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Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

RATIONALE

Desorption electrospray ionisation (DESI) is the ambient technique used for surface imaging. Despite its simplicity, the proper use of this technique is not easy, and usually leads to discouragement, especially in the case of biological sample measurements. Here, we present some tips and tricks which may be helpful during a complex process of ion source optimisation to achieve the desired results.

METHODS

Rat liver tissue as an example of a biological sample and a surface covered with rhodamine-containing marker were measured using a DESI ion source (OMNIspray source, Prosolia, Indianapolis, IN, USA) connected to a AmaZon ETD ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany).

RESULTS

The geometry of the ion source (nebulisation capillary angle, its distance to the surface, and to the MS inlet), and other settings like nebulising gas pressure, solvent flow and capillary voltage, were changed during the optimisation process. The results obtained for different parameters are presented.

CONCLUSIONS

Differences between the results and the method of optimisation for biological and non-biological samples were shown. The influence of different parameters on the quality of mass spectra was indicated. Optimal parameters for the tissue and non-biological sample analysis were suggested. Copyright © 2013 John Wiley & Sons, Ltd.

Desorption electrospray ionisation (DESI) belongs to the group of techniques focused on surface imaging and is being increasingly used in biological sciences, especially for lipid analysis.[1, 2] The basis of this technique appears to be very simple: a pneumatically assisted electrospray needle produces charged droplets of solvent, which are directed onto the surface to be analysed. A thin film of solvent is created on a surface, and thus the components located in the sample are dissolved. A new portion of charged droplets is responsible for releasing the secondary microdroplets with analytes from the surface. Finally, secondary droplets are evaporated by standard electrospray mechanism on their way to the inlet of the mass spectrometer.[3-6]

There are several parameters important for DESI ion source optimisation, which are responsible for spectra quality. Those parameters include the geometry of the ion source (nebulisation capillary angle, its distance to the surface, and to the inlet of the mass spectrometer), and other settings like nebulising gas pressure, solvent flow, and capillary voltage. Due to the multiplicity of parameters, optimisation of the DESI ion source is not an easy task.

Manuals provided by manufacturers are usually limited to ion source optimisation performed with the aid of well-known and easily ionisable standards (rhodamine, bradykinin). An excellent study of DESI ion source optimisation with such standards may be found in the article of Douglass et al.[7] A very valuable explanation of observed relationships is described there as well. Unfortunately, problems arise with the analysis of biological samples, especially tissue sections, cell culture monolayers, etc., which are much more complex. Judging from our previous results, it seems that different samples may need different DESI source settings. In some cases, even minor changes in geometry, voltages, solvent or gas flows may significantly influence the spectra quality acquired with the aid of DESI sources.

In the majority of cases, it is usually insufficient to imply an optimised DESI source using a standard for the analysis of complex biological samples. It could be very disappointing that, after careful optimisation using a standard compound, analysis of the real sample results in a virtually complete lack of meaningful mass spectra.

Based on our own experiences and an awareness of similar technical problems in many laboratories, our intention was to provide a set of advice on how to optimise the DESI ion source without wasting time with numerous unsuccessful trials. We focused on the parameters in order of their importance, and a range of their adjustments. All of the parameters were carefully tested on the OMNIspray source (Prosolia, Indianapolis, IN, USA) connected to an AmaZon ETD mass spectrometer (Bruker Daltonics, Bremen, Germany), but our recommendations may easily be adapted to other sources and mass spectrometers. During our study, two different samples were analysed: red permanent Staedtler marker as an example of standard, non-biological sample analysis (marker contains rhodamine), and rat liver tissue, as an example of a homogeneous biological sample.[8]

EXPERIMENTAL

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Rhodamine-containing marker

A microscopic sodium glass slide was covered with the rhodamine-containing marker (Staedtler, Germany). This marker is characterised by an intense peak from rhodamine 6G at m/z 4431+ on the DESI spectrum in the positive ion mode, and m/z 4411– in the negative ion mode. An additional characteristic peak derived from the marker for the negative ion mode is m/z 10011–.

Animal tissue

All experiments using animal tissues were performed in agreement with the appropriate Polish and European Council Directives (86/609/EEC), and were approved by the Local Ethics Committee.

Rat liver tissue was chosen for analysis due to its homogeneity.[8] We recommend this tissue for the initial setup as it is very homogenous and contains lipids that are characteristic of other tissues. It makes optimisation of the DESI source much closer to the more complex tissues compared to optimisation based on a single, highly concentrated standard. This tissue also allows for the scientist starting their work with DESI to gain their own experience and feelings on how adjustments to the parameters may influence the results.

Male Wistar rats (weight 150–200 g) obtained from a local distributor (HZL, Warsaw, Poland) were used in the experiments. Animals were decapitated and their livers were immediately isolated and frozen in liquid nitrogen, where they were stored until analysis. Before preparing the sections, the tissue was warmed up to ca. –20 °C and mounted on the cryotome head. The tissue was cut using a Cryotome FSE cryostat (Thermo Scientific, Cheshire, UK) into 35 µm thick sections and thaw-mounted on the sodium glass slide. Chromatograms for several peaks characteristic for lipids in the positive (e.g. 758.51+; 782.51+; 806.51+) and in the negative (e.g. 885.51–; 762.41–) ion modes were observed during analysis.

DESI ion source

The DESI ion source (OMNIspray, Prosolia, Indianapolis, IN, USA), with OEM software to control DESI stage movements, was combined with an AmaZon ETD mass spectrometer (Bruker-Daltonics, Bremen, Germany), which was operated using the Bruker TrapControl software.

During the optimisation process the tissue sections and glass with marker were scanned using a 2D moving stage. Optimisation steps and analysis of the sample should use the same scanning velocity. CH3OH/H2O solution (1:1, v/v) (MS grade, Sigma-Aldrich, Poznan, Poland), as recommended in other publications,[1] was selected as a standard solution for tissue analysis.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

To properly illustrate the results obtained, and at the same time to avoid overloading the text with different figures, we decided to choose the clearest examples for the positive and negative ion modes. Figures showing minute changes in the spectra depending on the parameters optimised and additional data are included in the Supporting Information.

Main settings

Below, several parameters are listed that we found to be important for optimisation:

  1. capillary protrusion from the DESI nozzle;
  2. pressure of the nebulising gas (nitrogen);
  3. solvent flow rate;
  4. capillary voltage;
  5. source geometry:
    1. nebulisation capillary angle;
    2. nebulisation capillary height over the scanned surface;
    3. distance between nebulisation capillary and mass spectrometer inlet;
    4. nebulisation capillary and mass spectrometer inlet geometry.

In particular, the first parameter has an influence on spray quality and is crucial for obtaining abundant and stable DESI mass spectra. Biological samples are very sensitive to changes in this parameter. Considering parameters 2 and 3, when the nebulising gas pressure or the solvent flow rate is too high, the sample may be destroyed. Small fragments of the tissue detached from the scanned surface may clog the inlet of the mass spectrometer and dramatically reduce the quality of the mass spectra. On the other hand, setting that are too low for those parameters may hamper good analysis, due to the impact on ion desorption from the surface.

Capillary protrusion settings

The range of capillary protrusion (parameter marked as 2 in Fig. 4) was from 0 to ca. 1 mm from the sprayer nozzle. Interestingly, for the standard substance (rhodamine) placed on the glass slide, this setting should be close to 1 mm but, for liver tissue (as for other tissues analysed in our laboratory), much better results were obtained for settings equal to 0.5 mm. This parameter is strongly connected to a Taylor cone formation. When the capillary protrudes too far, the Taylor cone cannot be properly formed. In contrast, when the capillary is hidden in the nozzle, the signals obtained are very unstable, even though they are quite intensive (see Fig. 1). This parameter might be very difficult to be appropriately set; the perfect setting for a particular sample type may not be suitable for another surface.

image

Figure 1. DESI analysis of liver tissue with various settings of the nebulising capillary (positive ion mode). Y axes for every scan have the same range, and clearly show changes in spectra intensities for various settings of the nebulising capillary protrusion. The average spectra presented were prepared after accumulation of spectra acquired from 4 min of analysis.

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Nitrogen gas pressure

We tested the pressure range from 8 to 14 bar. The pressure was measured on a two-stage gas pressure regulator (RBAz-3/Z, Perun, Poland) on the nitrogen bottle (Air Liquide local distributor, N2 purity 5.0). The gas delivery line was made from chemically inert polyamide tubing (i.d. 4.0 mm, length 2.0 m) with additional Teflon tubing (i.d. 1.0 mm, length 50 cm) connected directly to the DESI nozzle (i.d. ca. 150 µm). As the whole gas delivery line efficiency is significantly higher than gas consumption during Taylor cone formation, the backpressure in the system is generated mainly by the DESI nozzle; therefore, the nitrogen pressure may be used as a comparable measure of the delivered gas parameters. Judging from the spectra presented in Fig. 2, nitrogen pressure that is too high is responsible for poor spectrum quality. This may cause sample destruction and mass spectrometer inlet clogging due to the detachment of tissue fragments. On the contrary, nitrogen pressure that is too low does not allow for the proper drying of the sprayed solvent which, as a consequence, decreases signal intensity. The optimum nitrogen pressure for tissue analysis in the positive and negative ion modes was found to be 11 bar (see Fig. 2). For the rigid, dry and thin structures deposited on the surface (marker signs, other samples) it is possible to receive satisfactory results over the entire pressure range, but the sample should be observed during analysis to avoid any detachment of its parts.

image

Figure 2. Influence of the nitrogen pressure on spectra quality tested for three settings (positive ion mode). The average spectra presented were prepared after accumulation of spectra acquired from 4 min of analysis.

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Solvent flow rate

There is no agreement in the literature regarding what composition of solvent would be optimal for DESI. In fact, this is not surprising, as samples undergoing analyses with the aid of this ion source vary widely. We used a methanol/water solution (1:1, v/v) for our experiments as the most suitable solvent, easy to switch between positive and negative polarisations. Solvent flow rate was optimised in the range from 1 to 5 μL/min. Lower solvent flow rates (1–2 μL/min) allow for acquisition of good quality spectra from substances, creating thin films on the surfaces (like standard marker). With increasing thickness of the investigated layer, higher flow rates are needed. On the other hand, solvent overflow causes damage to the tissues, dislocation or removal of small tissue fragments and, as a result, unsuccessful analysis. In our tests, the best setting of this parameter was 1 μL/min for standard substance and 2 (up to 3) μL/min for tissue scanning (see Supplementary Fig. S1, see Supporting Information). It is recommended that the solvent flow rate is optimised along with the sheath gas pressure. The best conditions for the analyses were achieved when the entire sprayed solvent evaporated just after wetting the sample and there is no visible damage to the tissue after scanning. It is not uncommon that tissue samples look burred or scratched after exposure to solvent spray, with small droplets of the solvent visible on the sample. In such cases the solvent flow rate should be decreased without any changes to the sheath gas settings.

Capillary voltage

The capillary voltage (parameter marked as 5 in Fig. 4) was tested in the positive and negative ion modes. Voltage range was set from 2000 to 5000 V with polarisation being dependent on the mode. For the positive ion mode the best setting for both of the tested samples was –3000 V (see Fig. 3). In the negative ion mode, the best results were achieved close to the extremes: +4500 V to +5000 V (see Supplementary Fig. S2, Supporting Information). Both modes should therefore be optimised separately. Especially in the positive ion mode, it is not very difficult to induce electrospray analysis instead of DESI desorption. When the voltage between capillaries is too high, the Taylor cone formed from the nebulising cone goes directly to the inlet of the mass spectrometer and omits the scanned surface. It is not difficult to detect such a situation. Distinction between the DESI and ESI state relies on the observation of the voltage current between capillaries. Typically, voltage current between capillaries during DESI analysis is one to two orders of magnitude lower than for ESI spray. Additional parameters, which could be observed using a mass spectrometer equipped with an ion trap (as in the case of our instrument), are ion quantity and accumulation time inside the trap. In the typical ESI analysis, this ion injection time varies from dozens of microseconds to 10–20 milliseconds. The automatic gain control (AGC) algorithm does not allow for an enormous increase in the ion quantity inside the ion trap, which could result in spectra disruption from excessive space charge effects.[9] In DESI analysis, according to the relatively poor effectiveness of ion desorption from the surface, the ion trap needs much more time to acquire a sufficient number of ions to produce a reasonable spectrum. The time required is routinely longer than 50 milliseconds. Thus, observation of the acquisition time along with the quantity of ions forming a mass spectrum is a valuable diagnostic tool to distinguish between DESI and ESI behaviour of the ion source. When working with ion traps, we recommend setting the ion injection times to much higher values than for ESI spraying (up to 200–300 ms) along with the maximal quantity of ions that are safe for the instrument used. This guarantees acquisition of enough desorbed ions and the generation of high-quality mass spectra. In cases when ESI spray is accidentally formed instead of DESI, the active AGC algorithm will cut the time into values that are safe for the mass analyser. Detailed data describing AGC or equivalent algorithms with their application for various instruments can be found elsewhere.[10-12]

image

Figure 3. Influence of the capillary voltage on spectra quality (positive ion mode). The average spectra presented were prepared after accumulation of spectra acquired from 1 min of analysis.

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We are aware that DESI can be connected to various instruments, but we believe that the above comments could be easily adapted to other instruments. Independent of the DESI manufacturer, geometrical settings must be the same or very similar; however, tissue properties are independent of the type of instrument. Optimisation of the mass spectrometer settings should be performed in cases where other ion sources are installed.

Source geometry

Proper positioning of the nebulisation capillary in relation to the inlet of the mass spectrometer and the analysed surface is crucial to obtain good quality results. Depending on the source design, it is possible to manipulate the following parameters: angle of the nebulisation capillary (parameter 1, Fig. 4), its height above the scanned surface (parameter 3, Fig. 4), symmetry between capillaries (not marked), and the distance between them (parameter 4, Fig. 4). The latter parameter, which can usually be altered, is the distance between the scanned surface and the inlet capillary: this type of movement is accomplished by lifting the 2D moving stage, which simultaneously influences the distance between the nebulising capillary and the sample surface (param. 6, Fig. 4).

image

Figure 4. Geometry of DESI ion source. A – nebulisation capillary, B – MS inlet, C – scanned surface, 1 – capillary angle, 2 – capillary protrusion, 3 – capillary height, 4 – distance between capillaries, 5 – voltage between capillaries, 6 – MS inlet height.

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Nebulisation capillary angle

Regulation of the nebulisation capillary angle (parameter marked as 1 in Fig. 4) is another important issue to consider during tissue analysis. This angle can be changed across a wide range without the loss of spectra quality in cases of optimisation using a standard (like rhodamine), which is always present at high concentration. The range of regulation of this parameter was from 50° to 70°. We observed that for optimisation using tissue, the optimum for the negative ion mode was shifted toward slightly higher values (60°) than in the positive ion mode (58°). Modulation of the angle in the negative ion mode provides sharp and clear changes in the overall spectrum quality after reaching the optimal value (see Fig. 5). Analysis in the positive ion mode is not very sensitive to this parameter (see Supplementary Fig. S3, Supporting Information). What is interesting during the analysis of non-biological samples is that wide changes in this parameter are tolerable and do not significantly influence the spectra quality. For example, the parameter could be changed from 56° to 60° without loss, or even weakening, of the signal in the negative ion mode. Once again, this experiment clearly shows that even minor changes in various settings (e.g. capillary angle) may have a significant impact on spectra quality in the case of tissue sample analysis, whereas they are insignificant during rodamine standard optimisation. Moreover, changes in the angle of the nebulising capillary, like other changes described in the following paragraphs, induce changes in the whole geometry of the system. The nebulising capillary angle influences the geometry of the shape sprayed by the solvent. If the nebulising capillary is set closer to 90°, the sprayed area is more circular and a significant part of the secondary droplets, detached from the scanned surface, are not directed into the inlet of the mass spectrometer. After reduction of the angle, the sprayed area is more elliptical and the trajectories of the secondary droplets are directed towards the inlet. However, for values lower than 55–45°, the ESI spray overcomes the DESI effect, which should be taken into consideration when adjusting this angle.

image

Figure 5. Influence of the capillary angle on spectra quality (negative ion mode). The average spectra presented were prepared after accumulation of spectra acquired from 1 min of analysis.

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Nebulisation capillary height over the scanned surface

This is another parameter (parameter marked as 3 in Fig. 4) that is distinct for tissue and non-biological samples. We used a range from 1.5 to 4 mm for this parameter. The best effects for the standard sample were observed when the nebulising needle was close to the surface – in the negative ion mode, the best distance was 2.0 mm while it was 1.5 mm in the positive ion mode; this shows that even such small differences in this parameter are important. In the case of tissue analysis, it is recommended to shift the nebulising needle up to 3 mm (see Supplementary Figs. S4 and S5, Supporting Information). We realise that, especially for tissue imaging, spatial resolution of the produced images is lower, because spray droplets penetrate a wider area per scan. On the other hand, the sensitivity of the entire system is higher, due to the ions formed from the larger area. Additionally, the risk of tissue damage, as a consequence of the high flow solution impact, is lower than in cases where the capillary is located in close proximity to the sample surface. As in the case of flow rate and gas pressure settings, the operator should consider pros such as increased sensitivity of the system and cons such as risk of tissue damage, and reduced spatial resolution of the final image. As can be seen, the distance from the nebulising needle to the surface should be optimised along with the solvent flow rate, gas pressure, and the distance between capillaries, which is also important.

Nebulisation capillary and mass spectrometer inlet distance

This parameter (marked as 4 in Fig. 4) has to be tested from the longest distance to the shortest one, because, when the distance is too short, an electrospray is formed instead of the surface analysis mode. An increase in this distance from the shortest value to the longest may not inhibit electrospray formation; it is easier to turn off the voltage between capillaries or retract the DESI manipulator to stop the ESI spray, and then increase the distance. It is important to remember that moving the capillary toward the mass spectrometer inlet also changes its location toward scanned samples, as the only moving part of the capillaries/sample system is the nebulising capillary. Therefore, when the sample has small dimensions it should be inspected during optimisation to check wether the spray is still located on the sample surface during adjustment of the distance between the capillaries. Changing the capillary angle and location of the nebulising capillary towards the mass spectrometer inlet might cause confusion when the mass spectrum suddenly vanishes. In that case it is worth checking whether the spray is still wetting the tissue (or reference material). If not, the location of the moving stage with the sample on it must be corrected appropriately.

We tested distances ranging from 2 to 7 mm. For both ionisation modes, as well as for both samples tested, the best distance varied from 5 to 6 mm (see Fig. 6 and Supplementary Fig. S6, Supporting Information). It should be additionally taken into consideration that the distance may be simultaneously optimised, together with the voltage between capillaries. Too short a distance in combination with a high voltage may cause electrospray formation or even electric arc ignition. Other parameters, which should be optimised along with the distance setting, are: the angle of the nebulising capillary and the height of the mass spectrometer inlet capillary over the sample. In the latter setting, the general rule is that a very small value of this distance ensures that the mass spectra remain stable and sensitive. Judging from our own experience, spectra are generally better with values equal or lower than 1 mm.

image

Figure 6. Influence of the nebulisation capillary and mass spectrometer inlet distance on spectra quality (positive ion mode). The average spectra presented were prepared after accumulation of spectra acquired from 1 min of analysis.

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Nebulisation capillary and mass spectrometer inlet symmetry

The nebulising capillary is able to change its location to the left and right from the axis of symmetry connecting both capillaries. In our opinion, this parameter is of minor importance. We tested the range between –2 and +2 mm from the axis of symmetry after optimisation of the distance between capillaries, but this parameter was found to have a less significant influence on spectrum formation. This is probably caused by the fact that the spraying area of the sample surface is wide enough to acquire ions into the mass spectrometer inlet by the suction force, even if the capillaries are not symmetrically positioned.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Optimal parameters for DESI analysis are presented in Table 1. Differences in analysis between biological material, such as tissue, and synthetic samples, such as e.g. markers or other non-biological samples, are important and may cause confusion among users. Parameters of the DESI ion source that were optimised using the synthetic marker, producing highly abundant signals, cannot be applied to biological samples. Thus, as a result, such settings may not provide any results at all, or the spectra may be of poor quality. As can be seen from our results, the solvent flow, nebulisation capillary height or its angle should be adjusted depending on the sample type, as even minor differences (e.g. 1 mm shift) may lead to an unsuccessful measurement. Negative and positive ion modes also require separate optimisations. Even the sequence in which parameters should be adjusted seems to be important in the case of DESI measurements. We recommend applying the following order:

  1. Initial settings of the geometry of capillaries and moving stage from Table 1.
  2. Flow rate of the standard solvent and gas pressure.
  3. Voltage between capillaries avoiding ESI formation.
  4. Tuning geometry of the system (distance between capillaries, angle, and height above the sample).
  5. Second round of the voltage optimisation.
  6. When the signal is stable and satisfactory parameters are achieved, additional optimisation of the solvent composition may be performed.
Table 1. Optimised parameters of the DESI ion source for tissue and non-biological sample analysis
ParameterMarker positiveLiver tissue positiveMarker negativeLiver tissue negative
Capillary protrusion (mm)10.510.5
Gas pressure (atm)11111111
Solvent flow (μL/min)12–312–3
Capillary voltage (V)3000300040004500
Nebulisation capillary angle (°)56–585856–6060
Nebulisation capillary height (mm)1.53.02.03.0
Capillary distance (mm)6.05.05.05.0
MS inlet height (mm)0–10–10–10–1

Judging from our experience (data not shown), every type of tissue has its own optimal parameters; therefore, data from the table above should be treated as a good starting point for optimisation but cannot be taken for granted.

Tips and tricks

It is also worth taking care with the aspects of DESI analysis that are briefly described below:

  • During the optimisation process, the nebulising needle should be moving all the time over the sample. Otherwise, the constant elution of compounds from the exposed part of the sample will lead to the constant decrease in signal intensity, independent of the optimisation process.
  • All of the solvents should be at the highest available purity. Spectra obtained from this ion source are very sensitive to impurities in solvents, which may strongly affect the final results.
  • DESI may react slowly after changing the selected parameters. During the optimisation it is better to change a given setting within a specific range, and wait for at least 1 min in order to allow the source to stabilise. While choosing the best settings not only the parameters that produce the spectra of high intensity should be taken into consideration, but, more importantly, settings that provide reasonable stability in the following spectra should be selected. Certain adjustments are considered optimal, when a compromise between stability and sensitivity of the system has been achieved.

When the intensity of the spectra is dramatically low or an unacceptable instability of the system is observed:

  • It is recommended to be patient – DESI reacts quite slowly to any changes. Wait for at least 1 min for signal stabilisation.
  • When DESI imaging has been completed, remove the first line of the analysis from the final results. The first line usually has lower intensity scans than the subsequent lines, probably due to sample wetting. The following lines usually give stronger signals. DESI prefers stable conditions and comparison between dried and wetted lines predominantly leads to unpredictable results.
  • If the signal vanishes slowly, independently from operator exertions, it is recommended to check the inlet of the mass spectrometer as it may be clogged up with material removed from the sample by the overdosed solvent flow rate, along with high nebulising gas pressure.
  • At least once per day check the nebulisation capillary: it may be clogged, or the tip may have been damaged by the electric arc or by mechanical impact. It may also protrude from the optimal settings which can result in a sudden and difficult to diagnose loss of mass spectra quality.
  • After imaging of several tissue sections, a slow loss of sensitivity may be observed. In such cases, it is strongly recommended to remove the DESI interface, follow the recommended procedure for ion source cleaning (instrument manual) and start the analysis again.

Acknowledgements

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

The research was supported by the Foundation for Polish Science – POMOST Programme (POMOST/2011-3/1) co-financed by the European Union within European Regional Development Fund, The Polish National Science Centre 3744/B/H03/2011/40, and EuroNanoMed " META" 05/EuroNanoMed/2012.

REFERENCES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information
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rcm6755-sup-0001-figureS1toS6.docxWord 2007 document1545KSupporting info item

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