Mass spectrometry technology provides a clear image of the future of quantitative microscopy
"By the help of Microscopes, there is nothing so small as to escape our inquiry; hence there is a new visible World discovered to the understanding." Thus wrote Robert Hooke in his pioneering work Micrographia  published by the Royal Society in 1664. In this revolutionary book, Hooke described with excitement his discoveries using a simple light microscope, coining the word 'cell' to define the microscopic structures he saw in cork and plant samples. In this issue of Journal of Biology , Claude Lechene and colleagues describe a 21st century microscopy technology (Figure 1) that also reveals images we have never seen before.
Figure 1. Microscopy through the ages. (a) This illustration, from Robert Hooke's Micrographia , shows the plans for his lens-grinding machine and for his setup of the microscope. (b) Prototype of the NanoSIMS 50 (Cameca, France) used for MIMS technology.
Hooke was fascinated by the new vision of the world and the planets afforded by the lenses of the early light microscopes and telescopes of the 17th century. Ever since these discoveries, researchers have been gazing at the microscopic world and developing better and better instruments to do so. Over the centuries, the demanding needs of biologists have fuelled countless improvements in imaging technologies. For example, electron microscopy has become a standard instrument for high-resolution imaging (in the nanometer range) in biology, and scanning probe microscopy techniques provide three-dimensional images of atomic surfaces.
Quantitative imaging with mass spectrometry
Lechene, of Harvard Medical School and Brigham and Women's hospital in Boston, USA, knew exactly what requirements he was looking for in a quantitative imaging instrument. He was interested in using stable isotopes as tracers in biological samples. "To do that one has to be able to recognize them by mass spectrometry," explains Lechene (see the 'Background' box for explanations and definitions). "And there was no instrument to do so." During his studies in Paris, Lechene came across Georges Slodzian of the Université Paris-Sud in Orsay, a third-generation disciple of the French school of electron and ion optics. Slodzian's work on ion microscopy was a major input to the development of secondary-ion mass spectrometry (SIMS) , which is widely used in fields such as geochemistry, cosmology and materials sciences. "I needed an instrument that had high spatial resolution, the ability to detect several isotopes in parallel with high sensitivity and, at the same time, a mass resolution high enough to separate isobars like the ones found with nitrogen compounds," says Lechene.
The ability to look at multiple isotopes simultaneously was critical for assessing isotope ratios and normalizing one tracer isotope with respect to another; this is useful, for example, for distinguishing the isotope label from the endogenous atoms. The previous generation of instruments measured only one isotope at a time. Lechene's innovative vision and Slodzian's technical wizardry led to the development of multi-isotope imaging mass spectrometry (MIMS) (see 'The bottom line' box for a summary of the technology). "Lechene was uniquely placed to make this development," notes John Vickerman of Manchester University, UK. "He is deeply immersed in the life-sciences community and has a long-standing interest in SIMS instrumental developments. Slodzian is an ion physicist of enormous skill and reputation who has been responsible for the ion-optical design of a number of extremely successful SIMS instruments. The new instrument that Slodzian developed has the spatial resolving power of an electron microscope with the added capability of detailed differentiation of chemical constituents."
Lechene's demanding requirements were important because he was keen to do experiments using the 15N isotope. 15N had been used for the pioneering experiments of Schoenheimer , to demonstrate protein turnover, and by Meselson and Stahl , to confirm the semiconservative nature of DNA replication. The problem is that nitrogen atoms hardly ionize and must therefore be examined as cyanide (CN-) ions. Lechene needed a system that could distinguish between the different isobars, such as 12C15N- (mass 27) and 13C14N- (also mass 27) and other similar atomic clusters. Slodzian's instruments enabled both high spatial resolution and the high mass resolution necessary for separating isobars at high secondary-ion transmissions.
Once the instrument and the tracer strategies were in place, the remaining challenge was developing the functional software and computational know-how to analyze all the data. Each image pixel has an intensity that is a function of the number of ions with a given mass that are at the pixel address. Lechene likens an image of 256 × 256 pixels to an array of over 65,000 test tubes. So, when the researchers analyze 12C, 13C, 14N and 15N, it's as if each of those test tubes contains four radioactive compounds. The isotope ratios are then normalized with respect to each other and then the peaks are analyzed. "When I began it took me weeks, if not months, to do some of the calculations. And now it takes us minutes," says Lechene (see the 'Behind the scenes' box for a summary on the development of MIMS).
A plethora of applications
Lechene teamed up with biologists from different disciplines to demonstrate how MIMS could be applied to quantitative imaging of biological samples. The Lechene study  is full of examples looking at turnover of proteins, DNA and fatty acids and at subcellular localization. Although these are spectacular examples of the MIMS technique, many researchers agree that this is just the tip of the iceberg. "The labelling of the lymph node cells by 15N is really convincing and suggests that MIMS may be highly useful in immunology and cancer research," says Brad Amos of the MRC Laboratory of Molecular Biology in Cambridge, UK. "The paper shows that a remarkable amount of fine detail can be seen. This may turn out to be a key paper in the development of a really important imaging method."
"The most significant feature of this technique is that it opens up a whole new world of imaging; we haven't yet imagined all that we can do with it," says Peter Gillespie from the Oregon Health & Science University in Portland, USA. He agrees with Amos that the technology represents an imaging revolution. "The novelty of the technique means it will take some time for the details to be absorbed, [but it] sets a spectacular new standard for spatial resolution and detection of stable and radioactive compounds in cells." Vickerman is also enthusiastic about the applications: "This study is important in that it demonstrates across a range of demanding applications that SIMS can deliver unique information, inaccessible by other means."
But Vickerman adds a cautionary note about how widely MIMS technology will be applied in the future. "It is clear that the technique has great potential in medicine and biology, but there are two issues that have to be overcome: the conservative approach of much of the potential user community and the cost of the equipment." Gillespie agrees "Once commercial instruments are available, will they be affordable and easy enough to use that we will do many experiments with them? Or will they be like electron microscopes, where the expense of the instruments and the difficulty in operation means that relatively few people use them well?"
"It may take a long time (EM took a long time), but I am convinced that in 10–15 years this will be an easily accessible technique, with routine instruments in many departments of biological research," responds Lechene. A machine based on Slodzian's prototype is already sold by a French company called Cameca Inc., and Lechene notes that there are over a dozen around the world. At a cost of two million dollars they are beyond the budget of most laboratories. But Lechene is keen to point out that more and more mass spectrometry machines are being purchased. "After all, it's just the price of five electron microscopes, but it does so much more!"
Perhaps we should leave the last word to Hooke , whose prophecies echo through three centuries of improvements in microscopy: "Tis not unlikely, but that there may be yet invented several other helps for the eye, at much exceeding those already found, as those do the bare eye, such as by which we may perhaps be able to discover ... the figures of the compounding Particles of matter and the particular Schematisms and Textures of Bodies."
Lechene C, Hillion F, McMahon G, Benson D, Kleinfeld AM, Kampf JP, Distel D, Luyten Y, Bonventre J, Hentschel D, Park KM, Ito S, Schwartz M, Benichou G, Slodzian G: High resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.