gas cluster ion beams for secondary ion mass spectrometry

AC11CH02_Winograd ARI 3 May 2018 15:10

Annual Review of Analytical Chemistry

Gas Cluster Ion Beamsfor Secondary Ion MassSpectrometryNicholas WinogradDepartment of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802,USA; email: [email protected]

Annu. Rev. Anal. Chem. 2018. 11:29–48

First published as a Review in Advance onFebruary 28, 2018

The Annual Review of Analytical Chemistry is onlineat anchem.annualreviews.org

https://doi.org/10.1146/annurev-anchem-061516-045249

Copyright c© 2018 by Annual Reviews.All rights reserved

Keywords

bioimaging, cluster ion beams, phospholipids, instrumentation, moleculardepth profiling, molecular dynamics computer simulations

Abstract

Gas cluster ion beams (GCIBs) provide new opportunities for bioimagingand molecular depth profiling with secondary ion mass spectrometry (SIMS).These beams, consisting of clusters containing thousands of particles, ini-tiate desorption of target molecules with high yield and minimal fragmen-tation. This review emphasizes the unique opportunities for implementingthese sources, especially for bioimaging applications. Theoretical aspects ofthe cluster ion/solid interaction are developed to maximize conditions forsuccessful mass spectrometry. In addition, the history of how GCIBs havebecome practical laboratory tools is reviewed. Special emphasis is placed onthe versatility of these sources, as size, kinetic energy, and chemical compo-sition can be varied easily to maximize lateral resolution, hopefully to lessthan 1 micron, and to maximize ionization efficiency. Recent examples ofbioimaging applications are also presented.

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ANNUAL REVIEWS Further

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https://www.annualreviews.org/doi/full/10.1146/annurev-anchem-061516-045249


SIMS: secondary ionmass spectrometry, inwhich secondary ionsare generated fromprimary ionbombardment

INTRODUCTION

Since the discovery by J.J. Thompson over 100 years ago that charged particles could be emittedfrom surfaces (1), secondary ion mass spectrometry (SIMS) has become a mainstay for materialscharacterization. Currently, the technique is unique in its ability to provide surface-specific molec-ular information, to acquire spatially resolved mass spectra with submicron resolution, and to ac-quire in-depth composition of molecular solids with nanometer resolution. The imaging modalityis particularly interesting. Originally developed in the SIMS community using focused atomic ionbeams to define position (2), the method provides unprecedented chemically specific informationat each pixel. Moreover, by stacking images acquired at different sample depths, three-dimensional(3D) information is forthcoming. The SIMS community has retained an unusual buoyancythrough the decades, and the recent emergence of cluster ion beams to initiate desorption has had aremarkable effect. With multiple atoms comprising the primary ion, chemical damage to the sam-ple is reduced because each atom carries a smaller share of the incident kinetic energy. Molecularfragmentation is reduced during the desorption process, resulting in nearly fragment-free massspectra. Molecular depth profiling of complex materials has allowed subsurface information tobe acquired for the first time. When combined with imaging, 3D information on the nanoscale ispossible to achieve. Cluster projectiles have placed molecular SIMS spectral capabilities, when them/z < 3,000, nearly on par with other stimulated desorption techniques that do not havethese unique aspects (3–5). An excellent comprehensive treatise on this topic has recently beenpublished (6).

Although countless cluster projectile varieties have been proposed, evaluated, and espoused,the implementation of gas cluster ion beams (GCIBs) has a special allure. These clusters, createdduring supersonic expansion, generate clusters that consist of ∼1,000 to >10,000 componentatoms or molecules. To allow for beam focusing and to provide sufficient kinetic energy to initiatemolecular desorption, these beams are typically accelerated to >10 keV of kinetic energy. Becausekinetic energy divides equally among constituents (7), the kinetic energy of each particle is reducedto a value comparable to chemical bond strengths. Hence, molecular fragmentation, subsurfacedamage, and interlayer mixing are reduced. There is a fundamental difference between GCIBsand smaller cluster projectiles such as Bi3+ or C60

+, where the kinetic energy per atom rangesfrom a few hundred to a few thousand electron volts.

There are practical challenges associated with GCIBs that prevent them from dominatingSIMS laboratories. These beams are characterized by a distribution of cluster sizes that renderspulsing for time-of-flight (TOF)-SIMS problematic. Focusing to a submicron spot has not yetbeen routinely demonstrated, compromising the ability to acquire high spatial resolution chemicalimages. Moreover, ionization efficiency is often poor even though molecular desorption is highlyefficient. Because of these issues, most SIMS researchers utilize the GCIB as an effective erosionsource for molecular depth profiling but employ the smaller cluster sizes for spectral characteri-zation and imaging. As it turns out, however, none of the aforementioned challenges is withoutsolution. Instrumental developments now allow high-quality mass spectra to be acquired easilywith GCIBs, and sources focused to at least one micron are becoming available. The great flexi-bility of tuning the chemistry of the GCIB also has implications for enhancing ionization. In thisreview, prospects for the exclusive use of GCIBs as the ion source of choice for molecular SIMSexperiments are considered by examining each of these factors in detail.

THEORETICAL CONSIDERATIONS

The emergence of GCIB technology has enormously expanded the space available for projectilechoice. Variables include size, composition, energy, and incidence angle. How can we optimize

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the experiment? Several important properties need to be considered. First, the sample needs toreceive sufficient kinetic energy to initiate molecular desorption. Second, the kinetic energy mustbe low enough so as not to initiate collision-induced fragmentation. Third, there must be sufficientenergy provided to the system to allow ionization during desorption when precharged species arenot present. If there are no ions, there is no signal. Finally, because 3D imaging is an importantgoal, the depth resolution during erosion needs to be optimized by tuning either the kinetic energyor the angle of incidence. The basic challenge is, then, how to find the sweet spot that maximizesall of these demands. Is there enough parameter space to find some set of optimized conditions?Can there be a theory that leads us in the right direction?

To begin, a basic picture of the cluster-solid interaction is needed. This type of picture is mostvividly provided by molecular dynamics computer simulations, which for several decades haveprovided molecular-level information about the energy dissipation process (8–12). An examplethat illustrates the unique aspect of the GCIB when compared to other atomic or cluster ionprojectiles is shown in Figure 1. In this case, the substrate is an Ag(111) crystal, and the projectileis accelerated to a kinetic energy of 15 keV (13) (Ar872 at 15 keV moves at 104 m/s). Hence, theenergy per atom varies from 15,000 eV for Ga to 17.2 eV for Arn, where n is the number of atomsin the cluster; in this case, it is 872. Note that both Ga and Au3 penetrate deeply into the Ag crystal,leaving considerable damage and atomic mixing. The displacements are characterized by binarycollisions producing a collision cascade of atoms. For C60, the impact results in the formation ofa well-defined crater, much like that of a meteor striking the earth. This mesoscale phenomenonis not well described by a collision cascade because the diameter of the C60 molecule (0.7 nm) islarger than the interatomic spacing of the Ag crystal (0.23 nm). This observation has led to thedevelopment of analytical theories of particle emission based upon mesoscale fluid flow that aresuccessful for predicting a range of parameters (14, 15). For the GCIB bombardment, the crater isof similar diameter to C60, although the depth is much lower. In addition, Ar872 is large enough toblock emission of substrate atoms. Hence, most of the particles are emitted from the crater edge atoff-normal angles. The yield of substrate atoms is approximately one-third that of C60, presumablydue to this blocking effect and to the lower kinetic energy per particle in the cluster, E/n. It is thisparameter that may also affect ionization probability, which is generally lower for GCIBs thanfor C60. Many other graphical pictures and videos, acquired using molecular dynamics, have beenpublished over the last several years (9–12, 16–20).

With all of the variables associated with GCIBs, and with the possibility of generating clusterswith different chemistry (e.g., C60), there has been an effort to find universal relationships thatallow for a predictive model using a single equation. A successful approach involves plottingexperimentally measured Y/n versus E/n for Ar–GCIBs. Here, Y is the yield of neutral materialfrom a molecular solid in volume units (nm3), and E is the kinetic energy of the cluster (21–25).Such a plot is shown for a variety of materials in Figure 2. Note that the value of Y/n can varyover four orders of magnitude for organic solids and polymers, and even more for atomic solidssuch as Si, SiO2, and Au. The plot shows that different classes of materials tend to cluster aroundspecific regions. In general, the yield from atomic solids can be an order of magnitude lower thanthe yield from molecular solids, primarily due to cohesive energy differences. This effect makesthe characterization of hybrid materials consisting of metal and organic components challengingdue to differential sputtering effects. After classifying the experimental data, the information canbe fitted with an empirical equation, which then has some predictive value. From this log–logplot, the Y/n value has a higher slope below 10 eV/atom than above this value. This effect is dueto the fact that at higher E/n, the energy cannot efficiently flow away from the cluster impact sitefast enough, so the yield of material begins to level off with increasing E. Finally, it has recentlybeen shown that by plotting Y(E/U0), where U0 is the binding energy of the solid, versus E/n, the

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0 ps

3 ps

29 ps

Ga Au3 C60 Ar872

1 nm

2121 7878 317317 106106

Figure 1Molecular dynamics computer simulations for primary ions of varying sizes bombarding an Ag(111) crystalat normal incidence and at a kinetic energy of 15 keV. The layers are color coded to aid visual clarity (13).The first three rows show a cross section of the crystal, whereas the bottom row shows a top-down view togain insight into the size of the damaged area. The gray particles begin at a depth of 4.6 nm. The numbers inthe bottom left corner of the third row are approximate values for the number of Ag atoms ejected for eachprimary particle impact. The first row shows the initial state of the crystal, and the second and third rowsillustrate the time evolution of the damage after 3 ps and 29 ps. Figure adapted with permission fromReference 13. Copyright 2013, SurfaceSpectra Ltd.

behavior of different materials classes can be made to converge (25). The goal of attaining somesort of universally applicable result has nearly been achieved.

The previous discussion applies when the mass of the atom in the cluster does not change. Thesituation is less clear when trying to compare the behavior of C60 to Ar1000, for example (26, 27).These comparisons can be made by plotting the yield per nucleon of sample versus the energyper nucleon of the projectile (27). This result illustrates that the mass of the projectile is a keyparameter. It is interesting that as the energy per nucleon is increased from 0.5 to 200, the ejectedmass per nucleon increases by a factor of 200.

Collision-induced fragmentation of target molecules must also be considered when seeking tooptimize the size and energy of the GCIB. Using molecular dynamics computer simulations for Arclusters, Garrison and coworkers (28) have shown that fragmentation begins at ∼50 eV/atom for

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0.00001

0.0001

0.001

0.01

0.1

1.0

10

100

Y/n

E/n, eV/atom10 1001

Au

SiO2

Si

Polystyrene

PMMA

Polycarbonate

HTM-1

Irganox 1010

Figure 2A log–log plot of Y/n versus E/n (n is the number of atoms in the cluster) for a wide variety of materialsbombarded by Ar clusters of various sizes at a 45◦ angle of incidence. Solid lines are the predictions for theuniversal curve, proposed by Seah (24), whereas the other lines show the behavior of other materials asindicated. Note that the sputtering rate for organic material is 2–4 orders of magnitude greater than forelements or inorganic compounds. Moreover, the sputtering rate for a specific species can vary by more thantwo orders of magnitude, depending upon the E/n value. Figure adapted with permission from Reference 24.

Static SIMS: methodin which the dose iskept below damagethreshold

small molecules such as benzene and octane, and it begins at ∼10 eV/atom with longer entwinedmolecules such as β-carotene.

In the laboratory, GCIB clusters varying in size from several atoms to 10,000 atoms or morehave been synthesized. The yield of emitted secondary ions is found to be a function of boththe cluster size and the kinetic energy. How can we best organize all these variables to find thebest conditions for SIMS? The heart of the problem is illustrated in Figure 3, where the SIMSspectrum is examined as a function of cluster size. The results show that as the size is increased, themagnitude of the signal decreases in an exponential fashion. However, the number of fragmentsrelative to the number of molecular ions also decreases. A clear example of this effect has beenreported using a thin amino acid film as a model compound (29). An interesting dichotomy isthen apparent from Figure 3, where the ionization yield drops exponentially with decreasing E/nbeginning at ∼20 eV/atom. Maximization of sensitivity also introduces the risk of opening themolecule fragmentation channel. However, in the figure, there appears to be a sweet spot in the5–10 eV/atom range, where fragmentation is not too severe, and the ion yield drop-off is not toosignificant.

MOLECULAR DEPTH PROFILING

As noted in the Introduction, an important and unique aspect of cluster SIMS is the ability tointerrogate molecular thin films in depth by eroding the sample with the primary ion beam (30).This property is remarkable because this beam creates damage to the surface and subsurface of themolecular film via collision-induced processes, altering the chemical composition. In fact, duringthe early years of SIMS research using atomic ion beams, so much damage was created that the iondose had to be kept below 1% of the number of surface molecules to acquire enough data to evenproduce a mass spectrum, a modality referred to as static SIMS (31). For many years, however, ithas been recognized that the degree of damage is much less when employing cluster ion beams,


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1

10

102

103

104

500 1,000 1,500 2,000Cluster size, n

Inte

nsit

y of

[M +

H]+

0.1

1.0

5 10 15 20E/n, eV

[Fra

gmen

t]+ / [

M +

H]+

a b

Figure 3Response of a generic organic molecule to 5-keV Arn

+ bombardment. These graphs illustrate theconundrum associated with molecular ion yield and molecular fragmentation. Panel a shows the typicalvariation of the molecular ion yield as the cluster size is increased from Ar200 to Ar2000. Panel b shows howthe fragments for a variety of molecules change with cluster size. Note that both ordinates are plotted on alog scale and that values of E/n (n is the number of atoms in the cluster) between 5 eV and 10 eV tend tomaximize sensitivity and minimize fragmentation. The kinetic energy of the cluster is held constant at 5 keVin all cases. Figure adapted from Reference 29.

similar to what has been observed when diluting the molecule into a matrix. There are at least twofactors at play. There is a nonlinear increase in the sputtering yield when the sample is hit by manyparticles simultaneously, and the lower E/n value associated with clusters inherently yields lessdamage. The result is attainment of a steady-state composition where the damage created by theprimary cluster ion beam is efficiently removed and does not accumulate as the dose is increased.

Many different types of cluster beams, consisting of various sizes and composition, have beenespoused over the last several decades. Early experiments using giant glycerol clusters (32), carbonclusters (33), and SF5

+ (7) were particularly influential in demonstrating the advantages of clusterSIMS over the use of atomic projectiles. For the first time, it was shown that molecular ion signalspersisted as the sample was eroded, and the mass spectra exhibited much less fragmentation thanwith atomic ion beams. Acceptance was slow, however, due to technical challenges and the factthat these beams could not be focused very well. Without the focusing, the imaging modality socrucial to the SIMS technique is severely compromised.

Molecular depth profiling achieved a quantitative status with the emergence of the C60 source(34). With this probe, detailed studies aimed at elucidating the factors that lead to a successfuldepth profile were initiated. Trehalose thin films of several hundred nanometers were employedas a model (35). This species is attractive because smooth, uniform, and stable thin films are easilyprepared in the laboratory via spin coating onto an Si substrate. Atomic force microscopy (AFM)is also a valuable tool, as it confirms film uniformity, detects the formation of topography thatdistorts thickness measurements, and reveals the physical shape of the erosion crater. Typicaldepth profiles are shown in Figure 4. Cheng and coworkers (36) utilized these data to create anerosion dynamics model that allowed the molecular sputtering yield, damage cross section, depthresolution, and altered layer thickness to be computed. They found that by using a 20-keV beam,245 trehalose molecule equivalents per incident C60

+ molecules were removed, and interfacewidths of <10 nm could be measured. The model has been extended and made more sophisticatedin the ensuing years (37).

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0 100 200 300 400 500 600

0.0

0.25

0.50

0.75

319.11

56.94522.20890

[µm]

[nm]

Rela

tive

inte

nsit

y

Depth, nm

Figure 4Prototypical molecular depth profile of a 300-nm-thin film of trehalose spin coated onto an Si wafer usingC60

+ for erosion. The red points are the quasimolecular ions of trehalose at m/z 325, [M−OH]+, whereasthe black points represent the underlying Si signal. Note that the ion signals reach a steady state after a fewnanometers of erosion. The fluctuations near the film surface arise from matrix effects due to either surfaceimpurities or the buildup of chemical damage. After 300 nm of erosion, the trehalose signal decays to zero asthe Si surface is exposed. Atomic force microscopy (AFM) images are taken from the surface of the film,showing that the root mean square roughness is less than 3 nm after removal of the film, which shows theformation of the crater due to erosion by C60. Note that the AFM measurement confirms that the crater hasa flat bottom, indicating uniform erosion. Figure adapted with permission from Reference 36. Copyright2006, American Chemical Society.

Delta layer: anembedded layer that isthinner than the depthresolution of thetechnique

Irganox: a popularSIMS molecule thatexists in manynumbered forms; it is apolymer additive

The development of the GCIB as a dominant cluster type for molecular depth profiling hasan interesting history. The beams themselves were first produced at Kyoto University as a toolfor smoothing surfaces utilized in the semiconductor industry (38, 39). Several years later, thisgroup showed that they could be successfully applied to SIMS experiments (40). Although theresults were impressive, the broader SIMS community did not take up this technology due tothe complexity of the ion sources. As discussed below, the beams are produced by supersonicexpansion of a high-pressure Ar gas and require a great deal of vacuum pumping. However, inrecent years, the instrument companies have been able to manufacture a device that will fit ontostandard vacuum system flanges (41–43). With this convenience now available, the use of GCIBshas become widespread.

An important advance for molecular depth profiling with GCIBs emerged from a collaborationbetween the Kyoto group and the National Physical Laboratory (NPL) in the United Kingdom.(44). At the NPL, an especially stable organic multilayer sample was prepared, consisting of ul-trathin ∼2.4-nm delta layers of Irganox 3114 embedded between ∼46-nm layers of Irganox 1010.The procedure involves erosion with an Ar–GCIB and analysis of Irganox fragment ions usingBi3 or C60. These experiments are similar in spirit to the trehalose/C60 studies mentioned above.A structure such as this is extremely valuable for determining the depth resolution associated withthe erosion of the organic material and for allowing accurate determination of the sputtering yield.This round-robin sample was sent to 20 different laboratories to evaluate for reproducibility andfor the ability to provide quantitative information (45). The results were shown to be remarkably


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Normal depthprofile: defined as acondition whereby asteady-state signal isachieved during filmerosion

consistent, although significant corrections to the data are required to obtain accurate composi-tional information (46). In fact, this sample was also employed earlier to optimize conditions forachieving the best depth resolution and to test reproducibility of measurements in a laboratoryround-robin study using C60 as a projectile (47).

There are unique aspects associated with GCIB-depth profiling that need to be taken intoaccount. In a follow-up study to the trehalose depth profiling using C60 discussed above, Shenand coworkers (48) examined how molecular depth profiles are influenced by GCIB cluster sizeand kinetic energy by varying the projectile from 10 keV Ar4000

+ (E/n 2.5 eV) to 20 keV Ar1000+

(E/n 20 eV). Their results show that when E/n > 5 eV, normal depth profiles are obtained withrelatively high sputter yield. When E/n < 5 eV, however, unusual depth profiles are observedthat are characterized by ionization efficiency fluctuations and low, yet unchanging, sputteringrates. An observed signal increase, rather than steady-state formation, suggests that the ionizationprobability of the trehalose molecular ion is increasing, perhaps by interacting with fragmentspecies created in the near-surface region of the thin film. Hence, there is a conundrum: how tokeep high-quality depth profiles (higher E/n values) but minimize ionization fluctuations (lowerE/n values). These experiments suggest that the E/n value should be kept above but close to thethreshold value of 5 eV to minimize fragmentation and ionization artifacts.

EXPERIMENTAL ISSUES

The Mass Spectrometer

Instrumental requirements for GCIB-SIMS studies present a number of unique challenges. Tra-ditional TOF-SIMS machines are designed for maximum transmission so that mass spectra arerecorded before damage accumulation kills the acquisition (49). For molecular depth profilingexperiments with TOF-SIMS, analysis is typically done using a metal cluster (e.g., Bi3+) ion beamdelivered to the sample in the form of a pulse a few nanoseconds wide with a repetition rate of 1–10 kHz (50–52). The duty cycle, or the amount of time the beam is hitting the sample, is thereforeless than one part in 105. Erosion of the sample is carried out using a continuous GCIB during thetime when the Bi3+ beam is switched off. This temporal interleaving of beams is necessary becauseit is difficult to create short ion pulses with the GCIB, as there is generally a distribution of clustersizes in the beam. This configuration is potentially inefficient because most of the sample is lostduring erosion.

Largely because of these issues, new approaches for mass analysis have been under develop-ment that are better suited to the unique properties associated with cluster ion beam-induceddesorption. As instrument transmission is no longer the overriding consideration, SIMS instru-ments, which include aspects of the latest mass spectrometer designs, are now feasible. Manyof the new instruments utilize a continuous primary ion beam and acquire time information bymanipulating the secondary ion beam. An early approach involves modifying a QSTAR hybridmass spectrometer manufactured by Sciex to accept a C60 source (53–55). This hybrid instrumentchannels the secondary ion beam through a triple quadrupole for initial mass selection and anorthogonal TOF for mass analysis. Mass resolution of one part in 15,000 is achieved, exceedingwhat is possible using traditional TOF analyzers. At about the same time, Ionoptika in the UnitedKingdom constructed a similar device that utilizes a shaped field buncher to time focus a sectionof the secondary ion beam (56), which is then injected into a harmonic reflectron. This device isspecifically designed for cluster SIMS studies focusing on biological samples because it has exten-sive cryogenic sample-handling capabilities. The fact that it also utilizes a continuous GCIB forcreation of secondary ions allows high mass resolution and high spatial resolution to be achieved at

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MS/MS: tandem massspectrometry in whichthe molecular ion isselected for furtherfragmentation; themethod is useful forstructuredetermination

Wien filter:device consisting oforthogonal magneticand electric fields andused for velocityselection

the same time. Both of the above instruments implement tandem mass spectrometry (MS/MS), animportant capability that is missing in the original TOF-SIMS systems. Other groups are devel-oping Fourier transform mass spectrometer analyzers, which have ultrahigh mass resolution (57).Finally, a major new initiative led by researchers at NPL involves collaborations between two largeinstrument companies and a major pharmaceutical company to analyze the distribution of drugsin single biological cells. This device utilizes both a traditional TOF analyzer when high-speedacquisition is required and an orbitrap analyzer when high mass resolution is required (58, 59).Hence, with the emergence of cluster ion beams, and especially the GCIBs, we have entered agolden age of instrument development for SIMS.

Gas Cluster Ion Beam Sources

There is now a wide selection of commercially available GCIB sources for SIMS researchers, adevelopment that has been critical to the emergence of this technology (41–43, 60). As noted above,the original Kyoto source was unwieldy. Nonetheless, this group pursued the SIMS applicationby modification of one of these giant sources, and, in combination with computer simulations,demonstrated higher secondary ion signal, reduced topography upon sputtering, and reducedsubsurface chemical damage (61).

Given this potentially significant application for GCIBs, several instrument companies man-aged to produce greatly simplified sources that could be retrofitted to a standard vacuum chamber.A schematic diagram of a generic GCIB is shown in Figure 5. Neutral clusters are produced in theexpansion chamber where a high pressure of Ar gas is forced through a nozzle. The gas undergoesan adiabatic expansion, which allows formation of clusters of 1,000 to 10,000 atoms upon cooling.Various positions within the beam can be selected by moving the nozzle, after which time the

Gas inlet

Pump 1Pump 2

Gate valve

Lens 2

Scans

Beam bend

Mass filter

Lens 1

Ionization

Skimmers

Nozzle

Figure 5Generic diagram of a typical gas cluster ion beam source. The feed gas is introduced at the gas inlet (left). Supersonic expansion occurs asgas emerges from the nozzle. The large turbomolecular pump 1 evacuates this expansion chamber. A section of the beam is sampled by askimmer; the beam is ionized by electron impact and partially focused by lens 1. The turbomolecular pump 2 further reduces the pressure,typically to the range of 10−7 torr. The ion beam is then mass selected with a low-resolution Wien filter, followed by a slight beambend to remove the influence of any remaining neutral species. Finally, it is possible to add a second focusing lens for high-resolutionimaging and to include scanning plates to move the beam from pixel to pixel. Figure reproduced with permission from Ionoptika Ltd.


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beam is directed into a collimating skimmer. Ionization occurs in the next chamber by electronbombardment. The resulting ions are mass selected with a device such as a Wien filter. Typically,the Wien filter has a resolution of no more than 1 part in 50, which is adequate to remove thesmaller clusters but leaves a distribution of sizes at larger values. Finally, the beam enters a standardion optical column for focusing and rastering. The expansion chamber and the ionization chamberare separately pumped by large turbomolecular pumps to reduce collisions between the clustersand the background gas. Sources are now available with beam energies ranging from 10 keV to70 keV and can be focused to a spot size of a few millimeters down to less than 1 micron.

GAS CLUSTER ION BEAM CHEMISTRY

Change the Cluster Composition

An obvious strategy is to create projectiles with different components than Ar gas. We have foundthat it is straightforward to produce beams of CO2 clusters ranging in size from 1,000 to 12,000molecules simply by changing the feed gas from Ar to CO2 (62). This beam exhibits improvedproperties relative to Ar clusters for several reasons. Most importantly, CO2 clusters are morestable than Ar clusters and hence do not require as high a backing pressure. The binding energyof an Ar cluster is 0.065 eV/atom, whereas that of CO2 is 0.27 eV/atom. The CO2 GCIB is alsoeasier to focus, as background pressure in the source is generally lower, and metastable decay ofthe cluster as it travels down the column is less likely. At most kinetic energies, CO2 acts like anatom with m/z 44, because at up to 10 eV/atom, there is not enough energy to break the C–O bondto a significant degree. This source has great potential for high-resolution imaging experiments.

Many other strategies are now open for exploration. For example, it is a long-standing goalof the SIMS community to improve ionization probability (63), especially for high-resolutionimaging experiments in which the number of molecules available for detection becomes vanishinglysmall, requiring high sensitivity (i.e., an image pixel of 100 × 100 nm contains only 104 molecules).This goal is particularly important for GCIBs because the ionization probability is generally low,even though molecular fragmentation is greatly reduced. A primary mechanism of ionization fororganic molecules is the protonation of desorbed neutral molecules to form a protonated molecularion, [M+H]+. To aid in protonation, there has been interest in creating a GCIB consisting of(H2O)x

+ as a species potentially capable of increasing this ionization pathway. The Manchestergroup, in collaboration with Ionoptika, modified an existing GCIB to produce such a beam (64–66). They utilized steam at the gas inlet and added a heating system to the nozzle to preventice formation. The resulting beam consists of clusters of 1,000–10,000 H2O molecules. Theseresults are encouraging in that more than an order of magnitude enhancement is observed for avariety of molecules, including lipids, peptides, and pharmaceutical agents. These experiments arealso important because they demonstrate that chemically driven pathways are possible during thebombardment process.

Mixed Clusters

It is not difficult to produce clusters of mixed composition to optimize further secondary ionemission. We attempted to utilize CH4 as an intense source of protons (67). The idea is thatduring the bombardment, there are enough energetic processes involving electrons and smallfragments to allow production of protons. To avoid destroying the turbopumps with reactivegases, a mixture of 97% Ar and 3% CH4 was employed as the feed gas. The results of theseexperiments were not particularly successful because only a factor of four improvement of signal

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was observed, and the sputtering yield was nearly unchanged. Presumably, the energetics of theprocess are not adequate to break enough C–H bonds to make much of a difference to [M+H]+

formation.A second more successful approach involves dynamic reactive ionization (DRI) using hydrogen

chloride (HCl) as an example (68). By itself, the cluster will not have enough energy to break theH–Cl bond, and if broken, the dissociation products would be H and Cl atoms, which are unhelpfulfor ionization enhancement. Damage to the mass spectrometer by HCl gas is also problematic. Itwould be interesting to try a mixture of HCl and H2O, where dissociation into H3O+ and Cl− isexothermic, but technical reasons make this approach impractical.

An alternative strategy is to combine neutral HCl doped at the 5% level into an Ar–GCIBwith D2O adsorbed onto the target surface. The D2O layer should be thin enough to allowdetection of the target molecules but thick enough to form a solvent ice matrix for the HCl. Theamount of adsorption is controlled by adjusting the background pressure of D2O and by coolingthe sample to below 150 K. Deuterated water is useful for these studies, as it provides an easyway to clearly identify the source of protons. This arrangement is interesting because the acidicreactions will only occur near the point of cluster impact, precisely where they are needed toenhance [M+H(D)]+ formation. Initial experiments showed that the yield of D2O cluster ionsfrom the ice layer increases by a factor of 1,000 in the presence of HCl, confirming the basichypothesis associated with DRI (68). A recent example of this enhancement for a thin film oferythromycin is shown in Figure 6. When D2O is condensed onto the target, the quasimolecularion of erythromycin is enhanced by nearly a factor of ten.

A modified version of this protocol also allows molecular depth profiling to be performedwith enhanced sensitivity. In this situation, D2O is allowed to continuously condense onto acooled target surface by proper adjustment of the water vapor pressure in the analysis chamber.Optimization of this background pressure during erosion of the target allows a stable signal tobe produced. Using a trehalose film as the model, enhancement factors of [M+H]+ of more thantenfold are observed (69, 70).

These model studies reveal another interesting result that has significance for bioimagingstudies. For most real-world samples, the largest signal observed in the mass spectrum is typically[M+Na]+ due to the presence of adventitious salt. This complication can lead to large matrix ion-ization effects, making quantitative measurements tricky at best. Under DRI conditions, however,the [M+Na]+ channel is unaffected during water deposition, and the signal is rapidly overtakenby [M+H]+ (69, 70). In general, DRI provides a more uniform ionization efficiency betweendifferent molecules that is not observed under pure Ar cluster ion bombardment. These trendsare particularly noticeable in positive ion mode. The prospect of reducing these matrix effects isindeed an exciting one.

BIOIMAGING APPLICATIONS

Mouse Brain Sections and Lipidomics

As we have implied up to this point, mass spectra created from GCIBs yield less fragmentation thansmaller projectiles and allow molecular ions of a variety of biomolecules to be obtained with muchhigher sensitivity. Hence, it is plausible to assume that the GCIB will be a useful tool for bioimagingapplications, where the nature of the sample consists of a complicated array of fragile molecules.An early example (71) involves the imaging of a coronal mouse brain section using 20 keV Ar4000

+

focused to a 4–10-μm spot and covering the mass range of m/z 650 to 850. For this section, withresults shown in Figure 7, a 10-μm-thick slice was mounted onto indium tin oxide–coated glass


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0 1 2 3 4 5 0 1 2 3 4 5

Inte

nsit

y/co

unts

Interface maximum

1.5 × 107

1.0 × 107

5.0 × 106

0.0

1.5 × 107

1.0 × 107

5.0 × 106

0.0

9× signal increaseof [Mn* + H/D]+

[Mn* + H/D]+

Ion dose (1013 ions cm–2)

[Mn* + H/D]+

D2O cluster

[Fn* + H/D]+[Fn* + H/D]+

Erythromycin, C37H67NO13

[M + H]+ @ m/z 734.46

H3C

H3C

H3C

O

H3C

CH3

O

CH3

OHOH

HO

CH3

O

O

O

O

CH3

OH

CH3

OH

CH3

CH3

CH3N

OCH3

O

Figure 6Dynamic reactive ionization for a thin film of erythromycin doped into a biological lysate on an Si substrate.The left panel shows the depth profile in the absence of D2O condensed on the surface, whereas the right panelshows the increase in signal with deposited D2O. The red curve shows the intensity of the quasimolecular ion[M+H or D]+ as a function of primary ion dose. The primary ion in this case is Ar1500

+ doped with 5% HCl at20 keV. The blue curve represents the behavior of selected fragment ions (F) associated with erythromycin. Thedose required to reach the interface between the D2O overlayer and the erythromycin is indicated in the figure.The sample was held at 115 K to promote the condensation of D2O. The structure of erythromycin is shownin the bottom half of the figure. Top half of figure based on unpublished data from H. Tian and N. Winograd.

and analyzed directly without application of matrix or other pretreatment steps. The field of viewof a typical SIMS instrument is generally restricted to less than 1 mm2. Hence, to cover the entiretissue slice of 10 mm2, a series of up to 100 images is acquired by rastering the beam over 1 mm2,followed by mechanically moving the sample stage to a new position and stitching each tiled imagetogether to create the final result. In general, the total ion intensity in this mass range is at leastone order of magnitude greater than is found using smaller clusters. There is a rich distributionof glycerophosphocholines (GPCho) in the form of [M+H]+, [M+Na]+, and [M+K]+, as well asglucosylceramides and cholesterol. Note that the GPCho lipids are antilocalized with cholesterol.

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GPCho C32:0

GPCho C34:0

Cholesterol Color overlay image

5 mm

co

inhi

ce

740 760 780 8000.0

0.2

0.4

0.6

0.8

1.0

800.

5079

8.48

784.

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769.

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54

739.

41

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734.

52

741.

43

772.

48

m/z 734.5 [M + H]+ m/z 756.5 [M + Na]+ m/z 772.5 [M + K]+

m/z 762.5 [M + H]+ m/z 784.5 [M + Na]+ m/z 800.5 [M + K]+

m/z 369.3 [M + H–H2O]+

Figure 7Selected positive ion images of a mouse brain section using Ar1700

+ at 20 keV as a projectile. The top rowshows the distribution of GPCho C32:0 (the notation refers to the number of carbon atoms in the fatty acidchain and the number of double bonds). There are three images because intensity is observed for not onlythe protonated molecule, but also the sodiated and potassiated adducts. Similar results are shown in thesecond row, where the C34:0 species is presented. The bottom left image shows the distribution ofcholesterol. The remaining two images are overlays with GPCho C32:0 at m/z 734.5 in blue, GPCho C34:3at m/z 822.6 in turquoise, cholesterol at m/z 369.3 in yellow, and glucosyl ceramide (d18:1/16:0) at m/z722.5 in magenta. The major brain structures are indicated (ce, cerebellum; co, corpus callosum; hi,hippocampus; in, internal capsule). The last image shows a diacyl glycerol ion at m/z 534.3 in magenta andGPCho C36:3 at m/z 822.6 in turquoise. The positive ion secondary ion mass spectrometry spectrumcollected from the tissue in the mass range between m/z 720 and 810 is shown at the bottom. Figure adaptedwith permission from Reference 71. Copyright 2014, John Wiley and Sons.


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Stroma: connectivetissue and blood vessels

These results are consistent with similarly performed matrix-assisted laser desorption ionization[MALDI (72)] where a matrix is required, and desorption electrospray ionization [DESI (73)]imaging experiments, except that the spatial resolution is improved.

The higher sensitivity of GCIB-SIMS to various types of biomolecules has inspired othergroups to undertake more sophisticated studies aimed at unraveling important fundamental bio-logical processes related to disease (42, 74–77). Angerer and coworkers (42, 75, 76) have sought toimprove image quality by incorporating a prototype CO2-doped Ar–GCIB operating with Ar4000

at 40 keV. The higher kinetic energy and CO2 incorporation allow spatial resolution of less than4 μm to be achieved, which permits tackling the issue of lipid distributions in healthy and diseasedcells. Moreover, the higher kinetic energy produces higher secondary ion yields, suggesting thateven higher kinetic energies might be beneficial. The mass range amenable to study for imagingat this spatial resolution is from m/z 80 to a maximum of m/z 950, typically the upper limit forSIMS imaging studies. The results show that elevated levels of essential lipids are associated withinflammatory cells in the stroma of human breast cells but do not penetrate cancerous tumors. Theabsence of essential fatty acids in cancer cells is proposed to indicate that the de novo productionof fatty acids in combination with anaerobic glycolysis acts to protect against cell death. Theseresults are particularly important because the traditional method for examining changes in lipiddistributions involves creating a homogenate of many cells. This diversity masks the fact that cellswith different origins contain a heterogeneous cancerous microenvironment. The 40-keV Ar4000

source has also been employed to detect the drug methylphenidate in Drosophila brain tissue (78,79), which clearly represents an important conceptual advance for cluster SIMS.

It is now feasible to extend the mass range to m/z values of several thousand Daltons while re-taining impressive spatial resolution using special sample preparation protocols. We have shown,for example, that by properly treating a rat brain slice (80), low abundant cardiolipins and gan-gliosides are detected to m/z 2,000 and higher. These molecules represent an important classof mitochondria-specific phospholipids, which are important in cell death pathways (81). Theprojectile in this case is (CO2)3500

+ at 20 keV. The idea is to utilize a combination of enzy-matic and chemical treatments to the tissue slice. These treatments cross-link proteins to stabilizelipid location and remove phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids,generally present in excess amounts. The PC and PE components largely comprise the cell mem-brane, whereas the cardiolipin species exist within the interior of the cell. Removal of PC and PEcomponents is also likely to reduce matrix ionization effects associated with charge suppressionphenomena (82). With this protocol, it is possible to determine changes in the cardiolipin distri-butions associated with tissue that has undergone traumatic brain injury. The results show thatoxidizable polyunsaturated cardiolipin species are depleted after injury in distinct regions of thehippocampus. An example of this depletion is shown in Figure 8. This preliminary study suggeststhat GCIB-SIMS can provide an important complementary tool to MALDI imaging, which canalso detect cardiolipin species in tissue, but at a spatial resolution limited to ∼50 μm (83).

Three-Dimensional Imaging

An important goal of the GCIB-SIMS effort is to combine molecular depth profiling with high-resolution spatial imaging to create a 3D representation of the sample. The idea is to acquireimages during the depth profile and then stack them to obtain information in depth. For biologicalsamples, there have been a number of reported examples, particularly those involving single cells(84–86). The biggest issue that needs to be addressed involves the problem of differential erosionrates (87, 88). If structures inside a cell are removed more easily than other structures, the depth(z) scale will be in error. So far, numerical schemes for z-scale corrections have been proposed

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CO2-GCIB-SIMS

Controlledcortical impact

Depletion ofcardiolipin

m/z 1478.0 CL(74:7)

1,360 1,380 1,400 1,420 1,440 1,460 1,480 1,500 1,520 1,5400

CL (6

6:2)

CL (6

6:4)

CL (6

8:6)

CL (6

8:3)

CL (6

8:0)

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8:2) CL

(70:

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(70:

3)CL (7

0:4)

CL (7

2:7)

CL (7

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) CL (7

4:9)

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2:6) CL

(72:

4)CL (7

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)

CL (7

6:11

)CL

(76:

10)

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4:6)

m/z

CL (7

4:7)

CL (7

4:5)

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6:12

)

CL (7

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)CL

(78:

12)CL

(72:

3)

CL (7

2:8)

CL (7

2:9)

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0:5)

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0:6)

CL (7

0:7)CL

(68:

4)CL

(68:

5)

CL (7

4:4)

CL (6

6:3)In

tens

ity/

coun

ts

1 × 105

2 × 105

3 × 105

Figure 8CL images of traumatically injured rat brain hippocampus. The images were recorded in negative ion modeusing a (CO2)3500

+ beam at 20 keV. More than 40 CLs were identified. The top images consist of an opticalimage (H&E stain) of the brain slice, followed by the CL image at m/z 1,478. Note the depletion of CL inthe ipsilateral contusional cortex. The total ion mass spectrum acquired from the brain slice is shown in thelower panel, highlighting the mass range associated with CL. Figure adapted with permission fromReference 80. Copyright 2017, American Chemical Society. Abbreviations: CL, cardiolipin; GCIB, gascluster ion beam; SIMS, secondary ion mass spectrometry.

(85), with position-sensitive depth values determined using techniques such as AFM. It is alwayspossible to create 3D images using stacked serial tissue slices, although the depth resolution is onthe order of microns rather than nanometers.

Several recent studies have used the two-gun interleaved strategy to create 3D images, wherebythe erosion source is a GCIB, and the analysis beam is Bi3+. This approach has been employed todetect and locate an active metabolite in small glandular trichomes, which are found at the surfaceof leaves in certain rare plant species (89). Another study recently elucidated the 3D configuration


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of nuclei at the single-cell level by monitoring the adenine ion (90). At this point, however, thereare no examples of 3D images acquired using the GCIB alone, presumably due to the limitednumber of instruments capable of such measurements. It will be of interest going forward to makedirect comparisons between the two-gun and single-gun approaches (52).

CONCLUSION

In recent years, tremendous progress has been made in our understanding of basic SIMS phe-nomena. Such progress has led inexorably to the development of more sophisticated tools withwhich the community has continued to expand applications. Here, the unique properties of GCIBshave been highlighted, with the intent of inspiring increased use of these projectiles, particularlyin bioimaging experiments. Effective implementation of this tool requires significant changes toexisting SIMS instrumentation, although as noted in the review, these changes are well underway.It is fascinating to see how the instrument companies, through their creative efforts, have broughtthe GCIB technology to the user laboratory in very convenient packages. The interplay betweenexperiment and theory has been extraordinarily valuable in advancing applications.

Despite the flurry of recent progress, there are clearly areas that require further investigation.Central to every application is ionization efficiency, and the numbers suggest that several ordersmore sensitivity are there for the taking. The flexibility of GCIBs with respect to cluster size,kinetic energy, and chemical composition provide real pathways for more breakthroughs. Thefact that DRI, for example, minimizes the dreaded matrix ionization effect in biological tissue (69,70) is an observation that needs much closer attention.

Finally, in addition to ionization efficiency, improvements in the focusing power of GCIBsare still needed. One possibility in this regard is to design a source where the primary ion beamis accelerated to higher kinetic energies to overcome chromatic aberrations. Our laboratory hasimplemented a source that operates at 70 keV, yielding a spot size of less than 1 micron for clustersof CO2 up to sizes of 10,000. This higher energy also results in better ionization yields, as notedwhen comparing spectra acquired at 20 keV to those acquired at 40 keV (42).

SUMMARY POINTS

1. The emergence of GCIB sources provides game-changing advances for molecular SIMS.

2. Theories aimed toward unifying the behavior of the myriad of GCIB types are beingdeveloped successfully and have predictive value.

3. Ionization enhancement is possible using a wide range of new strategies owing to theflexibility of synthesizing GCIBs of different chemical composition.

4. A golden age of SIMS instrument development is underway.

5. Molecular depth profiling and imaging are best carried out using the GCIB alone, withoutthe need for interleaved ion sources.

6. Bioimaging applications are emerging rapidly, closing the mass range gap with MALDIwhile retaining submicron spatial resolution.

DISCLOSURE STATEMENT

The author is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of the review.

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ACKNOWLEDGMENTS

The author wishes to acknowledge Dr. Barbara Garrison for essential contributions to theseideas and for help in editing the manuscript. Dr. Hua Tian provided many of the figures and theunpublished data. It is difficult to single out the funding agencies that have allowed me to carryout this work. Over the years, the National Science Foundation, National Institutes of Health,US Department of Energy, US Office of Naval Research, and US Air Force Office of ScientificResearch have been generous contributors. Recent funding from Novartis Pharmaceuticals hasalso had a major impact on this work.

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AC11_TOC ARI 3 May 2018 12:3

Annual Review ofAnalytical Chemistry

Volume 11, 2018

Contents

Mass Spectrometry for Synthesis and AnalysisR. Graham Cooks and Xin Yan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Gas Cluster Ion Beams for Secondary Ion Mass SpectrometryNicholas Winograd � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �29

Relative and Absolute Quantitation in MassSpectrometry–Based ProteomicsJ. Astor Ankney, Adil Muneer, and Xian Chen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �49

Technologies for Measuring Pharmacokinetic ProfilesA.A. Heller, S.Y. Lockwood, T.M. Janes, and D.M. Spence � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �79

Interfacing Cells with Vertical Nanoscale Devices:Applications and CharacterizationAllister F. McGuire, Francesca Santoro, and Bianxiao Cui � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 101

Wearable and Implantable Sensorsfor Biomedical ApplicationsHatice Ceylan Koydemir and Aydogan Ozcan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 127

SERS Sensors: Recent Developments and a Generalized ClassificationScheme Based on the Signal OriginXin Gu, Michael J. Trujillo, Jacob E. Olson, and Jon P. Camden � � � � � � � � � � � � � � � � � � � � � � � 147

DNA Nanotechnology-Enabled Interfacial Engineering for BiosensorDevelopmentDekai Ye, Xiaolei Zuo, and Chunhai Fan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 171

DNA Electrochemistry and Electrochemical Sensors for Nucleic AcidsElena E. Ferapontova � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 197

Improving Lateral Flow Assay Performance UsingComputational ModelingDavid Gasperino, Ted Baughman, Helen V. Hsieh, David Bell,

and Bernhard H. Weigl � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 219

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AC11_TOC ARI 3 May 2018 12:3

Recent Advances and Trends in Microfluidic Platforms for C. elegansBiological AssaysFarhan Kamili and Hang Lu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 245

Fabrication and Use of Nanopipettes in Chemical AnalysisShudong Zhang, Mingzhi Li, Bin Su, and Yuanhua Shao � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 265

3D Printed Organ Models for Surgical ApplicationsKaiyan Qiu, Ghazaleh Haghiashtiani, and Michael C. McAlpine � � � � � � � � � � � � � � � � � � � � � � � 287

Analytical Chemistry in the Regulatory Science of Medical DevicesYi Wang, Allan Guan, Samanthi Wickramasekara, and K. Scott Phillips � � � � � � � � � � � � � � 307

(Multi)functional Atomic Force Microscopy ImagingAnisha N. Patel and Christine Kranz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 329

Nano-Enabled Approaches to Chemical Imaging in BiosystemsScott T. Retterer, Jennifer L. Morrell-Falvey, and Mitchel J. Doktycz � � � � � � � � � � � � � � � � � � 351

Single-Molecule Force Spectroscopy of Transmembraneβ-Barrel ProteinsJohannes Thoma, K. Tanuj Sapra, and Daniel J. Muller � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 375

Voltammetric Perspectives on the Acidity Scale and H+/H2 Process inIonic Liquid MediaCameron L. Bentley, Alan M. Bond, and Jie Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 397

Nanoscale Electrochemical Sensing and Processing in MicroreactorsMathieu Odijk and Albert van den Berg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 421

Electrochemical Probes of Microbial Community BehaviorHunter J. Sismaet and Edgar D. Goluch � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 441

Boron Doped Diamond: A Designer Electrode Material for theTwenty-First CenturySamuel J. Cobb, Zoe J. Ayres, and Julie V. Macpherson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 463

Recent Advances in Solid-State Nuclear MagneticResonance SpectroscopySharon E. Ashbrook, John M. Griffin, and Karen E. Johnston � � � � � � � � � � � � � � � � � � � � � � � � � � 485

Methods of Measuring Enzyme Activity Ex Vivo and In VivoYangguang Ou, Rachael E. Wilson, and Stephen G. Weber � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 509

Errata

An online log of corrections to Annual Review of Analytical Chemistry articles may befound at http://www.annualreviews.org/errata/anchem

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gas cluster ion beams for secondary ion mass spectrometry

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