Research Article Received: 30 January 2014
Revised: 27 March 2014
Accepted: 2 May 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1649–1657 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6940
Modifications to a commercially available linear mass spectrometer for mass-resolved microscopy with the pixel imaging mass spectrometry (PImMS) camera E. Halford1, B. Winter1, M. D. Mills2, S. P. Thompson2, V. Parr2, J. J. John3, A. Nomerotski4, C. Vallance5, R. Turchetta6 and M. Brouard1* 1
The Department of Chemistry, University of Oxford, The Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, UK 2 SAI Ltd, 9 Hadfield Street, Old Trafford, Manchester M16 9FE, UK 3 The Department of Physics, University of Oxford, Denys Wilkinson Building, Keble Road, Oxford OX1 3RH, UK 4 Physics Department, Brookhaven National Laboratory, Upton, NY 11973, USA 5 The Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA, UK 6 Rutherford Appleton Laboratory, Didcot OX11 0QX, UK RATIONALE: Imaging mass spectrometry is a powerful analytical technique capable of accessing a large volume of
spatially resolved, chemical data from two-dimensional samples. Probing the entire surface of a sample simultaneously requires a detector with high spatial and temporal resolutions, and the ability to observe events relating to different mass-to-charge ratios. METHODS: A commercially available time-of-flight mass spectrometer, designed for matrix-assisted laser desorption/ ionization (MALDI) analysis, was combined with the novel pixel imaging mass spectrometry (PImMS) camera in order to perform multi-mass, microscope-mode imaging experiments. A number of minor modifications were made to the spectrometer hardware and ion optics so that spatial imaging was achieved for a number of small molecules. RESULTS: It was shown that a peak width of Δm50 % < 1 m/z unit across the range 200 ≤ m/z ≤ 800 can be obtained while also achieving an optimum spatial resolution of 25 μm. It was further shown that these data were obtained simultaneously for all analytes present without the need to scan the experimental parameters. CONCLUSIONS: This work demonstrates the capability of multi-mass, microscope-mode imaging to reduce the acquisition time of spatially distributed analytes such as multi-arrays or biological tissue sections. It also shows that such an instrument can be commissioned by effecting relatively minor modifications to a conventional commercial machine. Copyright © 2014 John Wiley & Sons, Ltd.
Mass spectrometry (MS) is used in a number of applications, from the analysis of agricultural products and medical samples, to security screening.[1–3] Different applications have different requirements in terms of acquisition speed, range, accuracy, and resolution such that it would be impossible to create the ’perfect’ mass spectrometer which excels in all categories. However, by choosing realistic goals for a few parameters it is possible to concentrate on improving those characteristics which matter, in a systematic fashion. The field of mass spectrometry imaging (MSI) has been widely used to give additional information relative to onedimensional mass spectra. In areas such as pharmacological research, for example, spatial information is correlated to a given mass-to-charge ratio (m/z) resulting in a more informative dataset capable of showing both which
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* Correspondence to: M. Brouard, The Department of Chemistry, University of Oxford, The Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, UK. E-mail: [email protected]
metabolites are formed and where they are located.[4] Similarly, MSI has been used in forensic applications such as the chemical analysis of fingerprints tying incriminating compounds to a single identity, the study of heterogeneous catalysis, and even in the mapping of metabolites in wholebody rodent tissue sections.[5–7] MSI can be performed in two complementary modes: microprobe, and microscope. With conventional detector technologies, these modes correspond to either scanning the position on the sample, or scanning the m/z value.[8,9] For microprobe-mode MSI the position is held constant by selecting a single ’pixel’ on a sample with a focused ionising beam. An entire mass spectrum is acquired for this position before the next ’pixel’ is chosen, and another mass spectrum is obtained. The process is continued until each pixel in the image has an associated mass spectrum. Conversely, microscope-mode MSI holds the m/z value constant, and a defocused ionising beam is used to ablate ions from a large area of the surface. A position-sensitive detector then records the associated position of the analyte in question. An image can then be acquired for each m/z range of interest.
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Microscope-mode MSI has been heralded as advantageous as it allows spatial resolutions to be measured independently of the ionising beam size.[10,11] This means that large areas can be imaged at high spatial resolutions in a much shorter time than with the microprobe-mode where an increase in the target area or an increase in the spatial resolution results in a quadratic increase in the acquisition time. However, in microscope-mode imaging the trajectories of the analyte ions have to be controlled, which severely limits the range of techniques that can be used to increase the mass resolution of an experiment. Methods common to microprobe-mode MS, such as the use of reflectrons, or ion cyclotron resonance instrumentation, allow for mass resolution (m/Δm50 %) of the order of 105, but have not been shown to be compatible with microscope-mode imaging;[12] however, Suits and co-workers have shown well-resolved velocity-mapped images (those associated with the back-focal plane of the imaging system) using an instrument with a single-stage reflectron implying that spatial imaging should be possible.[13,14] It has also been shown that electrostatic analysers (ESAs) can be used to improve focusing in the time domain while retaining spatial information in microscope mode. This has been shown both in open arrangements, and in looped systems.[11,15,16] Microscope-mode MSI also increases the requirements on the instrumentation used both for the ionisation process and for ion detection. Ionising beams must be defocused yet remain homogeneous, while detectors must have high spatial, and temporal resolutions. An MCP-scintillator detector coupled with a framing CCD camera can achieve high spatial resolutions but more recently time-stamping pixel cameras and delay line detectors have been developed which can acquire images for multiple m/z ranges simultaneously.[8,17,18] The introduction of this multi-mass imaging technology means that a microscope-mode experiment no longer needs to involve scanning over the m/z coordinate giving a further marked decrease in acquisition time. Delay line detectors (DLDs) have response times of the order of hundreds of picoseconds but are limited by the number of ion events that can be detected: there is a deadtime within which ’simultaneous’ ion events cannot be distinguished. They have been used in stigmatic imaging by Heeren and co-workers, and more recently Awazu and co-workers.[17,19,20] The use of hexanode DLDs allows for a higher count rate during these experiments, although the fundamental limitation still exists that ions arriving within the dead-time (~10 ns) in the near vicinity of one another (~1 mm) cannot be resolved.[21] Awazu and co-workers propose that a spatial resolution of 12 μm with a mass resolution of 15,000 is achievable.[20] Time-stamping pixel detectors such as the Timepix or PImMS sensors allow for simultaneous acquisition of ion events during a single ToF cycle.[22–24] Only ion events are stored (in contrast to a framing camera, in which every pixel records data for a set length of time), and the coordinates of these voxels are transferred to a PC for processing after every laser shot. Heeren and co-workers have recently reported coupling the Timepix camera to a microscope-mode secondary ion mass spectrometer with great success, achieving a spatial resolution of ~5 μm with a clock cycle of 10 ns corresponding to a precision of 150 mDa for an ion with
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an m/z value of around 400.[18] Further to this, Heeren and co-workers have also shown that such a pixel detector can be used to detect species ranging up to 400 kDa using a MALDI source, and post-acceleration methods.[25] In previous work it has been shown that the novel PImMS camera could be applied to a simple, three electrode, velocitymap imaging (VMI) system, in order to perform spatial-map imaging experiments, and that spatial resolutions of 23 μm were obtainable simultaneously with a mass resolution of 67.[26] These experiments showed the advantage gained in using a time-stamping camera capable of multi-hit detection within each pixel: images correlated to multiple m/z values can be recorded simultaneously (i.e. within a single duty cycle) by each pixel.[27] This increases the possible throughput of an MSI experiment greatly; although, the mass resolution attained was insufficient for most practical applications. Here we describe the combination of the PImMS camera with a commercial linear mass spectrometer, the LT2 Plus (SAI Ltd, Manchester, UK).[28] An order of magnitude improvement is observed in the mass resolution over previous MSI experiments with the PImMS camera. This improvement in mass resolution is shown without a significant loss in the observed spatial resolution, while still imaging multiple analytes simultaneously.
THEORY AND SIMULATIONS In order to use the PImMS camera to its full potential it is important to keep any detector limitations in mind when optimising the ion optics that are going to be used. The current camera hardware housing the 72 × 72 pixel PImMS sensor has a maximum clock frequency of 80 MHz (corresponding to a 12.5 ns timing precision). Time information is stored in a 12-bit register giving possible time codes of 0 to 4095. In order to be able to resolve two neighbouring m/z peaks the ion optics need to have a sufficiently high mass resolution, and the peaks need to be separated in arrival time by a value that exceeds a minimum of one time code. Simulations were conducted using the SIMION 8.0 software package.[29] A replica of the five-electrode ion optic used in the LT2 Plus, consisting of an extraction lens coupled to an einzel lens, was modelled. The lens was followed by a 1.0 m flight tube terminating in a surface representing the front face of the detector. The ion optics were modelled at a resolution of 1.0 mm/grid unit, and a script, written in the Lua programming language, was used to control the pulsing of the ion optics during the ion trajectory runs. Ions were initialised at six points positioned on the repeller at 50 μm steps from the centre of the plate. At each position, 1000 ions were created with a normal distribution of speeds, and directions. The average speed was set at 500 ms1 with a FWHM of 500 ms1, and the average direction was set to the surface normal (azimuth, and elevation angle equal to zero) with a FWHM, in both the azimuth and the elevation distributions, of60°. These values are consistent with values reported for the MALDI process.[30–33] Further to these values, the assumption was made that only singly charged species would be produced in any great quantity and that any spread in creation time would be negligible.[34]
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Figure 1. A plot of the time separation, ΔtToF, between the edge of one m/z peak at 50% of the maximum and that of the next integer mass observed in SIMION simulations. The peaks have been covoluted with a 2.5 ns FWHM Gaussian function which represents the response time of the fast scintillator (detailed later). The four data sets represent the different accelerating potentials, and the constant values correspond to one, two, and three PImMS time codes.
To a first approximation, the time-of-flight for an analyte ion having been accelerated by a potential, V, equivalent to the potential applied to the repeller electrode, is proportional to the inverse of the square root of that potential: 1 tToF ∝ pffiffiffiffi V
(1)
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peak at around m/z 500. The pure ion optics resolution reaches a maximum value > 3000 and drops off rapidly either side of this. The response function reduces this limit greatly down to < 1500, although the reduction in resolution is less pronounced away from the peak. This indicates that the detector response time is the limiting factor for any ion optics with a resolution of much more than m/Δm50 % = 1000. For the range 200 ≤ m/z ≤ 800 both plots stay above the isotopic limit (i.e. Δm50 % < 1 m/z unit). Figure 2(b) shows the variation in spatial resolution as a function of the analyte m/z value. The spatial resolution is defined as the FWHM of a Gaussian fit to the distribution of final ion positions at the detector for a given starting spot. As the MALDI process produces ions with a given velocity, independent of their mass, there is an optimum focus formed for a specific m/z value. Here, a spatial resolution between 20 μm and 60 μm is achievable for the range 200 ≤ m/z ≤ 800 with the optimum value falling just below m/z 500. Centroiding A single ion incident on a set of MCPs generates 105-106 electrons which are then accelerated towards the scintillating screen. This illuminates a region on the screen which can trigger multiple pixels on the PImMS camera. All these triggered pixels are stored as individual events, despite only corresponding to one ion event. This “cluster” of events can spread across multiple pixel coordinates, (x, y), and over multiple time codes, τ. By centroiding the data set in (x, y, τ) all the events corresponding to a single ion arrival can be identified and the cluster can be reduced to a single precise coordinate. This procedure reduces the size of the data set as well as improving the mass, and spatial resolution.
Copyright © 2014 John Wiley & Sons, Ltd.
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By reducing V, the ion ToF is increased resulting in an increased gap between each m/z peak. Unfortunately, a lower accelerating potential increases the effect of chromatic blurring arising from any spread in the initial distribution of ion velocities. Figure 1 shows the time between the falling edge (at 50% of the maximum) of an m/z peak, and the rising edge of the next integer m/z peak as predicted by the SIMION simulations. The different curves arise from different values of the accelerating potential, V. Each curve was individually optimised to adjust for higher order focussing effects. At low mass we see that a lower accelerating potential has increased the separation of ion ToFs. As the mass of the analyte is increased the peaks get closer and, despite the ion optics having a sufficient mass resolution to distinguish integer mass steps, the camera binning process becomes the limiting factor. Figure 2 shows the mass dependence of the resolution achievable with the ion optics when a 2000 V acceleration potential is employed. The initial simulations did not include a correction for the PImMS timing precision. Figure 2(a) shows the mass resolution along with an adjusted resolution. This adjusted resolution includes a response function for the detector; that is, the response time of the MCPs, scintillator, and the PMT combined. The response function is modelled as a Gaussian function. It is assumed that the MCP response time is negligible (a few hundred picoseconds), and that the scintillator and PMT have response times with FWHM of ~2 and ~4.5 ns, respectively. This gives a full detector response time of ~5 ns (FWHM). Both the plots of the mass resolutions
Figure 2. A plot of the simulated mass resolution and spatial resolution achievable with the ion optics at an accelerating potential of 2000 V.
E. Halford et al. The centroiding process works by separating the PImMS data into individual experimental cycles (laser shots). Each of these shot data sets can then be searched to identify event clusters. The search starts in the first time code, τ = 1, looking through the entire pixel range. If an event is not found, the search proceeds to the next time code. Once an event has been found it is logged as the first event belonging to a cluster list containing NA = 1 events. This first event is therefore given the index nA = 1. Next, the (x, y, τ) coordinate of the first event is set as a reference point, (x0, y0, τ 0), and is removed from the shot data set to avoid double counting. The algorithm then searches for any further events neighbouring this “reference” (i.e. Δx = 1, 0, 1; Δy = 1, 0, 1; excluding Δx = Δy = 0), and extending into later time codes up to some set length (i.e. 0 ≤ Δτ ≤ Δτ max). Any new events are then added to the cluster list, increasing the value of NA, and are removed from the shot data set. Once all the events neighbouring the reference event have been found the reference point is redefined taking on the coordinate of the next event in the cluster list (i.e. index nA = 2). Any events neighbouring this new reference are now located in the same way as before, then being added to the cluster list and removed from the shot data set. This routine continues until all the events in the cluster list have been used as the reference point. The cluster list now contains all the events belonging to this cluster. The search can now continue stepping through the remainder of the shot data set to identify any further clusters. Each resultant list of events can later be reduced to single points which best approximate the (x, y, τ) coordinate of the original ion hits. This is trivial for τ as it is not possible for an event to be detected before it has occurred, so the earliest time code can be assumed to be the most accurate. The (x, y) coordinate is found by taking the spatial “centre-ofmass” and binning it to the original pixel array. It is possible to employ more complicated (x, y) centroiding options but with a low pixel count this is not often meaningful.[35] Clusters are easily defined in (x, y), as events are always connected, but the spread of clusters in t is less well defined. Events can be separated by multiple time codes. If Δτ max is set too low, there is a risk that events belonging to a cluster will be missed and will be allocated to another cluster. Conversely, if Δτ max is set too high, the clusters are more likely to overlap. In the work presented here a value of Δτ max = 7 is used.
that the beam spot diameter of 400 μm covered the full 800 μm diameter corresponding to the detector field-of-view. The maximum measured fluence was found to be 45 mJ/cm2. Initial samples were produced by electrospraying different solutions of organic dyes onto ITO-coated slides (25 mm × 25 mm × 1.1 mm; Oxford Research Solutions, Oxford, UK) using an electrosprayer built in-house. Solutions of Auramine O (3.3 mg/mL in 99.9% methanol), Crystal Violet (0.5 mg/mL in 99.9% methanol), Exalite 384 (saturated solution in 99.9% acetone), and Exalite 404 (saturated solution in 99.9% acetone) were prepared. All these solutions were applied to separate slides, save for the Crystal Violet solution which was sprayed onto the same slide as the Auramine O. The Crystal Violet was sprayed through a Ni grid (BM0080-02; Industrial Netting, Minneapolis, MN, USA[38]) with a pitch of 317.5 μm and a wire width of 33.5 μm. A flow rate of 70 – 80 μL/hr at a needle height of ~30 mm and a potential of 5500 V resulted in sample spots with radii of 5–10 mm. The ITO-coated glass slides were mounted in the repeller plate of the ion optics assembly. The repeller plate was further mounted on an (x, y) -translational stage allowing the user to choose the position of laser incidence. The LT2 Plus employs a delayed extraction technique which holds the extractor and repeller at the same potential (2000 V) until the switch time (370 ns), after which the extractor potential is reduced
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The LT2 Plus consists of three main sections: the ionisation source, the ion optics, and the detector. For the experiments presented here, the N2 laser (NL100, Stanford Research Systems Inc., Sunnyvale, CA, USA[36]) provided with the instrument was used. Lasing occurs at 337 nm, with a repetition rate of 20 Hz, and a pulse width of 3.5 ns (FWHM). The beam was guided into a 1.5 m long optical fibre (Schaefter + Kirchhoff, Hamburg, Germany[37]) with a 200 μm core diameter. Finally, the fibre was coupled to the experiment and defocussed onto the sample. By coiling and twisting the fibre while analysing the resulting beam profile it was possible, to some extent, to homogenise the beam, although certain mode structures were still present. To counteract this, the laser position was moved on the sample to average out the laser fluence. This further meant
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Figure 3. A comparison between the mass spectra obtained from different PImMS data processing levels, and that obtained using the PMT. (a) shows the raw data obtained from the PImMS camera, (b) and (c) show two successive levels of centroiding applied to the previous data, and (d) is a plot of the raw data obtained with the PMT. The data taken with the PImMS camera and with the PMT were obtained simultaneously over 3000 shots.
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(a)
(b)
(c)
(d)
Figure 4. Fits of the four mass spectra obtained when imaging the four different organic dyes. All the data sets have been post processed as in Fig. 3(c). The spectra in (a), (b), (c), and (d) are of Auramine O, Crystal Violet, Exalite 384, and Exalite 404, respectively.
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RESULTS Figure 3 shows various mass spectra extracted from a PImMS data set using increasing levels of centroiding plotted above the mass spectrum simultaneously obtained with the PMT. The data in Fig. 3(a) has only undergone a simple reduction in background noise, and so represents, as closely as possible, the “raw” data set. Figures 3(b) and 3(c) show two levels of (x, y, τ) centroiding applied to the “raw” data set. Figure 3(d) shows the mass spectrum obtained from the PMT simultaneously to the PImMS data.
Figure 5. A plot of the mass resolution predicted by SIMION simulations plotted with the values found experimentally. The experimental values are split between those obtained purely with the PImMS camera (blue circles), and those obtained using the PMT (red triangles). The reduced simulation values have a 5 ns convolution added to compensate for the combined response time of the detector assembly. The error bars represent an uncertainty of two standard deviations.
Copyright © 2014 John Wiley & Sons, Ltd.
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(by 230 V) to produce the required shallow extraction field.[39,40] After extraction, spatial focussing is optimised by an einzel lens (set to a positive potential of 995 V). These voltages and switching times were modified from their original values in order to optimise the mass resolution and spatial resolution simultaneously. All the results shown below were taken with these voltages applied. The original electron multiplier detector supplied with the LT2 Plus was exchanged for a pair of micro-channel plates in a chevron assembly with an active diameter of 25 mm, a pore pitch of 12 μm, a pore diameter of 10 μm, and a bias angle of 5° (Photonis, Sturbridge, MA, USA[41]). The MCP assembly was followed by a fast plastic scintillator (BC-408; Saint-Gobain Crystals, Hiram, OH, USA[42]). The response time of this scintillator is ~2.5 ns, and its emission peaks at 425 nm. The active area of the detector was 25 mm, corresponding to roughly 1.1 mm on the sample surface (with the predicted magnification × 22). A photomultiplier tube (PMT) (1P28; RCA, New York, NY, USA) and the PImMS camera were both coupled to the scintillating screen. The PImMS camera was focussed using a conventional c-mount camera lens (DO-5095; Navitar, Rochester, NY, USA[43]) with a 10 mm spacer to reduce the field of view to ~25 mm. The PMT was directly connected to an ADC board (1 GSa/s) supplied with the LT2 Plus, replacing the electron multiplier signal feed. The PImMS camera was connected, via an USB, to a computer running in-house Labview 2010 image acquisition and analysis software (National Instruments, Austin, TX, USA[44]). The software allows for complete control of the PImMS camera, as well as some on-the-fly analysis. A more complete analysis can then be performed after acquisition using a further suite of in-house programs (written in the C++ programming language). The camera housing the PImMS sensor (Aspect Systems, Dresden, Germany[45]) includes facilities to cool the sensor down to 15°, and a dry N2 line to keep the humidity low and thus prevent condensation on the sensor surface.
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Figure 6. A comparison between the images associated with (a): the Auramine O signal; and, (b): the Crystal Violet signal. These images were acquired simultaneously over 5000 shots taking a total of 250 s. Figure 3(b) shows the result of centroiding on the mass spectrum extracted from the “raw” data. The distribution is shifted towards lower mass, and a bimodal feature starts to appear. Figure 3(c) shows a further evolution of the centroiding process where clusters below a threshold cluster size are discarded. Here, a distinct bimodal distribution is apparent, and the similarity to the PMT spectrum is far improved over the original data set in Fig. 3(a). In order to ascertain the mass resolution of the experiment, the width at half the maximum of the mass spectral peaks must be measured (Δm50 %). This is done by fitting a Gaussian function to each peak. With such a discrete data set it is necessary to facilitate the fitting by assuming that the mass resolution of integer mass neighbours is approximately equal (i.e. Δm50 % ≈ Δ(m + 1)50 %) so that we can set the width σ of adjacent peaks to be equal. Using this approximation the spectrum in Fig. 3(c) is found to have a mass resolution of m/Δm50 % ≈ 936. The counterpart value found using the PMT spectrum (Fig. 3(d)) is m/Δm50 % ≈ 1260.
Before moving on to the analysis of a MALDI target it is simpler to analyse organic dyes which can be more easily prepared with well defined spatial distributions on the sample plate. These dyes persist for a long time which greatly facilitates the repetition of experiments during which other samples would only last for a few thousand laser shots. Figure 4 shows mass spectra obtained with the PImMS camera for the four organic dyes Auramine O, Crystal Violet, Exalite 384, and Exalite 404 (Figs. 4(a), 4(b), 4(c), and 4(d), respectively). All four data sets have undergone the same post-acquisition processing as in Fig. 3(c), and have been fitted using the same approximations as before. All four spectra show isotopic resolution (i.e. Δm50 % < 1 m/z unit), although at the highest m/z value of 658.5 (Exalite 404) this is less defined, as would be expected from the calculations shown in Fig. 1. The obtained mass resolutions determined from these plots are: m/Δm50 % ≈ 540 (Auramine O), 597 (Crystal Violet), 936 (Exalite 384), and 882 (Exalite 404). Figure 5 shows a plot of the previously acquired simulation values for mass resolution along with those obtained when analysing the dyes using both the PImMS camera (blue circles), and the PMT (red triangles). It is evident from the plot that, as expected, the PMT gives a far faster response than the PImMS camera. The simulated resolutions that have been convoluted with a 5 ns response time match the absolute values of the experimental data obtained with the PMT very well giving some insight into the response time of the scintillating screen in combination with the PMT. Figure 6 shows a comparison between the images correlated to the Auramine O peak and the Crystal Violet peak obtained using the PImMS camera. These images were obtained simultaneously as the Crystal Violet sample was applied first to the ITO-coated slide, in a grid pattern, after which the Auramine O was applied. The resultant image
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Figure 7. The mass spectrum of the PEG sample obtained with the PImMS camera. The two insets (a) and (b) show enlarged areas of the mass spectrum with accompanying Gaussian fits. The peaks in (a) correspond to [CH2(C2H4O)10OH + Na]+ (m/z 496), and to Exalite 384 (m/z 498) which was included as an internal calibrant. The peaks in (b) correspond to [CH2(C2H4O)13OH + Na]+ (m/z 628).
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Linear mass spectrometer for mass-resolved microscopy clearly shows a marked difference in the location of the two dyes. By taking a line profile (six pixels wide) across the image, an estimate of the magnification, and resolution can be obtained. In these images the magnification was found to be × 28.7. The resolution is equated to the rising distance of the present features from 20% to 80%, and was found to be 25.0 μm which is close to the simulated optimum resolution of ~ 20.0 μm.[46] All four dyes were found to be sensitive to laser ablation without the need for a MALDI matrix, and so all the previous experiments were conducted without any such matrix. To fully test the experiment a further sample was produced consisting of 100 μL of poly(ethylene-glycol) methyl ether (MAV = 550 Da) (PEG) in 5 mL of 2.65 mg/mL α-cyanohydroxycinnaminic acid (CHCA) in 50% methanol with 0.01% trifluoroacetic acid (TFA). This solution was applied to an ITO-coated glass slide via the dried droplet method before being analysed. Figure 7 presents an example of the full mass spectrum of the PEG solution. All the experimental settings were identical to those used for the organic dyes. The laser fluence at the sample was ~40 mJ/cm2, and the data was taken over ~5000 shots. The data has undergone the same level of postprocessing as with the dye experiments. Many more peaks are present in the spectra obtained under these conditions. The majority of the data (59%) was collected in the first register of the PImMS camera with the second, third, and fourth registers containing 17%, 13%, and 11% of the data, respectively. This implies that there is data that would have been missed without the mulit-hit pixel capacity of the PImMS camera, but also that there is still scope for higher count rates or more complicated spectra. Figure 7(a) shows a magnified area of the mass spectrum. There are two species present at this m/z value: the PEG species [CH2(C2H4O)10OH + Na]+ (m/z 496), and Exalite 384 (m/z 498) which was added as an internal calibrant. The two species are clearly separated and exhibit the isotopic
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DISCUSSION AND CONCLUSIONS It has been shown that a conventional mass spectrometer can be modified in order to perform spatially resolved mass spectrometry in microscope-mode. This offers the advantage of processing large quantities of data in parallel, whether it be multiple regions within a tissue section, or multiple samples set in an array. Accessing all the spatial and m/z data simultaneously puts a heavy performance requirement on all aspects of the experiment. In order to increase the experimental field-of-view a more powerful laser will be needed, and losses due to coupling the laser into the fibre will need to be reduced. Further homogenising of the beam profile will also be required if normalised images are to be produced. The currently used pulsing regime, delayed extraction, is limited by the upper bound of the switching time. The mass resolutions shown in Fig. 7 are encouraging but there is an upper limit to the obtainable m/z values. During the initial, field-free, expansion step spatial information is lost as ions travel orthogonally to the time-of-flight axis. By the time a field is finally applied this information is already lost. The post-extraction differential acceleration (PEDA) technique pioneered by Aoki and co-workers, and its extension (velocity correction) developed by Brouard and co-workers address this issue by controlling the trajectories of ablated ions from their creation rather than letting the ablation plumes expand into the vacuum.[47,48] The pulsed focussing then occurs further along the time-of-flight path. This can extend the range at which the peak width, Δm50 % < 1 m/z unit. The reduction in the accelerating potential applied to the repeller electrode means that the PImMS camera is used at its optimum. The timing precision of 12.5 ns is the limiting factor in the mass resolution measurements shown in the results section, but this can be reduced by improving the coupling between the detector and camera. The response speed of the PImMS sensor is dependent on the intensity of the illumination that it is detecting. A bright flash on a scintillating screen will trigger a large cluster of pixels, with a short response time, giving a more reliable signal. To this end, more efficient scintillators, such as those proposed by Brouard and co-workers, should improve the performance of the PImMS sensor.[49] The response time of the BC-408 scintillator used here is short enough (2.5 ns) that it should not affect the time resolution for ion detection but when further planned firmware improvements reduce the PImMS timing precision to 6.25 ns, there will be a demand for still faster scintillators.
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Figure 8. A plot of the mass resolution predicted by SIMION simulations plotted with the values found for the PEG sample experimentally. The experimental values are split between those obtained purely with the PImMS camera (blue circles), and those obtained using the PMT (red triangles). The reduced simulation values have a 5 ns convolution added to compensate for the combined response time of the scintillator and PMT. The error bars represent an uncertainty of two standard deviations.
resolution obtained with the organic dyes. Figure 7(b) shows a higher mass PEG species, [CH2(C2H4O)13OH + Na]+ (m/z 628), which also exhibits isotopic resolution. Figure 8 shows an overview of the mass resolutions obtained for the PEG sample spectra shown in Fig. 7. The resolutions obtained with the PImMS camera (blue circles) is presented along with resolutions obtained simultaneously, using the PMT (red triangles). These experimental points are compared with the simulation data presented earlier. The analyte peaks correspond to the polymer adduct ions [CH2(C2H4O)NOH + Na]+ where the assigned range covers 8 ≤ N ≤ 16.
E. Halford et al. In conclusion, we have shown that the PImMS camera, in combination with an off-the-shelf mass spectrometer, is capable of multi mass imaging with mass resolutions greater than the isotopic limit Δm50 % = 1 m/z unit over a range of 200 ≤ m/z ≤ 800, while still achieving an optimum spatial resolution of 25 μm. The simple linear geometry of the experiment lends itself to being used as a tabletop instrument for the high-speed analysis of multiplexed assays, or tissue sections with analytes in the small molecule range.
[13]
Acknowledgements
[14]
The support of the STFC through the Mini-IPS grant ST/J002895/1, the EPSRC via Programme grant No. EP/L005913/1, and the EU through grant FP7 ITN “ICONIC” (238671) is gratefully acknowledged. Elements of the work described here are detailed in the US Patent Application No. 20100294924 A1.
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Revised: 27 March 2014
Accepted: 2 May 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1649–1657 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6940
Modifications to a commercially available linear mass spectrometer for mass-resolved microscopy with the pixel imaging mass spectrometry (PImMS) camera E. Halford1, B. Winter1, M. D. Mills2, S. P. Thompson2, V. Parr2, J. J. John3, A. Nomerotski4, C. Vallance5, R. Turchetta6 and M. Brouard1* 1
The Department of Chemistry, University of Oxford, The Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, UK 2 SAI Ltd, 9 Hadfield Street, Old Trafford, Manchester M16 9FE, UK 3 The Department of Physics, University of Oxford, Denys Wilkinson Building, Keble Road, Oxford OX1 3RH, UK 4 Physics Department, Brookhaven National Laboratory, Upton, NY 11973, USA 5 The Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA, UK 6 Rutherford Appleton Laboratory, Didcot OX11 0QX, UK RATIONALE: Imaging mass spectrometry is a powerful analytical technique capable of accessing a large volume of
spatially resolved, chemical data from two-dimensional samples. Probing the entire surface of a sample simultaneously requires a detector with high spatial and temporal resolutions, and the ability to observe events relating to different mass-to-charge ratios. METHODS: A commercially available time-of-flight mass spectrometer, designed for matrix-assisted laser desorption/ ionization (MALDI) analysis, was combined with the novel pixel imaging mass spectrometry (PImMS) camera in order to perform multi-mass, microscope-mode imaging experiments. A number of minor modifications were made to the spectrometer hardware and ion optics so that spatial imaging was achieved for a number of small molecules. RESULTS: It was shown that a peak width of Δm50 % < 1 m/z unit across the range 200 ≤ m/z ≤ 800 can be obtained while also achieving an optimum spatial resolution of 25 μm. It was further shown that these data were obtained simultaneously for all analytes present without the need to scan the experimental parameters. CONCLUSIONS: This work demonstrates the capability of multi-mass, microscope-mode imaging to reduce the acquisition time of spatially distributed analytes such as multi-arrays or biological tissue sections. It also shows that such an instrument can be commissioned by effecting relatively minor modifications to a conventional commercial machine. Copyright © 2014 John Wiley & Sons, Ltd.
Mass spectrometry (MS) is used in a number of applications, from the analysis of agricultural products and medical samples, to security screening.[1–3] Different applications have different requirements in terms of acquisition speed, range, accuracy, and resolution such that it would be impossible to create the ’perfect’ mass spectrometer which excels in all categories. However, by choosing realistic goals for a few parameters it is possible to concentrate on improving those characteristics which matter, in a systematic fashion. The field of mass spectrometry imaging (MSI) has been widely used to give additional information relative to onedimensional mass spectra. In areas such as pharmacological research, for example, spatial information is correlated to a given mass-to-charge ratio (m/z) resulting in a more informative dataset capable of showing both which
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* Correspondence to: M. Brouard, The Department of Chemistry, University of Oxford, The Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, UK. E-mail: [email protected]
metabolites are formed and where they are located.[4] Similarly, MSI has been used in forensic applications such as the chemical analysis of fingerprints tying incriminating compounds to a single identity, the study of heterogeneous catalysis, and even in the mapping of metabolites in wholebody rodent tissue sections.[5–7] MSI can be performed in two complementary modes: microprobe, and microscope. With conventional detector technologies, these modes correspond to either scanning the position on the sample, or scanning the m/z value.[8,9] For microprobe-mode MSI the position is held constant by selecting a single ’pixel’ on a sample with a focused ionising beam. An entire mass spectrum is acquired for this position before the next ’pixel’ is chosen, and another mass spectrum is obtained. The process is continued until each pixel in the image has an associated mass spectrum. Conversely, microscope-mode MSI holds the m/z value constant, and a defocused ionising beam is used to ablate ions from a large area of the surface. A position-sensitive detector then records the associated position of the analyte in question. An image can then be acquired for each m/z range of interest.
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Microscope-mode MSI has been heralded as advantageous as it allows spatial resolutions to be measured independently of the ionising beam size.[10,11] This means that large areas can be imaged at high spatial resolutions in a much shorter time than with the microprobe-mode where an increase in the target area or an increase in the spatial resolution results in a quadratic increase in the acquisition time. However, in microscope-mode imaging the trajectories of the analyte ions have to be controlled, which severely limits the range of techniques that can be used to increase the mass resolution of an experiment. Methods common to microprobe-mode MS, such as the use of reflectrons, or ion cyclotron resonance instrumentation, allow for mass resolution (m/Δm50 %) of the order of 105, but have not been shown to be compatible with microscope-mode imaging;[12] however, Suits and co-workers have shown well-resolved velocity-mapped images (those associated with the back-focal plane of the imaging system) using an instrument with a single-stage reflectron implying that spatial imaging should be possible.[13,14] It has also been shown that electrostatic analysers (ESAs) can be used to improve focusing in the time domain while retaining spatial information in microscope mode. This has been shown both in open arrangements, and in looped systems.[11,15,16] Microscope-mode MSI also increases the requirements on the instrumentation used both for the ionisation process and for ion detection. Ionising beams must be defocused yet remain homogeneous, while detectors must have high spatial, and temporal resolutions. An MCP-scintillator detector coupled with a framing CCD camera can achieve high spatial resolutions but more recently time-stamping pixel cameras and delay line detectors have been developed which can acquire images for multiple m/z ranges simultaneously.[8,17,18] The introduction of this multi-mass imaging technology means that a microscope-mode experiment no longer needs to involve scanning over the m/z coordinate giving a further marked decrease in acquisition time. Delay line detectors (DLDs) have response times of the order of hundreds of picoseconds but are limited by the number of ion events that can be detected: there is a deadtime within which ’simultaneous’ ion events cannot be distinguished. They have been used in stigmatic imaging by Heeren and co-workers, and more recently Awazu and co-workers.[17,19,20] The use of hexanode DLDs allows for a higher count rate during these experiments, although the fundamental limitation still exists that ions arriving within the dead-time (~10 ns) in the near vicinity of one another (~1 mm) cannot be resolved.[21] Awazu and co-workers propose that a spatial resolution of 12 μm with a mass resolution of 15,000 is achievable.[20] Time-stamping pixel detectors such as the Timepix or PImMS sensors allow for simultaneous acquisition of ion events during a single ToF cycle.[22–24] Only ion events are stored (in contrast to a framing camera, in which every pixel records data for a set length of time), and the coordinates of these voxels are transferred to a PC for processing after every laser shot. Heeren and co-workers have recently reported coupling the Timepix camera to a microscope-mode secondary ion mass spectrometer with great success, achieving a spatial resolution of ~5 μm with a clock cycle of 10 ns corresponding to a precision of 150 mDa for an ion with
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an m/z value of around 400.[18] Further to this, Heeren and co-workers have also shown that such a pixel detector can be used to detect species ranging up to 400 kDa using a MALDI source, and post-acceleration methods.[25] In previous work it has been shown that the novel PImMS camera could be applied to a simple, three electrode, velocitymap imaging (VMI) system, in order to perform spatial-map imaging experiments, and that spatial resolutions of 23 μm were obtainable simultaneously with a mass resolution of 67.[26] These experiments showed the advantage gained in using a time-stamping camera capable of multi-hit detection within each pixel: images correlated to multiple m/z values can be recorded simultaneously (i.e. within a single duty cycle) by each pixel.[27] This increases the possible throughput of an MSI experiment greatly; although, the mass resolution attained was insufficient for most practical applications. Here we describe the combination of the PImMS camera with a commercial linear mass spectrometer, the LT2 Plus (SAI Ltd, Manchester, UK).[28] An order of magnitude improvement is observed in the mass resolution over previous MSI experiments with the PImMS camera. This improvement in mass resolution is shown without a significant loss in the observed spatial resolution, while still imaging multiple analytes simultaneously.
THEORY AND SIMULATIONS In order to use the PImMS camera to its full potential it is important to keep any detector limitations in mind when optimising the ion optics that are going to be used. The current camera hardware housing the 72 × 72 pixel PImMS sensor has a maximum clock frequency of 80 MHz (corresponding to a 12.5 ns timing precision). Time information is stored in a 12-bit register giving possible time codes of 0 to 4095. In order to be able to resolve two neighbouring m/z peaks the ion optics need to have a sufficiently high mass resolution, and the peaks need to be separated in arrival time by a value that exceeds a minimum of one time code. Simulations were conducted using the SIMION 8.0 software package.[29] A replica of the five-electrode ion optic used in the LT2 Plus, consisting of an extraction lens coupled to an einzel lens, was modelled. The lens was followed by a 1.0 m flight tube terminating in a surface representing the front face of the detector. The ion optics were modelled at a resolution of 1.0 mm/grid unit, and a script, written in the Lua programming language, was used to control the pulsing of the ion optics during the ion trajectory runs. Ions were initialised at six points positioned on the repeller at 50 μm steps from the centre of the plate. At each position, 1000 ions were created with a normal distribution of speeds, and directions. The average speed was set at 500 ms1 with a FWHM of 500 ms1, and the average direction was set to the surface normal (azimuth, and elevation angle equal to zero) with a FWHM, in both the azimuth and the elevation distributions, of60°. These values are consistent with values reported for the MALDI process.[30–33] Further to these values, the assumption was made that only singly charged species would be produced in any great quantity and that any spread in creation time would be negligible.[34]
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Figure 1. A plot of the time separation, ΔtToF, between the edge of one m/z peak at 50% of the maximum and that of the next integer mass observed in SIMION simulations. The peaks have been covoluted with a 2.5 ns FWHM Gaussian function which represents the response time of the fast scintillator (detailed later). The four data sets represent the different accelerating potentials, and the constant values correspond to one, two, and three PImMS time codes.
To a first approximation, the time-of-flight for an analyte ion having been accelerated by a potential, V, equivalent to the potential applied to the repeller electrode, is proportional to the inverse of the square root of that potential: 1 tToF ∝ pffiffiffiffi V
(1)
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peak at around m/z 500. The pure ion optics resolution reaches a maximum value > 3000 and drops off rapidly either side of this. The response function reduces this limit greatly down to < 1500, although the reduction in resolution is less pronounced away from the peak. This indicates that the detector response time is the limiting factor for any ion optics with a resolution of much more than m/Δm50 % = 1000. For the range 200 ≤ m/z ≤ 800 both plots stay above the isotopic limit (i.e. Δm50 % < 1 m/z unit). Figure 2(b) shows the variation in spatial resolution as a function of the analyte m/z value. The spatial resolution is defined as the FWHM of a Gaussian fit to the distribution of final ion positions at the detector for a given starting spot. As the MALDI process produces ions with a given velocity, independent of their mass, there is an optimum focus formed for a specific m/z value. Here, a spatial resolution between 20 μm and 60 μm is achievable for the range 200 ≤ m/z ≤ 800 with the optimum value falling just below m/z 500. Centroiding A single ion incident on a set of MCPs generates 105-106 electrons which are then accelerated towards the scintillating screen. This illuminates a region on the screen which can trigger multiple pixels on the PImMS camera. All these triggered pixels are stored as individual events, despite only corresponding to one ion event. This “cluster” of events can spread across multiple pixel coordinates, (x, y), and over multiple time codes, τ. By centroiding the data set in (x, y, τ) all the events corresponding to a single ion arrival can be identified and the cluster can be reduced to a single precise coordinate. This procedure reduces the size of the data set as well as improving the mass, and spatial resolution.
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By reducing V, the ion ToF is increased resulting in an increased gap between each m/z peak. Unfortunately, a lower accelerating potential increases the effect of chromatic blurring arising from any spread in the initial distribution of ion velocities. Figure 1 shows the time between the falling edge (at 50% of the maximum) of an m/z peak, and the rising edge of the next integer m/z peak as predicted by the SIMION simulations. The different curves arise from different values of the accelerating potential, V. Each curve was individually optimised to adjust for higher order focussing effects. At low mass we see that a lower accelerating potential has increased the separation of ion ToFs. As the mass of the analyte is increased the peaks get closer and, despite the ion optics having a sufficient mass resolution to distinguish integer mass steps, the camera binning process becomes the limiting factor. Figure 2 shows the mass dependence of the resolution achievable with the ion optics when a 2000 V acceleration potential is employed. The initial simulations did not include a correction for the PImMS timing precision. Figure 2(a) shows the mass resolution along with an adjusted resolution. This adjusted resolution includes a response function for the detector; that is, the response time of the MCPs, scintillator, and the PMT combined. The response function is modelled as a Gaussian function. It is assumed that the MCP response time is negligible (a few hundred picoseconds), and that the scintillator and PMT have response times with FWHM of ~2 and ~4.5 ns, respectively. This gives a full detector response time of ~5 ns (FWHM). Both the plots of the mass resolutions
Figure 2. A plot of the simulated mass resolution and spatial resolution achievable with the ion optics at an accelerating potential of 2000 V.
E. Halford et al. The centroiding process works by separating the PImMS data into individual experimental cycles (laser shots). Each of these shot data sets can then be searched to identify event clusters. The search starts in the first time code, τ = 1, looking through the entire pixel range. If an event is not found, the search proceeds to the next time code. Once an event has been found it is logged as the first event belonging to a cluster list containing NA = 1 events. This first event is therefore given the index nA = 1. Next, the (x, y, τ) coordinate of the first event is set as a reference point, (x0, y0, τ 0), and is removed from the shot data set to avoid double counting. The algorithm then searches for any further events neighbouring this “reference” (i.e. Δx = 1, 0, 1; Δy = 1, 0, 1; excluding Δx = Δy = 0), and extending into later time codes up to some set length (i.e. 0 ≤ Δτ ≤ Δτ max). Any new events are then added to the cluster list, increasing the value of NA, and are removed from the shot data set. Once all the events neighbouring the reference event have been found the reference point is redefined taking on the coordinate of the next event in the cluster list (i.e. index nA = 2). Any events neighbouring this new reference are now located in the same way as before, then being added to the cluster list and removed from the shot data set. This routine continues until all the events in the cluster list have been used as the reference point. The cluster list now contains all the events belonging to this cluster. The search can now continue stepping through the remainder of the shot data set to identify any further clusters. Each resultant list of events can later be reduced to single points which best approximate the (x, y, τ) coordinate of the original ion hits. This is trivial for τ as it is not possible for an event to be detected before it has occurred, so the earliest time code can be assumed to be the most accurate. The (x, y) coordinate is found by taking the spatial “centre-ofmass” and binning it to the original pixel array. It is possible to employ more complicated (x, y) centroiding options but with a low pixel count this is not often meaningful.[35] Clusters are easily defined in (x, y), as events are always connected, but the spread of clusters in t is less well defined. Events can be separated by multiple time codes. If Δτ max is set too low, there is a risk that events belonging to a cluster will be missed and will be allocated to another cluster. Conversely, if Δτ max is set too high, the clusters are more likely to overlap. In the work presented here a value of Δτ max = 7 is used.
that the beam spot diameter of 400 μm covered the full 800 μm diameter corresponding to the detector field-of-view. The maximum measured fluence was found to be 45 mJ/cm2. Initial samples were produced by electrospraying different solutions of organic dyes onto ITO-coated slides (25 mm × 25 mm × 1.1 mm; Oxford Research Solutions, Oxford, UK) using an electrosprayer built in-house. Solutions of Auramine O (3.3 mg/mL in 99.9% methanol), Crystal Violet (0.5 mg/mL in 99.9% methanol), Exalite 384 (saturated solution in 99.9% acetone), and Exalite 404 (saturated solution in 99.9% acetone) were prepared. All these solutions were applied to separate slides, save for the Crystal Violet solution which was sprayed onto the same slide as the Auramine O. The Crystal Violet was sprayed through a Ni grid (BM0080-02; Industrial Netting, Minneapolis, MN, USA[38]) with a pitch of 317.5 μm and a wire width of 33.5 μm. A flow rate of 70 – 80 μL/hr at a needle height of ~30 mm and a potential of 5500 V resulted in sample spots with radii of 5–10 mm. The ITO-coated glass slides were mounted in the repeller plate of the ion optics assembly. The repeller plate was further mounted on an (x, y) -translational stage allowing the user to choose the position of laser incidence. The LT2 Plus employs a delayed extraction technique which holds the extractor and repeller at the same potential (2000 V) until the switch time (370 ns), after which the extractor potential is reduced
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The LT2 Plus consists of three main sections: the ionisation source, the ion optics, and the detector. For the experiments presented here, the N2 laser (NL100, Stanford Research Systems Inc., Sunnyvale, CA, USA[36]) provided with the instrument was used. Lasing occurs at 337 nm, with a repetition rate of 20 Hz, and a pulse width of 3.5 ns (FWHM). The beam was guided into a 1.5 m long optical fibre (Schaefter + Kirchhoff, Hamburg, Germany[37]) with a 200 μm core diameter. Finally, the fibre was coupled to the experiment and defocussed onto the sample. By coiling and twisting the fibre while analysing the resulting beam profile it was possible, to some extent, to homogenise the beam, although certain mode structures were still present. To counteract this, the laser position was moved on the sample to average out the laser fluence. This further meant
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Figure 3. A comparison between the mass spectra obtained from different PImMS data processing levels, and that obtained using the PMT. (a) shows the raw data obtained from the PImMS camera, (b) and (c) show two successive levels of centroiding applied to the previous data, and (d) is a plot of the raw data obtained with the PMT. The data taken with the PImMS camera and with the PMT were obtained simultaneously over 3000 shots.
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(a)
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Figure 4. Fits of the four mass spectra obtained when imaging the four different organic dyes. All the data sets have been post processed as in Fig. 3(c). The spectra in (a), (b), (c), and (d) are of Auramine O, Crystal Violet, Exalite 384, and Exalite 404, respectively.
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RESULTS Figure 3 shows various mass spectra extracted from a PImMS data set using increasing levels of centroiding plotted above the mass spectrum simultaneously obtained with the PMT. The data in Fig. 3(a) has only undergone a simple reduction in background noise, and so represents, as closely as possible, the “raw” data set. Figures 3(b) and 3(c) show two levels of (x, y, τ) centroiding applied to the “raw” data set. Figure 3(d) shows the mass spectrum obtained from the PMT simultaneously to the PImMS data.
Figure 5. A plot of the mass resolution predicted by SIMION simulations plotted with the values found experimentally. The experimental values are split between those obtained purely with the PImMS camera (blue circles), and those obtained using the PMT (red triangles). The reduced simulation values have a 5 ns convolution added to compensate for the combined response time of the detector assembly. The error bars represent an uncertainty of two standard deviations.
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(by 230 V) to produce the required shallow extraction field.[39,40] After extraction, spatial focussing is optimised by an einzel lens (set to a positive potential of 995 V). These voltages and switching times were modified from their original values in order to optimise the mass resolution and spatial resolution simultaneously. All the results shown below were taken with these voltages applied. The original electron multiplier detector supplied with the LT2 Plus was exchanged for a pair of micro-channel plates in a chevron assembly with an active diameter of 25 mm, a pore pitch of 12 μm, a pore diameter of 10 μm, and a bias angle of 5° (Photonis, Sturbridge, MA, USA[41]). The MCP assembly was followed by a fast plastic scintillator (BC-408; Saint-Gobain Crystals, Hiram, OH, USA[42]). The response time of this scintillator is ~2.5 ns, and its emission peaks at 425 nm. The active area of the detector was 25 mm, corresponding to roughly 1.1 mm on the sample surface (with the predicted magnification × 22). A photomultiplier tube (PMT) (1P28; RCA, New York, NY, USA) and the PImMS camera were both coupled to the scintillating screen. The PImMS camera was focussed using a conventional c-mount camera lens (DO-5095; Navitar, Rochester, NY, USA[43]) with a 10 mm spacer to reduce the field of view to ~25 mm. The PMT was directly connected to an ADC board (1 GSa/s) supplied with the LT2 Plus, replacing the electron multiplier signal feed. The PImMS camera was connected, via an USB, to a computer running in-house Labview 2010 image acquisition and analysis software (National Instruments, Austin, TX, USA[44]). The software allows for complete control of the PImMS camera, as well as some on-the-fly analysis. A more complete analysis can then be performed after acquisition using a further suite of in-house programs (written in the C++ programming language). The camera housing the PImMS sensor (Aspect Systems, Dresden, Germany[45]) includes facilities to cool the sensor down to 15°, and a dry N2 line to keep the humidity low and thus prevent condensation on the sensor surface.
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Figure 6. A comparison between the images associated with (a): the Auramine O signal; and, (b): the Crystal Violet signal. These images were acquired simultaneously over 5000 shots taking a total of 250 s. Figure 3(b) shows the result of centroiding on the mass spectrum extracted from the “raw” data. The distribution is shifted towards lower mass, and a bimodal feature starts to appear. Figure 3(c) shows a further evolution of the centroiding process where clusters below a threshold cluster size are discarded. Here, a distinct bimodal distribution is apparent, and the similarity to the PMT spectrum is far improved over the original data set in Fig. 3(a). In order to ascertain the mass resolution of the experiment, the width at half the maximum of the mass spectral peaks must be measured (Δm50 %). This is done by fitting a Gaussian function to each peak. With such a discrete data set it is necessary to facilitate the fitting by assuming that the mass resolution of integer mass neighbours is approximately equal (i.e. Δm50 % ≈ Δ(m + 1)50 %) so that we can set the width σ of adjacent peaks to be equal. Using this approximation the spectrum in Fig. 3(c) is found to have a mass resolution of m/Δm50 % ≈ 936. The counterpart value found using the PMT spectrum (Fig. 3(d)) is m/Δm50 % ≈ 1260.
Before moving on to the analysis of a MALDI target it is simpler to analyse organic dyes which can be more easily prepared with well defined spatial distributions on the sample plate. These dyes persist for a long time which greatly facilitates the repetition of experiments during which other samples would only last for a few thousand laser shots. Figure 4 shows mass spectra obtained with the PImMS camera for the four organic dyes Auramine O, Crystal Violet, Exalite 384, and Exalite 404 (Figs. 4(a), 4(b), 4(c), and 4(d), respectively). All four data sets have undergone the same post-acquisition processing as in Fig. 3(c), and have been fitted using the same approximations as before. All four spectra show isotopic resolution (i.e. Δm50 % < 1 m/z unit), although at the highest m/z value of 658.5 (Exalite 404) this is less defined, as would be expected from the calculations shown in Fig. 1. The obtained mass resolutions determined from these plots are: m/Δm50 % ≈ 540 (Auramine O), 597 (Crystal Violet), 936 (Exalite 384), and 882 (Exalite 404). Figure 5 shows a plot of the previously acquired simulation values for mass resolution along with those obtained when analysing the dyes using both the PImMS camera (blue circles), and the PMT (red triangles). It is evident from the plot that, as expected, the PMT gives a far faster response than the PImMS camera. The simulated resolutions that have been convoluted with a 5 ns response time match the absolute values of the experimental data obtained with the PMT very well giving some insight into the response time of the scintillating screen in combination with the PMT. Figure 6 shows a comparison between the images correlated to the Auramine O peak and the Crystal Violet peak obtained using the PImMS camera. These images were obtained simultaneously as the Crystal Violet sample was applied first to the ITO-coated slide, in a grid pattern, after which the Auramine O was applied. The resultant image
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Figure 7. The mass spectrum of the PEG sample obtained with the PImMS camera. The two insets (a) and (b) show enlarged areas of the mass spectrum with accompanying Gaussian fits. The peaks in (a) correspond to [CH2(C2H4O)10OH + Na]+ (m/z 496), and to Exalite 384 (m/z 498) which was included as an internal calibrant. The peaks in (b) correspond to [CH2(C2H4O)13OH + Na]+ (m/z 628).
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Linear mass spectrometer for mass-resolved microscopy clearly shows a marked difference in the location of the two dyes. By taking a line profile (six pixels wide) across the image, an estimate of the magnification, and resolution can be obtained. In these images the magnification was found to be × 28.7. The resolution is equated to the rising distance of the present features from 20% to 80%, and was found to be 25.0 μm which is close to the simulated optimum resolution of ~ 20.0 μm.[46] All four dyes were found to be sensitive to laser ablation without the need for a MALDI matrix, and so all the previous experiments were conducted without any such matrix. To fully test the experiment a further sample was produced consisting of 100 μL of poly(ethylene-glycol) methyl ether (MAV = 550 Da) (PEG) in 5 mL of 2.65 mg/mL α-cyanohydroxycinnaminic acid (CHCA) in 50% methanol with 0.01% trifluoroacetic acid (TFA). This solution was applied to an ITO-coated glass slide via the dried droplet method before being analysed. Figure 7 presents an example of the full mass spectrum of the PEG solution. All the experimental settings were identical to those used for the organic dyes. The laser fluence at the sample was ~40 mJ/cm2, and the data was taken over ~5000 shots. The data has undergone the same level of postprocessing as with the dye experiments. Many more peaks are present in the spectra obtained under these conditions. The majority of the data (59%) was collected in the first register of the PImMS camera with the second, third, and fourth registers containing 17%, 13%, and 11% of the data, respectively. This implies that there is data that would have been missed without the mulit-hit pixel capacity of the PImMS camera, but also that there is still scope for higher count rates or more complicated spectra. Figure 7(a) shows a magnified area of the mass spectrum. There are two species present at this m/z value: the PEG species [CH2(C2H4O)10OH + Na]+ (m/z 496), and Exalite 384 (m/z 498) which was added as an internal calibrant. The two species are clearly separated and exhibit the isotopic
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DISCUSSION AND CONCLUSIONS It has been shown that a conventional mass spectrometer can be modified in order to perform spatially resolved mass spectrometry in microscope-mode. This offers the advantage of processing large quantities of data in parallel, whether it be multiple regions within a tissue section, or multiple samples set in an array. Accessing all the spatial and m/z data simultaneously puts a heavy performance requirement on all aspects of the experiment. In order to increase the experimental field-of-view a more powerful laser will be needed, and losses due to coupling the laser into the fibre will need to be reduced. Further homogenising of the beam profile will also be required if normalised images are to be produced. The currently used pulsing regime, delayed extraction, is limited by the upper bound of the switching time. The mass resolutions shown in Fig. 7 are encouraging but there is an upper limit to the obtainable m/z values. During the initial, field-free, expansion step spatial information is lost as ions travel orthogonally to the time-of-flight axis. By the time a field is finally applied this information is already lost. The post-extraction differential acceleration (PEDA) technique pioneered by Aoki and co-workers, and its extension (velocity correction) developed by Brouard and co-workers address this issue by controlling the trajectories of ablated ions from their creation rather than letting the ablation plumes expand into the vacuum.[47,48] The pulsed focussing then occurs further along the time-of-flight path. This can extend the range at which the peak width, Δm50 % < 1 m/z unit. The reduction in the accelerating potential applied to the repeller electrode means that the PImMS camera is used at its optimum. The timing precision of 12.5 ns is the limiting factor in the mass resolution measurements shown in the results section, but this can be reduced by improving the coupling between the detector and camera. The response speed of the PImMS sensor is dependent on the intensity of the illumination that it is detecting. A bright flash on a scintillating screen will trigger a large cluster of pixels, with a short response time, giving a more reliable signal. To this end, more efficient scintillators, such as those proposed by Brouard and co-workers, should improve the performance of the PImMS sensor.[49] The response time of the BC-408 scintillator used here is short enough (2.5 ns) that it should not affect the time resolution for ion detection but when further planned firmware improvements reduce the PImMS timing precision to 6.25 ns, there will be a demand for still faster scintillators.
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Figure 8. A plot of the mass resolution predicted by SIMION simulations plotted with the values found for the PEG sample experimentally. The experimental values are split between those obtained purely with the PImMS camera (blue circles), and those obtained using the PMT (red triangles). The reduced simulation values have a 5 ns convolution added to compensate for the combined response time of the scintillator and PMT. The error bars represent an uncertainty of two standard deviations.
resolution obtained with the organic dyes. Figure 7(b) shows a higher mass PEG species, [CH2(C2H4O)13OH + Na]+ (m/z 628), which also exhibits isotopic resolution. Figure 8 shows an overview of the mass resolutions obtained for the PEG sample spectra shown in Fig. 7. The resolutions obtained with the PImMS camera (blue circles) is presented along with resolutions obtained simultaneously, using the PMT (red triangles). These experimental points are compared with the simulation data presented earlier. The analyte peaks correspond to the polymer adduct ions [CH2(C2H4O)NOH + Na]+ where the assigned range covers 8 ≤ N ≤ 16.
E. Halford et al. In conclusion, we have shown that the PImMS camera, in combination with an off-the-shelf mass spectrometer, is capable of multi mass imaging with mass resolutions greater than the isotopic limit Δm50 % = 1 m/z unit over a range of 200 ≤ m/z ≤ 800, while still achieving an optimum spatial resolution of 25 μm. The simple linear geometry of the experiment lends itself to being used as a tabletop instrument for the high-speed analysis of multiplexed assays, or tissue sections with analytes in the small molecule range.
[13]
Acknowledgements
[14]
The support of the STFC through the Mini-IPS grant ST/J002895/1, the EPSRC via Programme grant No. EP/L005913/1, and the EU through grant FP7 ITN “ICONIC” (238671) is gratefully acknowledged. Elements of the work described here are detailed in the US Patent Application No. 20100294924 A1.
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