Nobel Laureate Roger Tsien Dies at 64 - NBC News Tsien died on Aug. 24 in Eugene, Oregon, according to a statement Wednesday from the university. UC San Diego Chancellor Pradeep Khosla said that Tsien apparently died while on a bike trail, the San Diego Union-Tribune reported, but the cause of death had not been determined.
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The Nobel Prize in Chemistry 2008
Osamu Shimomura, Martin Chalfie, Roger Y. Tsien
Q: What do elementary school pupils and Nobel Laureates have in common?
A: They both have to write autobiographical essays on command.
Ancestors and family
My father, Hsue Chu Tsien (1915–1997), came from the "scholar-gentry" class in Hangzhou, China, where "Tsien" (now more commonly transliterated as Qian) is quite a common surname. Apparently in 907 A.D., Qian Liu, my paternal ancestor 34 generations ago, established a kingdom around Hangzhou and fostered its growth through many civil engineering projects. This fiefdom prospered peacefully under the rule of Qian Liu and his successors until 978, when they surrendered to the Sung dynasty to avoid bloodshed*. I had thought that descent from Qian Liu was an obscure secret of our family, but this factlet somehow found its way onto Wikipedia through no fault of mine. Furthermore, this genealogy is hardly much of a distinction given that everyone in principle has 234 ancestors from 34 generations ago. 234 (about 17 billion) vastly exceeds the earth's population in the 10th century, so practically everyone, at least from that part of China, probably has Qian Liu as an ancestor, even if not so strictly through the Y chromosome. By far the most famous Tsien in modern times is Hsue Shen Tsien or Qian Xuesen, the aeronautical engineer who was deported from the U.S. during the McCarthy era and then became father of the ballistic missile program of the People's Republic1. He and my father were first cousins. Several other Chinese-American bioscientists, including Robert Tjian, now President of the Howard Hughes Medical Institute, and Shu Chien, a prominent bioengineer at UCSD, also have the same Chinese surname as mine and are likewise descended from Qian Liu, so we are distant relatives.
Dad too was excited by flight and airplanes, which were the cutting-edge technology of his day. In the 1930s he won a national scholarship (Tsinghua) to study in America. He went to MIT's mechanical engineering department, where he obtained a Master's degree for research on aircraft engines, including a proposal to boost the thrust during takeoff by injecting water into the exhaust to become steam. Before he could pursue any further studies in America, he had to return to China to serve in the Nationalist (Kuomingtang) Air Force. One of his best friends and fellow engineers, Yao Tzu Li, had an attractive and intelligent sister, Yi Ying Li, who had trained as a nurse at Peking Union Medical College, the most prestigious of Chinese medical institutions. My father courted her eagerly by letters even before they had ever met in person. When they finally did meet, she found him socially awkward and overly impressed with his own academic prowess2. Despite her lack of romantic feelings for him, she agreed to marry him, perhaps because she doubted her own prospects in wartime China. Their first son, Yongyou, was born in March 1945. Soon thereafter, Dad was ordered to go to the U.S. as a liaison officer to try to extract more military aid for the Chinese Air Force. He had to travel over the Himalayas to India and then by ship, zigzagging to avoid enemy submarines, so he did not arrive in the U.S. until the day that Japan's surrender was announced2. His mission was therefore futile, but he knew that China would be racked by postwar civil war. Somehow he used contacts in the Defense Department to arrange for Mom and Yongyou to come to the U.S. Such permission was not trivial, because the Chinese Exclusion Act forbidding immigration from China to the U.S. had been repealed only in 1943, at which time the national quota was set at just 105 immigrants per year and thousands were ahead on the waiting list.
After Mom and Yongyou arrived in America in January 1947, life was quite a struggle because Dad could not find a professional job as an aircraft engineer. Such employment at the major firms required a security clearance, which a Chinese citizen could not get. So he started a tiny export-import business in New York City and later an engineering consultancy firm in Westchester County, which yielded enough to live on but not to become prosperous. Nevertheless their next son, Yonglo or Louis, was born in October 1949. Around then, Yongyou started school and needed to pick an American name. He wanted to be "Dick", so the school officials explained to my parents that this was a nickname for "Richard". "Yongyou" was somehow transliterated as "Winyu" to become Richard's middle name in English.
According to Mom, she always planned to have three children, though this statement came many years after the fact. After two sons, even Dad was looking forward to a girl2, but in February 1952 they got me instead. Dad picked my Chinese name, Yongjian (transliterated Yonchien to become my middle name in English), but Dick insisted that my American name should be Roger. My mother later told me this was because Dick had a playmate at the time named Roger. Much later, perhaps when I was in college, I quizzed Dick about this mysterious namesake. Dick confessed that he actually named me after Roy Rogers, the famous cowboy actor. I mention all this to clarify the origins of the similarity between the names "Richard W. Tsien" and "Roger Y. Tsien", which has continually confused many scientists and their secretaries even up to now. I don't know why my parents chose two different transliterations for "Yong", but if they had not, Richard and I would be completely indistinguishable ("Tsien RY") in bibliographical databases.
Growing up: Home chemistry experiments
One of my earliest memories, probably from age 3 or 4, is of building a sand path at the beach across a strip of coarse pebbles that hurt my feet to cross. I loved to draw maps of imaginary cities with freeways vaulting over or tunneling under the surface streets. Perhaps these were the first signs of my future obsessions with bridge-building and activity-mapping. Some time in elementary school my parents bought a Gilbert chemistry set, but I didn't find it very interesting because the experiments seemed so tame. Then I discovered a book in the school library that had much better experiments and illustrations. Regrettably, I cannot now remember the book's name or author, though I hand-copied many sketches of its experiments into a notebook dated around 1960, now deposited in the Nobel Museum. Two experiments I remember best: 1) silica gardens, in which crystals of metal salts (e.g. CoCl2, NiSO4, CuSO4) dropped into a solution of sodium silicate would develop bright magenta, green, or blue gelatinous coatings from which vertically rising dendrites would sprout; 2) preparation of a strongly alkaline (0.5M NaOH or KOH) aqueous solution of dilute (~ 0.5 mM) potassium permanganate, which colored the liquid an intense purple. As this solution passed through a folded cone of filter paper, its color changed to a beautiful green, reflecting reduction of MnO4- to MnO42–, presumably by the cellulose. In November 2008, I reproduced this surprisingly little-known demonstration for Swedish television and Nobel Media as an example of what got me interested in chemistry. Both experiments reflect an early and long-lasting obsession with pretty colors.
Figure 1. Our family in 1960, just before moving to Livingston. From left: Richard (15), Louis (11), H.C. (my father), me (8), Yi Ying (my mother).
In 1959, Dad closed his consulting firm and started working for RCA's vacuum tube division in Harrison, NJ. Mom and Dad looked for a town with affordable homes, within convenient commuting distance, and with good public schools for the three of us. A photo from around then is Figure 1. They chose a new housing development in Livingston, NJ, but the developer refused to sell to us, saying that they could not permit Livingston to become a Chinatown, nor could they afford the likelihood that other customers would refuse to buy houses next to a Chinese family. My parents appealed to the Governor of New Jersey, Robert Meyner. His office sent a letter to the developers warning them that racial discrimination was illegal. Finally a compromise was reached: the developers sold us a house completely surrounded by houses that had already been sold. The problem for us kids was that Livingston has lots of rocks in its soil, left from the glaciers. My parents were determined to have a respectable American-style grassy lawn, which required removal of the rocks. We had to cart away not only our own stones but many from our neighbors, who had used the unoccupied leftover lot as a dumping ground, or so we believed. The many weeds in the lawn revealed a deep personality difference: Dad, as an impatient mechanical engineer, liked the instant solution of digging them up one by one from close enough to extirpate all the roots. I was an occasionally asthmatic hay fever sufferer, deeply afraid of pollen, so I advocated a chemical approach, sprinkling herbicide on the weeds from a safe distance. We tried my way once. The weeds slowly turned brown but eventually recovered. Dad declared the experiment a failure and went back to hand weeding. I still think about this result in relation to our current research on cancer therapy.
In 1960, RCA closed its vacuum tube division, presumably because semiconductors were replacing tubes, so Dad moved to Esso (later renamed Exxon) Research and Engineering. Esso provided much better projects and pay, so he stayed until his retirement in 1983. I believe some of the chemicals and glassware that enabled me to do the more interesting chemistry experiments were diverted from the company stockroom. Other supplies could be bought by mail order in those days with a parent's signature. Over the next 5 or 6 years I gradually did many of the classic experiments of inorganic chemistry in the basement of our house: preparing and burning H2 gas, preparing O2 and burning steel wool in it, preparing NH3 in a flask and watching it suck water up as a fountain inside the flask. I distilled HF from CaF2 + H2SO4 in plastic apparatus and was delighted to see its ability to etch glass. I electrolyzed molten NaOH using a step-down transformer and rectifier from a model train set, the nickel crucible as cathode, and a carbon rod salvaged from a dead flashlight battery as anode. I managed to get a few granules of very impure metallic sodium, which gave off a satisfying hiss when dropped into water. Pyrotechnics were naturally of great interest: I made and ignited gunpowder, ammonium dichromate volcanoes, and even a spectacular thermite reaction with powdered aluminum and chromium oxide. My most ambitious attempt was a multistep sequence aimed at synthesizing aspirin, for which I needed acetic anhydride, which had to be made from acetyl chloride, for which I needed phosphorus trichloride, for which I needed to burn red phosphorus in a stream of chlorine gas. I tried to do this reaction sequence in flasks with rubber stoppers (Figure 2), because I had no glassware with ground glass joints. The corrosive chemicals largely chewed up the rubber, so I did not get beyond acetyl chloride. Because I had no fume hood, I did the more dangerous experiments outdoors on a picnic table on the backyard patio. Looking back, I am appalled at how dangerous all this was for an unsupervised boy of 8 to 15, but it was also very good training in how to improvise equipment, plan and execute experiments, interpret confusing results, and decide how to do things better. These experiments made me confident enough that when I had to earn my first merit badge as a Boy Scout and was advised to pick something really easy, I chose Chemistry. Tougher merit badges like Hiking, with its requirement for a twenty-mile hike in one day, I got later.
Figure 2. Setup for preparing Cl2 and reacting it with red phosphorus, sometime in 1966? 1967, in our screened backyard patio. The leftmost flask contained KMnO4 to react with aqueous HCl added through the funnel controlled by a pinch clamp. The Cl2 was dried by passage through CaCl2 then directed onto P4 in the flask on the ring stand. Because no running water was available, the water to cool the PCl3 condenser was siphoned from the recycled milk jug and deposited into the waste can labeled "Hawaiian Punch". The receiver for PCl3 was immersed in ice in the thermos bottle. The alcohol lamp allowed auxiliary heating for the phosphorus. Note rubber stoppers everywhere.
Elementary school to high school;
Westinghouse science talent search
School was usually bearable but frequently boring. I really looked forward to days in winter when heavy snow would close school, so that I could spend the day sledding. I was terrible at ball games at school, such as football, soccer, basketball, and softball, because I was small, nonathletic, and two years younger than my classmates at an age when this makes a huge difference. But I was popular enough in high school to be elected student council treasurer by an overwhelming majority.
Mom tried hard to teach us Chinese after school, but as I got older I found these lessons increasingly tedious. I well understood spoken Chinese at a child's level (e.g. the Chinese for "Tidy your room!" is permanently etched into my brain) but was reluctant to speak it myself, due to the wish (all too common among children of immigrants) to distance myself from my parents' accents and intense pride in their ethnicity and traditions. Likewise they despaired over my refusal (like a "foreign devil") to eat most Chinese food, especially the most authentic dishes with the strongest tastes or smells, or prepared from exotic creatures.
My first exposure to a research environment was in a National Science Foundation-sponsored summer research program at Ohio University in 1967, where I was assigned to work in the laboratory of Prof. Robert Kline on the ambident coordination of thiocyanate (SCN–). The Pearson theory of hard and soft ligands and metals was new and fashionable at the time, so Prof. Kline wanted me to find out if thiocyanate could simultaneously bind with its "soft" sulfur to a soft metal and its "hard" nitrogen to a hard metal, e.g. PhHg–SCN–Cr(III). He hoped that the infrared absorbances of thiocyanate would tell us whether such bridging was taking place. I prepared a lot of amorphous precipitates of rather ill-defined composition and measured their infrared spectra. In the winter of 1967, my senior year at Livingston High School, I entered the Westinghouse Science Talent Search, the nationwide "science fair" competition. (This annual event still exists, though sponsorship was taken over by Intel in 1998.) For lack of any alternatives, I wrote up my Ohio University project, trying my best to draw some conclusions from a mess of dubious data. Prof. Kline largely disowned those conclusions, pointing out that my preparations had not given correct carbon, hydrogen, and nitrogen microanalyses. The 40 finalists were summoned to Washington DC in March 1968 for interviews and a public poster session. I remember being envious of my fellow finalists, who were much more adult and sophisticated. Also their projects and exhibits seemed much more exciting and explainable than mine. I felt intimidated by the senior judge, Glenn Seaborg, partly because of his commanding height, partly because he was chairman of the U.S. Atomic Energy Commission, partly because of his 1951 Nobel Prize for work in inorganic chemistry. The awards ceremony was very tense for us because the ten scholarship winners were announced in reverse order, forcing everyone to hope their name was called but as late as possible. I am still mystified how I won first prize despite the unsoundness of my project, and I retain a dislike for scientific competitions. Dad did his bit to keep me grounded: when I phoned home, his first comment was that it was a good thing I now had a $10,000 scholarship, because he had recently lost that amount on the stock market. One of the most satisfying compliments I received was that the developer who had not wanted to sell a house to Mom and Dad in 1960 now used my photo in one of their advertisements as evidence of the quality of the local school system.
Harvard
In April 1968 I had to choose between four colleges: Columbia, MIT, Caltech, and Harvard. Dad vetoed Columbia because of the student unrest that spring, and I did not mind because I wanted to get further away from New Jersey. I rejected MIT because Dick and Louis had both gone there and I was tired of being compared to them. The small size of Caltech's undergraduate class sounded attractive, but I finally decided against Caltech because Richard Feynman was no longer teaching introductory physics and because the music department was tiny and of negligible fame compared to Harvard's. Indeed Harvard did turn out to be a salutary experience on the whole. Friendships with classmates were crucial in helping me grow up. The student protests of spring 1969 and 1970 provided my first exposures to cannabis, police brutality, and participatory politics. The diversity of courses let me sample art history, visual design, economics, Colonial history, constitutional law, psychology, both music theory and chamber music performance, etc. Ironically, the worst courses were those intended to lead Harvard's elite chemistry majors into research careers. These required courses were so distasteful I abandoned chemistry. Looking for alternatives, I dabbled in molecular biology (taught by Walter Gilbert, who later shared a Nobel Prize for DNA sequencing), oceanography, relativistic quantum mechanics, and astrophysics. But what I finally chose was neurobiology, partly because the relationship between brain and mind seemed philosophically the most important problem in science, partly because David Hubel, John Nicholls, and Torsten Wiesel ran a course charismatically proselytizing undergraduates to become neuroscientists. Hubel and Wiesel were still doing the research on visual cortex that eventually won them the 1981 Nobel Prize in Medicine or Physiology. I asked Prof. Hubel if I could do a summer internship in their lab, but he told me they had no space for undergraduates and suggested that I apply to Nelson Kiang at the Massachusetts Eye and Ear Infirmary instead. In summer 1971, Kiang gave me intensive tutorials in auditory neurophysiology and an interesting project analyzing spike trains from the cochlear nucleus. I am still plugging away at neurobiological problems almost four decades later.
Cambridge
When I asked Hubel and Kiang for advice on where to apply to graduate school in neuroscience, their only point of agreement was that the top places were Cambridge, MA and Cambridge, UK. I felt it was time to leave Cambridge, MA to broaden my horizons, so I applied for a Marshall Scholarship to go to the other Cambridge. In early 1972, while still a senior at Harvard, I learned my application was successful, and that my Ph.D. supervisor would be a Dr. R. H. Adrian, whom I had never heard of. I phoned my brother Dick, who had just become an Assistant Professor at Yale after finishing his D. Phil. from Oxford on cardiac electrophysiology. Dick informed me that R. H. Adrian was one of Britain's most eminent skeletal muscle electrophysiologists, and son of E. D. Adrian, a Nobel Laureate in neurophysiology. Moreover R. H. Adrian had been the external examiner on Dick's D. Phil. degree. "But muscle is a backwater," I exclaimed. "I want to work on the brain." Dick assured me that Richard Adrian was a true British gentleman, who would let me work on a topic of my own choosing. So I decided to wait and see. After a summer intensively studying music at Fontainebleau, near Paris, I arrived in Cambridge in October 1972. At my first lunch in Churchill College, an aristocratic-looking don sat down opposite me and asked if I was Roger Tsien. I immediately realized he was Richard Adrian, because only someone who knew a member of my family could pronounce our surname correctly, as he just had. Within the first few minutes of our conversation, he asked "Is it true you think muscle is a backwater?" I had to admit the accuracy of the quotation. (I later found out that Dick had mischievously teased Adrian about this at a conference they had both attended that summer.) Adrian looked a bit pained at my confession, but immediately said that he would not object whenever I wanted to transfer to one of the real neurophysiologists in the department.
Thus began my Ph.D. training. I never did switch to another official supervisor, because I soon realized I did not enjoy doing conventional electrophysiology of the central nervous system. The traditional thesis project, basically following the paradigm so successfully employed by Hubel and Wiesel, was to drop an extracellular microelectrode into the brain of an anesthetized animal and record the activity of individual neurons while providing sensory stimuli. After several hundred such recordings, one could classify the different response patterns and write up a thesis and several publications. To me this seemed too much like ice fishing, i.e. cutting a hole in the ice covering a lake, dropping a fishing line into the opaque water beneath, and patiently waiting for a bite. The brain derives its power from trillions of neurons working in parallel, so I wanted to see lots of neurons simultaneously signaling to each other and processing information. Ideally one would stain the neurons with a dye that would visibly light up or change color whenever and wherever a neuron fired an action potential. A few commercially available dyes had indeed been found that responded to neuronal action potentials, but the optical responses were extremely tiny, e.g. a 10–4 or 10–5 change in fluorescence.
They were detectable only if thousands of action potentials driven by the investigator were averaged under highly simplified conditions3. Many orders of magnitude improvement would be necessary to detect endogenous signals in a complex brain. I rashly decided in winter 1972 that I would try to design and synthesize new dyes for the specific purpose of imaging neuronal activity. One strategy was to target the dye to the vicinity of sodium channels, which were believed to undergo large conformational changes as they generated action potentials. Another strategy was to create "electrochromic dyes" with large changes in dipole moment between ground and excited state, so that a change in neuronal membrane potential could shift the peak wavelengths of absorbance or fluorescence4. In either case I would have to learn organic synthesis, which I had hated in those Harvard chemistry courses and which nobody in the Physiological Laboratory could teach me. Fortunately, Dr. Ian Baxter, a junior faculty member in the Chemistry Department and a friend of a friend of Richard Adrian's, was intrigued by my idea for targeting sodium channels and agreed to supervise me unofficially. Baxter had no other students and had the time, kindness, and patience to look over my shoulder several times a day and show me the necessary techniques. I found to my own surprise that I could do and enjoy organic synthesis once it was for a biological purpose of my own choosing. I remained hooked on this type of research even though the molecule I synthesized proved incapable of binding sodium channels, even though Baxter soon left to become a careers counselor in the north of England, and even after other generations of my synthetic voltage sensors proved inferior to those found by other labs screening large numbers of commercially available dyes and their close analogs5.
My first glimmer of success required shifting to another biological target. Action potentials almost always generate large increases in intracellular calcium to exert any biological effect such as the release of neurotransmitters to excite or inhibit the next neuron in the pathway. In 1975 there was great excitement over the discovery that arsenazo III, a dye invented to measure heavy metals in nuclear waste, could also be used to monitor calcium in giant axons from squid neurons, though the signals from this dye were very small and somewhat ambiguous6. I felt that designing a dye to measure Ca2+ should be a far easier problem than designing a dye to track fast changes in neuronal membrane potential. Hundreds of dyes were already known in the chemical literature to respond to Ca2+, e.g. for determination of water hardness. The real problem was that inside cells, the free Mg2+ concentration is about four orders of magnitude higher than that of Ca2+, so that an intracellular Ca2+ indicator needs yet higher selectivity for Ca2+ over its sister ion Mg2+. No chemist had yet recognized the biological need for such a selective indicator. A colorless buffer called EGTA was the only synthetic molecule known to have the necessary Ca2+:Mg2+ selectivity7, but it had never been made into any sort of dye molecule. By doodling on paper and playing with molecular models, I saw a way to make EGTA into a very rudimentary dye molecule. I started on this brand new project without telling Richard Adrian, because any prudent supervisor would have told me I should be bringing older projects to closure rather than starting radically new ones. Fortunately, within a few weeks I managed to make a small, impure sample of the target molecule (much later given the acronym "BAPTA") and found that it had the expected optical response to Ca2+ combined with high Ca2+:Mg2+ selectivity8. After many more years and discoveries, better dyes descended from BAPTA** became the most popular way of seeing endogenous intracellular Ca2+ signals, screening for ligands and receptors linked to Ca2+ signaling, and imaging neuronal activity microscopically.10
After my Ph.D., I stayed in Cambridge as a postdoctoral Research Fellow at Gonville & Caius College. My change in focus towards Ca2+ signaling led me into collaboration with Dr. Timothy Rink, a new faculty member in the Physiological Laboratory, because Tim wanted to make Ca2+-selective electrodes from materials sent from Switzerland11. The directions for assembly were in German, which Tim could not read. I had learned to read chemistry papers in German, so I translated the instructions. Our collaboration started with these Ca2+-selective electrodes and continued with the biological testing and exploitation of my fluorescent indicators for Ca2+. Even more importantly, Tim and his wife Norma invited me to their Christmas party in 1976, where I first met their sister-in-law, Wendy. Soon I was spending every weekend visiting Wendy at her house in North London. When Tim and Norma found out several months later, they were quite astonished at the effectiveness of their entirely unintentional matchmaking. Wendy (Figures 3–4) is still the love of my life.
Berkeley
My fellowship at Gonville & Caius College was to end in late 1981, so in 1979–1980 I started looking for an independent position. Because of Wendy's residence in London, I applied to the National Institute of Medical Research in Mill Hill, but was rejected without an interview. This was not a good time to search for a research job in Britain, because of the austerity program of the new Thatcher administration. It was time to return to the U.S., yet I had almost no contacts and few publications. Almost all my applications were unsuccessful. Biological departments considered me a chemist, while chemistry departments rejected me as a biologist. Nowadays the application of chemistry to solve biological problems is a very fashionable subdiscipline dubbed "chemical biology", but in 1980 the only venue for such interdisciplinary efforts was in the pharmaceutical industry. Even there, individual scientists were typically either chemists or biologists, not both simultaneously.
Figure 3. Wendy with our dog, Kiri, in 2004.
Luck intervened. The Department of Physiology-Anatomy, University of California, Berkeley, had a vacant assistant professorship, for which the chair of the search committee was Terry Machen, whom I had gotten to know while he was on sabbatical in Cambridge. Also Berkeley had two faculty members, Richard Steinhardt and Robert Zucker, who were interested in Ca2+ signaling. These connections enabled me to get an interview at Berkeley. Fortunately, the fluorescent indicators for Ca2+ had finally progressed enough to enable the first direct measurements of cytosolic Ca2+ in lymphocytes, including the elevation due to mitogenic stimulation12,13. Now one could investigate Ca2+ signals in populations of small mammalian cells, whereas previous techniques required single cells large and robust enough to withstand microinjection. This prospect, together with the fact that my Ph.D. was in Physiology, convinced the Department to offer me the Assistant Professorship, which I accepted before I found out that Berkeley was suffering a financial crisis. The startup package to get my laboratory going in early 1982 was cut to just a few thousand dollars, and each item had to be justified as a replacement for obsolete instructional equipment. For example, to get me a UV lamp for viewing thin layer chromatography plates, an old microscope illuminator from the teaching lab had to be junked. More importantly, the Department had no resources to provide a fume hood, which I needed to continue synthesizing the Ca2+ indicators. Prof. Robert Macey, whose lab was next to mine, kindly donated an old fume hood including its irreplaceable ductwork extending to the roof of the building. For the remainder of my seven years at Berkeley, all our synthetic reactions took place in this single wooden fume hood, less than 4 feet wide, with wire netting embedded in the glass of the front window. The entire lab stank from chemicals in unvented storage cabinets, and became lachrymatory when reactions using excess ethyl bromoacetate had to be worked up outside the hood. I mention these austerities only to remind young scientists that some good research can be accomplished without lavish facilities and startup funds.
Despite these troubles, my time at Berkeley was scientifically quite productive, including collaborations with Machen14, Steinhardt15, Zucker16, and others. I recruited Drs. Grzegorz Grynkiewicz and Akwasi Minta, who synthesized much improved Ca2+ indicators (fura-2, indo-1, fluo-3)17,18 and a Na+ indicator (SBFI)19, all of which are still in use today. After the budget crisis eased, the Berkeley administration helped me buy a primitive image processor, which I painfully programmed20 to calculate images of the ratio of fluorescences at two alternating excitation wavelengths. Such real-time ratioing revealed Ca2+, Na+, and pH signals14 inside single living cells, often with unprecedented spatiotemporal resolution.
Moving to UCSD
However, I began to worry about being trapped in a career of imaging inorganic ions. I wanted to explore signals transmitted through more complex biochemicals such as cAMP (cyclic 3',5-adenosine monophosphate) and the wider, more fashionable world of macromolecular interactions. As my bargaining power grew, I also came to want a lab with enough fume hoods, vented storage cabinets, and small darkrooms for fluorescence microscopy to support my unusual combination of chemistry and biology, as well as a joint appointment in a Chemistry department and support from the Howard Hughes Medical Institute. None of these were possible in Berkeley, so in 1989 we moved south to the University of California, San Diego, where we still are. UCSD satisfied the above desires and was much younger, roomier, faster-growing, and less tradition-bound than Berkeley, which I felt more than compensated for its lesser fame. The highlights of the science started at UCSD are recounted in my Nobel lecture.
Figure 4. Wendy and I, dressed up for the Nobel Ceremony.
Conclusions
Writing this autobiography has reminded me how my career has been shaped by a strange mixture of chance and fateful predisposition. The use of chemistry to build biologically useful molecules is a form of engineering, so I did not escape the tradition set up by my father, uncles, and brothers. However, I avoided the mechanical, aeronautical, electrical, and computer specialties they chose, probably because like many youngest siblings21, I had to seek a distinct niche. But if I had not found Ian Baxter to re-instill my enjoyment of chemistry, perhaps I would have chosen yet another direction. My interest in imaging with multiple glowing colors also reflects visual interests from early childhood, which I have been lucky enough to align with a professional career. From a strictly biological point of view, our contributions have mainly been in the development of techniques. Man-made techniques do have a habit of becoming obsolete, whereas basic discoveries about how nature works should last forever. But truly fundamental insights such as those of Darwin or Watson & Crick are rare and often subject to intense competition, whereas development of successful techniques to address important problems allows lesser mortals to exert a widespread beneficial impact for at least a few years. Moreover, the same engineering approach is what creates new therapeutic strategies to alleviate disease, not just tools for our fellow researchers.
* The benevolent reign of these kings is commemorated in at least two immaculately maintained shrines, one in Lin'an, a medium-sized city in Zhejiang Province, the other constructed in 2002 on prime real estate on the famous West Lake at the center of Hangzhou. My mother, my wife, and I visited both shrines in 2004. My mother interpreted the prominence of these shrines as an attempt by the current Chinese regime to advertise a historical precedent for reunification with Taiwan.
** The invention of a generalizable structure that sensed Ca2+ with unprecedented selectivity was duly reported to the National Research Development Corporation, as required for work funded by the UK Science Research Council. Initially NRDC was enthusiastic enough to file a patent application, 42927/78, but the administrators soon decided that measuring intracellular Ca2+ was of negligible commercial value. They felt that the only possible use for biological Ca2+ measurements was in clinical assays in blood serum, an application with completely different performance criteria, so they abandoned the patent application. In principle I could have taken over the patent costs out of my own pocket, but the NRDC's estimate of the fees equaled about 20 years of a postdoctoral salary, so I did not try. Eventually, follow-up patent applications by the University of California covering narrower variations in molecular structure proved quite lucrative. A much more important example of the NRDC's conservatism9 was their failure to patent Milstein and Köhler's monoclonal antibodies, another Cambridge invention of the mid-1970's.
References
1. Chang, I. Thread of the Silkworm. Basic Books (1995).
2. Tsien, Y. Y. Unpublished memoirs. 2007.
3. Conti, F. & Tasaki, I., "Changes in extrinsic fluorescence in squid axons during voltage-clamp," Science 169, 1322–1324 (1970).
4. Platt, J. R. "Electrochromism, A Possible Change of Color Producible in Dyes by An Electric Field," J. Chemical Physics 34, 862–863 (1961).
5. Cohen, L. B. & Salzberg, B. M., "Optical measurement of membrane potential," Rev. Physiol. Biochem. Pharmacol. 83, 35–88 (1978).
6. Brown, J. E. et al., "Rapid changes of intracellular free calcium concentration: detection by metallochromic indicator dyes in squid giant axon," Biophys. J. 15, 1155–1160 (1975).
8. Tsien, R. Y., "New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures," Biochemistry 19, 2396–2404 (1980).
9. Milstein, C., "With the benefit of hindsight," Immunol. Today 21, 359–364 (2000).
10. Tsien, R. Y., "Monitoring Cell Calcium," chapter 2 in Calcium as a Cellular Regulator, Carafoli, E. & Klee, C. (eds.), pp. 28–54 (Oxford University Press, New York, 1999).
11. Ammann, D., Güggi, M., Pretsch, E. & Simon, W., "Improved calcium ion-selective electrode based on a neutral carrier," Analytical Letters 8, 709–720 (1975).
12. Tsien, R. Y., Pozzan, T. & Rink, T. J., "T-cell mitogens cause early changes in cytoplasmic free Ca2+ and membrane potential in lymphocytes," Nature 295, 68–71 (1982).
13. Tsien, R. Y., Pozzan, T. & Rink, T. J., "Calcium homeostasis in intact lymphocytes: cytoplasmic free Ca2+ monitored with a new, intracellularly trapped fluorescent indicator," J. Cell Biol. 94, 325–334 (1982).
14. Paradiso, A. M., Tsien, R. Y. & Machen, T. E., "Digital image processing of intracellular pH in gastric oxyntic and chief cells," Nature 325, 447–450 (1987).
15. Poenie, M., Alderton, J., Tsien, R. Y. & Steinhardt, R. A., "Changes in free calcium levels with stages of the cell division cycle," Nature 315, 147–149 (1985).
16. Tsien, R. Y. & Zucker, R. S., "Control of cytoplasmic calcium with photolabile tetracarboxylate 2-nitrobenzhydrol chelators," Biophys. J. 50, 843–853 (1986).
17. Grynkiewicz, G., Poenie, M. & Tsien, R. Y., "A new generation of Ca2+ indicators with greatly improved fluorescence properties," J. Biol. Chem. 260, 3440–3450 (1985).
18. Minta, A., Kao, J. P. Y. & Tsien, R. Y., "Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores," J. Biol. Chem. 264, 8171–8178 (1989).
19. Minta, A. & Tsien, R. Y., "Fluorescent indicators for cytosolic sodium," J. Biol. Chem. 264, 19449–19457 (1989).
20. Tsien, R. Y. & Harootunian, A. T., "Practical design criteria for a dynamic ratio imaging system," Cell Calcium 11, 93–109 (1990).
21. Sulloway, F. J., Born to Rebel: Birth order, family dynamics, and creative lives, Pantheon Books, New York (1996).
From Les Prix Nobel. The Nobel Prizes 2008, Editor Karl Grandin, [Nobel Foundation], Stockholm, 2009
This autobiography/biography was written at the time of the award and later published in the book series Les Prix Nobel/Nobel Lectures/The Nobel Prizes. The information is sometimes updated with an addendum submitted by the Laureate.
Roger Yonchien Tsien pioneered the use of light and colour to 'peek and poke' at living cells to see how they work. His most famous achievement, recognized by a share of the Nobel Prize in Chemistry in 2008, transformed biology: he developed a rainbow of probes, based on the jellyfish green fluorescent protein (GFP), to illuminate cell structure and function.
Holger Motzkau/Wikipedia/Wikimedia Commons
Roger died suddenly in a park near his home in Oregon on 24 August. He was born in New York in 1952 with science in his blood. His father's cousin was Tsien Hsue-shen (Qian Xuesen), architect of China's missile and space programme. Roger would combine his father's engineering talent with the medical interests of his mother, a nurse.
Roger had an early passion for chemistry. Despite his Chinese name (which means 'always healthy'), childhood asthma often kept him indoors, reading and drawing. He fought going to kindergarten until his teacher allowed him to bring in a favourite book: he picked All about the Wonders of Chemistry. From the age of eight, he performed increasingly complex and sometimes hazardous chemistry experiments at home. At 16, he went to Harvard University in Cambridge, Massachusetts (avoiding the Massachusetts Institute of Technology, where his father, uncles and brothers studied), and sampled many subjects. Ironically he found the chemistry courses “so distasteful” that he abandoned them for neurobiology.
Roger then spent nine years at the Physiological Laboratory at the University of Cambridge, UK. First he was a PhD student with the eminent muscle physiologist Richard Adrian; then he did a postdoc with one of us (T.J.R.). He emerged as an ingenious, largely self-taught synthetic chemist.
Much of Roger's early work was directed at imaging neural activity, by trying to develop tracers of sodium- or calcium-ion movements that support brain signalling. By 1980, he had invented quin2, a synthetic fluorescent dye that selectively binds to calcium, and had devised a clever way to sneak this dye and other probes into intact cells. This first practical probe for calcium found wide early use in studies of intracellular calcium signalling.
Amazingly, Roger struggled to find a faculty position because his work straddled disciplines. In 1982, he joined the physiology department at the University of California, Berkeley, where colleagues encouraged him to create more tools. First came superior calcium dyes, in particular fura2, which is strongly excited by different wavelengths of ultraviolet light before and after binding calcium. Capitalizing on this feature of fura2 (and indicators with similar optical properties), Roger and his group made it much easier to monitor calcium under challenging conditions, for example, across the width of a cell. His group also created valuable fluorescent sensors for pH and for sodium.
In 1989, facing resource constraints, Roger transferred to the University of California, San Diego (UCSD). Here he remained for the rest of his career. He wanted to make sensors that could be genetically encoded, allowing researchers to target specific cell types without having to inject a tracer. In the 1990s, he saw the potential of GFP. The protein had been isolated from jellyfish in the 1960s by Osamu Shimomura (who shared the 2008 Nobel) and cloned by Douglas Prasher in 1992. Martin Chalfie, who also shared in the Nobel, first used GFP to image living cells in 1994.
Roger's lab pioneered the development of GFP variants. Through a combination of rational design and random mutagenesis, they created dozens of bright fluorescent proteins of various colours based on GFP. Roger later produced longer-wavelength sensors based on red fluorescent proteins. He took great pleasure in naming probes after fruits such as the tomato, cherry and plum.
GFP variants are now ubiquitous in biological research. They can be used to bind with and track cancer cells, aid gene therapy, image mitosis, paint neurons in rainbow colours and spy on signalling in subcellular organelles such as mitochondria. They have even been used to make art.
Roger's group at UCSD developed many other optical probes, including fast-response sensors to measure electrical signals across cell membranes, and dyes for tracking proteins with a combination of light and electron microscopy. In recent years, he had two main projects: the design of fluorescent tracers to illuminate tumours during cancer surgery; and the storage of long-term memory by the pattern of holes in the perineuronal net that surrounds neurons in the brain.
Roger's trajectory helped to make it respectable, indeed fashionable, to spend a career inventing reagents and methods. He is named in more than 160 US patents, often as lead inventor. Although naturally keen to participate in the first application of his new tools, he was also generous in providing materials to other scientists.
Roger co-founded three biotech companies that capitalized on his inventions. He semi-seriously quipped to his wife Wendy that, apart from the potential human benefit, the main point of these companies was to provide suitable jobs for his postdocs.
Roger was a fine pianist and briefly considered a musical career. A gifted amateur photographer — a hobby in keeping with his passion for colour and imaging — he enjoyed holidays in the wild outdoors, often taking arduous treks, camera in hand.
Roger will be hugely missed by family, friends, colleagues and the many scientists who appreciated him as a brilliant enabler of scientific progress.
据妈妈说,她一直计划要三个孩子,但这种说法是多年以后我们家有三个孩子时她才说的。生了两个儿子后,虽然爸爸期待着要一个女孩,但在1952年2月,我来到了人间。爸爸给我起了我的中国名字永健(音译Yonchien成为我的英语中间名),但迪克坚持,我的美国名字应该是罗杰Roger。我母亲后来告诉我,迪克用他儿时的玩伴Roger的名字为我命名。后来,也许是当我在大学里,我询问了有关迪克这个神秘的名字命名。迪克交待,他用著名牛仔演员罗伊·罗杰斯(Roy Rogers)给我命名。我每次跟外人解释我俩名字的相似性渊源(Richard W. Tsien和Roger Y. Tsien)时,都得提到这一切,你可以想象许多科学家和他们的秘书都被我们的名字搞混了。我不知道为什么我的父母选择了两种不同的拼音来为我们中间的“永”字注音,但如果他们搞成一样的,那理查德和我会在文献数据库中完全没有区别(都是Tsien RY)。
1968年4月,我必须在四个学院之间进行选择:哥伦比亚大学,麻省理工学院,加州理工学院,和哈佛大学。爸爸否决了哥伦比亚,因为那年春天的学潮,我也不介意这一决定,因为我想离开新泽西州。我拒绝了MIT,因为迪克和路易斯都去那里了,我受够了与他们比来比去。加州理工学院的本科小班制度听起来很诱人,但我最后还是决定不去加州理工,因为理查德·费曼已经不再教物理学导论,也因为他们的音乐系非常小,与哈佛相比简直可以忽略不计。事实上,哈佛也变成我人生值得欢呼的经历。与同学的友谊是帮助我成长的关键。1969年和1970年春天那次学生抗议活动提供了我第一次接触大麻,警察暴力和参与政治的经历。课程的多样性让我接触到艺术史,视觉设计,经济学,殖民史,宪法学,心理学,以及音乐理论和室内乐演出,等等。讽刺的是,最坏的课程是那些旨在带领哈佛的精英化学专业的学生到科研事业的课程。这些必修课程是如此讨厌,让我最终放弃了化学。为了寻找替代品,我涉猎了分子生物学,海洋学,相对论量子力学,和天体物理学(由沃尔特·吉尔伯特主讲,他后来分享了DNA测序的诺贝尔奖)。但我最终还是选择了神经生物学,部分原因是大脑和心灵之间的关系似乎是哲学在科学中最重要的问题,另一部分是因为大卫胡贝尔,约翰·尼科尔斯和托斯滕·威塞尔(David Hubel, John Nicholls, and Torsten Wiese)领衔了一门课程,忽悠本科生成为神经科学家。胡贝尔和威塞尔仍然在做视觉皮层,最终他们赢得了1981年的诺贝尔医学奖或生理学研究。我问胡贝尔教授,我能否在他们的实验室做暑期实习,但他告诉我他们没有给本科生的空间了,并建议我申请马萨诸塞州眼耳医院的尼尔森•藏(Nelson Kiang)的实验室。在夏季1971年,藏教授给我一个神经生理学和有趣的项目,分析听觉耳蜗核的冲动序列。近四十年后,我仍然在神经生物学问题上刻苦钻研。
剑桥
当我问胡贝尔和藏教授在何处申请神经科学研究生学校的意见时,他们的分歧很大,只有一点达成了一致,那就是最顶尖的研究是马萨诸塞州剑桥市和英国的剑桥。我觉得是时候离开马萨诸塞州剑桥市以拓宽我的视野了,所以我申请了马歇尔奖学金去到了另一个剑桥。早在1972年,我,作为一个哈佛的大四学生,已经知道我的申请成功了,而我的博士导师将是一个叫做R. H.阿德里安博士(Dr. R. H. Adrian)的人,而他的大名我之前从来没有耳闻。我打电话给我的弟弟迪克,他从牛津大学心脏电生理专业获得博士学位后,刚刚成为耶鲁大学的助理教授。迪克告诉我,R. H.阿德里安是英国最杰出的肌肉电生理学家之一,而他的老爸E. D.阿德里安,是诺贝尔神经生理学奖得主。另外,R. H.阿德里安是迪克的博士外审评委之一。“但是肌肉是一潭死水,”我感叹道。“我想做大脑的工作。”迪克向我保证,理查德•阿德里安是一位真正的英国绅士,他愿意让我做我自己选择的课题。所以我决定以静观其变。经过一个夏天在巴黎附近的枫丹白露钻研音乐后,我于1972年10月到达剑桥。我在丘吉尔学院第一顿午餐,一位贵族打扮的绅士坐在我对面,问我是不是罗杰•钱永健。我马上意识到他一定是理查德•阿德里安,因为老外里面只有那些认识我的家庭成员的人才能正确地发音我们的姓,而他刚刚做到了。我们谈话的最初几分钟内,他问:“难道你真的觉得肌肉是一潭死水?”我不得不承认他这一评述的准确性。(我后来发现,那年夏天他们参加了同一个会议,在那儿迪克恶作剧地取笑了阿德里安这个研究。)阿德里安痛苦地看着我同意这一点,但马上说,今后无论何时我想转学到系里某一真正的神经生理学领域,他都不会反对。
He kept much in himself most of time - not with that pictured smile much, popping up some jokes sometimes both in his talks and in conversation. That's my impression in his presence. R.I.P.