The present invention relates to an ion source, to a mass
spectrometer system and to a method of matrix-assisted laser desorption ionization,
in the preferred embodiments to optical and spectroscopic systems, and more particularly,
but without limitation, to apparatus and a method of combined laser focusing and
spot imaging for matrix-assisted laser desorption ionization (hereinafter referred
to as MALDI).
Matrix-assisted ionization methods such as matrix-assisted
laser desorption ionization have proven useful in spectroscopic analysis of organic
and biologic compounds. In the MALDI technique, a sample is combined with an organic
matrix that co-crystallizes with the sample, and then deposited on a sample plate.
The sample plate may contain a large number of such samples, with each sample occupying
a small area on the surface of the plate. The sample plate is placed in a MALDI
ion source, where a laser beam directed at the sample vaporizes the matrix, and
ionizes the analyte compounds within the sample.
In a MALDI system, a laser beam is focused at a specific
target area on the sample plate containing a particular sample of interest. An imaging
device is setup to visualize the target area and the trajectory of the laser beam,
to locate the sample of interest and ensure it is in the target area, to confirm
that the laser beam is aligned correctly for impacting the sample in the target
area and also to view the interaction of the laser beam with the sample matrix.
In conventional MALDI sources, the laser beam that is used
to vaporize the sample and the optical radiation (usually visible light radiation)
which reflects off of the sample and is captured by the imaging device, follow separate
optical paths. In particular, the laser beam, which may comprise ultraviolet radiation,
is usually directed along a dedicated optical path separate from other optical paths.
Since the separation between these optical paths, misalignment errors, in which
the area on the sample plate surface viewed using the imaging device does not match
the target area impacted by the laser beam, can be difficult to avoid, with the
result that it is difficult to determine whether the laser beam is directed at the
sample of interest in the target area.
Moreover, employment of optical devices that can enhance
the resolution of such systems by allowing the viewing and the ionization of smaller
target areas and samples, such as powerful optical lenses that provide focusing
and magnification, is especially problematic in MALDI systems in which the laser
and visible optical paths are separate, since the employment of such devices in
either optical path (or both) can exacerbate the misalignment of the paths, or require
expensive and duplicative mechanisms for readjustment of the paths. Since increased
optical resolution can enhance the throughput and efficiency of MALDI sources, there
is a need for a MALDI system and method in which such misalignment problems are
not likely to occur, or are likely occur to a much more limited extent, enabling
the employment of optical devices that facilitate improved sample utilization and
The present invention seeks to provide an improved ion
source, mass spectrometer system and method of matrix-assisted laser desorption
ionization, as well as an improved MALDI.
In one aspect, the present invention provides an ion source
that comprises a sample plate for receiving a sample, a laser for producing laser
radiation to ionize the sample, a first optical element arranged so as to direct
the laser radiation along a first optical path towards the target area, and a second
optical element arranged along the first optical path to focus the laser radiation
onto the target area. The first and second optical elements are arranged such that
light that is reflected from the target area travels along the first optical path
through the first and second optical elements, the first optical element reflecting
the laser radiation along a first direction and transmitting the light reflected
from the target area that has traversed the first optical path in a second direction.
An imaging device for viewing the plate surface may be
arranged to receive the light that has been reflected from the target area and has
traversed the first optical path between the first and second optical elements in
the second direction.
In one embodiment, the ion source may further include a
third optical element arranged in the first optical path between the first and second
optical elements. The third optical element is arranged to reflect the laser radiation
directed in the first direction towards the second optical element and to direct
reflected light directed in the second direction towards the first optical element.
The first direction and the second direction may be perpendicular.
In one embodiment, the laser comprises ultraviolet (UV)
radiation and the third optical element comprises an ultraviolet (UV) mirror.
The first optical element may comprise a beam splitter
Advantageously, the laser radiation comprises ultraviolet
radiation and the second optical element comprises an ultraviolet (UV) lens.
The ion source preferably includes an optical filter element
arranged between the first optical element and the imaging device. The optical filter
element may comprise a polarization filter and/or an ultraviolet (UV) blocking filter.
There may be provided a lens element arranged adjacent
to the laser for focusing laser radiation and/or a lens element arranged adjacent
to the imaging device.
Preferably, there is provided an illumination device for
producing a light beam contacting a surface of the sample plate at a target area.
The illumination device may comprise a fibre optic light guide connected to a light
Advantageously, a lens element is arranged between the
optical fibre and the sample plate for focusing the light beam towards the target
The illumination device is preferably arranged with respect
to the sample plate such that it defines a grazing angle between the light beam
and the illuminated surface of the sample plate, the grazing angle being between
0 and 15 degrees.
In another aspect, the present invention provides a method
for matrix-assisted laser desorption ionization comprising directing ultraviolet
(UV) laser radiation along a first optical path to a target area to ionize a sample
in the target area, and capturing optical radiation reflected from the target area
that traverses the first optical path.
Preferably, the method includes focusing the ultraviolet
radiation traversing the first optical path onto the target area of the sample plate.
Advantageously, the method includes performing the focusing
using an ultraviolet (UV) lens arranged in the first optical path.
In an embodiment, the method includes focusing the ultraviolet
radiation at a target area below the surface of the sample plate.
The method may include producing an image of the target
area using the captured optical radiation.
According to another aspect of the present invention, there
is provided a mass spectrometer system as specified in claim 5.
Preferably, in the mass spectrometer the first optical
element comprises a beam splitter mirror.
The laser radiation from the mass spectrometer may comprise
ultraviolet radiation and the second optical element comprises an ultraviolet (UV)
The ion source of the mass spectrometer is preferably operated
at atmospheric pressure.
Embodiments of the present invention are described below,
by way of example only, with reference to the accompanying drawings, in which:
- FIG. 1 is a schematic illustration of an exemplary embodiment of a MALDI ion
source according to an embodiment of the present invention;
- FIG. 2 is a schematic illustration of another exemplary embodiment of a MALDI
ion source according to the present invention; and
- FIG. 3 is a schematic illustration of an exemplary mass spectrometer system
according to the present invention
It is to be understood that a reference to a singular item
herein includes the possibility that there is a plurality of the same items present.
More specifically, as used herein and in the appended claims, the singular forms
"a", "an", "said" and "the" may include plural referents unless the context clearly
FIG. 1 shows a schematic illustration of a first embodiment
of a MALDI ion source according to the present invention. The ion source 10 includes
a movable sample plate 15 having a surface containing one more spatially distinct
matrix-based analyte samples, an illumination device 20 that is positioned to cast
a light beam which contacts an area on the sample plate surface, and a laser source
30 that generates a beam of high-intensity coherent radiation directed onto a target
area on the sample plate surface. The ion source 10 also includes an imaging device
40 for imaging the area on the sample plate illuminated by the illumination device,
or a portion thereof. These elements are positioned with respect to one another
and further optical elements (described below) such that the laser radiation impinging
the target area of the sample plate, and light reflected by (or emitted from) the
sample plate and thereafter captured by the imaging device, partially share the
same optical path. It is noted that all of the above-described elements are not
necessarily (and generally are not) contained within an enclosed space or chamber.
For example, the imaging device and the laser source can both be positioned externally
to the chamber that contains the sample plate.
Referring again to FIG. 1, the illumination device 20 is
positioned adjacent to and distanced from the sample plate 15. The illumination
device 20 may be used to illuminate the sample plate 15 directly, or optical elements
such as an optical fibre 22 and/or a lens element 24 may be positioned adjacent
to the illumination device between it and the sample plate to enhance the directionality
and/or focus of the light emanating from the illumination device (hereinafter termed
"illumination radiation") prior to its reaching the surface of the sample plate.
A filter 28 may also be optionally included directly adjacent to the illumination
source 20 for filtering and/or polarizing the illumination radiation. In one embodiment,
the illumination source and associated optical is omitted and the target area is
illuminated with ambient light.
In an advantageous implementation, the illumination device
20 is arranged so that the illumination radiation contacts the sample plate surface
at a grazing angle of between 0 and 15 degrees as described in co-pending and commonly
U.S. Patent Application Serial No. 11/148,786
entitled "Ion Source Sample Plate Illumination System". It is emphasized
however, that this configuration is merely an advantageous implementation and should
not be regarded as limiting the scope of the teachings herein in any way.
Laser source 30 may be positioned so as to direct a laser
beam at an angle with respect to the direction of the illumination radiation when
the illumination is from a directed source. In the depicted embodiment, the laser
beam is approximately perpendicular to the illumination radiation, but this merely
represents one implementation and is also not to be regarded as limiting the scope
of the invention. The laser source 30 generates coherent radiation of an intensity
and frequency suitable for the vaporization of the matrix of the sample and the
subsequent ionization of the analyte molecules. In many spectroscopic applications,
ultraviolet radiation is found to have suitable photonic energy for the purposes
of matrix-assisted desorption and ionization.
The impact of the laser beam on the matrix causes vaporized
ions to flow off of the sample plate in a plume which are attracted by gas flows
and/or electrostatic forces present in the ion source 10 to a capillary 60. The
ions and any entrained gases are drawn through the capillary towards a mass analyzer
(not shown) by a pressure gradient.
A first optical element 32 is positioned between the laser
source 30 and the sample plate 15 in the initial path of the beam emitted from the
laser source. The first optical element 32 is semi-reflective and may comprise a
beam splitting mirror that can reflect a substantial portion of incident radiation
in the ultraviolet band and can also transmit a substantial portion of incident
radiation in the visible band. Suitable beam splitters are known in the optical
arts. A lens element 34 may be positioned adjacent to and in front of the laser
source to condition the laser beam on its initial path towards the first optical
element 32. The first optical element 32 may be oriented at an angle in a range
from 30 and 60 degrees to the initial path of the laser beam; in an advantageous
implementation the first optical element may be oriented at approximately 45 degree
to the path of the laser beam so that the incident laser beam is reflected in a
direction approximately perpendicular to its initial path. The laser beam reflected
off of the first optical element 32 travels along a 'first optical path' that extends
between the first optical element and a target area on the sample plate 15. The
direction of the reflected laser beam along the first optical path from the first
optical element 32 to the target area is hereby denoted as the 'first' direction
and the opposite direction which reflected optical radiation takes along the first
optical path from the target area to the first optical element 32 is denoted as
the 'second' direction. It is noted that while FIG. 1 shows the paths of the laser
beam travelling in the first direction and reflected light travelling in the second
direction along the first optical path as slightly spatially separated, this is
merely for illustrative purposes, and the laser radiation and optical radiation
A second optical element 38 is positioned in the optical
path further along in the first direction with respect to the first optical element
32, and may be positioned adjacent to the sample plate, depending on various optical
factors and parameters as is well known to those of skill in the art. In particular,
the 'working distance', which is the distance between the second optical element
38 and the target area on the sample plate, can be about 20 mm or greater. The second
optical element 38 is refractive and comprises one or more lens elements that are
effective with respect to the laser radiation, i.e., one or more ultraviolet lenses
if the laser comprises ultraviolet radiation. The second optical element 38 may
have high focusing and magnification power and may serve to focus the laser towards
a small target area on (or below) the sample plate for ionizing a selected sample
within the targeted area. The target area of the laser beam, through the focusing
power of the second optical element can be reduced to an area as small as 25 micrometers,
which can dramatically improve the sample resolution.
In the depicted embodiment, a third reflective optical
element 36, positioned between the first and second optical elements 32, 38, reflects
and redirects incident radiation. Preferably, the third reflective element 36 may
be effective in reflecting optical radiation in both the visible and ultraviolet
bands. The third optical element 36 allows for the convenient spacing of the arrangement
of the sample plate 15, illumination source 20, laser source 30 and imaging device
as described and depicted in FIG. 1.
Optical radiation travelling along the first optical path
in the second direction from the target area is reflected off of the third optical
element 36 towards the first optical element 32. A substantial portion of the optical
radiation is transmitted through the first optical element 32 towards imaging device
40. A filter element 42, which may comprise, for example, an ultraviolet blocking
filter and/or a polarization filter and an optical lens element 44 may be positioned
in between the first optical element 32 and the imaging device 40. The filter element
42 may block ultraviolet radiation and/or enhance the polarization of the optical
radiation that is transmitted from the first optical path through the first optical
element 32, removing extraneous radiation that can interfere with imaging. The optical
lens element 44 focuses transmitted optical radiation towards the light-detecting
elements of the imaging device 40.
The imaging device 40 may comprise any detection device
that is responsive to optical radiation including, for example, a camera, although
a camera that provides a digitized output is most readily employed, such as a charge-coupled
device (CCD) or a complementary metal-oxide semiconductor (CMOS) camera. The imaging
device may be coupled to a monitor outside of the ion source (shown in FIG. 3) for
In one embodiment, the optical radiation travelling along
the first optical path in the second direction may comprise fluorescence radiation
that is emitted from the target area in response to laser excitation, and the optical
elements 42, 44 and the imaging device 40 may be also selected for optimal transmission,
detection and observation of this phenomenon.
In terms of operation, the laser beam generated by the
laser source 30 is focused by lens element 34 and then reflected by the first optical
element 32 which redirects it at an angle along the first optical path in the first
direction. Along the first optical path, the laser beam is reflected by the third
optical element at an angle towards the target area on the sample plate 15. Along
the path to the target area, the laser beam passes through the second optical element
38 where it is focused, which reduces its diameter and eventually increases the
intensity of the beam before its impact at the target area.
The impact of the laser beam desorbs and vaporizes a substantial
portion of the matrix and analyte contained in the target area. Some of the molecules
of the matrix are also ionized by the laser beam; the matrix ions then ionize analyte
molecules by a process of charge transfer. The vaporized particles are released
as a plume and then the ions within the plume are guided electrostatically and/or
by gas flow towards the entrance of a capillary 60 that transports the ions towards
downstream stages of a mass spectrometer, including a mass analyzer. In addition,
if the matrix includes fluorescing compounds, the laser beam may excite such compounds
and they may emit fluorescence radiation in response to the laser excitation.
Simultaneously, the illumination radiation from the illumination
source is directed onto the sample plate 15 with the intention of illuminating the
area on the sample plate surface including the target area. The most important application
of the illumination is to locate the sample crystals in the target area; however,
the illumination also allows the impact of the laser beam on the sample to be captured
and recorded or viewed in real time via a monitor. As discussed above, the illumination
radiation may be filtered, guided and focused by optical elements 22, 24, 28 to
increase the focus and intensity of illumination at a small area of the surface
of the sample plate 15.
Illumination radiation is reflected, diffracted and/or
scattered from the surface of the sample plate 15 at or near the target area, and
a portion of this reflected optical radiation travels along the first optical path
in the second direction. Along the first optical path, the optical radiation is
focused by the second optical element 38, then reflected by the third optical element
36 towards the first optical element. A substantial portion of the optical radiation
is transmitted through the first optical element 32 towards the imaging device 40.
The optical radiation may be filtered and focused again by respective optical elements
42, 44 before reaching the imaging device 40.
According to this method, as long as the illumination radiation
encompasses the area in which the laser beam impacts the sample plate, a view of
the target area is captured by the imaging device because both the optical radiation
captured by the imaging device and the laser beam travel along the same optical
path and are modified by the same refractive optical element, i.e., the second optical
element 38 within that path. Conversely, as long as the imaging device 'views' the
sample of interest in the target area, the laser beam will be directed onto the
sample. For example, if the angle of the third optical element 36 is altered accidentally,
this alteration will alter the trajectory of the laser beam as it reflects off of
this element so that the target area of the laser beam will change. Equally, however,
any optical radiation reflected by the 'new' target area will have the same angular
trajectory as the laser beam as it travels from the surface of the sample plate
towards the third optical element 36, and then will be reflected by the altered
third optical element back towards the first optical element 32 towards the imaging
device 40. Thus, the MALDI source system of the present invention is self-correcting
in that the laser beam and the optical radiation travel along the same first optical
path, share common optics within that path, and automatically correspond to one
FIG. 2 illustrates another embodiment of the present invention
in which the third optical element is not employed. In this case the first optical
element 32 is oriented at about 45 degrees with respect to the laser beam so that
the laser beam is reflected directly towards the surface of the sample plate 15.
Thus, the first optical path in this case is the path from the first optical element
through the second optical element to the sample plate surface and there are no
intervening reflective elements between the first and second optical elements. Likewise,
reflected, scattered, diffracted or emitted optical radiation arising from the target
area of the sample plate 15 travels through the second optical element 38 directly
to the first optical element 32 in the opposite direction. In this embodiment, the
placement of the imaging device 40 differs from the first embodiment, and it is
rotated with respect to its position in the first embodiment in a clockwise direction
in a range between 20 and 70 degrees (depending on the angle of the first optical
element 32) to capture the optical radiation transmitted through the first optical
The system and method taught herein provide numerous conveniences
and advantages for performing MALDI. As noted above, misalignment errors are much
more easily avoided since the ion source contains one main optical path connecting
the first optical element with the target area. This eliminates visualization parallax
error. This is important for directing a laser accurately onto the target area.
In addition, use of one or more high magnification ultraviolet
lenses in the second optical element allows a much higher optical resolution to
be obtained as well as a convenient working distance of 20 mm or greater. The laser
beam can be focused using such lens elements to the degree that sub-portions of
a sample may be selected, or even portions located at some depth under the surface
of the sample target area. This may occur, for example, when it is desired to 'bombard'
crystalline structures embedded within a liquid matrix. The high magnification lenses
also allow very precise measurements of the depth and thickness of a sample as well
as precise size measurements comparable with the precision of motion of the sample
plate obtainable using state-of-the-art x/y stage motion control. Due to these technical
advantages, the number of target areas per sample plate can be increased by more
than a factor of ten. For example, sample plates having 96 sample areas are typically
used in MALDI ion sources; the improved laser and image focusing of the present
invention enables as many as 1536 sample areas to be deposited on a sample plate
and accurately targeted for ionization and imaging.
FIG. 3 schematically illustrates a mass spectrometer system
that employs a MALDI ion source described above with respect to FIG. 1. The mass
spectrometer 100 includes an ion source 10 and a mass analyzer 90 containing an
ion detector 92, connected by one or more intermediate chambers 80 (represented
in the figure by a single chamber) which may include one or more vacuum stages and
ion guides 82. An external monitor 70 may be coupled to the imaging element within
the ion source for observational purposes. However, it is again noted that several
of the elements depicted within the ion source enclosure in FIG. 3 can also be positioned
externally, such as the imaging device, the illumination device and the laser source.
A control system 110 may be coupled to the ion source 10,
and in particular may be coupled so as to receive input from the imaging device
and to transmit output control signals to the sample plate 15 within the ion source.
The control system may have stored algorithms for image recognition and automated
target acquisition, so that it can recognize from the image information captured
by the imaging device whether the target area on the sample plate includes the sample
of interest and can then (depending on the received input) transmit signals to adjust
the positioning of the sample plate in its plane in x and y directions using stage
motion control so that the sample crystals of interest in the target area may be
The mass analyzer 90 of the mass spectrometer 100 may comprise
a quadrupole, triple quadrupole, linear ion trap, three-dimensional ion trap, time-of-flight,
orbitrap, FT-ICR (Fourier transform ion cyclotron resonance) or other mass-to-charge
analyzer known in the art.
In use, if the MALDI ion source is used at atmospheric
pressure, the initial intermediate chamber 80 may be maintained at a pressure of
around two orders of magnitude below atmospheric and further intermediate chambers
are maintained at successively lower pressures. Mass analyzer 90 is generally maintained
at a pressure of about two to four order of magnitude below the intermediate chamber(s).
The ions generated in the ion source 10 enter the capillary and are swept into the
intermediate chamber 80, conditioned therein using ion guide(s) 82, and then transported
to the mass analyzer 90 where they are detected. The mass analyzer 90 determines
the m/z ratio of the ions, which may then be used to derive further information
about the samples from which the ions have been generated.
The disclosures in
United States patent application number 11/266,950
, from which this application claims priority, and in the abstract accompanying
this application are incorporated herein by reference.