The current focus on single-cell biology reflects the growing awareness among life scientists that all cells are not alike.
In the genomics world, methods such as scRNA-seq and Drop-seq allow researchers to probe cellular heterogeneity at the genetic level using next-gen DNA sequencing. Mass spectrometry imaging (MSI) does likewise for protein and metabolite studies.
MSI involves blasting a biological sample with either a laser or ion beam to map its molecular composition. The material is ‘ablated’ point by point from the sample surface and introduced into a mass spectrometer for identification based on molecular mass, producing an image in which each pixel is colored to reflect the molecules present.
The instruments used to do this typically come in two forms. Laser-based MALDI is a gentler technique, suitable for mapping protein distribution. SIMS (secondary ion mass spectrometry), which uses a tightly focused ion beam instead of a laser, provides sharper spatial resolution and the ability to dig down into the sample surface, allowing researchers to map metabolite abundance in 3D with subcellular precision. But traditionally, SIMS instruments have been limited in their ability to distinguish molecules of similar molecular weight, making definitive identifications difficult. Now a new mass spec system, described in the December issue of Nature Methods, promises to rectify that.
The 3D OrbiSIMS blends a traditional time-of-flight (TOF) SIMS mass spectrometer from German firm ION-TOF GmbH with an ultra-high-resolution, high-mass-accuracy Orbitrap analyzer from Thermo Fisher Scientific. Using the system, researchers can rapidly sample the metabolite content of a biological sample surface in high-speed TOF-SIMS mode, then revisit areas of interest using the slower, but higher mass-resolution Orbitrap for more confident identifications. The system achieves approximately micron-scale lateral resolution for biological samples, and nanometer-level depth resolution for 3D imaging.
(A separate study in the same issue, led by Mario Kompauer of Justus Liebig University in Germany, describes an advance on MALDI MSI that allows researchers to simultaneously map molecular composition and topology in 3D samples, such as leaves and nematode worms.)
Ian Gilmore, a senior fellow at the National Physical Laboratory in Middlesex, UK, led the 3D OrbiSIMS study alongside researchers from ION-TOF, Thermo, the University of Nottingham, UK, and GlaxoSmithKline. He says he conceived of the new design as a way to combine the strengths of TOF-SIMS and Orbitrap. “The Orbitrap is great for doing the high resolution and the high accuracy, but it’s too slow to do all of the imaging,” he explains. “And the TOF is the opposite — it’s really fast but not very accurate. So the concept of having a hybrid analyzer was, I thought, a good one.”
Indeed, the 3D OrbiSIMS solves a serious problem with SIMS MSI, says Nicholas Winograd, a long-time SIMS user at the Pennsylvania State University in University Park. Because TOF-SIMS devices have low mass resolution, Winograd explains, researchers often cannot be certain of what molecular species they’re seeing. The Orbitrap allows researchers to slam selected ions into gas molecules and measure the resulting fragments to provide more confident identifications. That “fills a major gap in the capabilities of traditional TOF-SIMS, and it makes the whole technology, I think, more in line with modern-day mass spectrometry,” Winograd says.
Gilmore’s team used the system to map metabolite abundance in mammalian cells and tissues, focusing on lipids, neurotransmitters, and a drug called amiodarone. “You can see, cell by cell, drug uptake and changes in the metabolic profile of the cell. And I just think that’s really cool,” he says.
Indeed, Gilmore says a key motivation for developing the system was the high cost of drug development, now estimated at over $2 billion. Using high-resolution MSI, drug developers could gain confidence that their drugs are getting where they are needed, and not where they are likely to cause side effects. “It’s all to do with this drug-attrition situation of trying to find out if you’ve got the right target … at a really early stage so you don’t have late-stage failures,” he says.
One thing the 3D OrbiSIMS cannot do, though, is measure proteins — the technique uses such high energy ion beams that the polypeptides shatter. One alternative, Gilmore suggests, would be to stain tissues with specially labeled antibodies tagged with rare-earth elements and reconfigure the instrument to detect those ions instead. Such a design, he explains, would allow users to correlate drug abundance with its protein target. And, he adds, it would open the door to “lots of different fancy labeling strategies that would give you many, many, many, many, many different possibilities for multiplexing.”
Others have explored similar strategies using different instrumentation. In 2014, Michael Angelo, a clinical fellow in the lab of Stanford University immunologist Garry Nolan, used a SIMS-based system called MIBI (multiplexed ion beam imaging) and lanthanide-labeled antibodies to perform MSI of 10 different proteins in human breast cancer tissues. Separately, Bernd Bodenmiller of the University of Zurich, a former postdoc in Nolan’s lab, and his team detected 32 molecules at once using a Fluidigm CyTOF mass cytometer modified for laser ablation.
Nolan, Angelo, and their colleagues formed a company called IonPath to commercialize MIBI for pathology applications. But Nolan also is using the system (called MIBIScope) in his lab to pursue basic research questions, including super-resolution (68-nm lateral resolution) imaging of chromatin.
“One of the really exciting things was we were able to label RNA with bromo-ribonucleoside and see the nascent transcripts as they’re being generated all across the entire chromosomes,” Nolan says. “We see these clouds of bubbles in the nucleus — it’s absolutely gorgeous.”