivated, unbleached molecules is depleted. Summing the molecular images across all frames results in a diffraction-limited image (E and F). However, if the location of each molecule is first determined by fitting the expected molecular image given by the PSF of the microscope [(G), center] to the actual molecular image [(G), left], the molecule can be plotted [(G), right] as a Gaussian that has a standard deviation equal to the uncertainty x,y in the fitted position. Repeating with all molecules across all frames (A' through D') and summing the results yields a superresolution image (E' and F') in which resolution is dictated by the uncertainties x,y as well as by the density of localized molecules. Scale: 1 x 1 µm in (F) and (F'), 4 x 4 µm elsewhere. [View Larger Version of this Image (56K GIF file)]

Multiple emitters within a single diffraction-limited region (DLR) have been isolated from one another by either spectral (15, 16) or temporal means, the latter exploiting the photobleaching (14, 17) or blinking (18) of the emitters. However, the number of emitters isolated per DLR (typically 2 to 5) has been too small to give resolution within the DLR that is comparable to existing superresolution techniques, and it is far from the molecular level. Here, we developed a method for isolation of single molecules at high densities (up to 10⁵/µm²) based on the serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivatable fluorescent protein (PA-FP) molecules (19–24) within a sample. We then applied the method to image specific target proteins in thin (50- to 80-nm) sections and near the surfaces of fixed cultured cells, resolving the most precisely localized molecules therein at separations (10 nm) approaching the molecular level.

The method and typical data subsets are shown in Fig. 1. Cultured mammalian cells expressing PA-FP–tagged target proteins were prepared by transient transfection, fixed, and processed on cover slips either as whole cells or in cryosections cut from a centrifuged pellet of cells (25). Such cover slips were then placed in a custom microscope chamber (fig. S1) designed to minimize thermal and mechanical drift (fig. S2) (25). They were continuously excited by a laser at a wavelength (_exc = 561 nm) near the excitation maximum of the activated form of the expressed PA-FPs. Finally, to minimize both autofluorescence and detector noise, they were imaged by total internal reflection fluorescence (TIRF) microscopy (13, 26) onto an electron-multiplying charge coupled device (EMCCD) camera that can detect single photons.

Initial image frames typically consisted of sparse fields of individually resolvable single molecules on a weaker background presumably dominated by the much larger population of PA-FP molecules still in the inactivated state. When necessary, excitation and thus bleaching was maintained until such sparse fields were obtained. Additional image frames were then captured until single-molecule bleaching resulted in a mean molecular separation considerably larger than that required for isolation (Fig. 1, A and C). At that point, we applied a light pulse from a second laser at a wavelength (_act = 405 nm) capable of activating the remaining inactive PA-FPs, at a duration and intensity chosen so that the overall density of activated PA-FPs was increased back to a higher, but still resolvable, level (Fig. 1, B and D). This process of photoactivation, measurement, and bleaching was then repeated (movie S1) for many cycles over 10⁴ to >10⁵ image frames (depending on the expression level and spatial distribution of the PA-FPs) until the population of inactivated, unbleached molecules was depleted. At typical frame rates of 0.5 to 1.0 s, between 2 and 12 hours were required to acquire a complete image stack that could be distilled to a single superresolution image containing 10⁵ to >10⁶ localized molecules. We continued to explore methods (such as brighter molecules, higher excitation power, and higher activation density) to speed this process.

When the xy frames from any such image stack are summed across time t, the molecular signals overlap to produce a diffraction-limited image (Fig. 1, E and F) similar to that obtained by conventional TIRF, in which all molecules emit simultaneously (fig. S3). However, when the data are plotted in a multidimensional volume xyt (Fig. 1, center), the signal from each molecule m is uniquely isolated and can be summed at each pixel and across all of the frames in which it appears. This result (Fig. 1G, left) is then fitted using a robust nonlinear least squares algorithm to an assumed Gaussian PSF of free center coordinates x_o, y_o (Fig. 1G, center) (25), yielding coordinates x_m, y_m for the location of the molecule, with a position uncertainty (_x,y)_m. Finally, each molecule is rendered in a new xy frame as a Gaussian of standard deviation (_x,y)_m (rather than the much larger standard deviation s of the original PSF), centered at x_m, y_m (Fig. 1, G, right, and A' to D') and normalized to unit strength when integrated over all xy space. Thus, the superresolution image obtained by summing the rendered Gaussians associated with all localized molecules in the original image stack (Fig. 1, E' and F') provides a probability density map where brightness is proportional to the likelihood that a PA-FP molecule can be found at a given location.

This technique, termed photoactivated localization microscopy (PALM), is capable of resolving the most precisely localized molecules at separations of a few nanometers. These represent the very brightest emitters (the much larger population of all isolated molecules exhibits a much broader range of photon counts; fig. S4). Thus, when rendering PALM images, a fundamental trade-off exists: Including fewer, but brighter, molecules results in higher localization and crisper images, but at a reduced molecular density giving less complete information about the spatial distribution of the target protein (fig. S5). Both parameters—localization precision and the density of rendered molecules—are key to defining performance in PALM. Their specific values for the images in Figs. 2, 3, 4 are given in table S1.

Fig. 2. Comparative summed-molecule TIRF (A) and PALM (B) images of the same region within a cryo-prepared thin section from a COS-7 cell expressing the lysosomal transmembrane protein CD63 tagged with the PA-FP Kaede. The larger boxed region in (B), when viewed at higher magnification (C) reveals smaller associated membranes that may represent interacting lysosomes or late endosomes that are not resolvable by TIRF. In a region where the section is nearly orthogonal to the lysosomal membrane, the most highly localized molecules fall on a line of width 10 nm (inset). In an obliquely cut region [(D), from the smaller boxed region in (B)], the distribution of CD63 within the membrane plane can be discerned. [View Larger Version of this Image (106K GIF file)]

Fig. 3. Comparative summed-molecule TIRF (A), PALM (B), TEM (C), and PALM/TEM overlay (D) images of mitochondria in a cryo-prepared thin section from a COS-7 cell expressing dEosFP-tagged cytochrome-C oxidase import sequence. Higher magnification PALM (E), TEM (F), and overlay (G) images within the box in (B) reveal that these matrix reporter molecules extend up to, but not into, the 20-nm outer mitochondrial membrane. The molecular distribution across two mitochondria along lines 1 and 2 in PALM image (E) are compared in (H) to the TEM signal along lines 3 and 4 in (F) across the same mitochondria. Scale bars: 1.0 µm in (A) to (D); 0.2 µm in (E) to (G). [View Larger Version of this Image (75K GIF file)]

Fig. 4. Examples of PALM imaging near the surfaces of whole, fixed cells. (A) A summed-molecule TIRF image of focal adhesions for a FoLu cell expressing dEos-tagged vinculin. (B) A magnified PALM view of the structure within a single adhesion over the region indicated by the box in (A), including apparent assembly of vinculin in a partial network (arrows). (C) A summed-molecule TIRF image near the periphery of a FoLu cell expressing tdEos-tagged actin. (D) A magnified PALM view of the actin distribution within the portion of the lamellipodium outlined by the box in (C). Inset, a further magnified view near the leading edge over the region indicated by the smaller box. (E and F) Summed-molecule TIRF and PALM images, respectively, of a COS-7 cell expressing the retroviral protein Gag tagged with dEos. The PALM image highlights voids (arrows labeled V), a higher density region (arrow R), and probable condensation at several points (arrows labeled P) into VLPs of 100- to 150-nm size (inset). [View Larger Version of this Image (109K GIF file)]

This performance is largely dictated by the photophysical characteristics of the PA-FPs. Longer photobleaching half-life leads to more photons per molecule, but for a given excitation intensity, it also requires longer data acquisition times between activation pulses to maintain an appropriate density of individually resolvable molecules. Higher excitation cross-sectional and fluorescence quantum efficiency can speed this process of signal extraction and bleaching, with the added benefit of increasing the molecular contrast relative to the autofluorescence background. Also vital is the contrast C(_exc) = ()_act/()_inact between the PA-FP in its activated and inactivated state at _exc, because this dictates the maximum molecular density beyond which the background from many weakly emitting inactivated molecules in a DLR dominates the signal from a single activated one. PA-FPs that remain activated until bleached ensure that all possible photons are extracted. Finally, PA-FPs less prone to blinking are desirable, given that it can be difficult to distinguish a single blinking molecule from multiple molecules that are serially activated and bleached in the same DLR (25).

Although we have demonstrated isolation and localization with both green [photoactivatable green fluorescent protein (PA-GFP) and Dronpa] and yellow [Kaede, Kikume Green-Red (KikGR), and Eos Fluorescent Protein (EosFP)] excitable PA-FPs, for imaging cellular structures we focused on tetrameric Kaede and the various oligomers of EosFP—the former for its somewhat higher brightness and the latter for their less perturbative effect on cellular structure and function. Each also exhibits high contrast relative to both the inactivated state and autofluorescence background at _exc = 561 nm.

We used PALM imaging to view intracellular structures in thin cryosections (25), akin to those used in transmission electron microscopy (TEM) but imaged under ambient conditions (Figs. 2 and 3). In Fig. 2, lysosomes in a COS-7 cell are visualized through expression of the lysosomal transmembrane protein CD63 fused to Kaede. Localization to the lysosome membrane was confirmed by comparative immunofluorescence labeling in similarly prepared samples (fig. S6). A TIRF image shows the outlines of the limiting membrane (Fig. 2A) but only hints at the intricate structure that is resolved by PALM, such as smaller associated membranes that may represent interacting lysosomes or late endosomes (Fig. 2, B and C). Indeed, in regions where the section plane is nearly orthogonal to the membrane, the most highly localized molecules fall on a line with an apparent width of 10 nm (inset, Fig. 2C), demonstrating that they are indeed fixed and that sample drift has been successfully mitigated (25). In other regions of the cryosection where the cut is more oblique to the lysosome, a wider, yet still sharply defined, swath of membrane is projected onto the image plane (Fig. 2D), permitting detailed investigation of the distribution of CD63 within the membrane plane.

In Fig. 3, PALM images of dEosFP-tagged cytochrome-C oxidase import sequence localized within the matrix of mitochondria in a COS-7 cell are compared with TEM images of the same mitochondria. The high degree of correlation between the two data sets validates the PALM imaging principle, and the sharpness of the mitochondrial edges (Fig. 3H) as viewed by PALM is far closer to that seen by TEM than that observed by diffraction-limited TIRF (Fig. 3A). Such comparative PALM/TEM imaging permits the nanometer-scale distribution of a specified protein to be determined in relation to the rest of the cellular ultrastructure at much higher molecular density than in immunolabeled TEM—more than 5500 molecules are localized in Fig. 3E, compared with the 20 or so particles typical in immunogold labeling of the mitochondrial matrix. Superposition of the PALM and TEM images (Fig. 3, D and G) also reveals that the matrix reporter molecules extend up to, but not into, the 20-nm outer mitochondrial membrane, underscoring the resolution capability of the technique. Correlated PALM/TEM does not have the added preparation steps and specificity issues associated with exogenous labels for combined fluorescence/EM such as fluorescein or resorufin arsenical helix binder (27). Finally, efforts are underway to establish dual-labeled PALM or PALM fluorescence resonance energy transfer, which would permit the relative distribution or regions of interaction between multiple proteins to be discerned at the nanometer level.

Thin sections are advantageous for PALM because they exhibit less autofluorescence than bulk samples, ensure that the PA-FPs are immobile, and permit the study of intracellular organelles that are inaccessible under TIRF excitation. However, demonstration of PALM on fixed cultured cells in phosphate-buffered saline (Fig. 4) is also notable both as a means to study proteins at or near the plasma membrane under minimally invasive conditions and as a precursor to eventual three-dimensional (3D) PALM imaging.

Confirmation that the nanometer-level resolution of PALM is retained under such conditions is given by the comparison of TIRF (Fig. 4A) and PALM (Fig. 4B) images of dEos-fused vinculin at focal adhesion regions (fig. S7) of a fox lung fibroblast (FoLu) cell to a cover slip. PALM reveals the heterogeneity within a selected attachment (box, Fig. 4A) and, in one subregion, suggests the partial assembly of a vinculin network (arrows, Fig. 4B). Similarly, a TIRF image (Fig. 4C) of tandem-dimer EosFP-fused actin in a cultured FoLu cell (fig. S8) shows both large cytoskeletal stress fibers and a lamellipodium, whereas PALM within the latter (Fig. 4D) reveals an increased concentration of actin at the leading edge. Under even higher magnification (inset, Fig. 4D), numerous short filaments are observed. These may be independent structures fixed in the process of assembly, or they may be part of a larger, continuous 3D network only partially revealed by the short extent of the evanescent excitation field.

In whole cells, PALM with TIRF excitation is well suited to studies of proteins bound to the plasma membrane, such as the dEosFP-fused Gag protein of human immunodeficiency virus 1 imaged by TIRF and PALM in Fig. 4, E and F, respectively. Gag, a retroviral protein that mediates the assembly of virus-like particles (VLPs), is revealed by PALM in various stages of organization: voids (arrows marked V), one high-density region (arrow R), and several tight clusters probably indicative of budding VLPs (arrows marked P, and magnified inset of Fig. 4F).

In the future, PALM should benefit from improvements in and additions to the palette of available PA-FPs, as well as from the discovery of means to modify the PA-FP environment to enhance photostability (13) and suppress blinking. Recently, we demonstrated photoactivation in PALM through ultraviolet-induced uncaging (28) of fluorophores (fig. S10) which, when combined with immunolabeling or other developing methods to achieve high-specificity intracellular protein labeling (27, 29), might offer a different avenue to improved localization precision and faster frame rates, given that a broad spectrum of high-brightness caged fluorophores is potentially available.

Algorithmically, additional well-localized molecules might be mined from the data if better means are found to unambiguously collate the multiple photon bursts from blinking molecules. Possible improvements to the fitting algorithm to achieve higher localization accuracy should also be explored. Although most of the observed molecules are well represented by a circularly symmetric Gaussian PSF, possible systematic position errors due to chromophore orientation, pixel nonuniformity, and chromatic aberration deserve closer attention. Perhaps most importantly, position error due to background nonuniformity within the molecular fitting window needs to be addressed, particularly when the number of inactivated molecules contributing to this background is high.

Experimentally, multiple angles and polarizations of TIRF excitation may eventually permit the precise determination of the xyz position and dipole orientation for fixed PA-FP molecules within the evanescent field. Standing wave TIRF could provide an excitation PSF of width _exc/6, improving localization precision for a given photon count. Bulk cellular autofluorescence complicates the extension of PALM to 3D, but the improved single-molecule sensitivity predicted for a proposed optical lattice microscope (30) may help. However, the most promising path to 3D may involve cryogenic PALM of vitrified cells, due to the narrow molecular line widths, large cross-sections, and improved stability expected (8). On the other hand, the ambient, TIRF-based PALM system demonstrated here has the advantage of simplicity, requiring only a TIRF-capable microscope with appropriate lasers, filters, and EMCCD camera, as well as basic acquisition, localization, and image rendering software. As such, it could be widely adopted in short order for the near-molecular resolution imaging of specified proteins for in vitro preparations and fixed cells.

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