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The place where we dwell 3rd edition pdf download,Reading and Writing about New York City

The NEW third edition of The Place Where We Dwell inspires an animated discussion and to sustain conversations through several defined themes such as city life, immigration, urban education, art and design, current issues, and the waterfront Web · The Place Where We Dwell 3Rd Edition PDF Book Details. Product details Publisher: Timber Press; Illustrated edition (February 7, ) Language: English Web · Place Where We Dwell: Reading and Writing about New York City | eBay. The Place Where We Dwell 3rd Edition Pdf Download Free; The Place Where We Dwell WebShowing all editions for 'The place where we dwell: reading and writing about New York City' Sort by: Format; All Formats (4) Print book (4) Refine Your Search; Year. (1) WebThe first edition ofPlace and Experienceestablished Jeff Malpas as one of the leading philosophers and thinkers of place and space and provided a creative and refreshing ... read more

Sign In. RETURN TO SEARCH RESULTS. The Place Where We Dwell: Reading and Writing about New York City Author s : Juanita But , Sean M Scanlan , Mark Noonan Edition: 3 Copyright: Pages: This product is for a printed text book only. OVERVIEW TABLE OF CONTENTS AUTHOR BIO Many outsiders might view New York City as inscrutable - a place too vast and complex to understand - never mind to live in. The Place Where We Dwell: Reading and Writing about New York City : Includes numerous essays that provide and demonstrate details and critical reflection that students could incorporate into their own writing. Examines the rhetorical strategy of comparison and contrast. Provides exemplary models of exposition, analysis, and argumentation and offer a broad context for student response. Offers a sampling of imaginative writing that both expands upon many of the themes found throughout the reader and offers readers imaginative visions of the urban experience.

Includes student-friendly pedagogical elements such as chapter introductions, author biographies preceding each selection, pre-reading questions. Discussion questions, writing tasks, a companion website, and more! SECTION I: Here is New York Chapter 1. Nooney, Domestic Photographs of Brooklyn Rem Koolhaas, Prediction Donald Reynolds, The Making of an Icon Mark Naison, From Doo Wop to Hip Hop: The Bittersweet Odyssey of African-Americans in the South Bronx Chapter 5. Current Issues James Parrott, As Incomes Gap Widens, New York Grows Apart Brian Paul, Affordable Housing Policies May Spur Gentrification, Segregation Margaret Morton, The Homeless Mark Berkey-Gerard, Youth Gangs Aarti Shahani, Legalization and De-Legalization Courtney Gross, Despite Setbacks, Bloomberg Plan Has Made New York Greener Benjamin Shepard, Fighting Police Brutality in Global Brooklyn Chapter 6.

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All rights reserved. Used under license. Redemption code will be sent out by email within 60 days of purchase. Limited quantities and while supply lasts. We reserve the right to replace titles in the offer for ones of equal or greater value. Certain titles may not be available to all consumers because of age restrictions. The Offer sponsor is Intel Corporation, Mission College Blvd. To participate you must create an Intel Digital Hub Account, purchase a qualifying product during the redemption period, enter a valid Master Key, and respond to a brief survey. Information you submit is collected, stored, processed, and used on servers in the USA. Intel, the Intel logo, and other Intel marks are trademarks of Intel Corporation or its subsidiaries in the U. Other names and brands may be claimed as the property of others. Bernard Lewis, who died on Saturday, adapted the essay below from a lecture delivered at Brandeis University on March 24, There is a well-worn platitude that we have all heard many times before: it is perfectly legitimate to criticize the actions and policies of the state of Israel or the doctrines of Zionism without necessarily being motivated by anti-Semitism.

The fact that this has been repeated ad nauseam does not detract from its truth. Not only do I accept it, but I would even take it a step further with another formulation that may perhaps evoke surprise if not shock: it is perfectly possible to hate and even to persecute Jews without necessarily being anti-Semitic. Unfortunately, hatred and persecution are a normal part of the human experience. Taking a dislike, mild or intense, to people who are different in one way or another, by ethnicity, race, color, creed, eating habits—no matter what—is part of the normal human condition. We find it throughout recorded history, and we find it all over the world. It can sometimes be extraordinarily vicious and sometimes even amusing.

Bernard Lewis, FBA 31 May — 19 May was a British American historian specialized in Oriental studies. He was also known as a public intellectual and political commentator. Lewis was the Cleveland E. Dodge Professor Emeritus of Near Eastern Studies at Princeton University. Pdf Man Myth Matthew Hussey Get The Guy Autocad Xforce Keygen 64 Bit Download Introduction To The Practice Of Statistics Third Edition Pdf Torrent Copytrans Crack Electrolux Time Manager 6. Not long after World War II, the Danes were seething with resentment against two of their neighbors: the Germans, for having occupied them, and the Swedes, for having stood by with unhelpful neutrality.

A Danish saying current at the time was: What is a Swede? A German in human form. Another double-barreled insult, this one from the British army in the late s, when it was concerned about two different groups of terrorists: What is an Arab? A toasted Irishman. I quote these not in any sense with approval or commendation, but as examples of the kind of really nasty prejudice that is widespread in our world. Anti-Semitism is something quite different. It is marked by two special features. One of them is that Jews are judged by a standard different from that applied to others.

We see plenty of examples of this at the present time. But there too one has to be careful. There can be different standards of judgment on other issues too, sometimes even involving Jews, without anti-Semitism or without necessarily being motivated by anti-Semitism. For instance, in mid-September in Spain, five terrorists convicted of murdering policemen were sentenced to death. European liberal opinion was outraged that in this modern age a West European country should sentence people to death. Unheard of! There was an outcry of indignation, and strong pressures were brought to bear on the Spanish government.

But in the Soviet Union and its satellite states during the same period, vastly greater numbers were being sentenced to death and executed; and, in Africa, Idi Amin was slaughtering hundreds of thousands, a large part of the population of Uganda. Hardly a murmur of protest in the Western world. The lesson is very clear. Right-wing governments General Francisco Franco was still in charge are not allowed to sentence offenders to death; left-wing governments are. A further implication: slaughter of or by white people is bad; slaughter of or by people of color is normal. Similar discrepancies may be found in responses to a number of other issues, as for example the treatment of women and of ethnic or other minorities.

These examples show that even a wide disparity of standards of judgment is not necessarily in itself evidence of anti-Semitism. There may be other elements involved. For example, the comparison is sometimes made between the world reaction to the massacre of Palestinians by Lebanese Christian militiamen at Sabra and Shatila in September , where some people were killed, and the massacre earlier in the same year in Hama in Syria, where tens of thousands were killed. On the latter, not a dog barked. The difference, of course, was in the circumstances. In both cases the perpetrators were Arab, but in the case of Sabra and Shatila, because of the dominant Israeli military presence in the region, there was a possibility of blaming the Jews. In Hama, this possibility did not exist; therefore the mass slaughter of Arabs by Arabs went unremarked, unnoticed, and unprotested. This contrast is clearly anti-Jewish. In a different way, it is also anti-Arab.

We see other instances of differing standards and methods of judgment nearer home and in a perhaps less alarming form. We hear a great deal, for example, about the Jewish lobby and the various accusations that are from time to time brought against it, that those engaged in it are somehow disloyal to the United States and are in the service of a foreign power. The Jewish lobby is, of course, not the only lobby of its kind. Consider three others: the Irish, Greek, and Armenian lobbies. The other special feature of anti-Semitism, which is much more important than differing standards of judgment, is the accusation against Jews of cosmic evil. Complaints against people of other groups rarely include it. This accusation of cosmic, satanic evil attributed to Jews, in various parts of the world and in various forms, is what has come to be known in modern times as anti-Semitism.

In the Western world, anti-Semitism has gone through three clearly distinct phases. Some people have written and spoken about anti-Semitism in antiquity, but the term in that context is misleading. We do indeed find texts in the ancient world attacking and denouncing Jews, sometimes quite viciously, but we also find nasty remarks about Syrians, Egyptians, Greeks, Persians, and the rest. There is no great difference between the anti-Jewish remarks and the ethnic and religious prejudices expressed against other peoples, and on the whole the ones against Jews are not the most vicious. The Syrian-born Roman historian Ammianus Marcellinus, for example, speaking of the Saracens, remarks that they are not to be desired either as friends or as enemies. Polytheism was essentially tolerant, each group worshiping its own god or gods, offering no objection to the worship of others. Indeed, one might have been willing to offer at least a pinch of incense to some alien god, in courtesy as a visitor or, even at home, in deference to a suzerain.

Only the Jews in the ancient world insisted—absurdly, according to the prevailing view of the time—that theirs was the only god and that the others did not exist. This gave rise to problems with their neighbors and their various imperial masters, notably the Romans. It sometimes provoked hostile comments and even persecution, but not the kind of demonization that has come to be known as anti-Semitism. The tendency was rather to ridicule the Jews for their faceless, formless god in the clouds and for such absurd and barbarous customs as circumcision, the rejection of pig-meat, and, most absurd of all, the Sabbath.

Several Greek and Roman authors noted that because of this comic practice the Jews were wasting one-seventh of their lives. Demonization, as distinct from common or garden-variety prejudice or hostility, began with the advent of Christianity and the special role assigned to the Jews in the crucifixion of Christ as related in the Gospels. Christianity started as a movement within Judaism, and the conflict between Christians and Jews had that special bitterness that often makes conflicts within religions more deadly than those between religions. The rejection of that message by the Jewish custodians of the Old Testament was especially wounding. An important concern of the early Christians was not so much to blame the Jews as, for understandable reasons, to exculpate the Romans.

Jewish guilt and Roman innocence, the two interdependent, became important parts of the Christian message, first to Rome and then beyond, with devastating effect on popular attitudes toward Jews, especially at Easter time. For many centuries, hatred and persecution of Jews, and the ideology and terminology used to express them, were grounded in religion. Then came the phase when religious prejudice was discredited, seen as not in accord with the ideas of the Enlightenment. It was seen as bigoted; worse, as old-fashioned, out-of-date. That meant new reasons were needed for hating Jews. They were found. The process of change began in Spain when large numbers of Jews—and also Muslims—were forcibly converted to Christianity. With a forcible conversion there was inevitably some doubt, especially among the enforcers, as to the sincerity of the converts. And this doubt was well grounded, as we know from the phenomenon of the Marranos and the Moriscos, the sometimes dubious converts from Judaism and Islam.

Thus the practice arose of examining the racial origins of the so-called new Christians. We even find statutes in 16th-century Spain about purity of blood, la limpieza de sangre. Only people who could prove Christian descent for a specified number of generations could be accepted as genuine Christians. This is where the racial form of anti-Semitism began. Aryan included languages as diverse as Sanskrit, Persian, and, by extension, Greek, Latin, and most of the languages of Europe. Semitic, similarly, brought together Syriac, Arabic, Hebrew, and Ethiopic. The people who advocated this bigotry spurned religious prejudice because they saw themselves as modern and scientific.

Their hostility to Jews, they claimed, was based on observed and documented racial otherness and inferiority. This discrediting of racism left a vacancy, an aching void. This is where the third phase of anti-Semitism arises, which for want of a better term we might call political-cum-ideological Judeophobia. Religious prejudice? This is political and ideological, and it provides a socially and intellectually acceptable modern disguise for sentiments that go back some 2, years. Turning from the Christian to the Islamic world, we find a very different history. If we look at the considerable literature available about the position of Jews in the Islamic world, we find two well-established myths.

Both are myths. Like many myths, both contain significant elements of truth, and the historic truth is in its usual place, somewhere in the middle between the extremes. There are certain important differences between the treatment, the position, the perception of Jews in the pre-modern Islamic world and in the pre-modern and also modern Christian worlds. The story of a golden age of complete equality is, of course, nonsense. No such thing was possible or even conceivable. Indeed, among Christians and Muslims alike, giving equal rights or, more precisely, equal opportunities to unbelievers would have been seen not as a merit but as a dereliction of duty. But until fairly modern times there was a much higher degree of tolerance in most of the Islamic lands than prevailed in the Christian world. For centuries, in most of Europe Christians were very busy persecuting each other; in their spare time, they were persecuting Jews and expelling Muslims—all at a time when, in the Ottoman Empire and some other Islamic states, Jews and several varieties of Christians were living side by side fairly freely and comfortably.

The comparison has often been made between the Cold War of the 20th century and the confrontation between Christendom and Islam in the 15th, 16th, and 17th centuries. In many ways the comparison is a good one. This was tolerance and no more than that. Tolerance is by modern standards an essentially intolerant idea. Tolerance means that I am in charge. I will allow you some though not all of the rights and privileges that I enjoy, provided that you behave yourself according to rules that I will lay down and enforce. That seems a fair definition of tolerance as usually understood and applied. It is, of course, an intolerant idea, but it is a lot better than intolerance as such, and the limited but substantial tolerance accorded to Jews and other non-Muslim communities in the Muslim states until early modern times was certainly vastly better than anything that was available in Christendom.

Prejudices existed in the Islamic world, as did occasional hostility, but not what could be called anti-Semitism, for there was no attribution of cosmic evil. And on the whole, Jews fared better under Muslim rule than Christians did. This is the reverse of what one might expect. The Prophet had more encounters with Jews than with Christians, so we find more negative statements about Jews than about Christians. The biography of the Prophet records armed clashes with Jews, and in those encounters it was the Jews who were killed. Muslims could therefore afford a more relaxed attitude toward Jews in the ensuing generations. The other advantage for Jews was that they were not seen as dangerous. Christianity was recognized as a rival world religion and a competitor in the cosmic struggle to bring enlightenment and with it, inevitably, domination to all humanity. This cosmic competition had important consequences. Local Christians were dangerous in that they were a potential fifth column for the Christian powers of Europe, the main adversary of the Islamic world.

Jews were not suspected of being pro-Christian. On the contrary, they were seen as being reliable and even useful. It was not merely tolerance or good will—though these were essential preconditions—that led the Ottoman sultans to admit so many Jewish refugees from Spain, Portugal, Italy, and elsewhere. Jews, especially those of European origin, were active in trade and industry, and from many documents in the Ottoman archives it is clear that they were valued as a revenue-producing asset. They were not just permitted; they were encouraged and even on a few occasions compelled to settle in Ottoman lands, especially in newly conquered provinces. Obviously, this is not equality, but it is not anti-Semitism in any sense of the word either.

We do of course find expressions of prejudice against the Jews, as against any group of people that are different, but their general attitude was of amused, tolerant superiority. An interesting difference in hostile stereotypes can be found in anecdotes, jokes, and the like. The main negative quality attributed to Jews in Turkish and Arab folklore was that they were cowardly and unmilitary—very contemptible qualities in a martial society. A late Ottoman joke may serve to illustrate this. The story is that in , at the time of the Balkan war, when there was an acute threat to the Ottoman Empire in its final stages, the Jews, full of patriotic ardor, decided that they, too, wanted to serve in the defense of their country, so they asked permission to form a special volunteer brigade.

Permission was given, and officers and ncos were sent to train and equip them. Once the Jewish volunteer brigade was armed, equipped, and trained, ready to leave for the front, they sent a message asking if they could have a police escort, because there were reports of bandits on the road. This is a very interesting human document. Is it hostile? Not really. It shows a sort of amused tolerance, at once good-humored and contemptuous, that may help us to understand the bewilderment and horror at the Israeli victories in and after. We have some vivid descriptions at the time of the expectations and reactions of We will utterly destroy them. We will sweep them into the sea. There would be no problem at all dealing with half a million Jews. It was then an appalling shock when five Arab armies were defeated by half a million Jews with very limited weaponry. It remains shameful, humiliating.

This was mentioned at the time and has been ever since. The Western form of anti-Semitism—the cosmic, satanic version of Jew hatred—provided solace to wounded feelings. It came to the Middle East in several stages. The first stage was almost entirely Christian, brought by European missionaries and diplomats. Its impact was principally on the local Christian minorities, where we find occasional recurrences of the previously little known blood libel. In the 15th and 16th centuries this had indeed been explicitly rejected in orders issued by Ottoman sultans. It was now revived on a massive scale. The first major case was the Damascus blood libel in This kind of anti-Semitism continued to grow, at first on a small scale, during the 19th and early 20th centuries, with a limited response.

At the time of the Dreyfus Affair in France, Muslim opinion was divided, some against Dreyfus, some supporting him. A prominent Muslim thinker of the time, the Egyptian Rashid Rida, wrote defending Dreyfus and attacking his persecutors, accusing them not of fanaticism, since they had no real religious beliefs, but of prejudice and envy. Despite this response, one consequence of the affair was the first translation into Arabic of a batch of European anti-Semitic writings. Then came the Third Reich, with connections to the Arab world and, later, to other Muslim countries. It is interesting that the common image of the Germans pursuing the Arabs is the reverse of what happened. The Arabs were pursuing the Germans, and the Germans were very reluctant to get involved. Sharonov, A. Wide-Field Subdiffraction Imaging by Accumulated Binding of Diffusing Probes. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proceedings of the National Academy of Sciences of the United States of America , 50 , CODEN: PNASA6 ; ISSN: National Academy of Sciences.

A method is introduced for subdiffraction imaging that accumulates points by collisional flux. It is based on targeting the surface of objects by fluorescent probes diffusing in the soln. Because the flux of probes at the object is essentially const. over long time periods, the examn. of an almost unlimited no. of individual probe mols. becomes possible. Each probe that hits the object and that becomes immobilized is located with high precision by replacing its point-spread function by a point at its centroid. Images of lipid bilayers, contours of these bilayers, and large unilamellar vesicles are shown. A spatial resoln. The ability of the method to effect rapid nanoscale imaging and spatial resoln. below Rayleigh criterion and without the necessity for labeling with fluorescent probes is proven. Schnitzbauer, J. Super-Resolution Microscopy with DNA-PAINT. Schnitzbauer, Joerg; Strauss, Maximilian T.

Nature Protocols , 12 6 , CODEN: NPARDW ; ISSN: Nature Publishing Group. techniques have begun to transform biol. and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topog. DNA-PAINT offers an easy-to-implement approach to localization-based super-resoln. microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled 'imager' oligonucleotides to their complementary target 'docking' strands creates the necessary 'blinking' to enable stochastic super-resoln. Using the programmability and specificity of DNA mols. as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resoln.

techniques, making them compatible with virtually any single-mol. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise mol. counting and ultra-high, mol. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample prepn. image reconstruction and post-processing such as drift correction, mol. counting qPAINT and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in d. Schueder, F. Multiplexed 3D Super-Resolution Imaging of Whole Cells Using Spinning Disk Confocal Microscopy and DNA-PAINT.

Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT. Schueder Florian; Woehrstein Johannes B; Strauss Maximilian T; Grabmayr Heinrich; Jungmann Ralf; Schueder Florian; Woehrstein Johannes B; Strauss Maximilian T; Grabmayr Heinrich; Jungmann Ralf; Lara-Gutierrez Juanita; Beliveau Brian J; Saka Sinem K; Sasaki Hiroshi M; Yin Peng; Lara-Gutierrez Juanita; Beliveau Brian J; Saka Sinem K; Sasaki Hiroshi M; Yin Peng. Nature communications , 8 1 , ISSN:. Single-molecule localization microscopy SMLM can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography DNA-PAINT with spinning disk confocal SDC hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells.

We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 μm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation. Uno, S. A Spontaneously Blinking Fluorophore Based on Intramolecular Spirocyclization for Live-Cell Super-Resolution Imaging. A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging. Uno, Shin-nosuke; Kamiya, Mako; Yoshihara, Toshitada; Sugawara, Ko; Okabe, Kohki; Tarhan, Mehmet C. Nature Chemistry , 6 8 , CODEN: NCAHBB ; ISSN: localization microscopy is used to construct super-resoln. images, but generally requires prior intense laser irradn. and in some cases additives, such as thiols, to induce on-off switching of fluorophores.

These requirements limit the potential applications of this methodol. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramol. spirocyclization reaction. Optimization of the intramol. nucleophile and rhodamine-based fluorophore electrophile provide a suitable lifetime for the fluorescent open form, and equil. between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-mol. localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resoln.

imaging of microtubules in live cells for up to 1 h. Lee, J. Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging. Biophysical Journal , 8 , CODEN: BIOJAU ; ISSN: Cell Press. There is no confocal microscope optimized for single-mol. imaging in live cells and superresoln. fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, the authors have developed a, to the authors' knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability 1. Full compatibility of the microscope with conventional cell-imaging techniques allowed the authors to do single-mol. imaging with a great ease at arbitrary depths of live cells. With the new microscope, the authors monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresoln.

fluorescence images of microtubules of COS-7 cells at depths in the range μm from the surface of a coverglass. Vandenberg, W. Diffraction-Unlimited Imaging: From Pretty Pictures to Hard Numbers. Cell Tissue Res. Diffraction-unlimited imaging: from pretty pictures to hard numbers. Cell and tissue research , 1 , ISSN:. Diffraction-unlimited fluorescence imaging allows the visualization of intact, strongly heterogeneous systems at unprecedented levels of detail. Beyond the acquisition of detailed pictures, increasing efforts are now being focused on deriving quantitative insights from these techniques. In this work, we review the recent developments on sub-diffraction quantization that have arisen for the various techniques currently in use.

We pay particular attention to the information that can be obtained but also the practical problems that can be faced, and provide suggestions for solutions or workarounds. We also show that these quantitative metrics not only provide a way to turn raw data into hard statistics but also help to understand the features and pitfalls associated with sub-diffraction imaging. Ultimately, these developments will lead to a highly standardized and easily applicable toolbox of techniques, which will find widespread application in the scientific community. Mortensen, K. Optimized Localization Analysis for Single-Molecule Tracking and Super-Resolution Microscopy. Methods , 7 , — , DOI: Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nature Methods , 7 5 , CODEN: NMAEA3 ; ISSN: The authors optimally localized isolated fluorescent beads and mols.

imaged as diffraction-limited spots, detd. the orientation of mols. and present reliable formulas for the precision of various localization methods. Both theory and exptl. data showed that unweighted least-squares fitting of a Gaussian squanders one-third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, whereas max. Lleres, D. Quantitative Flim-Fret Microscopy to Monitor Nanoscale Chromatin Compaction in Vivo Reveals Structural Roles of Condensin Complexes. Cell Rep. Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes.

Lleres, David; Bailly, Aymeric P. Cell Reports , 18 7 , CODEN: CREED8 ; ISSN: How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quant. FRET Forster resonance energy transfer -based fluorescence lifetime imaging microscopy FLIM approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 HP1 and SETDB1 H3-lysine-9 methyltransferase homologs in vivo.

Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I addnl. controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes. Datta, R. Fluorescence Lifetime Imaging Microscopy: Fundamentals and Advances in Instrumentation, Analysis, and Applications. Oleksiievets, N. Wide-Field Fluorescence Lifetime Imaging of Single Molecules. A , , — , DOI: Oleksiievets, Nazar; Thiele, Jan Christoph; Weber, Andre; Gregor, Ingo; Nevskyi, Oleksii; Isbaner, Sebastian; Tsukanov, Roman; Enderlein, Joerg.

Journal of Physical Chemistry A , 17 , CODEN: JPCAFH ; ISSN: American Chemical Society. Fluorescence lifetime imaging FLIM has become an important microscopy technique in bioimaging. The two most important of its applications are lifetime-multiplexing for imaging many different structures in parallel, and lifetime-based measurements of Forster resonance energy transfer. There are two principal FLIM techniques, one based on confocal-laser scanning microscopy CLSM and time-correlated single-photon counting TCSPC and the other based on wide-field microscopy and phase fluorometry. Although the first approach CLSM-TCSPC assures high sensitivity and allows one to detect single mols. The second allows, in principal, high frame rates by orders of magnitude faster than CLSM , but it suffers from low sensitivity, which precludes its application for single-mol.

Here, the authors demonstrate that a novel wide-field TCSPC camera LINCam25, Photonscore GmbH can be successfully used for single-mol. This is due to the virtually absent background and readout noise of the camera, assuring high signal-to-noise ratio even at low detection efficiency. The authors performed single-mol. FLIM of different red fluorophores, and the authors use the lifetime information for successfully distinguishing between different mol. Finally, the authors demonstrate single-mol. metal-induced energy transfer MIET imaging which is a first step for three-dimensional single-mol. localization microscopy SMLM with nanometer resoln. Bowman, A. Electro-Optic Imaging Enables Efficient Wide-Field Fluorescence Lifetime Microscopy. Electro-optic imaging enables efficient wide-field fluorescence lifetime microscopy. Nature communications , 10 1 , ISSN:.

Nanosecond temporal resolution enables new methods for wide-field imaging like time-of-flight, gated detection, and fluorescence lifetime. The optical efficiency of existing approaches, however, presents challenges for low-light applications common to fluorescence microscopy and single-molecule imaging. We demonstrate the use of Pockels cells for wide-field image gating with nanosecond temporal resolution and high photon collection efficiency. Two temporal frames are obtained by combining a Pockels cell with a pair of polarizing beam-splitters. We show multi-label fluorescence lifetime imaging microscopy FLIM , single-molecule lifetime spectroscopy, and fast single-frame FLIM at the camera frame rate with 10 3 5 times higher throughput than single photon counting.

Finally, we demonstrate a space-to-time image multiplexer using a re-imaging optical cavity with a tilted mirror to extend the Pockels cell technique to multiple temporal frames. These methods enable nanosecond imaging with standard optical systems and sensors, opening a new temporal dimension for wide-field low-light microscopy. Zhao, M. Parallel Excitation-Emission Multiplexed Fluorescence Lifetime Confocal Microscopy for Live Cell Imaging. Express , 22 , — , DOI: Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging. Optics express , 22 9 , ISSN:. We present a novel excitation-emission multiplexed fluorescence lifetime microscopy FLIM method that surpasses current FLIM techniques in multiplexing capability. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes.

The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community. Super-Resolution Imaging with Small Organic Fluorophores. The authors performed fluorescence quenching expts. with various Alexa Fluor and ATTO dyes and different electron donors, with the intention of finding reducing agents that selectively quench the energetically slightly stabilized triplet state but leave the singlet state unaffected. The authors found that reducing agents with thiol groups, such as MEA, DTT, and GSH, selectively quench the triplet state of rhodamine and oxazine derivs.

depending on the pH value of the aq. buffer used. Since most thiols RSH have a pKa SH and the reducing species is the thiolate anion RS- , the redn. efficiency of compds. potential with a decrease in pH value. Building on the result that millimolar concns. of thiol compds. in aq. allow reversible, facile switching of small org. fluorophores, the authors performed super-resoln. imaging of the cytoskeletal network of fixed cells with an exemplary selection of eight different org. For immunofluorescence imaging of microtubules, COS-7 cells were stained with primary antibodies and then with fluorescently labeled secondary antibodies. Sauer, M. Single-Molecule Localization Microscopy in Eukaryotes. Chemical Reviews Washington, DC, United States , 11 , CODEN: CHREAY ; ISSN: A review. fluorescence imaging by photoactivation or photoswitching of single fluorophores and position detn. localization microscopy, SMLM provides microscopic images with sub-diffraction spatial resoln.

This technol. has enabled new insights into how proteins are organized in a cellular context, with a spatial resoln. approaching virtually the mol. A unique strength of SMLM is that it delivers mol. This allows quant. access to cellular structures, for example, how proteins are distributed and organized and how they interact with other biomols. Ultimately, it is even possible to det. protein nos. in cells and the no. of subunits in a protein complex. SMLM thus has the potential to pave the way toward a better understanding of how cells function at the mol. In this review, the authors describe how SMLM has contributed new knowledge in eukaryotic biol. data extd. from SMLM images. Bates, M. Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes. Science , , , DOI: Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with a spatial resoln. of 20 to 50 nm. Multicolor super-resoln. imaging, however, remains a challenging task.

Here, the authors introduce a family of photoswitchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy STORM. Each probe consists of a photoswitchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photoactivation 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-resoln.

STORM image. Using this approach, the authors demonstrate multicolor imaging of DNA model samples and mammalian cells with to nm resoln. This technique will facilitate direct visualization of mol. interactions at the nanometer scale. Lampe, A. Multi-Colour Direct STORM with Red Emitting Carbocyanines. Cell , , — , DOI: Biology of the Cell , 4 , CODEN: BCELDF ; ISSN: Background information. Single mol. methods have become important tools to study nanoscale structures in cell biol. However, the complexity of multi-color applications has prevented them from being widely used amongst biologists. Direct stochastic optical reconstruction microscopy dSTORM offers a simple way to perform single mol. imaging without the need for an activator fluorophore and compatible with many conventionally used fluorophores. The search for the ideal dye pairs suitable for dual-color dSTORM has been compromised by the fact that fluorophores spectrally apt for dual-color imaging differ with respect to the optimal buffer conditions required for photoswitching and the generation of prolonged non-fluorescent OFF states.

We present a novel variant of dSTORM that combines advantages of spectral demixing with the buffer compatible blinking properties of red emitting carbocyanine dyes, spectral demixing dSTORM SD-dSTORM. In contrast to previously published work, SD-dSTORM requires reduced laser power and fewer imaging frames for the faithful reconstruction of super-resolved biol. In addn. available rather than custom-made probes and does not rely on potentially error-prone cross-talk correction, thus allowing reliable co-localization. SD-dSTORM presents a significant advance towards user-friendly single mol. localisation-based super-resoln. microscopy combining advantages of state-of-the-art methodologies to perform fast, reliable and efficient multi-color dSTORM.

Bossi, M. Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species. Nano Lett. Bossi, Mariano; Folling, Jonas; Belov, Vladimir N. Nano Letters , 8 8 , CODEN: NALEFD ; ISSN: By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single mol. level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resoln. using only two detection color channels. The applicability of the method to biol. specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable mol. species at a time opens up a new class of functional imaging techniques. Zhang, Z. Ultrahigh-Throughput Single-Molecule Spectroscopy and Spectrally Resolved Super-Resolution Microscopy. Methods , 12 , — , DOI: Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy.

Nature Methods , 12 10 , CODEN: NMAEA3 ; ISSN: in labeled cells in minutes, which consequently enabled spectrally resolved, 'true-color' super-resoln. The method, called spectrally resolved stochastic optical reconstruction microscopy SR-STORM , achieved cross-talk-free three-dimensional 3D imaging for four dyes 10 nm apart in emission spectrum. Excellent resoln. was obtained for every channel, and 3D localizations of all mols. were automatically aligned within one imaging path. Hershko, E. Multicolor Localization Microscopy and Point-Spread-Function Engineering by Deep Learning. Express , 27 , — , DOI: Multicolor localization microscopy and point-spread-function engineering by deep learning. Optics Express , 27 5 , CODEN: OPEXFF ; ISSN: Optical Society of America. Deep learning has become an extremely effective tool for image classification and image restoration problems.

Here, we apply deep learning to microscopy and demonstrate how neural networks can exploit the chromatic dependence of the point-spread function to classify the colors of single emitters imaged on a grayscale camera. While existing localization microscopy methods for spectral classification require addnl. optical elements in the emission path, e. Furthermore, we show how deep learning can be used to design new phase-modulating elements that, when implemented into the imaging path, result in further improved color differentiation between species, including simultaneously differentiating four species in a single image. Gómez-García, P. Excitation-Multiplexed Multicolor Superresolution Imaging with fm-STORM and fm-DNA-PAINT.

Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT. Gomez-Garcia, Pablo A. Proceedings of the National Academy of Sciences of the United States of America , 51 , CODEN: PNASA6 ; ISSN: Recent advancements in single-mol. microscopy have made it possible to visualize biol. structures with unprecedented spatial resoln. the spatial coorganization of these structures within cells under physiol. and pathol. conditions is an important biol. This goal has been stymied by the current limitations of carrying out superresoln.

microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresoln. imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be detd. based on the fluorophore's response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresoln. DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore d. per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as "frequency multiplexed DNA-PAINT"' or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresoln. imaging, which we term fm-STORM.

Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. Jungmann, R. Multiplexed 3D Cellular Super-Resolution Imaging with DNA-PAINT and Exchange-PAINT. Methods , 11 , — , DOI: Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Jungmann, Ralf; Avendano, Maier S. Nature Methods , 11 3 , CODEN: NMAEA3 ; ISSN: fluorescence microscopy is a powerful tool for biol. research, but obtaining multiplexed images for a large no. of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides DNA-PAINT, a variation of point accumulation for imaging in nanoscale topog. for simple and easy-to-implement multiplexed super-resoln. imaging that achieves subnm spatial resoln.

in vitro on synthetic DNA structures. We also report a multiplexing approach Exchange-PAINT that allows sequential imaging of multiple targets using only a single dye and a single laser source. We exptl. demonstrate ten-color super-resoln. imaging in vitro on synthetic DNA structures as well as four-color two-dimensional 2D imaging and three-color 3D imaging of proteins in fixed cells. Wade, O. Wade, Orsolya K. Nano Letters , 19 4 , CODEN: NALEFD ; ISSN: Optical super-resoln. techniques reach unprecedented spatial resoln. down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of mol.

species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resoln. microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally exptl. demonstrate plex super-resoln. imaging within minutes. Balzarotti, F. Nanometer Resolution Imaging and Tracking of Fluorescent Molecules with Minimal Photon Fluxes. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Balzarotti, Francisco; Eilers, Yvan; Gwosch, Klaus C. We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity min. of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision.

In our expts. In superresoln. only 6 nm apart. MINFLUX tracking of single fluorescent proteins increased the temporal resoln. and the no. of localizations per trace by a factor of , as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromols. in living cells and beyond. Gwosch, K. Minflux Nanoscopy Delivers 3D Multicolor Nanometer Resolution in Cells. Methods , 17 , — , DOI: MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells.

Confocal laser-scanning microscopy CLSM is one of the most important microscopy techniques for biology and medicine. Its fundamental purpose is to provide so-called optical sectioning and thus to enable the recording of three-dimensional images, which is impossible to achieve with conventional wide-field microscopy. Its disadvantage, when compared to wide-field microscopy, is its inherently slow image acquisition speed because image formation is realized by sequentially scanning single or multiple foci over a sample. This also limits its overall light throughput small dwell time per scan position , which is one reason why CLSM was nearly never used for single-molecule localization based super-resolution microscopy single molecule localization microscopy or SMLM , such as photoactivation localization microscopy PALM , 1 direct stochastic optical reconstruction microscopy dSTORM , 2,3 or points accumulation for imaging in nanoscale topography PAINT.

There are only a few exceptions, all using faster alternatives to a CLSM and a camera-based detection. One where a spinning-disk CLSM was employed for PAINT, exploiting the superior out-of-plane light rejection of a CLSM that is so important for reducing background from freely diffusing dyes in PAINT, 6 one where a spinning-disk CLSM was employed for STORM with self-blinking dyes, where it was used for reducing excitation intensity 7 and one where a custom line-scan confocal microscope was used for dSTORM deep inside the sample. First, single-focus CLSM uses single-point detectors which can be operated in single-photon counting mode Geiger mode and thus provides shot-noise limited detection, in contrast to emCCD or sCMOS cameras used in conventional wide-field SMLM, with their read-out, thermal, and electronic noise.

Second, when using Geiger mode for light detection, CLSM records the positions of single-photon detection events in a quasi-continuous, nonpixelated way, thus avoiding pixel size to affect single-molecule localization accuracy. FLIM is widely used for lifetime-based FRET and environment sensing applications. Additionally, the lifetime information introduce the option to colocalize different molecular species that differ only by their lifetime while having similar excitation and emission spectra, 15 thus efficiently circumventing all problems connected to chromatic aberration that hinder many multicolor SMLM methods. Several solutions to the chromatic aberration problem have been proposed in the past. For example, activation-based multicolor STORM entirely removes chromatic aberrations at the cost of relatively high crosstalk. The fluorescence signal of the different molecules is separated spectrally, and ratiometric fluorescence measurements are used for spectral demixing and co localizing different kinds of molecules.

One step further in this direction was the implementation of spectrally resolved SMLM, where full spectra are measured and used for sorting different molecules and their localizations. Another clever solution is Exchange-PAINT, 24 which sequentially images different targets with the same dye but uses different DNA-tags for directing the dye to different targets decorated with complementary DNA-strands. Similarly, barcoding PAINT 25 exploits the different binding kinetics of imager and docking strands for distinguishing between different target sites. Because one uses the same dye for all the different structures, chromatic aberrations do not impact the SMLM results, but the prize is increasing image acquisition time, ca. linearly increasing with the number of different targets one wants to resolve.

Finally, the recently introduced MINFLUX 26 allows for super-resolution imaging with a few nanometers accuracy and can be used for chromatic-aberration free multicolor imaging. In this work, we present the realization of SMLM with a time-resolved CLSM using single-photon avalanche-diodes SPADs for detection, and a rapid laser-scanning unit for excitation beam scanning. This unit enables us to record images with reasonable acquisition speed as required for efficient SMLM. Our approach combines all the advantages of CLSM with those of SMLM: axial sectioning, shot-noise limited single-photon detection, pixel-free continuous position data, and fluorescence lifetime information acquired by CLSM with the exceptional spatial resolution and single-molecule identification of SMLM. At first, we demonstrate the feasibility of using CLSM for fluorescence lifetime SMLM FL-SMLM by imaging labeled fixed cell samples by combining CLSM with two of the most widely used variants of SMLM, dSTORM for imaging microtubules in human mesenchymal stem cells and DNA-PAINT for imaging cellular chromatin in COS-7 cells.

To demonstrate the fluorescence lifetime multiplexing capability of FL-SMLM, we record images of polymer beads that are surface-labeled with two different dyes and two cellular targets microtubules and clathrin in COS-7 cells. Our results show that confocal laser-scanning FL-SMLM has great potential for many applications, extending the dimensions of fluorescence super-resolution microscopy by fluorescence lifetime. Confocal laser-scanning SMLM measurements were carried out on a custom-built time-resolved confocal microscope equipped with a fast laser scanner; see Figure 1a more details in the Material and Methods.

Single fluorescence photons were detected with a single-photon avalanche diode SPAD , and photon detection events were correlated in time to excitation pulses time-correlated single-photon counting or TCSPC with high-speed electronics. For data analysis, recorded photons were converted into a stack of intensity images, always combining 10 subsequent scans into one image to minimize distortions by those labels that are switching while being scanned. As shown in Figure S8, slower scanning leads to many incomplete PSFs whereas fast scanning without frame binning does not provide enough photons for a precise localization.

The stack of intensity images is then used to localize single molecules and to identify switching events. These localizations were subsequently used to reconstruct a super-resolved image similar to conventional wide-field dSTORM. Taking full advantage of our TCSPC detection, fluorescence lifetimes of each localized molecule were determined by pooling all photons associated with it and fitting the resulting lifetime histogram with a monoexponential decay function. To increase the number of photons per localization, identical localizations in subsequent frames were merged. Using this lifetime information, super-resolved fluorescence lifetime images were reconstructed. Figure 1. Measurement and data evaluation. a Schema of the confocal setup: The pulsed nm excitation light is converted to circular polarization with a quarter wave plate QWP , passes through a single mode fiber SMF , is reflected by a dichroic mirror DM into a galvometeric laser scanner and focused by the objective.

The collected fluorescent emission from the sample is descanned, passes the DM, and is focused on the pinhole PH then on the single-photon detector APD using the lenses L1, L2 and L3. The long pass filter LP and the band-pass filter BP are blocking scattered excitation light. b The scanning SMLM data analysis from raw data to reconstruction was done in an extended version of TrackNTrace. To make the data analysis for confocal laser-scanning FL-SMLM widely available, the complete data processing pipeline was integrated into a Matlab-based GUI app, see Figure1b. This app builds on the existing open-source framework TrackNTrace 28 and was now extended for processing also TCSPC data.

The app supports FLIM data and comes with a dedicated plugin that pools photon detections for each localized molecule and executes lifetime fits. The data visualizer offers filtering of localizations, drift correction with redundant cross-correlation RCC , 29 and reconstruction of super-resolved lifetime images. More details can be found in the methods section. To demonstrate the applicability of confocal laser-scanning SMLM for biological samples, we performed dSTORM of microtubules in fixed immunolabeled hMS cells. Alexa is a benchmark dye for dSTORM due to its outstanding photostability, brightness, and optimized blinking behavior. A Gaussian fit of the fluorescence lifetime histogram obtained from the fitted lifetime values of all identified molecules in the region of interest gives a mean value of 1. To better compare the performance of confocal laser-scanning dSTORM with conventional wide-field dSTORM, we imaged the same sample that we used for FL-SMLM with a custom-built wide-field setup.

The comparison is shown in Figure S2. To quantify the relative performance of both techniques, we determined single microtubule cross sections, and we estimated their diameter to be 64 nm fwhm for confocal laser-scanning dSTORM, and 53 nm fwhm for conventional wide-field dSTORM. The apparent size of microtubules in both cases is larger than their actual value, which we attribute to the extra size of the secondary immunolabels. We also calculated Fourier Ring Correlation FRC maps using the NanoJ-SQUIRREL plugin see Figure S1. The localization precision was estimated following modified Mortensen's equation, 10,33,34 and an average value was found to be 8. In summary, we find that both approaches show a similar performance, which demonstrates that confocal laser-scanning dSTORM can be a promising and versatile super-resolution technique adding the important fluorescence lifetime dimension to the picture.

Furthermore, the sectioning capabilities of the confocal imaging system offers the possibility to image dense 3D structures. To illustrate this, we have recorded z -stacks of images of Alexa labeled tubulin in COS-7 cells see Figure S2. Figure 2. Confocal dSTORM and DNA-PAINT imaging in cells. a—c Confocal laser-scanning dSTORM images of tubulin filaments in hMS cells labeled with Alexa d—f Confocal laser-scanning DNA-PAINT images of cellular chromatin in COS-7 cells utilizing DNA-labeled ATTO a, d Example of a single frame during acquisition. b, e Corresponding diffraction-limited and super-resolved images. c, f Lifetime histograms, based on individual single-molecule localizations. We applied our approach also to perform confocal laser-scanning DNA-PAINT.

DNA-PAINT is a recently developed and highly promising alternative to dSTORM, because it circumvents the inherent photobleaching limitations of dSTORM by employing dye-labeled imager DNA-strands that reversibly bind to targets of interest that are themselves labeled with complementary target DNA-strands. For DNA-PAINT and PAINT in general , optical sectioning is critical for efficiently suppressing fluorescent background from freely diffusing imager strands. To demonstrate confocal laser-scanning DNA-PAINT, we imaged histone H2B in COS-7 cells Figure 2d—f that was fused to mTagBFP which was subsequently high-affinity labeled with DNA docking strands using FluoTag-Q anti-TagBFP nanobodies.

ATTO was used for DNA-PAINT because of its high brightness and low unspecific binding to both coverslip surface and cell organelles. Although we reduced the concentration of imager strands, as compared to conventional DNA-PAINT, 24 by an order of magnitude to 0. A typical single frame from a recorded movie is shown in Figure 2d, and Figure 2e presents a comparison between super-resolved PAINT and conventional CLSM. The average localization precision of the reconstructed confocal laser-scanning DNA-PAINT image was found to be 18 nm. For more detailed information and comparison with conventional DNA-PAINT see Figure S3. The corresponding lifetime distribution for all localized molecules is shown in Figure 2f.

There, we find a lifetime value for ATTO that is longer than the reported value for free dye, which we attribute to its conjugation to single-stranded imager DNA. On our wide-field microscope, we could not detect single switching events when focusing at the center of the beads. In contrast, it was possible to localize switching molecules Figure 3a with the CLSM, to determine their fluorescence lifetimes, and to reconstruct a fluorescence-lifetime dSTORM image Figure 3b. The image shows two beads that are labeled with two different dyes, namely Alexa having an average fluorescent lifetime of 1. Details on data processing and the resulting lifetime histogram can be found in the Materials and Methods section and Figure S5. Figure 3. Lifetime-based multiplexed dSTORM imaging of polymer beads labeled with two different dyes. a Typical frames from a recorded movie including single-molecule localizations.

b Histogram of photon arrival times TCSPC for two indicated localizations. Lifetime is determined with a monoexponential fit blue line. c Super-resolved image reconstruction including lifetime information. The two different lifetime values for molecules on both beads reveal that the beads are labeled with different fluorophores Alexa and ATTO The obtained "ringlike" structures reflect the optical sectioning capability of confocal imaging. To enable fluorescence-lifetime multiplexing dSTORM, it is crucial to use buffer conditions which are compatible with all fluorophores. This includes control of sufficient on- and off-switching rates while guaranteeing high dye brightness. Typically, a primary thiol is used to enhance off-switching rates. The standard buffer for Alexa additionally contains GLOX to create an oxygen-depleted environment which reduces permanent photobleaching. We found that an oxygen-depleted environment results in low on-switching rates for ATTO , probably due to a long-living nonfluorescent reduced form.

To validate fluorescence-lifetime multiplexing SMLM on biological samples, we performed dSTORM imaging of Alexa labeled β-tubulin and ATTO labeled clathrin in COS-7 cells. Thus, two different targets are labeled with two spectroscopically similar dyes with different fluorescence lifetimes. To improve lifetime contrast and brightness of both dyes all dual-label dSTORM cell images were done in D 2 O instead of PBS buffer and with the same thiol concentration as for the bead imaging. For this, the probability that a reference TCSPC decay curve produces that of a localized molecule is calculated and the molecule classified according to the reference that yields the highest probability.

The two reference patterns for each sample, shown in Figure 4a,f, are obtained by measuring samples containing only one species and normalizing the compounded single molecule TCSPC curves. Bayesian pattern matching is, from a statistical point of view, the optimal method for classification and has several advantages: it uses all the detected photons, it does not assume that fluorescence decays are monoexponential, and it is fit-free and therefore fast and stable. Corresponding dSTORM images for both targets are shown in Figure 4d.

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