Cosmic Chemistry for Cytometry


  • Attila Tárnok

    Corresponding author
    1. Department of Pediatric Cardiology, Cardiac Centre, University of Leipzig, Leipzig, Germany
    2. Translational Centre for Regenerative Medicine (TRM), Universität Leipzig, Germany
    • Dept. Pediatric Cardiology, Cardiac Centre, University Leipzig, Strümpellstr. 39, 04289 Leipzig, Germany
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  • The work presented in this paper (editorial) was made possible by funding from the German Federal Ministry of Education and Research (BMBF, PtJ-Bio, 0315883).

Traditional elements such as Hydrogen, Helium, and some other chemical elements initially originate from the big bang at the beginning of our universe. In a later phase Helium and higher atomic weight elements were and still are generated by nuclear fusion in the sun and other stars. In most of the stars, this process is going to carbon, in extremely large stars even to the element iron. The genesis of heavier elements, however, requires another astro chemical process, namely neutrino capture in nucleosynthesis by the atoms in the so called s-process in small stars or the r-process in the exotic supernovae. These elements are then distributed by solar wind all over the universe and are eventually captured be protoplanets and planets. Therefore, many heavier elements can be or appear to be scarce on our planet and are therefore termed rare earth elements or metals. Most of these rare earth metals belong in the periodic system of elements to the group Lanthanoids or Lanthanides. Their name rare earth does not necessarily mean they have low abundance but can be interpreted as a lack of appropriate ores for their commercial exploitation. In fact, some of the so called rare earth elements are more abundant in the crust of the earth than copper, gold or other metals. Apart from the stellar processes of cosmic chemistry, rare earth elements can also be produced at high costs on earth by nuclear fusion in accelerators or are generated and released at atomic bomb explosions.

Rare earth elements recently became of high relevance and their commercial value vastly increased by the invention and introduction of new technologies and devices. As examples, laser technology requires Scandium (Sc), Yttrium (Y), Neodym (Nd), Samarium (Sm), Dysprosium (Dy), Holmium (Ho) or Erbium (Er); fuel cells: Sc, Lanthan (La); energy saving bulbs: Y, Terbium (Tb), Thulium (Tm), Ytterbium (Yb); LED/LCD and plasma monitors: Y, Europium (Eu), Gadolinium (Gd), Tm, Yb; (permanent) magnets: Praseodym (Pr), Nd, Sm, Tb, Dy, Ho and the list goes on. But why is it relevant describing astro-chemical phenomena and atom physics in cytometry?

The central goal of cytometry is to unravel the complexity of biology and to identify and analyze the extremely diverse phenotype and function of individual cells in complex cell systems (1). With the tools we have presently at hand this is an ever challenging task. It includes increasing the number of measured detecting molecules such as antibodies labeled from the plethora of fluorescent dyes that are excited at different wavelengths (multiple lasers in a flow cytometer, FCM) and emitting at different colors (multiple detectors or spectral analysis (2)). Cutting edge for maximum color FCM are to date the OMIPs such as OMIP-011 presented by Lachmann and colleagues from Chieti, Italy (this issue, page 549) applying eight colors. Unfortunately, it seems that there is an upper limit of around 20 for combining various fluorochromes in order to analyze complex cytomes and tell unequivocally one from the other. One reason for this limitation is the broad emission band of organic fluorochromes as well as sometimes a broad excitation band, resulting in multiple excitations at different laser wave lengths. This in turn results in a complex spectral overlap between the emissions of different dyes rendering it ever more complex to distinguish between various dyes when more colors are combined. To make it even more complicated, spectral interference can happen in a theoretically unforeseeable way and has to be tediously tested in laboratory experiments (3).

One potential solution is to optimize the fluorochromes so that their emission and excitation bandwidths are reduced. An excellent example is demonstrated by Guryev and colleagues from San Jose and Davis, California, USA (this issue, page 627). The authors optimized AMCyan fluorescent protein and developed a new fluorescent protein variant: AmCyan100, by site directed mutagenesis. Their newly isolated fluorescent molecule is excitable in the near UV but not at 488nm like AmCyan. This makes it easier to combine the fluorescent protein with 488nm excitable fluorochromes like FITC. In addition, with a Stokes shift of >100nm, AmCYan100 may be more easily combined with another near UV excited dye exhibiting a lower Stokes shift.

Additionally, more laser lines may be added to a flow cytometer to allow for more specific excitation of various fluorochromes and the introduction of new dyes not excited at all or excited below their absorption maximum by the presently available laser lines in commercial instruments. White color lasers seem to be a good alternative to introduce more laser lines and quickly change from one excitation color to the other (4). Rongeat and colleagues from Montpellier and Limoges, France (this issue, page 611) introduce a new super continuum white laser source into an FCM system for advanced excitation with potentially multiple laser lines from one laser for polychromatic flow cytometry. This system allows specific selection of the appropriate single or multiple laser lines by excitation filters and to optimize them in an experiment for the selected fluorochromes, not the other way round.

Some rare earth metals may help and have already been applied for adding new parameters to increase the number of available detecting molecules in cytometry. The advantages of using lanthanides in cytometry are several-fold. They are virtually non-existent in biological systems such as cells, so their presence can exclusively be assigned to a specific uptake or labeling. Several of them (such as Eu) produce fluorescence with a very high fluorescence life-time. This can be measured by time gated luminescence analysis which was for example done by time-gated luminescence microscopy (5) and can also be applied to flow cytometry (6). Thus, applying lanthanides can provide an additional new marker to detect biomolecules in FCM with hardly any cross-contamination by fluorescence signals in the time-gated detection channel.

So why not go one step further, taking advantage of the lack of rare earth metals in cells and measuring their presence on the single-cell level by the appropriate detection technique: time-of-flight mass spectrometry by which the atomic mass of every atom in a cell and its abundance is exactly determined? That this approach is feasible was lately demonstrated by the group of Tanner (7). But what are the advantages? There are 17 lanthanides with different atomic masses in the lanthanide series of the periodic system of elements that can be potentially used to distinguish between 17 different targeting molecules such as antibodies labeled with them. In addition, there are several stable isotopes of each lanthanide! This means ∼100 different isotopes are available to label antibodies and other targets which can be unequivocally distinguished from each other because the isotopes have just one mass without (ideally) any variance. The consequence is: Clear discrimination of each isotope (dye) and no problems with spectral overlap.

That more than 30 different epitopes can be detected and quantified using mass cytometry is now presented by Behbehani and colleagues from Stanford, California USA (this issue, page 552). The authors present data to pinpoint the cell cycle phases system wide in the whole cellular immune system of the peripheral blood as well as of bone marrow leukocytes in a single measurement using simultaneously up to 24 antibodies against cell surface antigens and 7 to detect different phases of the cell cycle. The authors used Indium and 13 different lanthanides (La, Pr, Nd, Sm, Eu, Gd, Dy, Tb, Ho, Er, Tm, Yb, Lu) and from many of them several isotopes were applied (Nd: 7 isotopes, SM:4, Er: 5). In addition, cells were incorporating the Iodine127 isotope labeled deoxyuridine that is built into the DNA of S-phase cells yielding up to 32 markers. The presence of these markers was quantified on a single cell level unraveling the different cell cycle phases in combination with the appropriate cell phenotypes without the need of further cell separation or hypothesis making. To summarize, mass cytometry can now be regarded as an integral part of the cytometry family of technologies. Thus, I propose the general term MCM as an abbreviation for Mass Cytometry in analogy to FCM for flow cytometry.

An open question remains: is MSM equivalent to traditional detection by FCM in regard to detection sensitivity and quantitation of molecule numbers per cell? Here, the international research group of Wang and colleagues from Gaithersburg, Bethesda (National Institute of Standards and Technology, NIST), and Silver Spring, Maryland, USA; Toronto and Richmond, Ontario, Canada and Herfordshire, United Kingdom (this issue, page 567) provides important answers. The group raised the issue regarding which types of cells may be used as reference biomarkers for calibrating antigen quantification by fluorescent microbeads using FCM and MCM. The good news is that their results demonstrate an equivalence of both methods. Pre-fixation of the cells with para-formaldehyde prior to labeling of surface antigens reduces the number of detected surface antigens.

And this brings me to another potential advantage of MCM: Autofluorescence and the effect of fixatives. In the case of MCM, autofluorescence is not measured. This is in contrast to the autofluorescence of intrinsic cellular compounds in FCM and image cytometry affecting the lower limit of detection for the specific signal and requiring sometimes sophisticated methods for separation from the signal by mathematical methods such as spectral deconvolution (8). Unfortunately, in order to stabilize the binding of an antibody post-fixation of the specimen is a standard preanalytical procedure which increases intrinsic fluorescence. Therefore, in FCM fixation is limited to cross-linking agents that produce the lowest level of autofluorescence. Glutaraldehyde and many other aldehydes that have been used as fixatives for many generations are due to their high autofluorescence induction are inappropriate. Now, with MCM, any fixative can be applied because fluorescence is not an issue anymore and probably there are better fixatives than para-formaldehyde.

In MCM, clearly only members of the lanthanide group can be used as molecular labels for proteins and nucleic acids that are stable isotopes but not radioactive elements. Therefore, Promethium (Pm), the only short lived radioactive element, is not applicable for obvious reasons. Generally, rare earth metals are mined from ores in mixtures with other metals and then isolated by elaborate chemical and physical processes. The separation of the different isotopes of a certain element is then done by (expensive and tedious) high-technology methods such as gas centrifugation, among others. But that is not the real problem. More critical is that the existence of such ores is very limited and often restricted to certain regions of the globe and that the science of cytometry competes for them with major commercial enterprises such as smart phone and flat screen producers. Today, China is the major rare earth metal producer (∼90%) and restricts export of these metals. Furthermore, the USA and Europe import nearly 100% of rare earth metals for their industries. Other new sources, such as the deep sea, asteroid mining, and Afghanistan (no comment) (9) may arise as new resources in the future. But there is light at the end of the tunnel. A few months ago in the state of Saxony, Germany, not very far from the city of Leipzig, an ore with relevant amounts of the rare earths Cer (Ce), La, Pr, Eu, and Y was discovered.