LU505964B1

LU505964B1 - Post-ionization laser module for AP-MALDI ion source

Info

Publication number: LU505964B1
Application number: LU505964A
Authority: LU
Inventors: Mathieu Gerard; Maureen Feucherolles; Olivier Bouton; Gilles Frache
Original assignee: Luxembourg Inst Science & Tech List
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2025-06-30
Also published as: WO2025140913A1

Abstract

A post-ionization laser module (30) for docking an atmospheric-pressure MALDI ion source (10) comprises a pulsed post-ionization (PI) laser (32), a synchronisation device with a delay generator, for controlling a timing of laser pulses of the post- ionization laser with respect to desorption events in the MALDI ion source, and an optical system (46, 48, 50, 52) for directing the laser pulses on an optical path from the post-ionization laser into a desorption plume (26) of the MALDI ion source. In a further aspect, the invention relates to an AP-MALDI ion source (10), comprising a target holder (12) for mounting a sample target (16), a pulsed desorption laser (14) for irradiating the sample target and creating desorption events, and a post-ionization laser module (30).

Description

DESCRIPTION

Post-ionization laser module for AP-MALDI ion source

Background of the Invention

[0001] The invention generally relates to laser-induced post-ionization for matrix- assisted laser desorption ionization (MALDI) mass spectrometry. More specifically, the invention proposes a post-ionization laser module for an atmospheric-pressure MALDI (AP-MALDI) ion source.

[0002] MALDI is considered as a “soft” ionization technique, i.e., an ionization technique that allows creating ions from molecules with no to little fragmentation. Laser post-ionization has been used in vacuum MALDI ion sources, operating at pressures between ultra-high vacuum and intermediate pressure of about 1 to about 10 mbar.

[0003] WO 2010/085720 A1 relates to a method and apparatus for ionizing a neutral

MALDI desorption plume, and in particular, for efficiently measuring the ionized MALDI desorption plume when post-ionization techniques are combined with a medium- pressure MALDI-ion mobility orthogonal time-of-flight mass spectrometry (MALDI-IM- oTOFMS) instrument. Tissue-sample MALDI ions are separated by IM-oTOFMS according to their chemical family. After separation, the MALDI ions are directly compared to the ions created by post-ionizing the co-desorbed neutral molecules with a second laser that is delayed by a few hundred microseconds.

[0004] US 2022/285142 A1 discloses an apparatus to generate ions from sample material deposited on a substrate that is at least partially transparent to electromagnetic waves. The apparatus, which operates at pressures below 100 hPa, e.g., at 1-10 hPa, comprises a support device which has a holder for the substrate, a desorption/ionization unit comprising a desorption device and an ionization device, and an extraction device. The desorption device desorbs deposited sample material from a desorption site on the substrate using at least one energy burst. The ionization device irradiates the desorbed sample material above the substrate using electromagnetic waves passing through the substrate, before encountering the desorbed sample material, at a location which corresponds to the desorption site. The extraction device

Is arranged and designed to extract ions from the desorbed sample material and transfer them into an analyzer.

[0005] US 2011/049352 A1 discloses a desorption/ionization source operated under ambient conditions for direct analysis of samples on a surface. The source comprises of a laser desorption system and a UV/electrospray combined ionization system. A UV lamp, in particular a vacuum UV (VUV) lamp with shorter than 200 nm wavelength, is used for ionization.

[0006] The paper “MALDI-2 at Atmospheric Pressure — Parameter Optimization and

First Imaging Experiments”, by M. Niehaus et al., J. Am. Soc. Mass Spectrom. 2020, 31, 2287-2295, reports about the implementation of the laser post-ionization technique to an atmospheric-pressure MALDI ion source.

Summary of the Invention

[0007] In a first aspect, the invention relates to a post-ionization laser module for docking an atmospheric-pressure MALDI ion source. The laser module comprises a pulsed post-ionization (PI) laser, a synchronisation device with a delay generator, for controlling a timing of laser pulses of the post-ionization laser with respect to desorption events in the MALDI ion source, and an optical system for directing the laser pulses on an optical path from the post-ionization laser into a desorption plume of the MALDI ion source.

[0008] In a further aspect, the invention relates to an AP-MALDI ion source, comprising a target holder for mounting a sample target, a pulsed desorption laser for irradiating the sample target and creating desorption events, and a post-ionization laser module, the post-ionization laser module including a pulsed post-ionization laser, a synchronisation device with a delay generator, for controlling a timing of laser pulses of the post-ionization laser with respect to the desorption events, and an optical system for directing the laser pulses on an optical path from the post-ionization laser into a desorption plume of the MALDI ion source.

[0009] The post-ionization laser module may be conditioned as a standalone module for retrofitting an existing AP-MALDI ion source. The PI laser module may be conditioned as a box mountable and/or removable, as a whole, from the atmospheric- pressure MALDI ion source. The laser module may be conditioned as a box with one or more removable or openable walls. Additionally, or alternatively, the laser module may be equipped with one or more connectors matching with corresponding mounting points on the AP-MALDI source.

[0010] The laser module may comprise one or more interlocks for deactivating the PI laser when the box is opened. Deactivation may include turning the PI laser off or blocking its beam. When turning the PI laser off is not desired, e.g., to avoid warmup times, a beam shutter may be used to block the PI laser beam. In this case, the laser itself and the optical path up to the beam shutter is preferably placed into an internal enclosure configured to confine the optical power when the shutter is closed.

[0011] The optical system may comprise a focussing lens or mirror for focussing the laser pulses of the PI laser into the desorption plume of the MALDI ion source. The optical system may comprise a beam expander in the optical path between the PI laser and the focussing lens or mirror for widening the laser pulses prior to focussing. The beam expander could be realised as an optical telescope. Such a telescope could be implemented as a refractive telescope or a reflective telescope or as a telescope including a combination of refractive and reflective optical elements.

[0012] The beam expander may comprise an monodirectional beam expander, e.g., a telescope with cylindrical lenses and/or mirrors, or an anamorphic prism pair.

[0013] The synchronisation device may comprise one or more photodiodes for detecting the desorption events and/or the laser pulses of the post-ionization laser.

[0014] The PI laser module may comprise a controller configured to trigger the post- ionization laser at a set repetition rate when the desorption events in the MALDI ion source are paused, e.g., when the desorption laser of the MALDI ion source is stopped or temporarily deactivated. The set repetition rate may, e.g., be a user-set repetition rate or a “learned” repetition rate corresponding to the repetition rate of the desorption events in the MALDI ion source in a certain time window preceding the occurrence of the pause. The PI laser module may comprise a beam shutter controlled to close the

Pl beam path when the desorption events are paused and to open the PI beam path when the desorption events are resumed.

[0015] The synchronisation device may be configured to control the timing of the laser pulses of the PI laser with respect to the desorption events based upon an external trigger signal depending on the desorption events, and wherein the controller is configured to produce a substitute trigger signal when the external trigger signal drops out. The synchronisation device may comprise one or more photodiodes for detecting the desorption events and/or the laser pulses of the post-ionization laser. The external trigger signal could thus be provided by a photodiode arranged to detect the desorption laser pulses. Alternatively, the clock signal of the desorption laser of the MALDI ion source could be used as the external trigger signal. The substitute trigger signal may have the same repetition rate as the external trigger signal.

[0016] In the present document, the verb “to comprise” and the expression “to be comprised of” are used as open transitional phrases meaning “to include” or “to consist at least of”, not excluding the presence of further features or components. Unless otherwise implied by context, the use of singular word form is intended to encompass the plural, except when the cardinal number “one” is used: “one” herein means “exactly one”. Ordinal numbers (“first”, “second”, etc.) are used herein to differentiate between different instances of a generic object; no particular order, importance or hierarchy is intended to be implied by the use of these expressions. Furthermore, when plural instances of an object are referred to by ordinal numbers, this does not necessarily mean that no other instances of that object are present (unless this follows clearly from context). When this description refers to “an embodiment’, “one embodiment’, ‘embodiments’, etc., this means that the features of those embodiments can be used in the combination explicitly presented but also that the features can be combined across embodiments without departing from the invention, unless it follows from context that features cannot be combined.

Brief Description of the Drawings

[0017] By way of example, preferred, non-limiting embodiments of the invention will now be described in detail with reference to the accompanying drawings, in which:

Fig. 1: is a schematic illustration of an AP-MALDI ion source with a PI laser module docked to it.

Fig. 2: shows an optical image of mouse liver sections (a) and molecular images obtained by AP-MALDI (b), (c) and (d), with (left-hand sections) and without (right- hand sections) laser post-ionization.

Detailed Description

[0018] Fig. 1 schematically shows an AP-MALDI ion source 10 retrofitted with a PI laser module 30. The AP-MALDI ion source 10 comprises a target holder 12, a pulsed laser 14 (“desorption laser”) for ablation and desorption of a MALDI target 16, optical components, such as, e.g., one or more mirrors 18 and/or lenses 20, for guiding and focussing the desorption laser beam 22 onto the target 16. The target 16 comprises a carrier plate coated with the matrix material having the analyte(s), e.g., a peptide sample, embedded therein or a matrix-coated tissue section. Opposite the target 16, the ion source 10 comprises a capillary extender 24, for directing the ionized species to spectrometer (not shown). The target holder 12 comprises a motorized XY -stage for moving the target 16 under the laser focus.

[0019] The laser module 30 comprises a pulsed post-ionization (PI) laser 32, a laser controller 34 and optical components for guiding the pulsed PI laser beam 36 into the desorption plume 26 of the MALDI ion source 10. The laser controller 34 comprises an external or internal synchronisation device featuring a delay generator, for controlling the timing of the PI laser pulses with respect to desorption events in the MALDI ion source 10.

[0020] The synchronisation device may comprise or be connected to one or more photodiodes 28 arranged so as to detect the desorption events and/or the laser pulses of the Pl laser. Each time a desorption laser pulse hits the MALDI target 16, the one or more photodiodes 28 provide a voltage or current pulse. The one or more photodiodes 28 thus provide an (external) trigger signal each time a desorption event occurs. The synchronisation device uses this trigger signal as a reference to control the timing of the Pl laser pulses, in particular the delay between the pulse train of the desorption laser pulses and the pulse train of the PI laser pulses. It should be noted that the controller 34 of the PI laser 32 may be interfaced with the controller (not shown) of the desorption laser 14. In particular, the controller 34 may be provided with the clock signal of the desorption laser 14. The delay between the pulse train of the desorption laser pulses and the pulse train of the PI laser pulses may be set by a user, e.g., via an HMI (human machine interface) of the laser controller 34. The controller 34 may be configured to produce a substitute trigger signal when the external trigger signal drops out, so as to maintain the PI laser pulse train when there are no desorption events.

This feature keeps the PI laser 32 running in a stable state and avoids warm-up or reinitialization times of the PI laser 32 when desorption events reoccur. The controller may be configured to monitor the delay between the clock signal of the desorption laser 14 and the desorption events. When the trigger signals corresponding to the desorption events drop out while the clock signal of the of the desorption laser 14 remains active,

the controller 34 may be configured to use the clock signal of the desorption laser 14 as the substitute trigger signal in such as way as to maintain the delay between the clock signal of the desorption laser 14 and the clock signal of the PI laser 32 substantially constant. The controller 34 may further be configured to turn the PI laser 32 off when the desorption laser 14 is turned off.

[0021] As shown in the illustrated embodiment, the PI laser module 30 may be configured as a standalone module that may be mounted on the AP-MALDI ion source and removed from it when no longer needed. The Pl laser module 30 may be configured as a box with one or more walls 38 that a user may remove or open to gain 10 access to the interior of the PI laser module 30.

[0022] The Pl laser module 30 may be mounted on the AP-MALDI ion source 10 by means of a mounting shoe 42 fixed on the AP-MALDI ion source 10 cooperating with corresponding connector rails 44 fixed on the PI laser module 30. The mounting shoe 42 may comprise a stop or a catch that defines the proper mounting position of the PI laser module 30, and, optionally, secures it in that position. In other embodiments, the

Pl laser module 30 may be mounted on the AP-MALDI ion source 10 by screws or other connectors matching with corresponding mounting points on the AP-MALDI source 10.

[0023] The PI laser module 30 may comprise one or more interlocks 40 for deactivating the PI laser 32 when the box is opened, i.e., when one or more of its walls 38 are open or removed. Additionally, or alternatively, PI laser module 30 may comprise one or more interlocks cooperating with the mounting shoe 42 and the connector rails 44 for deactivating the PI laser when the Pl laser module 30 is not completely in the mounting shoe 42, i.e., when the PI laser module 30 is not in the correct mounting position. Deactivation may be effected by turning the Pl laser off or controlling a shutter to block the PI laser beam 36. The use of a beam shutter may be preferred when warm-up or reinitialization times of the Pl laser 32 are not desired.

[0024] The optical system of the illustrated PI laser module 30 includes several mirrors 46, 48, 50, a beam expander 52 and a focussing element 54 (in the illustrated embodiment, the focussing element is a lens but in a different optical setup, it would also be possible to use a focussing mirror). As in the illustrated embodiment, the beam expander 52 may comprise a telescope. Various telescope configurations exist and could be used in the context of the invention. In the illustration, the telescope comprises a defocussing lens 56 to widen the PI laser beam 36 and a focussing lens 58 to collimate the expanded beam. After the beam expander 52, the PI laser beam 36 passes the focussing lens 54 and is then reflected on mirror 50, which is arranged and oriented so as to direct the PI laser beam 36 parallel to the surface of the target 16 into the desorption plume 26 of the AP-MALDI ion source 10. An aperture 60 is provided in the wall of the AP-MALDI ion source 10 for passing the PI laser beam into the interior of the AP-MALDI ion source 10. The adjacent wall 62 of the PI laser module 30 has therein a corresponding aperture 64.

[0025] According to a preferred embodiment, the beam expander is a monodirectional beam expander, i.e., a beam expander that expands the PI laser beam in one transversal direction. Consequently, an initially (nearly) Gaussian PI laser beam is transformed into an elliptical beam. In the waist, the beam diameter is thus smaller in the direction with the greater diameter in the far field. The monodirectional beam expander is preferably oriented in such a way that the beam waist in the transversal direction parallel to the target surface is smaller than the beam waist in the transversal direction orthogonal to the target surface. It should be noted that the beam expander in this case needs to be oriented taking into account the optical path between the beam expander and the target and the orientation of the target relative to the beam. A monodirectional beam expander could, e.g., comprise a telescope with cylindrical lenses or mirrors, or an anamorphic prism pair.

[0026] The Pl laser module 30 preferably comprises a communications interface 66, which may include a serial port, a parallel port, a USB port, a network interface, and/or a wireless communications interface, etc., allowing a user to configure the controller 34 and/or the synchronization device via a connected electronic device, such as, e.g., acomputer, a laptop computer, a tablet computer, a smart phone, or the like.

Example

[0027] A compact post-ionization module for the AP-MALDI UHR ion source from

Masstech Inc. was developed. This AP-MALDI ion source may be switched between

MALDI and LC/MS-MS configurations in a matter of seconds. A hole has been machined in the lower part of the source, below the mass spectrometer inlet, to allow the Pl laser beam to enter the AP-MALDI chamber along the sample holder.

[0028] The Pl laser module was equipped with a diode-pumped passively Q-switched solid-state Nd:YAG laser (FQSS 266-Q4, CryLaS, Germany) operating at the wavelength of 266 nm. The maximal pulse repetition of the PI laser is 1 kHz. The PI laser and the desorption laser of the AP-MALDI ion source were synchronized by using the external trigger signal with a controlled delay. A synchronization device including a delay generator was connected between the controller of the desorption laser and the controller of the Pl laser. The effective delay between the laser pulse trains was monitored by two photodiodes (one monitoring the desorption laser, the other monitoring the PI laser) connected to the synchronization device and to an external digital oscilloscope. The synchronization was carried out by a software program running on the synchronization device that allows the user to select the delay between the two laser pulse trains. Due to the type of the Pl laser (passively Q-switched laser), the internal delay between the external trigger and the release of a laser pulse was not stable immediately after startup (during a few seconds) but was constant thereafter.

To reduce these startups to a minimum, the synchronization device was configured to maintain the train of clock pulses for the Pl laser with the same rate and timing when the external trigger pulses dropped out. This way, the Pl laser was kept operating when there were no desorption events in the AP-MALDI ion source, and restarts of the PI laser could be avoided, in particular during the times the MALDI target was moved to anew position, e.g., between consecutive line scans. As soon as the external trigger reappeared, the synchronization device synchronized to the external trigger and clocked the PI laser with the delay previously set and stored.

[0029] To decrease the beam size at the point of focus, and decrease the depth of focus, the laser beam was shaped with a Galilean telescope, composed of a plano- concave cylindrical lens (LK4351 from Thorlabs, f = -8 mm) and a plano-convex cylindrical lens (LJ4878 from Thorlabs, f= 75 mm), followed by a plano-convex focusing lens (#48-288 from Edmund Optics, f = 150 mm). All optical components (lenses and mirrors) were selected in accordance with the wavelength of the PI laser.

The telescope lenses, the focusing lens and the last mirror bringing the PI laser beam infront of the mass spectrometer inlet were mounted on manual micro-positioners and goniometer stages, respectively. After alignment of the PI laser beam, the PI laser beam intersected the desorption plume in its focus at a distance of approximately 500 um from the MALDI target.

[0030] The dimensions of the PI laser module were 29 cm x 17.5 cm x 16.5 cm. The

Pl laser module was attached to the AP-MALDI source with four screws.

[0031] The AP-MALDI ion source equipped with the PI laser module according to the example was connected to a mass spectrometer (Orbitrap Exploris™ 480 from Thermo

Fisher Scientific Inc.) and several experiments were conducted.

[0032] AP-MALDI experiments were carried out using a standard mixture of deuterated lipids in a suitable matrix. The target was analyzed without post-ionization and with post-ionization. The settings of the AP-MALDI ion source were the same for both analyses and were kept optimal for post-ionization experiments. It was found that post-ionization significantly enhanced the signal intensities for certain classes of lipids (improvement by factors >20), or allowing the detection of other classes of lipids, which could not be detected without laser post-ionization.

[0033] These findings were confirmed in another experiment where an AP-MALDI ion source equipped with the PI laser module according to the example was used for molecular imaging of mouse liver sections. Fig. 2 shows (a) an optical image of the mouse liver sections that were subjected to the AP-MALDI experiments. The section on the left was analyzed with laser post-ionization switched on whereas the section on the right was analyzed under the same conditions, except that laser post-ionization was switched off. Fig. 2 (b) shows the signal at m/z = 764.522, identified as PC(35:6),

Fig. 2(c) the signal at m/z = 947.6530, identified as TG(57:12), and Fig. 2(d) the signal at m/z = 943.6254, identified as PI(40:0). It can be observed (Fig. 2(b)) that laser post- ionization produces significant signal enhancement, which leads to an improved visualization of the distribution of molecules within the tissue under investigation. As evidenced by Fig. 2(c) and (d), AP-MALDI with post-ionization allows detecting a higher number of species (and their distribution within the tissue) than AP-MALDI without post-ionization.

[0034] While specific embodiments and examples have been described herein in detail, those skilled in the art will appreciate that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure.

Accordingly, the particular arrangements disclosed are meant to be illustrative only and not limiting as to the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalents thereof.

Claims

1. A post-ionization laser module for docking an atmospheric-pressure MALDI ion source, comprising: a pulsed post-ionization laser, a synchronisation device with a delay generator, for controlling a timing of laser pulses of the post-ionization laser with respect to desorption events in the MALDI ion source, an optical system for directing the laser pulses on an optical path from the post- ionization laser into a desorption plume of the MALDI ion source.

2. The post-ionization laser module as claimed in claim 1, conditioned as a box with one or more removable or openable walls.

3. The post-ionization laser module as claimed in claim 2, comprising one or more interlocks for deactivating the post-ionization laser when the box is opened.

4. The post-ionization laser module as claimed in any one of claims 1 to 3, wherein the optical system comprises a focussing lens or mirror for focussing the laser pulses of the post-ionization laser into the desorption plume of the MALDI ion source.

5. The post-ionization laser module as claimed in claim 4, wherein the optical system comprises a beam expander in the optical path between the post- ionization laser and the focussing lens or mirror for widening the laser pulses prior to focussing.

6. The post-ionization laser module as claimed in claim 5, wherein the beam expander comprises an monodirectional beam expander, e.g., a telescope with cylindrical lenses or mirrors, or an anamorphic prism pair.

7. The post-ionization laser module as claimed in any one of claims 1 to 6, wherein the synchronisation device comprises one or more photodiodes for detecting the desorption events and/or the laser pulses of the post-ionization laser.

8. The post-ionization laser module as claimed in any one of clams 1 to 7, comprising a controller configured to trigger the post-ionization laser at a set repetition rate when the desorption events in the MALDI ion source are paused.

9. The post-ionization laser module as claimed in claim 8 wherein the synchronisation device is configured to control the timing of the laser pulses of the post-ionization laser with respect to the desorption events based upon an external trigger signal depending on the desorption events, and wherein the controller is configured to produce a substitute trigger signal when the external trigger signal drops out.

10. The post-ionization laser module as claimed in claim 9, wherein the substitute trigger signal has the same repetition rate as the external trigger signal.

11. An atmospheric-pressure MALDI ion source, comprising a target holder for mounting a sample target, a pulsed desorption laser for irradiating the sample target and creating desorption events, and a post-ionization laser module, the post-ionization laser module including a pulsed post-ionization laser, a synchronisation device with a delay generator, for controlling a timing of laser pulses of the post-ionization laser with respect to the desorption events, and an optical system for directing the laser pulses on an optical path from the post-ionization laser into a desorption plume of the MALDI ion source.

12. The atmospheric-pressure MALDI ion source as claimed in claim 11, wherein the post-ionization laser module is conditioned as a box removable as a whole from the atmospheric-pressure MALDI ion source, the box comprising one or more removable or openable walls.

13. The atmospheric-pressure MALDI ion source as claimed in claim 12, wherein the post-ionization laser module comprises one or more interlocks for turning the post-ionization laser off when the box is opened.

14. The atmospheric-pressure MALDI ion source as claimed in any one of claims 11 to 13, wherein the optical system comprises a focussing lens or mirror for focussing the laser pulses of the post-ionization laser into the desorption plume of the MALDI ion source.

15. The atmospheric-pressure MALDI ion source as claimed in claim 14, wherein the optical system comprises a beam expander in the optical path between the post-

ionization laser and the focussing lens or mirror for widening the laser pulses prior to focussing.

16. The atmospheric-pressure MALDI ion source as claimed in claim 15, wherein the beam expander comprises an monodirectional beam expander, e.g., a telescope with cylindrical lenses or mirrors, or an anamorphic prism pair.

17. The atmospheric-pressure MALDI ion source as claimed in any one of claims 11 to 16, wherein the synchronisation device comprises one or more photodiodes for detecting the desorption events and/or the laser pulses of the post-ionization laser.

18. The atmospheric-pressure MALDI ion source as claimed in any one of claims 11 to 17, wherein the post-ionization laser module comprises a controller configured to trigger the post-ionization laser at a set repetition rate when the desorption events in the MALDI ion source are paused.

19. The atmospheric-pressure MALDI ion source as claimed in claim 18, wherein the synchronisation device is configured to control the timing of the laser pulses of the post-ionization laser with respect to the desorption events based upon an external trigger signal depending on the desorption events, and wherein the controller is configured to produce a substitute trigger signal when the external trigger signal drops out.

20. The atmospheric-pressure MALDI ion source as claimed in claim 19, wherein the substitute trigger signal has the same repetition rate as the external trigger signal.