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Title:
DEVICE AND METHOD FOR HIGHLY LOCALIZED MASS SPECTROMETRIC ANALYSIS AND IMAGING
Document Type and Number:
WIPO Patent Application WO/2006/089449
Kind Code:
A2
Abstract:
A device and a method for localized mass spectrometric analysis of a sample surface (204) are disclosed. The device comprises an optical system, preferably a scanning near-field optical microscopy (SNOM) setup (2), which serves to ablate sample atoms/molecules from the sample surface with high spatial resolution, preferably at atmospheric pressure. These are transferred to an ion trap (410) by suitable transfer means (5), advantageously through a transfer tube by action of a pressure difference. The atoms/molecules are ionized, preferably by chemical ionization, and accumulated in the ion trap (410). After a sufficient number of ions have been accumulated, they are analyzed by a mass analyzer (3), preferably of the time-of-flight type.

Inventors:
ZENOBI, Renato (Kürbergstrasse 3, Zürich, CH-8049, CH)
SCHMITZ, Thomas (Feldgüetliweg 83, Feldmeilen, CH-8706, CH)
SETZ, Patrick (Neugasse 91, Zürich, CH-8005, CH)
Application Number:
CH2006/000117
Publication Date:
August 31, 2006
Filing Date:
February 23, 2006
Export Citation:
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Assignee:
EIDGENÖSSISCHE TECHNISCHE HOCHSCHULE ZÜRICH (Rämistrasse 101, Zürich, CH-8092, CH)
ZENOBI, Renato (Kürbergstrasse 3, Zürich, CH-8049, CH)
SCHMITZ, Thomas (Feldgüetliweg 83, Feldmeilen, CH-8706, CH)
SETZ, Patrick (Neugasse 91, Zürich, CH-8005, CH)
International Classes:
H01J49/42
Domestic Patent References:
WO2002084577A12002-10-24
WO2001078106A22001-10-18
Foreign References:
FR2797956A12001-03-02
US6204500B12001-03-20
US20040183009A12004-09-23
Other References:
DE SERIO M ET AL: "Looking at the nanoscale: scanning near-field optical microscopy" TRAC, TRENDS IN ANALYTICAL CHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 22, no. 2, February 2003 (2003-02), pages 70-77, XP004413151 ISSN: 0165-9936
Attorney, Agent or Firm:
DETKEN, Andreas (Isler & Pedrazzini AG, Postfach 6940, Zürich, CH-8023, CH)
Download PDF:
Claims:

Patent claims

1. Device for localized mass spectrometric analysis of a sample surface, comprising a light source (201) for generating light for ablating sample atoms and/or molecules from said sample surface (204), said light having a predetermined wavelength or having a distribution of wavelengths whose mean is a predetermined wavelength; - field localization means (202, 203) for concentrating said light into a field region with lateral dimensions in the range of said predetermined wavelength; field positioning means (211 , 212) for positioning said field localization means (202, 203) and said sample surface (204) relative to each other; an ion trap (4) for storing said sample atoms and/or molecules in ionized form; transfer means (5) for transferring said sample atoms and/or molecules from said sample surface (204) to said ion trap (4); and - a mass analyzer (3) coupled to said ion trap (4) for analyzing said sample atoms and/or molecules.

2. Device according to claim 1 , further comprising vacuum generating means (330, 430) for creating a vacuum pressure in a region within said ion trap (4), characterized in that said sample surface (204) is at a pressure higher than said vacuum pressure, preferably at ambient pressure.

3. Device according to claim 1 or 2, characterized in that said transfer means (5) comprise a transfer tube (511 ) for transferring said sample atoms and/or molecules from a first end disposed near said sample surface (204) to a second end by the effect of a pressure difference between said first and second ends.

4. Device according to claim 3, characterized in that said transfer tube (511 ) comprises a capillary portion (512) near said first end having an inner diameter (d) which is smaller than an inner diameter (D) of a por- tion of said transfer tube (511) near said second end.

5. Device according to claim 3 or 4, characterized in that said transfer means (5) comprises heating means (513, 515, 517) for heating said transfer tube.

6. Device according to one of claims 3 to 5, characterized in that said transfer means (5) comprise an intermediate vacuum chamber (516) and tube means (520) connecting said intermediate vacuum chamber (516) to said ion trap (4), and that said second end of said transfer tube (511) is disposed in said intermediate vacuum chamber.

7. Device according to one of claims 3 to 6, further comprising tube positioning means (501 , 510), preferably a micromanipulator, for positioning said first end of said transfer tube (511 ) relative to said sample surface (204) and/or relative to said field localization means (203).

8. Device according to one of the preceding claims, characterized in that said transfer means (5) provide a predetermined direction of entry of said sample atoms and/or molecules into said ion trap (410), that said ion trap (410) provides a predetermined direction of exit of said sample atoms and/or molecules in ionized form into said mass spectrometer (3), and that said direction of entry and said direction of exit are not col- linear.

9. Device according to one of the preceding claims, characterized in that said field localization means comprise an optical probe, preferably an optical fiber, having a sub-wavelength aperture, said optical probe being

coupled to said light source.

10. Device according to one of the preceding claims, characterized in that said field localization means comprise a dielectric or conducting tip and means for guiding said light from said light source to said tip.

11. Device according to one of the preceding claims, characterized in that said mass analyzer (3) is a time-of-flight mass spectrometer.

12. Device according to one of the preceding claims, characterized in that the device further comprises ionizing means adapted for ionizing said sample atoms and/or molecules after entry into said ion trap (4).

13. Device according to claim 10, characterized in that said ionizing means comprise gas supply means (422, 423) for supplying an chemical ionization gas to said ion trap (4) and auxiliary ionizing means (440) for at least partially ionizing said chemical ionization gas, wherein said chemical ionization gas in ionized form is adapted for transferring charge to said sample atoms and/or molecules by chemical ionization.

14. Device according to one of the preceding claims, characterized in that said field positioning means comprise at least one piezo stage (212) and a tuning fork (211).

15. Method for localized mass spectrometric analysis of a sample surface (204), preferably with a device according to one of the preceding claims, comprising the following steps:

(a) Providing a light source (201 ), for generating light having a predetermined wavelength or having a distribution of wavelengths whose mean is a predetermined wavelength, and field localization means (203) for concentrating said light into a field region with lateral dimensions in the range of said predetermined wavelength;

(b) Positioning said field localization means (203) relative to said sample surface (204) such that a first impact spot on said sample surface is disposed within said field region;

(c) Ablating sample atoms and/or molecules from said first impact spot by operating said light source;

(d) Transferring at least some of said sample atoms and/or molecules into an ion trap;

(e) At least partially ionizing said sample atoms and/or molecules in said ion trap; (f) Accumulating said sample atoms and/or molecules in ionized form in said ion trap;

(g) Transferring said sample atoms and/or molecules in ionized form from said ion trap into a mass spectrometer; (h) Determining the mass distribution of said sample atoms and/or molecules in ionized form in said mass spectrometer;

(j) Moving said field localization means and said sample surface relative to each other such that a second impact spot on said sample surface different from said first impact spot is disposed within said field region; (k) Repeating steps (c) to (j) for a desired number of times.

Description:

Device and Method for Highly Localized Mass Spectrometric Analysis and

Imaging

Field of the invention

The present invention generally relates to the field of mass spectrometry. In par- ticular, the present invention relates to a device and a method for localized mass spectrometric analysis of a sample surface.

Background of the invention

In materials science as well as in the biological sciences, it is often desirable to perform localized chemical analysis and imaging of a sample surface with high spatial resolution. This need has increased with the emergence of applications in nanoscience and nanotechnology, such as molecular electronics. A variety of methods have been proposed for this purpose. One such method is localized laser desorption mass spectrometry (LDMS). In localized LDMS, an intense, highly focused laser beam is applied to the sample surface, whereby the surface spot hit by the laser beam is partially vaporized and ionized (this process is well known as "laser desorption and ionization", LDI). The resulting ions are transferred to a mass spectrometer by applying electric fields and analyzed therein. By scanning the laser beam over the sample surface, information about the local chemical composition is obtained (B. Spengler, M. Hubert, J. Am. Soc. Mass Spectrom. 13 (2002) 735-748).

In order to overcome certain limitations of direct LDI, in particular poor vaporization of nonvolatile compounds and poor efficiency of ionization, it has been pro- posed to combine localized LDMS with a technique called matrix-assisted laser desorption ionization (MALDI). MALDI allows the intact detection by mass spectrometry of high molecular weight, labile compounds. For a reference on

MALDI, see (F. Hillenkamp et al., Anal. Chem. 63 (1991 ) 1193 A).

Another variant of localized LDMS has been proposed in WO 01/15191. A focused laser beam acts to ablate neutral sample molecules from a sample sur- face in a special ablation chamber. These neutral species are transferred by a gas stream into the ionizer of a mass spectrometer of the ion-trap kind.

However, these methods can only achieve a limited spatial resolution due to the need for focusing the laser beam. In ordinary optical microscopy, resolution is limited by the diffraction limit. For the UV laser wavelengths commonly used, the theoretical resolution limit is in the range of 200 nm or more. In practice, however, resolutions of hardly better than 1 micrometer have been achieved.

A better lateral resolution may be obtained by secondary ion mass spectrometry (SIMS). In SIMS, a high-energy primary ion beam is used to irradiate a sample surface. By the impact of the primary ions, secondary ions are liberated from the sample surface, transferred to a mass spectrometer by application of electric fields, and mass analyzed. The achievable spatial resolution is primarily determined by the spot size of the primary ioh beam. In practice, the spot size can be brought down to approximately 100 nanometers, and therefore SIMS can offer a better spatial resolution than localized LDMS. However, in SIMS the sample must be investigated in ultrahigh vacuum, and the ionization process in SIMS leads to a relatively high degree of undesired fragmentation of the sample molecules, which further increases with molecular size. Therefore, SIMS is only of limited usefulness for localized chemical analysis, especially when applied to biological materials.

Recently it has become possible to perform mass spectrometry in an imaging mode, using a specially configured instrument and so-called stigmatic ion imag- ing. (Luxembourg SL, Mize TH, McDonnell LA, Heeren R.M.A., Anal. Chem, 76 (2004) 5339). This approach allows the recording of ion images in a fast, parallel fashion. However, the resolution that has been demonstrated is 1 μm or

more, and the technique is limited to samples being compatible with vacuum.

In order to overcome some of these disadvantages, it has been suggested to perform laser ablation by a scanning near-field optical microscopy (SNOM) setup and combine this with mass spectrometry. SNOM is the optical analog of scanning tunneling microscopy (STM) and atomic force microscopy (AFM). Using aperture probes, a resolution of « 50 nm becomes possible. It has been shown that with high transmission SNOM probes (Stδckle et al., Appl. Phys. Lett. 75 (1999) 160-162; Stόckle et al. J. Microsc. 194 (1999) 378-382), it is possible to effect laser ablation of organic surfaces with a spatial resolution of approximately 70 nm (Dutoit et al., J. Phys. Chem. B 101 (1997) 6955-6959).

The proof of principle that LDMS using SNOM probes was possible was given in (Kossakovski et al., Ultramicroscopy 71 (1998) 111-115) and US 6,080,586. Kossakovsi and co-authors placed an entire SNOM setup immediately in the region of the ion source of a time-of-flight (TOF) mass spectrometer. Practical problems inherent in this design limited the sensitivity, mass spectrometric performance, ease of operation, and in particular, the spatial resolution (to > 1 micrometer) in the setup described by Kossakovski et al. Furthermore, the sample had to be introduced into a region of high vacuum (in the range of 10 "6 mbar) needed for proper operation of the mass spectrometer which is a major limitation for the analysis of biological samples.

Some of these limitations were overcome in (R. Stόckle et al., Anal. Chem. 73 (2001), 1399-1402). Laser ablation through a SNOM probe was performed at ambient pressure, and neutral analyte molecules ablated by the SNOM tip were transferred into a quadrupole mass spectrometer (QMS), where they were ionized by electron impact ionization. In addition to providing a much simplified setup and easier access to the sample, sensitivity (approximately 1 - 2 amol) and spatial resolution (approximately 170 nm) were improved. A severe limitation of this setup, however, was the inability to scan the QMS during the transit time of the laser ablation products through the ionizer, which is in the range of

100 to 200 milliseconds.

Summary of the invention

It is therefore an object of the present invention to provide an improved device for localized mass analysis which combines high spatial resolution, high sensitivity and high mass resolving power. It is a further object of the present invention to provide a method for local mass analysis with these advantages.

These objects are achieved by a device with the features of claim 1 and a method as set forth in claim 11.

Advantageous embodiments of the invention are laid down in the dependent claims.

Thus, the device of the present invention comprises a setup for providing a localized light field for ablating/desorbing sample atoms and/or molecules at an impact spot on the sample surface. This setup comprises a light source, preferably a laser, more preferred a pulsed laser (having a wavelength in the UV 1 VIS or IR range), for generating light. The light either has a single predeter- mined wavelength (in the preferred case of a coherent light source like a laser) or a - preferably narrow - distribution of wavelengths with a mean wavelength corresponding to a predetermined wavelength. The device further comprises field localization means for concentrating the light to a field region with lateral dimensions on the range of the predetermined wavelength. In other words, the field localization means serve for generating a localized optical field which is essentially confined to a region on the sample surface whose lateral dimensions are on the range of the predetermined wavelength. The term "lateral dimensions on the range of a predetermined wavelength" is to be understood as meaning that the intensity of the light field falls off to below 50% of the peak intensity within a lateral range of less than ten times, better five times the predetermined wavelength, more preferred less than two times the wavelength, even more preferred less than the wavelength itself, most preferred less than 50% of the

wavelength. There are different ways of achieving this goal, e.g. by a lens arrangement, by a confocal microscopy setup etc.

In a preferred embodiment, the field localization means comprise a scanning near-field optical microscopy (SNOM) setup. In this case, the lateral dimensions of the field region are preferably less than two times the predetermined wavelength, more preferred less than the predetermined wavelength itself. Different kinds of SNOM setups are known. In one possible embodiment, the field localization means comprise an optical probe, preferably an optical fiber, coupled to the light source, the probe having a probe tip with an aperture which is smaller than the predetermined wavelength in all lateral directions, i.e., a so-called sub- wavelength aperture. In another embodiment (the so-called apertureless approach), the field localization means comprise a dielectric or conducting (generally metallic) tip, which serves as a means for localized field enhancement. This tip is irradiated by the light from the light source. The light may be focused to the tip by any conventional means (e.g., a lens arrangement).

Positioning means are provided for positioning the field localization means relative to the sample surface. In particular, these means are preferably adapted to position the field localization means and the sample surface relative to each other such that the sample surface is within the field region of the narrowly concentrated light field generated by the field localization means. Preferably, the positioning means are adapted to position the field localization means and the sample surface relative to each other at a distance which is smaller than the predetermined wavelength. The region of the sample surface irradiated by the concentrated light field will be called the impact spot of the light. Irradiation of the impact spot with the light field will lead to highly localized ablation (desorp- tion) of atoms and/or molecules from the sample surface, in neutral form, in (partially) ionized form or both. In the context of the present document, the term "atom" refers to an atom in an arbitrary electronic state (neutral or singly or multiply charged/ionized, positively or negatively). Likewise, the term "molecule" refers to a molecule in an arbitrary electronic state.

While the field localization provides for high lateral spatial resolution, the amount of sample atoms and/or molecules ablated during each laser pulse event is extremely small, often only in the attomole range. Therefore, efficient means for collecting the atoms and/or molecules and sensitive means for analyzing them are required.

The invention therefore suggests to further provide an ion trap for storing the sample atoms and/or molecules ablated from the surface in ionized form, trans- fer means for transferring the sample atoms and/or molecules from the sample surface, specifically, from the vicinity of the impact spot, to the ion trap, and an additional mass analyzer coupled to the ion trap for analyzing the sample atoms and/or molecules. By providing an ion trap, atoms and/or molecules ablated from the sample surface can be collected and accumulated efficiently and can be analyzed once a sufficient number of atoms and/or molecules have accumulated. This overcomes the sensitivity problems known in the prior art.

Advantageously, the device is adapted for keeping the sample at a pressure different from, specifically, higher than, the pressure within the ion trap and/or the mass spectrometer. To this end, the device further comprises vacuum generating means (usually one or more suitable pumps, preferably diffusion pumps) for creating a vacuum within the mass spectrometer and within the ion trap. The levels of vacuum (residual pressures) may be different in the mass spectrometer region, the ion trap region, and in the transfer means. Preferably the device is adapted for keeping the sample at ambient pressure. This allows even the analysis of live biological samples, which is impossible with any methods requiring the sample to be in vacuum.

Transfer of the sample atoms and/or molecules from the vicinity of the impact spot into the ion trap is advantageously achieved by "sucking" the sample atoms and/or molecules in the form of a gas stream into the ion trap. To this end, the transfer means advantageously comprise a transfer tube extending from a

first end disposed near the sample surface (specifically, the impact spot) to a second end. Transfer occurs by the effect of a positive pressure difference between the first and second ends. In addition, an electromagnetic field may be applied to the transfer tube for accelerating and guiding atoms and/or molecules in ionized form into and/or within the transfer tube.

For minimizing the associated vacuum leak while still efficiently collecting ablated atoms and/or molecules, the transfer tube preferably comprises a capillary portion near its first end having an inner diameter which is significantly smaller than an inner diameter of a portion of the transfer tube near its second end, preferably smaller by a factor of at least 10, more preferred at least 50. In absolute numbers, the inner diameter of the capillary portion is preferably in the range of 25 to 400 micrometers.

In order to minimize adsorption of the sample atoms and/or molecules to the inner walls of the transfer tube, the transfer means advantageously comprise heating means for heating the transfer tube.

Further provisions for enrichment of sample atoms/molecules with respect to the ambient gas during transfer are possible by the construction of the transfer tube and its interface to the vacuum of the analyzing unit. Specifically, in an advantageous embodiment, the transfer means comprise an intermediate vacuum chamber and tube means connecting said intermediate vacuum chamber to the ion trap, and the second end of the transfer tube is disposed in the intermediate vacuum chamber. Among other advantages, such a construction enables a better control of the flow of the gas containing the analyte molecules, and a better control of the associated vacuum leak of the ion trap. In an advantageous embodiment, a skimmer cone with a central through-hole is disposed in the intermediate vacuum chamber, wherein the through-hole is connected to the tube means which connect the intermediate vacuum chamber to the ion trap.

Additionally, a "push-pull" method may be applied, meaning that another capil-

lary opposite the (suction) capillary of the transfer tube provides a continuous flow of an auxiliary gas (e.g., nitrogen), directing the ablated material towards the suction capillary.

For positioning the inlet end (first end) of the transfer tube close to the impact spot of the laser beam on the sample surface, the device preferably comprises tube positioning means, preferably a micromanipulator, for positioning the first end of the transfer tube relative to the sample surface and/or relative to the field localization means. Preferably, the distance from the first end of the transfer tube to the impact spot (or to the field localization means) is of the same order as the inner diameter of the first end. Advantageously this distance is less than three times the diameter of the first end of the transfer tube, since the effect of active suction is limited in distance to only two to three times of the diameter of a capillary.

Preferably, the mass spectrometer is a time-of-flight (TOF) mass spectrometer. The TOF mass spectrometer may advantageously be of the linear or of the re- flectron type. TOF mass spectrometers are preferred since they provide for high sensitivity and short repetition rates for spectral acquisition (high duty cycles).

In order to avoid undesired direct transmission of sample atoms and/or molecules from the impact spot on the sample surface into the mass spectrometer without previous storage in the ion trap, the direction of entry of the sample atoms and/or molecules into the ion trap and the direction of exit of the ionized sample atoms and/or molecules into the mass spectrometer are preferably not collinear. Specifically, for a TOF mass spectrometer, the long axis of the mass spectrometer (in other words, the drift direction of the ionized atoms and/or molecules in the mass spectrometer) and the direction of entry of sample atoms and/or molecules into the ion trap are preferably not collinear. Preferably, these directions subtend an angle of at least 30 degrees, more preferred at least 60 degrees. It is most preferred that this angle is 90 degrees, i.e., that the sample atoms and/or molecules enter the ion trap in a direction that is perpendicular to

the direction of transfer of the ionized sample atoms and/or molecules into the mass spectrometer.

Preferably, the sample atoms and/or molecules are transferred into the ion trap in neutral or partially ionized form and ionized at least partially (preferably as efficiently as possible) after they have entered the ion trap. To this end, the device advantageously comprises ionizing means adapted for ionizing the sample atoms and/or molecules after entry into the ion trap.

A particularly high efficiency of ionization may be achieved by employing chemical ionization (Cl) directly in the storage region of the ion trap. To this end, the ionizing means preferably comprise gas supply means for supplying a chemical ionization gas to the ion trap and auxiliary ionizing means, e.g., an electron source, for at least partially ionizing the chemical ionization gas. The chemical ionization gas may be ionized within the ion trap or externally. The chemical ionization gas in ionized form is adapted for transferring charge to the sample atoms and/or molecules by chemical ionization.

For achieving precise positioning of the probe tip relative to the sample surface in direction of the long axis of the probe, the field positioning means advantageously comprise a piezo stage and a tuning fork. The tuning fork is advantageously excited at or near its eigenfrequency, and its damping is monitored. A measure of the damping is employed for monitoring the distance of the tip end to the sample surface.

In principle, any type of ion trap may be used. A cylindrical or hyperbolic RF ion trap is preferred.

The inventive method comprises the following steps: (a) Providing a light source, for generating light having a predetermined wavelength or having a distribution of wavelengths whose mean is at a predetermined wavelength, and field localization means for concentrating said

light into a localized optical field having an intensity distribution which is essentially confined to a field region with lateral dimensions in the range of said predetermined wavelength;

(b) Positioning said field localization means relative to said sample surface such that a first impact spot on said sample surface is disposed within said field region;

(c) Ablating sample atoms and/or molecules from said first impact spot by operating said light source;

(d) Transferring at least some of said sample atoms and/or molecules into an ion trap;

(e) At least partially ionizing said sample atoms and/or molecules in said ion trap;

(f) Accumulating said sample atoms and/or molecules in ionized form in said ion trap; (g) Transferring said sample atoms and/or molecules in ionized form from said ion trap into a mass spectrometer;

(h) Determining the mass distribution of said sample atoms and/or molecules in ionized form in said mass spectrometer;

(j) Moving said field localization means and said sample surface rela- five to each other such that a second impact spot on said sample surface different from said first impact spot is disposed within said field region;

(k) Repeating steps (c) to G) for a desired number of times.

For imaging applications this will be performed with as great a speed as possi- ble. In imaging applications, each mass distribution recorded by the mass analyzer or mass information derived thereof is correlated with the lateral position of the field localization means for which the mass distribution has been determined. Additionally, the mass spectra may be correlated with additional data obtainable from the SNOM directly and/or through other observations. Such additional data may comprise, e.g.:

- the position of the field localization means in the direction perpendicular to the sample surface (depth information), leading to topographic images

of the mass distribution with a possible resolution in the sub-micrometer range; - absorption characteristics of the surface, as obtainable by observation of transmitted light; - fluorescence characteristics of the surface, as obtainable by observation of fluorescence light (shifted in wavelength).

Brief description of the drawings

The invention will be described in more detail in connection with an exemplary embodiment illustrated in the drawings, in which:

Fig. 1 shows a highly schematic view of a device according to the present invention;

Fig. 2 shows a schematic detail view of a scanning near-field optical microscope; Fig. 3 shows a schematic perspective sectional view of a time-of-flight mass spectrometer coupled to an ion trap; Fig. 4 shows a schematic side sectional view of a time-of-flight mass spectrometer coupled to an ion trap;

Fig. 5 shows an enlarged schematic perspective sectional view of the ion trap;

Fig. 6 shows an enlarged schematic side sectional view of the ion trap;

Fig. 7 shows a schematic view of a differentially pumped transfer system and a micromanipulator coupled to the transfer system; Fig. 8 shows a schematic partial side sectional view of a transfer tube; Fig. 9 shows a flow diagram of a process according to the present invention; and

Fig. 10 shows a mass spectrum obtained with a device according to the present invention.

Detailed description of preferred embodiments

Fig. 1 shows a schematic diagram of a device according to a preferred embodiment of the present invention. The device comprises several key units inter-

faced with each other: a SNOM setup 2 for highly localized laser desorption or ablation; an ion trap system 4 comprising an ionizer 440 and a trap 410; a transfer system 5 for transferring desorbed atoms or molecules into the ion trap system 4; and a TOF mass spectrometer 3 interfaced with the ion trap system 4. These units are controlled by a common control unit 1. In the following, the device will be described in more detail, followed by a description of its operation.

The control unit 1 comprises a computer 100 with CPU and storage means, an input device (keyboard) 110 and a visual output device (monitor) 111. The inter- face between the computer and the units to be controlled may be achieved in any of the usual ways, e.g., by an I/O card present in the computer 100 providing for external analog or digital input and output lines, by Ethernet connection, by serial bus connection etc.

The SNOM setup 2 comprises a laser 201 (in the UV 1 VIS or IR wavelength range) for generating a pulsed laser beam, which is coupled into an optical fiber 202. The fiber has a tapered tip 203 with a sub-wavelength aperture facing a sample surface 204. The fiber 202 may be positioned relative to the sample surface 204 and scanned over the surface. To this end, it is connected to a tuning fork 211 which is mounted on a first piezo stage 212. The sample itself is mounted on a second piezo stage 205 for lateral scanning. The whole SNOM setup 2 is kept at ambient pressure, which provides for easy access and handling of the setup as well as for great versatility and allows analysis of delicate samples (i.e. live cells).

Fig. 2 shows a more detailed, yet schematic view of the SNOM setup 2. For achieving correct positioning of the fiber 202 relative to the sample 204 in the direction of the fiber axis (henceforth called the z direction), the tuning fork 211 is employed. The tuning fork 211 is excited at or near its eigenfrequency by a transducer 213 driven by an oscillator unit 222, and the damping of the tuning fork by shear forces is monitored in a position control unit 221 via pickup transducers (not shown in Figs. 1 and 2) and an amplifier 214. When the tip 203 ap-

proaches the sample surface 204 to within atomic dimensions, shear forces strongly increase, leading to a strong increase in damping of the tuning fork 211. The damping signal is used in the position control unit 221 , which drives the piezo stages 205 and 212. In this way, precise positioning in the z direction may be achieved. Operation of such a tuning-fork mechanism is well-known in the art.

A CCD camera (not shown in Figs. 1 and 2) allows observation of the sample during scanning and analysis. The SNOM is mounted on an inverted micro- scope which allows simultaneous acquisition of emission spectra of the sample (i.e., fluorescence) as a complementary imaging contrast.

Referring again to Fig. 1 , the ion trap system 4 comprises a vacuum chamber which contains the actual ion trap 410 and the ionizer 440. The trap 410 is op- erated by a trap operating unit 401 , while the ionizer is operated by a HV power supply 402 and a filament power supply 403. A gas reservoir in the form of a gas cylinder 421 contains an chemical ionization gas, which may be fed into the vacuum chamber by means of a valve 422 and a gas supply line 423. An appropriate vacuum is maintained in the vacuum chamber by means of a pump 430.

The TOF mass spectrometer 3 comprises a vacuum chamber which contains a flight tube 310 and a detector 320. A pump 330 maintains high vacuum, typically below 10 '5 mbar, in the vacuum chamber of the mass spectrometer 3.

Figs. 3 and 4 illustrate the setup of the ion trap system 4 interfaced with the mass spectrometer 3 in more detail. For simplicity, only the most important of those parts are shown in a schematic way which are situated inside the respective vacuum chambers. Figs. 5 and 6 show the region of the ion trap on a mag- nified scale.

With reference to Figs. 5 and 6, the ion trap is a radiofrequency (RF) ion trap as

it is well known in the art. It comprises a central metallic ring electrode 416 whose interior defines the storage region of the trap. On both sides of the ring electrode, axial end plates 411 , 413 are disposed, which are electrically insulated from the ring electrode 416. In the ring electrode 416, two radial bores are provided. Through the first of these bores, an inlet tube 520 is guided into the interior of the ring electrode 416, while the second bore serves to guide the supply line 423 for the chemical ionization gas into this region. The ion trap chamber can also be filled with a buffer gas for collisional cooling (i.e. Helium, Argon) in order to optimize system performance. The first (left) end plate 411 , which is the end plate more distant from the mass spectrometer, has a central opening 412 for admitting an electron beam into the interior of the central ring electrode 416. The second (right) end plate 413, which is the end plate facing the mass spectrometer, also has an opening 414, which serves to allow the ions stored in the trap to exit the trap.

An electron beam 444, whose spatial distribution is illustrated in Fig. 6, is generated by an ionizer 440, comprising an electron source in the form of a filament (not shown in Figs. 3 to 6) and plates 441 , 442, 443 for accelerating, gating and focusing the electrons released from the filament.

Now referring to Figs. 3 and 4, the mass spectrometer comprises a flight tube 310 which is closed off towards the ion trap by a front end plate 311. Ions are accelerated into the flight tube by a HV potential on a pressure orifice 312 in the front end plate 311 and drift through the flight tube 310 as an ion beam 340. At the far end of the flight tube 310, they are guided through a pair of focusing (steering) plates 313, 314 before hitting a dual MCP detector assembled in a Chevron stack 320 (i.e., the direction of the channel bias angle in the first MCP is opposite to the one in the second MCP). The ion extraction region of the mass spectrometer is formed by the end plates 411 , 413 of the ion trap 410.

Since vibrations, as they are typically associated with turbo-molecular pumps, are detrimental to the proper operation of the SNOM setup 2, the pumps 330

and 430 shown in Fig. 1 are preferably pumps with a low level of vibration, such as oil diffusion pumps. The inlet tube 520 constitutes a leak of the vacuum chamber of the ion trap system 4 towards the outside environment, and the pressure orifice 312 constitutes a leak of the vacuum chamber of the mass spectrometer 3 towards the vacuum chamber of the ion trap system 4. Therefore, the pumps need to have a sufficient capacity to keep the required level of vacuum despite these leaks. To this end, they are preferably mounted in a loca- ' tion close to the respective vacuum chamber (directly below each chamber) and have a pipe connection of a sufficiently large diameter. For generating a back- ing vacuum, a backing pump such as a rotary pump may be provided; however, this pump should be kept at a certain distance from the setup and physically well-separated from it, e.g., in a different room. Because the backing pump does not need to obtain a high level of vacuum (a residual pressure of, say, below 0.1 mbar will often be sufficient), the associated need of longer piping pre- sents no limitation.

The level of vacuum to be attained in the vacuum chamber of the TOF mass spectrometer is in the high vacuum range, e.g., below 10 "5 mbar, preferably below 10 ~6 mbar, in order to avoid collisions between residual gas molecules and the ions to be analyzed when they drift through the flight tube. In the ion trap system, however, requirements are less stringent, and a vacuum level of better than 10 ~2 mbar is sufficient. Preferably the pressure in the ion trap region is higher than in the TOF region since ion traps exhibit more favorable operation characteristics at elevated pressures. These levels can be attained by the use of standard oil diffusion pumps despite the presence of the above-mentioned leaks.

In a simplified embodiment, only one single vacuum pump might be employed for both vacuum chambers. This pump is then connected to the vacuum cham- ber of the mass spectrometer 3. Through the pressure orifice 312, it serves at the same time for evacuating the vacuum chamber of the ion trap system 4. Because of the incoming gas through the inlet tube 520 and the relatively small

diameter of the pressure orifice 312, the level of vacuum in this chamber will automatically be somewhat worse than in the TOF mass spectrometer. This, however, is tolerable in view of the less stringent requirements in this region.

Now referring to Figs. 7 and 8, the transfer system 5 is explained. The transfer system comprises a capillary 512 mounted in a transfer tube 511. The transfer tube may be closed by means of a valve 514. The transfer tube 511 leads into a tee piece 516 (e.g., DN 25 ISO-KF) which serves as an intermediate vacuum chamber of the transfer system. A vacuum is generated by a backing pump 530 (merely symbolized by an arrow in Fig. 7). This pump, with a flow capacity of preferably at least around 10 liters per second, generates a vacuum in the range of approximately 0.01 - 1 mbar. This is sufficient to suck atoms and/or molecules present at the tip of capillary 512 (on the left of Fig. 7) into the tee piece 516. This arrangement of capillary 512, transfer tube 511 and intermedi- ate vacuum chamber (tee piece) 516 may be regarded as a first stage of the transfer system 5.

The tee piece 516 is closed off towards the ion trap system 4 by a flange 519 (e.g., DN 100 ISO-K). A skimmer cone 518 is mounted on the flange 519. It has a central through-hole which leads into an inlet tube 520 leading, in turn, into the central ring electrode 416 of the ion trap 410. The atoms/molecules which have been sucked into the tee piece 516 are enriched with respect to the ambient air by passing through the skimmer cone 518. They are sucked into the through-hole of the skimmer cone 518 and into the inlet tube 520 by virtue of the pressure difference between the tee piece 516 and the vacuum chamber housing the ion trap 410. Thus, the atoms/molecules are transferred into the ion trap 410. The skimmer cone 518 with its through-hole and the inlet tube 520 may be regarded as a second stage of the transfer system 5.

The long axis of the inlet tube 520 extends in a perpendicular direction to the long axis of the flight tube 310 of the mass spectrometer. Thereby it is avoided that sample atoms/molecules entering the trap through the inlet tube 520 are

immediately sucked through the pressure orifice 312 into the mass spectrometer 3 by the pressure difference present between the ion trap system 4 and the mass spectrometer 3, before ionization and storage in the ion trap 410 have occurred. In addition, the high kinetic energy of the incoming atoms/molecules is slowed down by this perpendicular arrangement, which enhances the capability to efficiently trap them.

Fig. 8 shows a schematic partial side sectional view of an end portion of the transfer tube 511 with the capillary 512 mounted in it. The transfer tube is ta- pered at its end. The capillary 512 is attached to inside of the transfer tube 511. The inner diameter D of the transfer tube is approximately 1 to 5 mm, while the inner diameter d of the capillary is in the range of 25 to 400 micrometers. There is a factor of at least approximately 10 between these inner diameters. To minimize wall adsorption of the molecules/atoms, the transfer tube is made of AT steel, a stainless steel specially coated in that sense to suppress adsorption of the molecules to be transported. This successfully avoids fractionated sampling of the analyte. The capillary 512 may be welded into the transfer tube 511 , or it may be held in the transfer tube by other suitable means, e.g., a fixing bushing on the transfer tube into which the capillary is inserted, facilitating re- placement of the capillary.

To further minimize wall adsorption, the transfer system is heated. To this end, the capillary 512, the transfer tube 511 and the inlet tube 520 are surrounded by electric heating elements 513, 515, 517 and 521 , as they are well known in the art.

If the analyte atoms and/or molecules resulting from the ablation event are already (partially) ionized, the transfer system may additionally comprise means for applying electric voltages (e.g., between the tip of the capillary 512 and the sample holder) for accelerating and/or steering ionized species and thus efficiently transporting them into the ion trap.

In summary, by providing a differentially pumped two-stage transfer system which is additionally heated, a high transfer efficiency is achieved.

Operation of the device is illustrated in the flow diagram of Fig. 9.

In a first step 901 , the device is prepared for operation. In particular, it is ensured that a proper vacuum is maintained in the vacuum chamber of the mass spectrometer 3, and that the vacuum chamber of the ion trap system 4 is filled with the chemical ionization gas at a suitable pressure. The sample is placed onto the sample holder of the SNOM setup 2. The optical fiber 202 is positioned relative to the sample surface such that its tip 203 faces a first spot on the sample surface which is to be analyzed. This positioning is achieved by appropriately operating the piezo stages 205 and 212. In this way, the distance between aperture of the fiber tip and the sample surface may be precisely adjusted to below the near-field range of the aperture. Here, the near-field range is defined as less than the wavelength of the laser, preferably less than 20% of the wavelength; typically only a few nanometers distance are employed.

In step 902, a strong laser pulse is generated by the laser 201. This laser pulse is coupled into the fiber 202. Through the aperture of the fiber tip 203, the laser beam illuminates the sample spot on the surface. By interaction of the laser light with the surface matter, atoms and/or molecules at the sample spot are brought into the gas phase (i.e., ablated or desorbed).

In step 903, these atoms/molecules are sucked into the ion trap 410 by means of the transfer system. A pressure difference is present between the end of the capillary 512 which faces the sample spot and the end of the transfer tube 511 in the intermediate vacuum chamber (tee piece) 516. Likewise, a pressure difference is present between the tip of the skimmer cone 518 and the end of the inlet tube 520 which is near the ion trap 410. By the pressure differences, sample atoms/molecules are continuously sucked from the vicinity of the impact spot through the capillary 512 and the transfer tube 511 into the intermediate

vacuum chamber 516 and from there into the ion trap 410. If ionized species are to be transferred, they can additionally be focused and steered by an electrical field (potential between sample support and capillary) to increase the collection efficiency.

Once the neutral atoms/molecules have entered the ion trap, they are ionized by chemical ionization (Cl) in step 904. To this end, a chemical ionization gas (Cl gas) is present in the ion trap 410 at a suitable pressure. Since the capillary acts as a leak for the vacuum chamber of the ion trap, it is important that the pump 430 has sufficient capacity to maintain a proper vacuum level in the chamber, i.e., to efficiently remove the incoming gas. Since in this process the pump will also continuously remove some of the Cl gas, it is furthermore important that the Cl gas is replenished in sufficient amounts that a suitable partial pressure of the Cl gas is maintained during the ionization step 904. For ioniza- tion, the Cl gas is bombarded by electrons from the ionizer 440 and is thereby ionized. The CI gas then transfers charge to the sample atoms/molecules entering the ion trap 410 through the transfer tube 511. The appropriate partial pressure of the Cl gas depends on its exact nature. Suitable pressure ranges for chemical ionization are well known in the art.

For trapping the ionized sample atoms/molecules within the trap (step 905), the trap is operated by applying an AC voltage in the RF frequency range on the central ring electrode 416. The principles of operation of such an RF trap are well known in the art. The parameters of operation are chosen such that the trap is operated in a mass-selective manner resulting in that only ions with a mass in a predetermined mass window are efficiently held in the trap. It is well known in the art how such a mass selectivity may be obtained. By choosing the low mass cut-off higher than the mass of the Cl gas ions, only the ionized sample atoms/molecules are stored while Cl gas ions are not held in the trap. Just by the way of example, if the Cl gas is methane (M = 16), the low mass cut-off is chosen to be, say, 30 amu. If desired, it is also possible to ionize and trap the Cl gas before the laser ablation is performed; in this case an Cl gas with a mass

above the low mass cut-off is used. Suitable parameters for either trapping or rejecting the chemical ionization gas can be chosen for operation of the ion trap.

When the sample atoms/molecules which have resulted from the initial laser pulse arrive in the ion trap 410, the arrival times inevitably spread over a certain time range, which is typically in the range of 100 to 200 msec. For collecting as many of the atoms/molecules liberated from the sample surface as possible, the atoms/molecules, after having been ionized, are accumulated and held in the trap for a certain amount of time in step 905.

Once a sufficient number of sample atoms/molecules have accumulated in the ion trap 410, they are extracted and accelerated from the ion trap 410 by means of a voltage pulse applied to the end plates 411 and 413 of the trap, accelerated further by a constant HV potential on the pressure orifice 312 and transferred into the flight tube 310 of the TOF mass spectrometer 3 (step 906). Alternatively, more atoms/molecules may be collected by further laser pulses, thereby repeating steps 902 to 905 a certain number of times. In the extraction step, the ion trap 410 serves as an initial ion accelerator (ion extraction device) for the mass spectrometer 3, i.e., it takes over functions which normally would be as- sociated with the mass spectrometer 3. Still, the main acceleration occurs by the potential difference to the end plate 311 of the mass spectrometer containing the pressure orifice 312.

Once the ionized atoms/molecules have entered the flight tube 310, they drift along the flight tube 310 and hit the detector 320. Since the ionized atoms/molecules have all been accelerated through the same potential difference, they all have the same energy per unit of charge. Consequently, their drift velocities and, as a result, their drift times, depend on their mass. The detector 320 is of the well-known multi-channel plate (MCP) type, yielding electrical sig- nals which depend on the rate of ions hitting the detector. The distribution of impact events on the detector 320 over time is recorded by the MS operating unit 301 , from which a mass distribution is determined (step 907).

Subsequently, the fiber tip is moved to the next desired sample spot (step 908), and the procedure is repeated, starting from step 902. In this way, a region of the sample surface may be systematically scanned, and an image of the chemi- cal composition of the surface may be obtained.

A specific embodiment was set up in the laboratory as given in Table 1 and operated with parameters as given in Table 2.

Table 1: Parameters of a preferred embodiment

Table 2: Operational parameters of a preferred embodiment

In this specific embodiment, all voltage potentials and pulses that are required for the operation of the ion trap 4 and the mass spectrometer 3 are supplied by the MS operating unit 301. The unit also acquires the electrical signal of the MCP detector 320 with high time resolution. The unit parameters and timing are controlled by the central control unit 1 , to which the acquired raw data is also sent for further processing.

The MS operating unit 301 can be designed as an integrated unit, comprising all the power supplies, high-voltage RF generator, timing circuits, hardware acqui- sition and communication boards and a real-time operating software, or a group of different stand-alone units may be provided, such as digital delay pulse generators, different power supplies, high-voltage pulsers/switches, a RF exciter and high-voltage RF amplifier and a digital oscilloscope.

In the idle system state, the MS operating unit is configured to operate the ring electrode of the ion trap at a constant RF frequency and a given RF amplitude while the axial end plates 411 and 413 are kept at ground potential and no electrons of the ionizer 440 are allowed to pass into the interior of the ion trap.

Each MS acquisition cycle starts as soon as the MS operating unit 301 receives a trigger signal from the control unit 1 (at the same time the laser 201 is also triggered by the control unit). First, electrons are now allowed to pass into the ion trap by gating the plate 441 in order to ionize the Cl gas in the interior of the trap which will ionize the incoming sample molecules. All ionized species having a mass which is within the ion trap mass window for the set parameters (RF frequency and amplitude) will be stored in the ion trap. After a set amount of time, typically in the range of 3 - 1000 ms, electrons are no longer steered into the ion trap by changing the potential on the plate 441 back to the initial value. After another set amount of time, typically in the range of 1 - 10 ms, a trigger is sent to the RF generator which turns off the RF output (i.e., by gradually decreasing the RF amplitude to 0 Volts within a RF cycle). The phase of the RF signal is constantly being monitored in order to start the clamp-down of the RF

amplitude at a defined phase angle. This also allows to synchronize the HV extraction pulses on the axial end plates 411 , 413 with the RF phase and amplitude. The time of extraction in respect to the RF signal is a critical parameter in ion trap operation as it has a large influence on the obtained ion yield. An opti- mized extraction is typically achieved at phase angles between 90 - 140°. If the ion trap is operated in the positive ion mode, the extraction voltage on the plate 411 is switched in 200 ns from ground to a positive voltage, and on plate 413 from ground to a negative voltage. The time when the extraction is initiated also serves as the start time for the acquisition of the time-of-flight MS signal. After all the ions have reached the detector 320, acquisition is stopped, the MS time signal transferred to the control unit 1 , and all operating parameters of the MS operating unit are restored back to the idle state.

Fig. 10 shows a mass spectrum obtained with a setup according to the present invention. Intensity at the MCP is recorded as a function of the mass/charge ratio m/z. The sample consisted of anthracene (M=178). The mass spectrum exhibits a main peak at m/z=178 (i.e., at a ratio of mass to charge of 178 in atomic units), showing that primarily anthracene molecules are ablated by the laser at the impact spot, are transferred successfully by the transfer system into the ion trap, are ionized (singly charged) and stored in the ion trap, and are finally transferred into the mass spectrometer.

It is to be understood that the foregoing description refers to specific preferred embodiments, and that various modifications are possible without leaving the scope of the present invention.

In particular, while in the above-described embodiment the sample is kept at ambient pressure, the sample may equally well be kept at any different pressure, including reduced pressures.

While the above-described SNOM setup comprises an etched and metallized optical fiber, different kinds of optical probes with sub-wavelength apertures

may be used, specifically, hollow optical waveguides of the capillary type. The SNOM setup may be different from the one presently described, as long as it allows to generate a localized optical field with preferably sub-micrometer lateral resolution. Specifically, in an apertureless approach, a dielectric or conducting (e.g., metallic or metallized) tip or AFM cantilever is provided near the sample surface, and a laser beam is directed at the tip by conventional focusing techniques. The field in the immediate vicinity of the tip will be strongly enhanced by the presence of the tip, thus leading to strong localization of the optical field.

The transfer system of the above-described embodiment comprises a transfer tube through which the sample atoms/molecules are transported by a gas stream, preferably in neutral form. It is also possible to adjust the laser operating parameters (pulse energy, etc.) in a way that ions are produced directly during the ablation event. In this case the transport can be assisted by electromag- netic fields, specifically, by applying an electric field between the sample holder and the end of the transfer capillary facing the sample surface. While the setup of the transfer system as described above in connection with Fig. 7 is preferred, the transfer system may be simplified by leaving away the intermediate vacuum chamber (tee piece) 516 with its skimmer cone.

In the above-described embodiment, the sample atoms/molecules are ionized by chemical ionization. While this method is preferred due to the high efficiency of the method, different methods are possible, including direct ionization of the sample atoms/molecules by an electron beam, a laser beam or by impact on a heated surface. Ionization of the ablated neutrals is also possible before they enter the ion trap by different means, i.e. by a laser beam directed perpendicular through the transfer tube or by photoionization (i.e. with UV light). If chemical ionization is employed, any suitable Cl gas may be used, including methane, propane, isobutane, water, ammonia, helium, hydrogen, and many other well described in the prior art.

Instead of a cylindrical ion trap, any other type of ion trap, as they are well-

known in the art, may be used, including other types of quadrupolar RF ion traps.

While a TOF mass spectrometer is preferred for mass analysis, other types of mass analyzers may be used, as they are well known in the art. Mass analyzers which are generally of the TOF type are however preferred due to their achievable high mass range, ease and speed of operation (especially if compared to mass analyzers of the ion-trap kind) and high detection efficiency. In particular, instead of a linear TOF mass spectrometer, a TOF mass spectrometer of the reflectron type may be used, which may lead to improved mass resolution by a refocusing of an initial spatial spread of the ions to be analyzed. A further advantage of TOF mass spectrometers is that the ion trap may serve as the ion extraction region of the mass spectrometer, such that no further specific means for this purpose are needed.

List of symbols and abbreviations AFM Atomic force microscopy amu atomic mass unit

HV High voltage I. D. Inner diameter

I/O Input/Output

IR Infrared

LDMS Laser desorption mass spectrometry

LDI Laser desorption and ionization M Atomic/molecular mass

MALDI Matrix-assisted LDI

MCP Multi-channel plate

ME-SIMS Matrix-enhanced SIMS

MS Mass spectrometery QMS Quadrupole mass spectrometer

RF Radio frequency

SIMS Secondary ion mass spectrometry

SNOM Scanning near-field optical microscopy

STM Scanning tunneling microscopy

TOF Time of flight

UV Ultraviolet VIS Visible (wavelength range)

List of reference signs

1 Control unit

100 Computer 110 Keyboard

111 Monitor

Scanning near-field optical microscopy setup

201 Laser

202 Optical fiber

203 Fiber tip

204 Sample

205 Piezo stage

211 Tuning fork

212 Piezo stage

213 Transducer

214 Amplifier

215 Sample holder

221 Position controller

222 Oscillator unit

3 Time-of-flight mass spectrometer

301 MS operating unit

310 Flight tube

311 Front end plate

312 Pressure orifice

313 Accelerating/focusing plate

314 Focusing plate

320 Detector

330 Vacuum pump

340 Ion beam

4 Ion trap system

401 Trap operating unit

402 HV power supply

403 Filament power supply

410 Cylindrical ion trap

411 First end plate

412 Electron inlet opening

413 Second end plate

414 Ion outlet opening

415 Auxiliary plate

416 Ring electrode

421 Gas reservoir

422 Valve

423 Gas supply line

430 Vacuum pump

440 Ionizer

441 Filament

442, 443 Accelerating/focusing plates

5 Transfer system

501 Controller

510 Micromanipulator

511 Transfer tube

512 Capillary

513 Heating element for capillary

514 Valve

515 Heating element for transfer tube

516 Tee piece

517 Heating element for transfer tube

518 Skimmer cone

519 Flange

520 Inlet tube

521 Heating element for inlet tube

530 Vacuum pump

d Inner diameter of capillary

D Inner diameter of transfer tube

901 Positioning step

902 Laser ablation step

903 Transfer step

904 Ionization step

905 Storage step

906 Injection step

907 Mass analysis step

908 Repositioning/scanning step




 
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