New Instrumental Developments in Cluster ToF-SIMS

Open Access
Carado, Anthony James
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
December 03, 2009
Committee Members:
  • Nicholas Winograd, Dissertation Advisor
  • Nicholas Winograd, Committee Chair
  • Philip C. Bevilacqua, Committee Member
  • David Lawrence Allara, Committee Member
  • Robert Allen Schlegel, Committee Member
  • c60
  • cluster tof-sims
  • orthogonal tof-sims
Time-of-flight secondary ion mass spectrometry is a surface analysis technique that utilizes a high energy primary ion beam which, upon collision with the sample, causes the ejection of secondary ions from the sample. These ions are then separated based on their mass in a time-of-flight mass analyzer. By rastering a highly focused beam at many locations on a sample and recording a mass spectrum at each of these locations, chemical specific images can be created of the sample. While extremely powerful in principle, this technique has several significant challenges. The two most persistent and problematic are low sensitivity and sample fragmentation upon impact of the primary ion. Exciting progress in addressing these issues was brought about by development of cluster projectiles such as Aun+, SF5+, Bin+, and C60+. These larger projectiles spread their energy over a wider surface area and have been shown through simulation and fundamental studies to increase the number of secondary ions ejected per impact as well as to better remove any damage that occurs during the impact. This leads to the inference that a simple way to collect more secondary ions is to impact the sample with more primary ions. Unfortunately, the ToF-SIMS instrumentation has not necessarily advanced in step with primary ion sources and so the true utility of cluster projectiles is not being fulfilled. In this work, we report on the addition of a 20 keV C60+ primary ion source to a commercial MALDI/ESI mass spectrometer by Applied Biosystems / MDS Analytical Technologies, the QStar XL mass spectrometer. This instrument is equipped with an orthogonal ToF which allows the primary ion source to operate in a continuous fashion as opposed to short, widely spaced pulses typical of traditional instrumentation. This mode of operation allows the delivery of several orders of magnitude more primary ion fluence. In addition, the QStar XL is a multiple quadrupole mass spectrometer which enables tandem mass spectrometry experiments (MS/MS) to aid in ion identification through collision induced dissociation fragmentation analysis. Aside from the differing geometry of the ToF mass analyzer, the most unusual aspect of the QStar XL is the fact that it operates at significantly higher pressures, at least surrounding the sample, than typical high vacuum mass spectrometers. Two different gas inlets result in different pressures at the sample. In collisional cooling mode, the pressure at the sample is ~1 Torr. This high pressure is required to reduce the fragmentation of electrospray and MALDI generated secondary ions. Unfortunately, utilizing this gas inlet resulted in a significant loss of SIMS generated secondary ions, likely due to scattering, and so proved to be unfeasible. Using the alternate gas inlet at Q0, N2 pressure at the sample is ~5 x 10-3 Torr. The vast majority of the experiments described in this thesis are using the Q0 gas configuration which results in collisional focusing, but not collisional cooling. Chapter two deals with the experimental considerations of operating a high voltage, high velocity, primary ion source on an instrument that requires high pressures for operation. Efforts to shield the primary ion beam from high gas include incorporating a differential pumping sleeve and a small aperture nose cone that not only restricts the amount of gas leaking in to the beam column, but also reduces the distance with which the primary ion beam needs to travel in high pressure before reaching the sample. These efforts result in a beam column pressure of less than 1x 10-6 Torr while the sample region is at normal operating condition of 5 x 10-3 Torr. Consequences of the high pressure on the primary ion beam characteristics do not seem serious. Beam spot size vs. pressure was measured with an atomic force profilometer on a thin-film of the peptide gramicidin S, and no significant broadening of the beam profile was seen for pressures ranging from < 5 x 10-4 to 8 x 10-3 Torr N2. Additionally, spectra obtained from this prototype instrument do not differ substantially from the those obtained from the high vacuum pulsed beam instrument with regards to degree of sample fragmentation. It is likely that were the C60+ ions fragmenting significantly due to collisions with N2, the sample spectra would show more fragmentation. In fact, there is some evidence that higher pressures may be preventing fragmentation through collisional cooling; a phenomenon that occurs when low energy ions transfer some of their thermal energy to neutral N2 molecules. This instrument is designed to take advantage of this when analyzing MALDI and electrospray generated ions, but evidence of it occurring with SIMS generated ions has not previously been seen. The QStar XL operates with negligible extraction voltages and so the use of high gas pressures near the sample and a decreasing pressure gradient through the quadrupoles to the mass analyzer facilitates ion transmission through collision focusing. Similar to collisional cooling, collisional focusing occurs when low energy secondary ions collide with neutral N2 molecules resulting in a restriction of their transverse motion which creates a beam of ions. The requirement of this instrument to operate under collisional focusing conditions is demonstrated on several molecules in chapter 2 and 3; however it should be noted that collisional focusing is required for all analytes. In chapter 3, comparisons to traditional SIMS instrumentation as well as to MALDI generated data from the QStar XL are made. Orthogonal ToF designs introduce a secondary ion loss that is not present in axial ToF designs, and so secondary ion efficiencies (secondary ions detected/primary ion impact) were of great interest. It was found that the secondary ion efficiencies from SIMS generated data from the QStar XL were similar to the high vacuum pulsed SIMS instrument in this lab. Secondary ion efficiencies for both instruments for indium and gramicidin S are listed in table 3-1. Orthogonal time-of-flight mass analyzers decouple the secondary ion ejection event from the mass analysis. This leads to more tightly bunched secondary ions reaching the detector resulting in superior mass resolution. An example of the superior mass resolution of this instrument is demonstrated on digitonin, a detergent utilized to solubilize membrane proteins, precipitate cholesterol, and permeabilize cell membranes. In addition, the spectra acquired for this molecule with SIMS, matrix enhanced SIMS and MALDI are compared. There are some limits to this prototype instrument, primarily in the area of beam size. In chapter 4, it is shown how this instrument can be used in tandem with a high spatial resolution pulsed beam mass spectrometer to improve sub-cellular imaging. MS/MS data of cholesterol taken with the QStar XL led to the discovery of a more prominent cholesterol fragment than is typically used to map cholesterol. The efficacy of using this fragment as an indicator of cholesterol was tested by doping one set of cells with cholesterol and comparing the fragment m/z+ 147, among others, to an undoped cell population. This fragment showed an increase that followed the pseudomolecular ion, m/z+ 369 which is commonly chosen as a cholesterol indicator, but at higher intensities. In addition, previous single cell imaging experiments were revisited and it was shown that by mapping m/z+ 147, better image contrast was achieved. Despite the imaging limitations, exciting results were achieved in imaging tissue and large single cells. Chapter 5 reports rat brain imaging, single cell sea snail neuron imaging, as well as a demonstration of the ability of MS/MS to differentiate the structural isomers leucine and isoleucine. Large samples such as rat brain sections present some unique challenges for mass spectral imaging. Traditional ToF-SIMS mass spectrometers have very small fields of view, making imaging of large samples difficult or impossible. MALDI instruments, such as the QStar XL, have the means to image large samples, but usually only at poor spatial resolution. In this experiment, the unique abilities of a primary ion source coupled to a MALDI mass spectrometer are demonstrated by imaging a rat brain at successively smaller fields of view; from 10 mm to 0.5 mm. Large single cell analysis is possible despite the spatial resolution limitation. Imaging of 350 µm sea snail neurons is demonstrated revealing subcellular localization of cholesterol and vitamin E. In addition it is shown that MS/MS spectra of vitamin E can be obtained from a single cell.