MİKROSKOPTA DEVRİME NOBEL (20 Ekim 2014 / Milliyet / Prof. Dr. Murat Tuzcu)

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1 MİKROSKOPTA DEVRİME NOBEL (20 Ekim 2014 / Milliyet / Prof. Dr. Murat Tuzcu) Bu yıl Kimya Nobeli yüzlerce yıldır kullandığımız mikroskobu tek tek molekülleri gösterebilecek kadar güçlendiren 3 bilim insanına verildi. Bu buluş, hücrelerimizin çalışmasını daha iyi anlamamızı sağlayacak hastalıklarla mücadelemizi kolaylaştıracak. Yeryüzünde biri insan gözünün gördüğü, diğeri göremeyeceğimiz kadar küçük iki dünya var desek yanlış olmaz. Dörtyüz yıl öncesine kadar görünmez dünyanın var olduğu bile bilinmiyordu. Gözle görünmeyen cisimlerin ve mikroskobik denilen yaratıkların keşfedilmesine yol açarak bambaşka bir dünyanın farkına varmamızı sağlayan gelişme mikroskopun icadıdır. Merceğin tarihi eski İnsanoğlu eski çağlardan beri küçük cisimleri büyüterek incelemek istemiştir. Romalılar iki bin yıl önce camların şekline ve kalınlığına göre farklı özellikleri olduğunu keşfettiler. Daha sonra ortası kalın, kenarları ince olan camların öte tarafındaki cisimleri olduğundan daha büyük gösterdiğini şaşırarak gördüler. Milattan sonra ilk yüzyılda ünlü Romalı filozof Seneka bir yazısında Harfler çok küçükse cam tutarak okuyun tavsiyesinde bulundu. O devirde bir merceği büyüteç olarak kullanmış olduğu anlaşılıyor. Merceğin varlığı bilinse de insanlığın hizmetine sunulması için bin yıldan uzun bir süre geçmesi gerekti. 13 üncü yüzyılda İtalya da gözlük yapımı başlayana kadar mercek yaygın olarak kullanılmadı. Ünlü fizikçi İbn-i Heysem Bir eski Irak Dinarı üstünde resmi görülen İbn-i Heysem 965 te Basra da doğdu. Uzun yıllar Mısır'da daha sonra Endülüs de çalıştı. Optik, astronomi, fizik, geometri, felsefe konularında birçok eseri vardır. Optik alanındaki gelişmeler Avrupalı bilim insanlarına atfedilse de, 10 ve 11. yüzyıl da yaşamış Arap bilim insanlarının optik biliminin gelişmesine büyük katkıları olmuştur. Bu âlimlerin başında Basralı İbni-Heysem gelir. Batılıların Alhazen dedikleri bilim adamı 11'inci yüzyılın başında yazdığı Kitab ül-menazir adlı eserinde, mercekleri ve aynaları kullanarak ışığın, yansıması, kırılması ve odaklanması konularında yaptığı deneyleri anlatır. Avrupa da rönesanstan sonra çalışmaları yoğunlaşan bilim insanları İbn-i Heysem in fizik alanındaki buluşlarından çok yararlanmışlardır.

2 İlk mikroskop Yanda görülen ilk mikroskop farklı özellikleri olan merceklerin art arda yerleştirildiği, iç içe geçen 3 borudan ibaretti. İncelenen cismi 10 kat büyütebilen bu ilk optik mikroskop yüzyıllar için de geliştirildiyse de temel özellikleri 20 inci yüzyılın ortasında kullanılan mikroskoptan (yanda) çok farklı değildi. Daha sonra mikroskobi alanında büyük gelişmeler kaydedildi. Rönesans ile uyanan Avrupa da İbn-i Heysem gibi bilim insanlarının buluşlarına dayanarak gözlük yapmak mümkün oldu. Gözlük yapan ustalar optik alanında edindikleri tecrübeleri kullanarak mikroskobu geliştirdi. İlk mikroskop Hollanda ve Almanya da 16 ncı yüzyılın sonunda imal edildi. Güneş dünyanın etrafında değil, dünya güneşin etrafında dönüyor diyen ünlü Galile, teleskobun yanı sıra mikroskopla da ilgilendi. Kendi adıyla anılan bir mikroskop imal etti. Işığın merceklerden ve aynalardan geçirilerek cisimleri olduklarından çok daha büyük gösteren mikroskoba optik mikroskop deniyor. Yirminci yüzyılın ikinci yarısında sağlanan gelişmeler giderek daha küçük cisimlerin görüntülenmesini sağladı. Lâkin, optik mikroskobun, ışığın dalga boyuna bağlı olarak aşamayacağı bir çözünürlük sınırının olduğu biliniyordu. Yeni mikroskoplar

3 Optik mikroskobun gelip dayandığı siniri aşmak için ışık yerine incelenen cisimden saçılan elektronların kullanıldığı mikroskoplar geliştirildi, Bu aletlerle nanometre düzeyinde cisimler görünür hale getirildiyse de bu teknolojiyi kullanmak için canlı hücrelerin öldürülüp özel olarak hazırlanması gerekiyordu, Canlı hücreyi mikroskopta iyice büyütüp inceleyebilmek için istenen cisimleri özel bir boyayla işaretleme yöntemi geliştirildi. Amaç tüm hücreyi boyamak değildi. Hücre içindeki molekülleri boyayıp nerede olduklarını ve hareketini saptamaktı hedeflenen. Bunun için hücrenin içine girip istenen moleküllere bağlanan özel maddeler üretildi. Üzerlerine tutulan belli dalga boyundaki lazeri emip çeşitli renklerde ışıldayan maddelerdi bunlar. Bu yöntem çok başarılı oldu. Ama optik mikroskobun sınırlılığı yine engel oluşturdu. Boyanan maddeleri, örneğin molekülleri ve proteinleri tek tek görmek mümkün olmuyordu. Bir mikroskop uzmanın yaptığı benzetmedeki gibi, birçok karıncanın bulunduğu bir topluluk diğer cins karıncalardan ayrılabiliyordu ama tek tek karıncaları görmek mümkün olmuyordu. Çünkü optik mikroskop moleküller arasında 200 nanometrelik bir mesafe yoksa iki ayrı cismi birbirinden ayırt edemiyordu. Gerçi 200 nanometre oldukça küçük bir mesafe. Bir saç teli bu mesafenin 500 katı. Ama hücrelerin işleyişinin temel taşı olan moleküllerin çoğunun boyu birkaç nanometreyi geçmiyor. Kimya Nobeli 3 optikçiye 2014 kimya Nobel ödülü, optik mikroskopun aşılamaz denilen sınırını aşarak nanometre düzeyinde görüntüleme yapılmasını sağlayan üç bilim adamına verildi. Mikroskobi alanında devrim denebilecek bu buluşlar, molekül düzeyinde görüntülemeyi mümkün kılarak canlı bir hücrede daha önce görmediğimiz ayrıntıları görebilmemizi sağladı. Nobel e layık görülen bilim adamları buluşlarıyla, aşılması mümkün olmayan fizik kanunlarının etrafından dolanmanın mümkün olduğunu gösterdiler.

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5 Exciting laser beam/uyaran lazer ışını Quenching laser beam/karartan lazer ışını A-) Hücrenin küçük bir bölümü standart optik mikroskopla, floresan boyama ve özel lazer kullanarak incelendiğinde 200 nanometreden küçük bir ayrıntı görülemez. 1-) Nobel ödülü getiren buluşta floresan boyanmayı uyaran lazerin yanı sıra birkaç nanometre dışındaki tüm alanı karartan ikinci bir lazer kullanıldı. 2-) Tarama sonunda ortaya çıkan resimde moleküllerin yerlerini, birbirleriyle ilişkilerini, hareketlerini saptama imkânı doğdu. 3-) İki lazer ışını eş zamanlı kullanılıp tüm alan taranınca binlerce küçük resimden oluşan birkaç nanometre çözünürlükte bir resim elde etmek mümkün oldu. Kaynak: nobelprize.org Elliiki yaşındaki Romanya doğumlu bir Alman vatandaşı olan Stefan Hell, Almanya daki ünlü Max Planck Enstitüsünde çalışıyor. Kısaca İngilizce adının baş harflerinden oluşan STED olarak bilinen yüksek çözünürlüklü mikroskopun temelini oluşturan kavramı bundan 20 yıl önce bulmuş. Yeni yöntemde bir lazer yerine iki lazer kullanarak başarıya ulaşmış. Bir hücreye odaklanmış olan mikroskoptan çıkan lazer ışını, üstüne düştüğü alanda özel maddelerle işaretlenmiş molekülleri uyararak ışıldatır. Mikroskoptan yollanan daha geniş ve farklı karakterli olan başka bir lazer hüzmesinin görevi ışıldamaları söndürmektir. Sadece bir veya birkaç nanometrelik bir alan dışındaki her şeyi söndüren ikinci lazer, geriye aydınlanmış çok küçük bir alan bırakır. Bu işlem binlerce kere tekrar edilip hücrenin her yanı tarandığında, ortaya çözünürlülüğü birkaç nanometre olan bir resim çıkar. Nobel komitesi başkanının deyişiyle eskiden bir karınca yığınını görürken şimdi karıncaları hatta bir karıncanın bacağını görmek mümkün oldu.

6 Yepyeni ufuklar açıldı Nobel ödülü alan bilim insanlarının geliştirdiği yöntemlerle çekilen bu resimde, bir kanser hücresinin büyümek için nasıl çoğaldığı görülüyor. Hücre kromozomlarını ikiye bölerek üremesini başlatıyor. Kaynak Betzig laboratuvarı. Hücrelerimiz işlevlerini ürettikleri küçük protein molekülleri aracılığıyla gerçekleştirirler. Birbirleriyle haberleşmeleri, karşılaştıkları olumlu veya olumsuz bir duruma karşı tepkilerini zincirleme bir protein reaksiyonuyla oluştururlar. Bu yıl Nobel ödülü alan bilim insanlarının buluşları sayesinde canlı hücrelerin içindeki molekülleri görmemiz, hareketlerini izlememiz mümkün olacak. Böylece kanser gibi birçok hastalığı daha iyi anlamamız ve onlarla mücadelede başarı kazanmamız kolaylaşacak. Aynı hedefe başka yoldan ulaştılar

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8 The principle of single-molecule microscopy / Tek molekül Mikroskobu Hücrenin küçük bir bölümü zayıf bir lazerle aydınlatıldığında moleküllerin birkaçı ışını emip, renkli olarak ışıldar. Single fluoresant protein/ Lazerle ışıldayan tek protein Hafifçe ışıldayan moleküller bilgisayar yardımıyla aslına sadık kalarak parlak hale getirilir. The distance between each protein > 0.2 μm / İki molekül arasındaki mesafe 200 nanometreden fazladır. Tek tek çekilen resimler üst üste konup bilgisayar marifetiyle tek bir resim haline getirilince, ortaya moleküllerin ve oluşturdukları proteinlerin yüksek çözünürlük resmi çıkar. Kaynak: nobelprize.org Nobel ödülünü kazanan diğer iki bilim insanı da optik mikroskobun sınırını aşma hedefine başka bir yol izleyerek ulaştılar. Nanometre düzeyinde görüntü sağlayan bir diğer mikroskop yöntemi geliştirdiler. ABD de California Üniversitesi nin San Diego yerleşkesinde çalışan W. Moerner, inceleyeceği hücreye çok az miktarda boya verip çok zayıf bir lazerle kısa bir uyarı gönderirse bir birine çok yakın olmayan birkaç molekülü görüntüleyebileceğini keşfetti. Bu buluştan yıllar sonra Eric Betzig adlı 54 yaşındaki Amerikalı bilim adamı bu yöntemi geliştirdi. Her seferinde farklı birkaç molekülün resmini çekti. Sonra bilgisayar yardımıyla bu fotoğrafları üst üste koyarak tek bir resim elde etti. Böylece gerçekte birbirine sadece birkaç nanometre uzakta olan molekülleri önce ayrı ayrı resimleyip sonra bir araya getirerek müthiş ayrıntılı bir fotoğraf elde etmeyi başardı.

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10 THE NOBEL PRIZE IN CHEMISTRY 2014 POPULAR SCIENCE BACKGROUND How the optical microscope became a nanoscope Eric Betzig, Stefan W. Hell and William E. Moerner are awarded the Nobel Prize in Chemistry 2014 for having bypassed a presumed scientific limitation stipulating that an optical microscope can never yield a resolution better than 0.2 micrometres. Using the fluorescence of molecules, scientists can now monitor the interplay between individual molecules inside cells; they can observe disease-related proteins aggregate and they can track cell division at the nanolevel. Red blood cells, bacteria, yeast cells and spermatozoids. When scientists in the 17th century for the first time studied living organisms under an optical microscope, a new world opened up before their eyes. This was the birth of microbiology, and ever since, the optical microscope has been one of the most important tools in the life-sciences toolbox. Other microscopy methods, such as electron microscopy, require preparatory measures that eventually kill the cell. Glowing molecules surpassing a physical limitation For a long time, however, optical microscopy was held back by a physical restriction as to what size of structures are possible to resolve. In 1873, the microscopist Ernst Abbe published an equation demonstrating how microscope resolution is limited by, among other things, the wavelength of the light. For the greater part of the 20th century this led scientists to believe that, in optical microscopes, they would never be able to observe things smaller than roughly half the wavelength of light, i.e., 0.2 micrometres (figure 1). The contours of some of the cells organelles, such as the powerhouse mitochondria, were visible. But it was impossible to discern smaller objects and, for instance, to follow the interaction between individual protein molecules in the cell. It is somewhat akin to being able to see the buildings of a city without being able to discern how citizens live and go about their lives. In order to fully understand how a cell functions, you need to be able to track the work of individual molecules. ABBE S DIFFRACTION LIMIT (0.2 µm) ant hair mammalian cell bacterium mitochondrion virus protein small molecule Nobel Prize is a registered trademark of the Nobel Foundation. 1 mm 100µm 10µm Figure 1. At the end of the 19th century, Ernst Abbe defined the limit for optical microscope resolution to roughly half the wavelength of light, about 0.2 micrometre. This meant that scientists could distinguish whole cells, as well as some parts of the cell called organelles. However, they would never be able to discern something as small as a normal-sized virus or single proteins. Abbe s equation still holds but has been bypassed just the same. Eric Betzig, Stefan W. Hell and William E. Moerner are awarded the Nobel Prize in Chemistry 2014 for having taken optical microscopy into a new dimension using fluorescent molecules. Theoretically there is no longer any structure too small to be studied. As a result, microscopy has become nanoscopy. 1µm 100 nm 10 nm 1nm

11 The story of how Abbe s diffraction limit was circumvented runs in parallel tracks; two different principles are rewarded, which have been developed independently of each other. We begin in 1993, in a student apartment in South-western Finland, where Stefan Hell had a brilliant idea as he was leafing through a textbook in Quantum Optics. Youthful revolt against Abbe s diffraction limit met with scepticism Ever since getting his Ph D from the University of Heidelberg in 1990, Stefan Hell had been looking for a way to bypass the limitation that Ernst Abbe had defined more than a century earlier. The thought of challenging such an established principle was tantalizing. But senior scientists in Germany had met his enthusiasm with scepticism, and hence Stefan Hell had taken refuge in the cold North. A professor at the University of Turku who was working on fluorescence microscopy had offered him a position on his research team. Hell was convinced that there had to be a way of circumventing Abbe s diffraction limit, and when he read the words stimulated emission in the book on Quantum Optics a new line of thought took shape in his mind: At that moment, it dawned on me. I had finally found a concrete concept to pursue a real thread. Such was his comment in let us delve into his idea. The solution: a nano-sized flashlight scanning over the sample In Turku Stefan Hell worked on so-called fluorescence microscopy, a technique where scientists use fluorescent molecules to image parts of the cell. For instance, they can use fluorescent antibodies that couple specifically to cellular DNA. Scientists excite the antibodies with a brief light pulse, making them glow for a short while. If the antibodies couple to DNA they will radiate from the centre of the cell, where DNA is packed inside the cell nucleus. In this manner, scientists can see where a certain molecule is located. But they had only been able to locate clusters of molecules, such as entangled strands of DNA. The resolution was too low to discern individual DNA strings. Think of being able to see a roll of yarn without being able to follow the thread itself. When Stefan Hell read about stimulated emission, he realized that it should be possible to devise a kind of nano-flashlight that could sweep along the sample, a nanometre at a time. By using stimulated emission scientists can quench fluorescent molecules. They direct a laser beam at the molecules that immediately lose their energy and become dark. In 1994, Stefan Hell published an article outlining his ideas. In the proposed method, so-called stimulated emission depletion (STED), a light pulse excites all the fluorescent molecules, while another light pulse quenches fluorescence from all molecules except those in a nanometre-sized volume in the middle (figure 2). Only this volume is then registered. By sweeping along the sample and continuously measuring light levels, it is possible to get a comprehensive image. The smaller the volume allowed to fluoresce at a single moment, the higher the resolution of the final image. Hence, there is, in principle, no longer any limit to the resolution of optical microscopes. Developing the first nano-flashlight in Germany Stefan Hell s theoretical article did not create any immediate commotion, but was interesting enough for Stefan Hell to be offered a position at the Max Planck Institute for Biophysical Chemistry in Göttingen. In the following years he brought his ideas to fruition; he developed a STED microscope. In 2000 he was able to demonstrate that his ideas actually work in practice, by, among other things, imaging an E. coli bacterium at a resolution never before achieved in an optical microscope (figure 3). 2(7) THE NOBEL PRIZE IN CHEMISTRY 2014 THE ROYAL SWEDISH ACADEMY OF SCIENCES

12 Figure 2 The principle of STED microscopy Regular optical microscope STED microscope Exciting laser beam 2 3 The final image gets a resolution that is much better than 0.2 micrometre. In a regular optical microscope, the contours of a mitochondrion can be distinguished, but the resolution can never get better than 0.2 micrometres. Exciting laser beam 1 Quenching laser beam 2 In a STED microscope, an annular laser beam quenches all fluorescence except that in a nanometre-sized volume. The laser beams scan over the sample. Since scientists know exactly where the beam hits the sample, they can use that information to render the image at a much higher resolution. The STED microscope collects light from a multitude of small volumes to create a large whole. In contrast, the second principle rewarded, single-molecule microscopy, entails the superposition of several images. Eric Betzig and W. E. Moerner (who always has been called by his initials, W. E.) have independently of each other contributed different fundamental insights in its development. The foundation was laid when W. E. Moerner succeeded in detecting a single small fluorescent molecule. Figure 3. One of the first images taken by Stefan Hell using a STED microscope. To the left, an E. coli bacterium imaged using conventional microscopy; to the right, the same bacterium imaged using STED. The resolution of the STED image is three times better. Image from Proc. Natl. Acad. Sci. USA 97: W. E. Moerner first to detect a single fluorescent molecule In most chemical methods, for instance measuring absorption and fluorescence, scientists study millions of molecules simultaneously. The results of such experiments represent a kind of typical, average molecule. Scientists have had to accept this since nothing else has been possible, but for a long time they dreamt of measuring single molecules, because the richer and more detailed the knowledge, the greater the possibility to understand, for instance, how diseases develop. Therefore, in 1989, when W. E. Moerner as the first scientist in the world was able to measure the light absorption of a single molecule, it was a pivotal achievement. At the time he was working at the IBM research centre in San Jose, California. The experiment opened the door to a new future and inspired many chemists to turn their attention to single molecules. One of them was Eric Betzig, whose achievements will be covered below. THE NOBEL PRIZE IN CHEMISTRY 2014 THE ROYAL SWEDISH ACADEMY OF SCIENCES 3(7)

13 Eight years later Moerner took the next step towards single-molecule microscopy, building on the previously Nobel Prize-awarded discovery of the green fluorescent protein (GFP). Molecular-sized lamps turning on and off In 1997 W. E. Moerner had joined the University of California in San Diego, where Roger Tsien, Nobel Prize Laureate to be, was trying to get GFP to fluoresce in all the colours of the rainbow. The green protein was isolated from a fluorescent jelly-fish and its strength lies in its ability to make other proteins inside living cells visible. Using gene technology scientists couple the green fluorescent protein to other proteins. The green light subsequently reveals exactly where in the cell the marked protein is positioned. W. E. Moerner discovered that the fluorescence of one variant of GFP could be turned on and off at will. When he excited the protein with light of wavelength 488 nanometres the protein began to fluoresce, but after a while it faded. Regardless of the amount of light he then directed at the protein, the fluorescence was dead. It turned out, however, that light of wavelength 405 nanometres could bring the protein back to life again. When the protein was reactivated, it once again fluoresced at 488 nanometres. Moerner dispersed these excitable proteins in a gel, so that the distance between each individual protein was greater than Abbe s diffraction limit of 0.2 micrometres. Since they were sparsely scattered, a regular optical microscope could discern the glow from individual molecules they were like tiny lamps with switches. The results were published in the scientific journal Nature in By this discovery Moerner demonstrated that it is possible to optically control fluorescence of single molecules. This solved a problem that Eric Betzig had formulated two years earlier. Tired of the academy but obsessed by Abbe s diffraction limit. Just like Stefan Hell, Eric Betzig was obsessed by the idea of bypassing Abbe s diffraction limit. In the beginning of the 1990s he was working on a new kind of optical microscopy called near-field microscopy at the Bell Laboratories in New Jersey. In near-field microscopy the light ray is emitted from an extremely thin tip placed only a few nanometres from the sample. This kind of microscopy can also circumvent Abbe s diffraction limit, although the method has major weaknesses. For instance, the light emitted has such a short range that it is difficult to visualize structures below the cell surface. In 1995 Eric Betzig concluded that near-field microscopy could not be improved much further. In addition, he did not feel at home in academia and decided to end his research career; without knowing where to go next, he quit Bell Labs. But Abbe s diffraction limit remained in his mind. During a walk a cold winter day a new idea came to him; might it be possible to circumvent the diffraction limit by using molecules with different properties, molecules that fluoresced with different colours? Inspired by W. E. Moerner, among others, Eric Betzig had already detected fluorescence in single molecules using near-field microscopy. He began to ponder whether a regular microscope could yield the same high resolution if different molecules glowed with different colours, such as red, yellow and green. The idea was to have the microscope register one image per colour. If all molecules of one colour were dispersed and never closer to each other than the 0.2 micrometres stipulated by Abbe s diffraction limit, their position could be determined very precisely. Next, when these images were superimposed, the complete image would get a resolution far better than Abbe s diffraction limit, and red, yellow and green molecules would be distinguishable even if their distance was just a few nano- 4(7) THE NOBEL PRIZE IN CHEMISTRY 2014 THE ROYAL SWEDISH ACADEMY OF SCIENCES

14 metres. In this manner Abbe s diffraction limit could be circumvented. However, there were some practical problems, for instance a lack of molecules with a sufficient amount of distinguishable optical properties. In 1995 Eric Betzig published his theoretical ideas in the journal Optics Letters, and subsequently left academia and joined his father s company. Lured back to microscopy by green fluorescent proteins For many years Eric Betzig was entirely disconnected from the research community. But one day a longing for science sprang to life again, and returning to the scientific literature he came across the green fluorescent protein for the first time. Realizing there was a protein that could make other proteins visible inside cells revived Betzig s thoughts of how to circumvent Abbe s diffraction limit. The real breakthrough came in 2005, when he stumbled across fluorescent proteins that could be activated at will, similar to those that W. E. Moerner had detected in 1997 at the level of a single molecule. Betzig realized that such a protein was the tool required to implement the idea that had come to him ten years earlier. The fluorescent molecules did not have to be of different colours, they could just as well fluoresce at different times. Surpassing Abbe s limit by superimposing images Just one year later, Eric Betzig demonstrated, in collaboration with scientists working on excitable fluorescent proteins, that his idea held up in practice. Among other things, the scientists coupled the glowing protein to the membrane enveloping the lysosome, the cell s recycling station. Using a light pulse the proteins were activated for fluorescence, but since the pulse was so weak only a fraction of them started to glow. Due to their small number, almost all of them were positioned at a distance Figure 4 The principle of single-molecule microscopy The blurred images are processed using 2 probability theory in order to render them much sharper. The distance between each protein > 0.2 µm Microscope 1 A weak light pulse activates a fraction of all the fluorescent proteins. The distance between them is greater than Abbe s diffraction limit of 0.2 micrometres. They glow until bleached, at which point the procedure is repeated on a new subgroup of proteins. When all images are 3 superimposed a high resolution totality appears, wherein individual proteins can be discerned. Single fluorescent protein High-resolution image THE NOBEL PRIZE IN CHEMISTRY 2014 THE ROYAL SWEDISH ACADEMY OF SCIENCES 5(7)

15 from each other greater than Abbe s diffraction limit of 0.2 micrometres. Hence the position of each glowing protein could be registered very precisely in the microscope. After a while, when their fluorescence died out, the scientists activated a new subgroup of proteins. Again, the pulse was so weak that only a fraction of the proteins began to glow, whereupon another image was registered. This procedure was then repeated over and over again. When Betzig superimposed the images he ended up with a super-resolution image of the lysosome membrane. Its resolution was far better than Abbe s diffraction limit. An article published in Science in 2006 subsequently presented the ground-breaking work. Figure 5. The centre image shows lysosome membranes and is one of the first ones taken by Betzig using single-molecule microscopy. To the left, the same image taken using conventional microscopy. To the right, the image of the membranes has been enlarged. Note the scale division of 0.2 micrometres, equivalent to Abbe s diffraction limit. The resolution is many times improved. Image from Science 313: The laureates are still mapping the innermost secrets of life The methods developed by Eric Betzig, Stefan Hell and W. E. Moerner have led to several nanoscopy techniques and are currently used all over the world. The three Laureates are still active researchers in the large and growing community of scientists spearheading innovation in the field of nanoscopy. When they direct their powerful nanoscopes toward the tiniest components of life they also produce cutting-edge knowledge. Stefan Hell has peered inside living nerve cells in order to better understand brain synapses. W. E. Moerner has studied proteins in relation to Huntington s disease. Eric Betzig has tracked cell division inside embryos. These are just a few of many examples. One thing is certain, the Nobel Laureates in Chemistry 2014 have laid the foundation for the development of knowledge of the greatest importance to mankind. 6(7) THE NOBEL PRIZE IN CHEMISTRY 2014 THE ROYAL SWEDISH ACADEMY OF SCIENCES

16 LINKS AND FURTHER READING Additional information on this year s Prizes, including a scientific background article in English, may be found at the website of the Royal Swedish Academy of Sciences, and at They also include web-tv versions of the press conferences at which the awards were announced. Information on exhibitions and activities related to the Nobel Prizes and the Prize in Economic Sciences may be found at Articles Nair, P. (2012) QnAs with W. E. Moerner, Proc. Natl. Acad. Sci. USA 109(17):6357 Deffke, U. (2011) Outsmarting optical boundaries, Gewin, V. (2006) Eric Betzig, group leader, Janelia Farm Research Campus, Howard Hughes Medical Institute, Leesburg, Virginia, Nature 440:578 Howard Hughes Medical Institute, Biography Eric Betzig, Lectures and interviews (video) Betzig, E. (2012) Day 2 Distinguished Lecture by Dr. Eric Betzig at MF Symposium, watch?v=uqmll8-eaxk Interview with Betzig, E. (2013) Eric Betzig Sequence, Interview with Hell, S. W. (2013) Stefan Hell Sequence, Interview with Moerner, W. E. (2013) Alumni Achievement Award: W.E. Moerner THE LAUREATES ERIC BETZIG U.S. citizen. Born 1960 in Ann Arbor, MI, USA. Ph.D from Cornell University, Ithaca, NY, USA. Group Leader at Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. STEFAN W. HELL German citizen. Born 1962 in Arad, Romania. Ph.D from the University of Heidelberg, Germany. Director at the Max Planck Institute for Biophysical Chemistry, Göttingen, and Division head at the German Cancer Research Center, Heidelberg, Germany. www3.mpibpc.mpg.de/groups/hell WILLIAM E. MOERNER U.S. citizen. Born 1953 in Pleasanton, CA, USA. Ph.D from Cornell University, Ithaca, NY, USA. Harry S. Mosher Professor in Chemistry and Professor, by courtesy, of Applied Physics at Stanford University, Stanford, CA, USA. Science Editors: Måns Ehrenberg and Sven Lidin, the Nobel Committee for Chemistry Text: Ann Fernholm Illustrations: Johan Jarnestad/The Royal Swedish Academy of Sciences Editor: Ann Fernholm The Royal Swedish Academy of Sciences THE NOBEL PRIZE IN CHEMISTRY 2014 THE ROYAL SWEDISH ACADEMY OF SCIENCES 7(7)