R.I.P.
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
Roger Y. Tsien - Biographical
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.
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| 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.
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.
Copyright © The Nobel Foundation 2008
Roger Tsien died on 24 August 2016.
~~~~~~~~~~~
Roger Yonchien Tsien (1952–2016)
- Journal name:
- Nature
- Volume:
- 538,
- Page:
- 172
- Date published:
- DOI:
- doi:10.1038/538172a
- Published online
Creator of a rainbow of fluorescent probes that lit up biology.
Subject terms:
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.
MLA style: "Roger Y. Tsien - Biographical". Nobelprize.org. Nobel Media AB 2014. Web. 2 Sep 2016.
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钱永健自传
Roger Y. Tsien- Biographical
翻译先生自传，谨以缅怀先生。您所发明的GFP和钱氏果园，照亮几代科学家的道路。
译者：席鹏（北京大学）
问：小学生和诺贝尔奖获得者有什么共同点？
答：他们都被逼着写自传。
钱永健(Roger Y. Tsien,1952-2016)，杰出生物化学家。
图 1 钱氏果园Tsien’s fruityard，多彩的荧光染料世界。
祖先和家庭
我的父亲钱学榘（1915-1997），来自中国杭州的士大夫阶层，在那里“钱”（Tsien，现在比较常用拼音为Qian）是一个相当常见的姓氏。在公元907年，我34代以前的父系祖先钱镠，在杭州周边建立国度（吴越国），并修建了许多民生工程。这封地钱镠和他的继任统治下的和平繁荣，直到978，他们投降了宋朝以避免战火中生灵涂炭。我原以为钱镠的后裔是我们家族的一个不起眼的小秘密，但这个微妙的事实被人们挖出来并放在了维基百科上（并不是我干的）。何况，这个也并不能让我和别人有多么不同，因为（考虑数学的话），每个人之前的34代人，总共是234之多。 234换算过来大约是170亿，大大超过了10世纪的地球人口数目。因此，中国几乎所有人都有可能是以钱镠作为祖先，即使不那么严格地通过Y染色体进行传递。而到目前为止，最有名的钱姓家族成员是钱学森，他在麦卡锡时代期间被美国驱逐出境，然后成为中华人民共和国的导弹之父。他和我父亲是堂兄弟。其他几位华裔美国生物学家包括钱泽南，现任霍华德休斯医学研究所的主任，和钱煦，加州大学圣地亚哥分校杰出的生物工程师。以及和我一样的钱姓中国人，他们与我一样，和钱镠大有联系，所以都是我的远房亲戚。
爸爸也热爱飞行和飞机，这是属于他那个时代的高科技。在20世纪30年代，他获得了国家奖学金（清华大学）得以在美国留学。他去了麻省理工学院机械工程系，在那里，他对飞机发动机进行了系统的研究，提出将水注入排放的废气中变成蒸汽，以提高起飞过程中的推力，并获得硕士学位。还没等他继续在美国进行下一步的研究，他就不得不回到中国，在国民党空军服役。我父亲最好的朋友和工程师同事李懿遥（Yao Tsu Li）有一个秀外慧中的妹妹叫李懿颖，谁曾受训于中国最负盛名的医疗机构----北京协和医学院担任护士。我的父亲甚至在他们还没有见面，就急切地写信给她，向她表达爱意。而当他们终于见面后，她发现他不擅言辞与社交，而且陶醉于自己的学术当中。尽管她发现这个男人缺少浪漫，她还是答应嫁给他，或许是因为她对自己在战火纷飞的中国的发展前景并不看好。我的哥哥，他们的第一个儿子永佑，在婚后不久出生于1945年3月。那时，父亲奉命赴美国担任联络官，努力为中国空军争取更多的军事援助。他不得不翻越喜马拉雅山到印度，然后乘船，迂回前进以躲避敌人的潜艇，所以他并没有到达美国，直到日本宣布投降那天。他的努力全都打了水漂。但他知道，中国将在战后饱受内战的折磨。不知怎的，他用在国防部的关系，为妈妈和永佑安排来美国。拿到这一许可真是太不容易了，因为排华法案禁止从中国到美国移民，直到1943年才被废除。那个时侯，每年可以有105个人可以拿到移民资格，而当时名单上有数千人排在我家前面。
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）。
长大：家庭化学实验
我的一个最早的记忆，大概是3、4岁，就是在海边建一条跨过粗石块的沙路，我的脚在越过那些粗石块时被弄伤了。我喜欢画画假想城市的地图，地面街道上面或者下面有高速公路或隧道穿过。这些或许是我未来执迷于桥梁建设和活动映射的第一个迹象。在小学的一段时间我的父母买了吉尔伯特化学集，但我并没有觉得它很有趣，因为那些实验显得那么逊。后来我在学校图书馆发现了一本书，有较好的实验和插图。遗憾的是，我现在不记得这本书的名字或作者，虽然我手抄了实验的许多草图，上面的日期是1960年。这个笔记本目前存放在诺贝尔博物馆。我记忆最深的两个实验是：1）二氧化硅的花园，将金属盐晶体（如氯化钴，硫酸镍，硫酸铜）投进硅酸钠溶液，会长出鲜洋红，绿色，或蓝色的胶装包衣，从垂直上升树突上长出新芽； 2）制备强碱（0.5M 氢氧化钠或氢氧化钾），该有色液体的强烈紫稀（约0.5毫米）高锰酸钾的水溶液。作为这种溶液通过滤纸的折叠锥体通过，它的颜色变为一种惊艳的绿色，反映了从MnO4-到MnO42-的还原，估计是通过纤维素。2008年11月，我在瑞典电视台和诺贝尔媒体前重做了这两个令人惊讶且鲜为人知的演示实验，来解释为什么化学反应让我如此着迷。这两个实验反映了我对那些动人的颜色的早日和持久的痴迷。
图 2 我们一家1960年，在搬到利文斯顿前的合影。左起：Richard, Louis, 我爸爸，我，我妈妈。
1959年，爸爸关闭了他的咨询公司，并开始在新泽西州的哈里森为RCA的真空管分部工作。爸爸妈妈找到了一个小镇，那里房价经济，又在方便通勤的距离，并有很好的公立学校让我们兄弟三个人上学。图1是当时的一张照片。他们在新泽西州的利文斯顿选择了一个盖新房的开发商，但人家不肯把房子卖给我们，说他们不能容许利文斯顿成为唐人街，而且如果有了华人，那其他美国人就可能不会买这里的房子了。我的父母向新泽西州州长罗伯特·梅恩（Robert Meyne）发起请愿。他的办公室致函开发商，警告他们种族歧视是非法的。最后的妥协达成：开发商卖给我们的房子由已经被卖了房子完全包围。而我们三个孩子面临的问题是，利文斯顿有很多岩石埋在它的土壤中，那是冰川时代遗留下的。我的父母打定主意要一个体面的美国式的草地草坪，这就需要去除那些院子里的岩石。我们必须用车拉走很多石头，，不仅我们自己的，还有从我们的邻居那儿来的，当时这块未被占用的土地就成了大家的垃圾场，至少我们是这么觉得的。在草坪上的杂草处理上透出浓重的个性差异：爸爸，作为一个不耐烦的机械工程师，希望将杂草一棵一棵连根拔起。而我则是一个偶尔的哮喘型花粉症患者，深深地害怕花粉，所以我提了一种化学方法，在安全距离上施用杂草除草剂。我们试过一次我的方法。杂草慢慢地变成褐色，但最终又长回来了。爸爸宣布实验失败，又回到手工除草。我仍然在思考这个结果和我们目前对癌症治疗的研究之间的关系。
1960年，RCA关闭了真空管部门，大概是因为半导体取代了真空管，所以爸爸换到了埃索Esso（后更名为埃克森Exxon）研究和工程公司工作。埃索提供更好的项目和工资，所以他在那儿一直呆到他1983年退休。我相信一些能够让我我做更有趣的化学实验的化学品和玻璃器皿是从他们公司库房转移过来的。在那些日子里，其他得用品可以通过邮购的方式获得，用父母的签名购买。在接下来的5到6年，我在我们家的地下室逐渐做了许多无机化学的经典实验：制备和燃烧氢气，制备O2和用氧气燃烧钢丝，在烧瓶中制备NH3，看它吸水向上，在烧瓶内形成喷泉。我在塑料设备中从氟化钙+H2SO4中蒸馏HF，并非常开心地看到它腐蚀玻璃的能力。我从一套火车模型中弄到了降压变压器及整流器，然后将镍坩埚为阴极，和从废手电筒电池弄下来的碳棒作为阳极，用电解法熔融氢氧化钠。我设法得到非常不纯的金属钠，在它落入水中时，发出了令人满意的丝丝声。烟火自然是我极大的兴趣点：我制作并点燃了火药，做了重铬酸铵火山实验，甚至做了铝粉和氧化铬产生的壮观的铝热反应。我最雄心勃勃的尝试是一个多步骤的顺序，旨在合成阿司匹林，为此我需要醋酸酐，这不得不从乙酰氯制成，为此我需要三氯化磷，为此我需要在氯气流中烧红磷。我试着在连续的橡胶塞烧杯中做这个实验（图2），因为我没有磨口玻璃接头的玻璃器皿。该腐蚀性化学品把橡胶腐蚀了，所以我没有获得乙酰氯后面的产物。由于我没有通风橱，我在后院的一个烧烤架上做更为危险的实验。回想起来，这一切对于我这样一个8到15岁的无监督的男孩的疯狂行为感到震惊。但它同时也是一个很好的锻炼，让你学会如何高效地搭建设备，规划和实施你的实验，解释令人混乱的结果，并决定如何把事情做得更好。这些实验让我有足够的信心，当我必须赚取我作为童子军的第一个成就徽章时，我义无反顾地选择了化学。一些更严厉的成就徽章，如远足，需要在一天内进行二十英里的徒步旅行，我后来也得到了。
图 3 制备氯气的装置并将其与红磷的反应（1966-1967年），在我们自家的后院有幕天井中。最左边的烧瓶包含高锰酸钾与盐酸水溶液反应，通过捏夹子控制的漏斗加料。氯气流经氯化钙干燥，然后引导到P4中的环支架上的烧瓶中。因为没有自来水可用，水冷却的PCl 3冷凝器从回收牛奶罐虹吸并存入标有“夏威夷鸡尾酒”的废罐头瓶中。对PCL3的接收浸没在暖瓶里面的冰中。在酒精灯辅助加热的磷。注意到处都是橡胶瓶塞。
小学到高中：西屋科学天才搜索
学校通常是可以忍受的，但生活经常十分无聊。我期待着天冬天的时候下大雪关闭学校，这样我就可以花一天时间玩雪橇。我在学校球类，如足球，篮球，垒球表现非常糟糕，因为我年纪小，不擅长运动，比我的同学要小一两岁，这是一个巨大的差异。但是，我在高中人气很足，以高票当选学生会的会计。
妈妈极力在放学回家后教我们中文，但我越长达，越发现这些经验教训十分乏味。我能够以儿童水平极好地理解中文口语（如中文的“清理你的房间！”被永久地刻入我的大脑中），但是却不愿意说出来，我自己，由于心愿（移民的儿童中太常见了）从我父母的口音上，并在他们强烈的的种族和传统自豪感上，与自己刻意拉开距离。同样，我对吃中国菜的抵制让他们感到绝望（就像一个“洋鬼子”），尤其是最正宗的菜肴，具有强烈的味道或气味的食物，或那些杂七杂八的动物肉的做法。
我第一次接触到的科研环境是在1967年，俄亥俄大学国家科学基金会资助的暑期研究计划，在那里我被分配在罗伯特·克莱因（Robert Kline）教授的实验室工作的硫氰酸盐（SCN-）。皮尔逊理论的软硬配体和金属的结合是一个全新的时髦理论，所以克莱恩教授要我找出，是否硫氰酸可以同时与它的“软”硫磺绑定到某种软金属，以及“硬”氮到某种硬金属上，如PhHg-SCN-铬（Ⅲ）。他希望硫氰酸盐的红外线吸收率会告诉我们，这样桥接是否正在发生。我准备了很多界限不清的无定形沉淀，并测量了它们的红外光谱。在1967年的冬天，我在利文斯顿高中年级时，我参加了西屋科学奖，一个全国范围的科学竞赛赛事。（这个年度的盛事现在依然存在，虽然赞助于1998年被英特尔替代），由于缺乏任何替代方案，我写了我在俄亥俄大学的项目，尽我所能从可疑数据的混乱得出一些结论。克莱恩教授在很大程度上否认这些结论，指出我的准备工作没有给予正确的碳，氢，氮微量分析。 40名入围者被召集到华盛顿，在1968年3月进行采访和公开海报会议。我记得我被同时入围的决赛选手羡慕，他们比我更年也更复杂。同时他们的项目和展品似乎比我更令人兴奋和易于解释。我感觉自己被高级法官，格伦·西博格（Glenn Seaborg）吓倒，部分原因是因为他的职位的高度，部分原因是因为他是美国原子能委员会的主席，还有部分是因为他获得1951年诺贝尔奖的无机化学的工作。颁奖典礼是对我们每个人来说都是非常紧张的，因为十个奖学金获奖者以相反的顺序宣布，迫使每个人都希望自己的名字被念到，但越晚越好。我依然不明白我如何获得的一等奖，尽管我的项目的不够周全，而且我对科学比赛这件事儿看不顺眼。爸爸的一句话让我能够不飘在天上：当我打电话给家里，他的第一个意见是，这是一件好事啊，我现在有$ 10,000的奖学金，因为他最近在股市上输了不少钱。其中我收到的最满意的恭维是，那个不想卖给我爸妈房子的开发商，现在用我的照片在广告中为当地教育质量的证据之一。
哈佛
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。
就这样，我开始了我的博士训练。我从来没有换过导师，因为我很快就意识到我不喜欢做传统中枢神经系统的电生理。传统的论文课题基本上遵循由胡贝尔和威塞尔成功地采用的范例，是一个细胞外微电极植入麻醉动物的脑并记录单个神经元的活性，同时提供感官刺激。经过几百个这样的记录，人们可以对不同的反应模式分类，并发表一篇论文和一些出版物。对我来说，这似乎太像冰钓，也就是在冰湖上切一个洞，投下鱼线放入不透明的水下，并耐心地等待鱼来咬钩。大脑拥有调动数万亿并行工作的神经元的能力，所以我想看到大量的神经元信号是如何同时彼此交互和处理信息的。理想情况下，人们会对神经元进行染色，当神经元发射动作电位时，染料明显亮起或改变颜色。一些市售的染料确实已经发现能够反映神经动作电位，但它们的光学反应是极其微小的，例如荧光的10-4或 10-5的变化。
只有在高度简化条件下，数千个由研究者驱动的动作电位求平均时，信号才能被检测到。为了检测在一个复杂的脑源性信号，需要提升好几个的数量级的改进。在1972年的冬天，我贸然决定我会尽力为成像特定目的神经元活动而设计并合成新的染料。一个策略是靶向染料钠通道，它被认存在于产生动作电位经受大的构象变化附近。另一种策略是打造“电致变色染料”，在偶极矩基态和激发态之间产生巨大变化，从而使神经细胞膜电位的变化可能会改变吸光率或荧光的峰值波长。在这两种情况下，我都不得不学习有机结合，这正是我所恨透的哈佛的那些化学课程，而我们现在的生理实验室已经无人能教我了。幸运的是，化学系的一个新来的教师，大三学生教员伊恩·巴克斯特博士（Ian Baxter），是理查德·阿德里安的一个朋友的朋友，对我提出的靶向钠通道很感兴趣，并同意非官方地成为我的导师。巴克斯特没有其他的学生，因此有时间、善良和耐心，每天数次，看看我的进展，并告诉我必要的技术。我发现我自己都感到惊讶，那就是我开始可以享受有机合成，当我为我自己选择的一个生物目标奋斗的时候。我仍然对这种类型的研究大呼过瘾，即使我合成的分子被证明不能结合钠通道，尽管巴克斯特很快就离开了剑桥，成为英格兰北部的职业顾问，甚至哪怕我合成电压传感器的等被证明不如那些由其他实验室筛选大量市售染料及其非常近似的结构时。
我如果想获得一丝的成功，需要转移到另一个生物目标。为产生任何生物效应，动作电位几乎总是产生大量增加的细胞内钙，如神经递质，以激发或抑制该途径的下一个神经元的释放。在1975年人们因为发现偶氮胂III而非常兴奋，它原一种为了测量在核废料的重金属而发明的染料，人们发现它也可以用于监测从乌贼神经元巨轴突所释放的钙，虽然从该染料的信号是很小的，而且有点含糊不清。我认为，设计某种染料来测量钙流，应该比设计染料来追踪在神经元膜电位的快速变化更为容易。众所周知，化学文献中数百种染料可以和钙离子发生反应，例如测定水的硬度。真正的问题是，在细胞内，游离Mg2+的浓度大约比Ca2+高4个数量级，从而使细胞内的Ca2+指示剂需要更高的选择性来超过它的姐妹离子Mg2+。而化学家还没有认识到这种有选择性的标记物的生物方面的需求。一种叫做EGTA的无色缓冲液是已知的Ca2+:Mg2+的选择性上合成的唯一分子，但它从未被制作成任何类型的染料分子。通过涂鸦在纸上和玩分子模型，我想到了一个方法，使EGTA变成非常简陋的染料分子。我开始没有告诉理查德•阿德里安这个全新的项目，因为任何谨慎的导师都会告诉我，我应该把旧的项目结束，而不是开始全新的。幸运的是，在几个星期内我成功地得到目标分子的一小块不纯样品（后给出的缩写“BAPTA”），并发现它具有对Ca2+的预期光学响应，同时具有极高的Ca2+:Mg2+选择性。经过许多年和发现，从BAPTA 后裔发展出的更好的染料成为最流行的方式，来观察内源性细胞内Ca2+信号，筛选链接到的Ca2+信号配体和受体，并用于在显微镜下成像神经元活动。
获得博士学位后，我留在剑桥冈维尔与凯斯学院（Gonville & Caius College）作为博士后研究员。我向Ca2 +信号的变化的研究重点转移，促进了我与蒂莫西•瑞克（TimothyRink）博士，他是生理实验室的一个新教员。蒂姆想用一些从瑞士发来的材料中，开发钙离子选择性电极。但说明书是德语写的，蒂姆无法阅读。当时我已经学会读德文的化学论文，所以我翻译了说明。我们的合作开始于这些钙离子选择性电极，并延伸到我的钙离子荧光指示剂的生物测试和开发中。更重要的是，1976年蒂姆和他的妻子诺玛邀请我参加他们的圣诞晚会，在那里我第一次见到他们的妻妹（sister in law），温迪。很快，每到周末，我就去她在北伦敦的房子看她。当蒂姆和诺玛发现了数个月后，他们对自己完全无意的牵线搭桥的效果感觉相当惊讶。温迪（图4，5）仍然是我一生的挚爱。
图 4 Wendy和我们的狗Kiri，2004.
幸运之神眷顾了我。加州大学伯克利分校的生理学与解剖学系，有一个助理教授职位空缺。招聘委员会主席被特里•玛沁（TerryMachen），当时刚好在剑桥休年假，我刚好认识了他。同时，伯克利有两个教师，理查德·斯坦哈特（Richard Steinhardt）和罗伯特·朱克（Robert Zucker），对Ca2+信号感兴趣。这些连接使我能够获得伯克利分校的应聘机会。幸运的是，钙离子的荧光指示剂终于敏感到能够直接测量淋巴细胞胞质钙离子的浓度，包括由于促有丝分裂而带来的提升。现在，人们可以研究Ca2 +信号在小的哺乳动物细胞群体，而先前的技术需要个头非常大、非常强壮的单细胞，足以承受显微注射。这一前景，与我在生理学获得博士学位的事实放在一起，说服了系里给我助理教授的职位，而我欢乐地接收了这个offer之后，才发现伯克利正遭受金融危机。我的实验室在1982年初的启动经费被缩减到只有几千美元，而每个采购的项目必须是合理的替代陈旧的教学设备。例如，为了给我订购一台紫外灯用于观看薄层色谱板，从教学实验室的旧显微镜照明必须报废。更重要的是，该系没有资源，提供通风橱，而我继续合成钙指标需要用到它。罗伯特·麦西教授（Robert Macey），他的实验室在我旁边，惠赠我一套老通风柜，包括其不可替代的延伸到建筑物的屋顶管道系统。对于我的其余的在伯克利任教的七年间，我们所有的合成反应发生都是在这个木通风柜中完成的，它不到4英尺宽，铁丝网深深嵌入前窗的玻璃中。整个实验室因为储物柜没有盖子而充斥着刺鼻的味道，当使用过量溴乙酸乙酯反应必须在通风橱外完成时，整个实验室就成了一个大的催泪瓦斯。提到这些苦日子，只为了提醒年轻的科学家，一些好的研究可以用不奢华的设施和启动资金来完成。
尽管有这些曲折，我在伯克利的科研卓有成效，包括和玛沁、斯坦哈特、朱克，以及其他人的合作。我招了Grzegorz Grynkiewicz和AkwasiMinta，他们合成了大幅改进的钙流染料(fura-2, indo-1,fluo-3)和钠流染料(SBFI)，所有这些被沿用至今。在经济危机缓解后，伯克利帮我买了一个简陋的图像处理器，我痛苦地编程控制它来计算在两个交互激发的波长情况下荧光的比值。这一实时比值揭示了Ca2+, Na+, 和pH 信号在单个活细胞中的变化，而且常常是以前所未有的高时空分辨率的。
搬到UCSD
然而，我开始担心被困在成像无机离子的职业生涯当中。我想探索通过更复杂的生物如cAMP信号（cyclic 3',5-adenosine monophosphate）和更广泛的、更时尚的生物大分子相互作用的世界。我议价能力的增长，让我也想要一个实验室有足够的通风柜，通风柜，小暗室荧光显微镜来支持我的不寻常的化学与生物学的结合，以及在化学系和霍华德休斯医学研究所联合任命。在伯克利，上述条件没有一个是可能的。所以在1989，我们搬到南部的加利福尼亚大学，圣地亚哥，直到现在。UCSD满足了我以上的奢求，而且它更年轻，更宽敞，成长更快的，与伯克利比起来更不受传统的束缚，我比较之后感觉这些优势足以弥补它名气小的劣势。我诺贝尔演讲中详述了我在UCSD的科学亮点。
图 5 Wendy和我，一起正装出席诺贝尔颁奖会。
结论
写这个自传提醒了我，我的职业生涯是如何被机会和命运塑造成这样的奇怪的混合物。利用化学来建立生物有用的分子是一种工程，所以我没有逃脱我的父亲，叔叔和兄弟建立的传统范式。然而，我避免了他们选择的机械、航空、电气和计算机专业，可能是因为像许多年轻的同胞们一样，我不得不寻求一个独特的生态位。但是，如果我没有Ian Baxter重新灌输我享受化学，也许我会选择另一个方向。我对多发性色彩的成像的兴趣也反映了童年早期的视觉兴趣，让我有幸能够将兴趣与职业相结合。从严格的生物学的角度来看，我们的贡献主要是在技术的发展。人造技术确实有一种将被淘汰的趋势，而关于自然如何工作的基本发现应该永远持续下去。但真正的根本性洞见，如达尔文和沃森与克里克是极其罕见的，经常饱受激烈的竞争。而一旦能解决重要的问题，成功的技术发展使得凡人至少产生几年的广泛的有利影响。此外，同样的工程方法是创造新的治疗策略，以减轻疾病，而不仅仅是我们科研人员的工具。
原文地址：
https://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/tsien-bio.html
本文欢迎转载，请注明及出处。因作者水平有限，错漏之处在所难免，欢迎留言指正！
http://blog.sciencenet.cn/blog-499502-1000464.html
上一篇：人性善恶讨论的终极答案
2 李颖业 李胜文
发表评论 评论 (4 个评论)
- [4]李胜文
- 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.
- [3]shatan
- 有一个秀外慧中的妹妹叫李懿颖，谁曾受训于中国最负盛名的医疗机构----北京协和医学院担任护士
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此处的“who”应该译为“她”，而不是“谁”。
- [2]席鹏
- 改了，多谢提醒！
- [1]李颖业
- 他和我的父亲是亲表兄弟
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英语里表兄弟和堂兄弟都是cousin，这里应该译成堂兄弟
