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SCIENCE & TECHNOLOGYPICTURING BIOLOGYMulti-isotope imaging MASS SPECTROMETRYanswers biological questionsCELIA HE NRY ARNAUD , C&EN WASHINGTONCOURTESY OF CLAUDE LECHENEMULTI-ISOTOPE IMAGING mass spectrometry,or MIMS, is proving to beabout more than just pretty pictures. It’salso a powerful way to answer biologicalquestions.The method, developed by Claude Lecheneand coworkers at Harvard MedicalSchool over the past two decades, is allowingscientists to track the synthesis of DNAand proteins in whole organisms—bacteria,fruit flies, mice, and even humans.“The three-dimensional images are trulymind-blowing,” says Nicholas Winograd ,a mass spectrometry imaging expert atPennsylvania State University.Lechene and his coworkers build theseimages from samples that have undergonenormal metabolic processes in the presenceof stable-isotope tracers. They gathersuch samples by feeding organisms metabolitessuch as amino acids or nucleosidesenriched in stable isotopes such as nitrogen-15.Organisms use these metabolites asbuilding blocks as they replenish constituentsin their cells. The researchers then calculateincorporation of the stable isotopesby comparing the isotopes’ abundance insamples to their natural abundance.15N is particularly useful for tracingthe synthesis of new DNA and proteins.Researchers can use other stable isotopes,such as oxygen-18, carbon-13, or deuterium,to track biological molecules, but 13 Cand deuterium are less useful for whole-organismstudies because of their ubiquity inbiological molecules. In addition, more 15 Nand 13 C compounds are commercially availablethan 18 O compounds, Lechene notes.Although the method uses nanometerscalesecondary ion mass spectrometry(SIMS) to detect these stable isotopes,Lechene hesitates to call it mass spectrometry.The instrument detects only up toseven masses simultaneously. “The methodis more ion microscopy,” he says.Those ions are generated by bombardinga sample with abeam of cesium ions.The cesium ions breakmolecules into atomicand polyatomic fragments,which evaporateand are ionizedto form so-calledGUT REACTION A15N- 14 N image ofthe mouse smallintestine showsthe incorporationof 15 N-labeledthymidine in DNA.secondary ions. The secondary ions arethen guided through a series of ion opticsto the mass spectrometer. In his biologicalapplications, Lechene detects nitrogen,both as 14 N and 15 N, with the cyanide ion,CN – . By moving the tightly <strong>focus</strong>ed cesiumion beam from point to point and drillingthrough the sample layer by layer, Lechenecan construct 3-D maps of 15 N incorporation.He has achieved spatial resolution of30 nm and depth resolution of 10 nm.“Since 1995, Lechene has produced thehighest quality images in the field,” saysWinograd, who uses conventional time-offlight(TOF) SIMS for biological imaging.“Trouble is, he almost never publisheshis work. In 2007, he published a Sciencepaper on nitrogen fixation which bleweveryone in the field away [DOI: 10.1126/science.1145557 ]. Many of us in the communitysure wish we could see more of it.”NOW THE COMMUNITY is getting itschance: Lechene published two highprofileMIMS papers last month. In oneexample, he and his colleagues used MIMSto reveal slow protein turnover in the stereociliaof mouse hair cells, which despitethe name are actually found in the innerear ( Nature, DOI: 10.1038/nature10745 ).These stereocilia help hair cells convertsound waves into nerve impulses. Earlierexperiments by other researchers used actinlabeled with green fluorescent proteinand suggested that all the actin in hair cellsis replaced within two or three days. Thosestudies were done with hair cells removedfrom infant mice and then grown in cultureswith high levels of actin.The MIMS analysis, in contrast, wasperformed with adult mice and revealedsomething very different. “We could seethat hair cells themselves were turningover relatively fast,” Lechene says, “but thestereocilia were lagging for a long periodof time.” The only part with high turnoverwas the tip, the portion of the stereociliaresponsible for transducing mechanicalmotion into auditory nerve impulses. “Thisfinding was contrary to the dogma that stereociliaturn over very fast,” he says.The use of stable-isotope labeling allowedLechene to study the cells undernormal conditions. “We have demonstratedthat we can study protein turnoverin volumes that are smaller than anythingthat has ever been done, in conditions thatdon’t disturb the system,” he says. “Thereis nothing that approaches this kind ofmeasurement in biology.”Lechene has also used MIMS to investigatea long-standing question aboutstem cell division ( Nature, DOI: 10.1038/“Since 1995, Lechene has produced the highest quality imagesin the field. Trouble is, he almost never publishes his work.”WWW.CEN-ONLINE.ORG 23 MARCH 2012