U.S. patent application number 13/656284 was filed with the patent office on 2013-04-25 for mass spectrometer with maldi laser system.
This patent application is currently assigned to Bruker Daltonik GmbH. The applicant listed for this patent is Bruker Daltonik GmbH. Invention is credited to Andreas Haase, Jens Hoehndorf.
Application Number | 20130099112 13/656284 |
Document ID | / |
Family ID | 47225444 |
Filed Date | 2013-04-25 |
United States Patent
Application |
20130099112 |
Kind Code |
A1 |
Haase; Andreas ; et
al. |
April 25, 2013 |
MASS SPECTROMETER WITH MALDI LASER SYSTEM
Abstract
The invention relates to a mass spectrometer comprising a laser
system for mass-spectrometric analyses with ionization of analyte
molecules in a sample by matrix-assisted laser desorption. A mass
spectrometer with a pulsed UV laser system produces a spatially
distributed spot pattern with peaks of uniform energy density on
the sample, increasing thereby the degree of ionization for analyte
ions as compared to conventional spot patterns. The spot pattern
with peaks of uniform energy density can be produced by homogeneous
illumination of a pattern generator, for example a lens array. The
homogeneous illumination can be generated by a low-cost
beam-shaping element, which does not act on the UV beam but on the
original infrared beam, in conjunction with changes to the beam
cross-section and beam profile brought about by the nonlinear
conversion crystals. This beam shaping not only produces a beam
profile which illuminates the pattern generator homogeneously with
low losses, but at the same time increases the efficiency of the
frequency multiplication and the lifetime of the conversion
crystals so that cost savings are achieved because less laser
energy is required and the lifetime is increased.
Inventors: |
Haase; Andreas; (Bremen,
DE) ; Hoehndorf; Jens; (Bremen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bruker Daltonik GmbH; |
Bremen |
|
DE |
|
|
Assignee: |
Bruker Daltonik GmbH
Bremen
DE
|
Family ID: |
47225444 |
Appl. No.: |
13/656284 |
Filed: |
October 19, 2012 |
Current U.S.
Class: |
250/282 ;
250/288 |
Current CPC
Class: |
H01S 5/005 20130101;
H01J 49/164 20130101; G01N 2001/045 20130101; H01S 5/0092
20130101 |
Class at
Publication: |
250/282 ;
250/288 |
International
Class: |
H01J 49/40 20060101
H01J049/40 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 19, 2011 |
DE |
102011116405.0 |
Claims
1. A mass spectrometer comprising a laser desorption ion source
having a laser system for the ionization of a sample by
matrix-assisted laser desorption, comprising: a pulsed solid-state
laser system; conversion crystals for increasing the frequency, and
a pattern generator in the frequency-increased laser beam, wherein
a beam-shaping optical device is located between the solid-state
laser system and the conversion crystals, which converts the pulsed
laser beam having a Gaussian profile into an approximately
rectangular beam with approximately homogeneous energy density.
2. The mass spectrometer of claim 1, wherein the solid-state laser
system generates a pulsed infrared beam and the conversion crystals
increase the frequency of the laser light by a factor of three
resulting in a UV laser wavelength in the range between about 300
and 450 nanometers.
3. The mass spectrometer of claim 1, wherein the beam-shaping
optical device comprises a lens operating with refraction.
4. The mass spectrometer of claim 1, wherein the beam-shaping
optical device comprises an diffractive optical device.
5. The mass spectrometer of claim 1, comprising two nonlinear
conversion crystals to increase the beam frequency, operating with
walk-off compensation.
6. The mass spectrometer of claim 1, comprising a pattern generator
generating a spot pattern with one of 4, 7, 9, 16, 19, and 25
spots.
7. The mass spectrometer of claim 6, wherein the pattern generator
comprises a lens array.
8. The mass spectrometer of claim 6, comprising a telescope and an
optical lens system, which image the spot pattern onto the
sample.
9. The mass spectrometer of claim 8, wherein the spots on the
sample have diameters of about 10 micrometers at most.
10. The mass spectrometer of claim 1, wherein the laser system is
configured to generate a sequence of laser light pulses with a
pulse rate in the range of about 1 to 10 kHz.
11. The mass spectrometer of claim 1, further comprising a
rotatable mirror system located between the pattern generator and
the telescope, which can be used to control the spot positions on
the sample.
12. A laser system for the ionization of a sample by
matrix-assisted laser desorption in a mass spectrometer, with a
pulsed solid-state laser system, conversion crystals to increase
the frequency, and a pattern generator in the frequency-increased
laser beam, comprising a beam-shaping optical device between the
solid-state laser system and the conversion crystals, which
converts the pulsed laser beam having a Gaussian profile into an
approximately rectangular beam with approximately homogeneous
energy density.
13. A method for the ionization of a sample by matrix-assisted
laser desorption in a mass spectrometer, comprising: providing a
sample with analyte molecules; and ionizing the analyte molecules
with a laser system for the ionization of a sample by
matrix-assisted laser desorption in a mass spectrometer, with a
pulsed solid-state laser system, conversion crystals to increase
the frequency, and a pattern generator in the frequency-increased
laser beam, comprising a beam-shaping optical device between the
solid-state laser system and the conversion crystals, which
converts the pulsed laser beam having a Gaussian profile into an
approximately rectangular beam with approximately homogeneous
energy density, and subsequently measuring the ionized analyte
molecules mass-spectrometrically.
Description
PRIORITY INFORMATION
[0001] This patent application claims priority from German Patent
Application No. 10 2011 116 405.0 filed on Oct. 19, 2011, which is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to a mass spectrometer comprising a
laser system for mass-spectrometric analyses with ionization of
analyte molecules in a sample by matrix-assisted laser
desorption.
BACKGROUND OF THE INVENTION
[0003] During the past twenty years, two methods have become
established in the mass spectrometry of biological macromolecules:
ionization by matrix-assisted laser desorption (MALDI), and
electrospray ionization (ESI). The biological macromolecules to be
analyzed are termed analyte molecules below. In the MALDI method,
the analyte molecules are generally prepared on the surface of a
sample support in a solid, polycrystalline matrix layer, and are
predominantly ionized with a single charge, whereas in the ESI
method they are dissolved in a liquid and are ionized with multiple
charges. The two methods have made it possible to conduct
mass-spectrometric analysis of biological macromolecules for
genomic, proteomic and metabolomic investigations; their inventors,
John B. Fenn and Koichi Tanaka, were awarded the Nobel Prize for
chemistry in 2002.
[0004] In a MALDI sample preparation, there are 10.sup.3 to
10.sup.5 times as many matrix molecules as analyte molecules, and
the matrix molecules form a polycrystalline layer in which the
analyte molecules are integrated in the interior of the crystals or
at their grain boundaries, largely without coming into contact with
other analyte molecules. The matrix substance is selected in such a
way that its molecules can absorb the ultraviolet light of the
laser pulse, on the one hand, and can protonate the analyte
molecules on the other. In the prior art, a small illumination area
(called "spot" in the following), with a diameter of around 50 to
200 micrometers, on the prepared MALDI sample is briefly irradiated
with a laser pulse, that is strongly absorbed by the matrix
molecules. The pulsed irradiation converts surface matrix material
from the solid state into the plasma phase in only a few
nanoseconds, during which many matrix molecules (or their
fragments) are thermally ionized. The analyte molecules are usually
ionized by being protonated or deprotonated in reactions with
matrix molecules or matrix ions in the dense plasma. The plasma
cloud expands into the vacuum in a few hundred nanoseconds, while
all the molecules are continuously accelerated by friction in the
expanding plasma, and undergoes strong adiabatic cooling in the
expanding process. At some point in time during the expansion, the
gas molecules cease to be in contact with each other: thereafter
the ionization state of the molecules in the plasma cloud is
frozen. The degree of ionization of the analyte molecules in
conventional MALDI is reported to amount to only around 10.sup.-4.
The analyte ions are predominantly singly charged. This process is
a "soft ionization" because the analyte molecules are ionized as
molecular ions without suffering breaking of bonds.
[0005] The ionization of the analyte molecules by the matrix is a
function of the energy density in the laser spot, and this function
is extremely nonlinear (according to several concurring literature
references, the degree of ionization increases with the sixth to
seventh power of the energy density). The first analyte ions appear
at an energy density threshold of around 10 millijoules per square
centimeter. At around 100 millijoules per square centimeter, at
least a million times more ions are created; but this energy
density constitutes an upper limit for a soft ionization, beyond
which spontaneous fragmentations of the analyte molecules occur.
The setting of the optimum energy density is critical because, on
the one hand, the mass resolution of the time-of-flight mass
spectrometer depends on the energy density and, on the other hand,
only a maximum of around a thousand analyte ions may be produced
per laser pulse. Otherwise the saturation limit of the ion detector
system, which is usually equipped with an 8-bit DAC, is exceeded.
For the analyses, it is important that individual ions can also be
detected with certainty; with a maximum of a thousand ions in the
strongest ion signal and a measuring rate of four gigahertz, the
ions of the strongest ion signal are distributed over several
measuring intervals in such a way that saturation is just avoided,
but individual ions still generate detectable signals. Since a
doubling of the energy density increases the degree of ionization
of the analyte molecules by at least a factor of 2.sup.6=64, this
optimum energy density is only a factor of about 2.5 to 3.0 above
the energy density threshold where the first ions appear. Total
energy and energy density must be kept constant to about one
percent; this keeps fluctuations in ion generation and in the
degree of ionization at around 6 to 7 percent. A further increase
in the energy density by a factor of 3 would be able to increase
the degree of ionization for analyte molecules to more than 10
percent, but would hopelessly oversaturate the ion detector system.
Furthermore, increasing the energy density causes a simultaneous
increase in the number of "metastable" ions; these are ions that
decay on their way to the ion detector and cannot reach the ion
detector for ion-optical reasons. If the number of metastable ions
becomes too high, the degree of ionization can still increase, but
the number of detectable ions cannot.
[0006] The MALDI process is complex, and is affected by numerous
factors, some of which are interdependent. Since the MALDI method
was first published in 1988, many parameters have been investigated
and varied. In spite of this, the processes in the matrix and in
the vaporization cloud which lead to the ionization of the analyte
molecules are still not completely understood and remain the
subject of intensive research, see for example the paper by K.
Dreisewerd, Chem. Rev. 103 (2003), 395-425: entitled "The
Desorption Process in MALDI".
[0007] The chemical parameters of the MALDI process, for example
the type of matrix substances, the concentration ratio between
matrix and analyte molecules, and the preparation conditions, have
been thoroughly researched. For analyte molecules of different
chemical substance classes, such as proteins or nucleic acids, over
a hundred different chemical matrix substances have been
elucidated, such as sinapic acid, DHB (2,5-dihydroxybenzoic acid),
CHCA (.alpha.-cyano-4-hydroxycinnamic acid) or HPA
(3-hydroxypicolinic acid), which affect the MALDI process in
different ways and can be used for different purposes. The matrix
substances are usually aromatic acids; the aromatic ring gives them
a strong absorption capacity in the wavelength range between 330
and 360 nanometers, and as acids they can easily donate a
proton.
[0008] A MALDI sample can be prepared in a number of different
ways, for example with "dried droplet" preparation, or the more
preferable thin-layer preparation. In "dried droplet" preparation,
the matrix substance is dissolved together with the analyte
molecules in a solvent before being applied to a sample support and
then dried. This preparation distributes analyte molecules
extremely inhomogeneously in the matrix crystal complexes, however,
and can scarcely be used for quantitative analyses. With thin-layer
preparation, on the other hand, the matrix substance is applied to
the sample support without analyte molecules and is dried to give a
thin polycrystalline matrix layer only a few micrometers thick.
This thin matrix layer has a high absorptivity for peptides and
proteins. A drop of an aqueous solution containing analyte
molecules is then applied to the thin matrix layer; the drop
spreads quickly over the whole thin layer, and the analyte
molecules are uniformly absorbed. The water can even be removed.
After final drying, special measures can be applied to partially
redissolve the matrix layer, and the analyte molecules can be
embedded uniformly in the matrix layer during the subsequent
drying.
[0009] As far as the physical parameters of the MALDI process are
concerned, investigations have so far chiefly focused on examining
how ionization and fragmentation are influenced by the temporal
duration of the laser pulses, the intensity in the laser spot and
the wavelength of the pulsed laser beam. Spontaneous fragmentations
primarily occur only at high energy densities in the first
nanosecond, for example; in contrast, most metastable ions are
produced by irradiations with durations of longer than three
nanoseconds.
[0010] Nowadays, commercially available MALDI mass spectrometers
are predominantly equipped with pulsed laser systems in the
ultraviolet spectral range (UV). Due to its limited lifetime, the
low-cost nitrogen laser, with a wavelength of .lamda.=337 nm, has
mostly been replaced by frequency-tripled Nd:YAG lasers, with a
wavelength of .lamda.=355 nm, in high-quality MALDI mass
spectrometers. The Nd:YAG laser is based on a YAG crystal
(yttrium-aluminum-garnet: Y.sub.3Al.sub.5O.sub.12) doped with
neodymium ions. In the Nd:YAG laser, the frequency of the strongest
laser line, which appears at a wavelength of 1064 nanometers, is
first doubled to the second harmonic frequency in a first nonlinear
optical conversion crystal, producing green light, and then
converted into the stated UV wavelength of the third harmonic
frequency in a second nonlinear conversion crystal by mixing
fundamental wavelength and second harmonic frequency. So-called
phase matching must be fulfilled in both crystals, which is
achieved by precise temperature control of the crystals to better
than 0.1 degree Kelvin. For this purpose, each of the crystals is
enclosed in an appropriately controlled oven. As is usual, the
second conversion crystal is arranged in such a way that it
compensates for the walk-off of the green light with respect to the
fundamental wavelength in the first nonlinear conversion crystal as
far as possible. The duration of the laser pulses used in the MALDI
method is typically between 3 and 8 nanoseconds in the UV.
[0011] When the nitrogen lasers were replaced with Nd:YAG lasers,
the degree of ionization of the analyte molecules surprisingly
dropped dramatically, which initially could not be explained.
Investigations by the applicant showed that the lower degree of
ionization was connected with the transition from an erratic beam
structure which is constantly changing over time in the beam
profile of the nitrogen laser to an unmodulated and constant
Gaussian profile of the Nd:YAG laser. The spatially and temporally
modulated beam structure of the nitrogen laser was generated by
lightning-like discharges in the nitrogen gas imaged onto the
sample, and form and position of these discharges changed from shot
to shot. The solid-state laser, in contrast, delivered a circular
beam with a Gaussian profile. The ion yield from a Gaussian profile
spot with a diameter of around 100 micrometers was extremely low
and had to be compensated for by increasing the energy density; but
this caused a deterioration in the mass resolution and an enormous
increase in sample consumption, partly because liquefied matrix
material was splashed away during the desorption process. It was
shown that a spot diameter of only five to ten micrometers improved
the degree of ionization; but, kept below the fragmentation limit
for the energy density, it supplied too few ions per laser shot.
Suitable measures were therefore employed to generate a structured
beam profile, which was imaged onto the sample as a spot pattern
with around ten intensity peaks, each roughly 6 to 10 micrometers
in diameter. This pattern generation led to a dramatic improvement.
It was possible to achieve an increase in the degree of ionization
of analyte molecules by a factor of 100 without saturation of the
ion detector system and with extremely low sample consumption. The
structured beam profile for the spot pattern can be generated by
many means, such as the introduction of phase disturbances for
transversely coherent beams (using crumpled plastic films, for
example). The most uniform spot patterns are generated by
commercially available lens arrays made of silica glass. The method
of beam generation and the corresponding laser systems have been
described in U.S. Pat. No. 7,235,781, which is hereby incorporated
by reference.
[0012] When generating the spot pattern, it is important that all
the individual spots have as nearly as possible the same energy
density. If, for example, a square lens array made up of nine
lenses is irradiated by a laser beam with Gaussian profile, the
central lens will produce a spot with higher energy density due to
the maximum in the Gaussian profile. If the energy density is
increased here by 50 percent, the degree of ionization increases by
more than a factor of 10, which makes the other spots of the
pattern insignificant for the production of ions. If all the spots
are to have approximately the same ionization, only a small,
central part of the Gaussian profile of a greatly expanded laser
beam can be used for the illumination of the lens array. The light
in the remaining part of the Gaussian profile, by far the largest
part of the painstakingly generated UV light, must be destroyed
with the aid of diaphragms or other measures. Furthermore, the
adjustment of the laser beam transverse to the pattern generator is
very demanding.
[0013] When commercial MALDI time-of-flight mass spectrometers were
in their infancy, laser pulse rates of 20 to 50 Hz were used
because the nitrogen lasers could not be operated with higher pulse
rates without dramatically reducing the number of shots during
their life. A good mass spectrum is comprised of a several hundred
to a thousand individual spectra; the acquisition of a good,
low-noise mass spectrum from a thousand individual spectra thus
took around 20 seconds. The switch to solid-state lasers soon
allowed pulse rates of 1000 hertz and acquisition times of around
one second. With the introduction of imaging mass spectrometry on
thin tissue sections with several tens of thousands of pixels and a
summed-up mass spectrum for each pixel, the desire for higher
acquisition rates was soon voiced, but this requires laser systems
with far greater power. In principle, the limit for the laser pulse
rate in time-of-flight mass spectrometers is around 10 kilohertz
because the 100 microseconds available for the acquisition of a
single flight time spectrum are just about sufficient. In order to
achieve laser pulse rates of 10 kilohertz, it is advisable to be
careful with the laser energy and not destroy most of the UV light
produced, because otherwise the high power demands would make the
laser systems far too expensive and complex.
[0014] There is a need for a mass spectrometer with a low costs
laser system for the ionization of a sample which can be operated
with particularly high pulse rate, allows the ionization yield to
be increased, has a long lifetime and can be operated with low
energy consumption.
SUMMARY OF THE INVENTION
[0015] A mass spectrometer with a laser desorption ion source
includes a pulsed solid-state laser system, conversion crystals to
increase the frequency, and a pattern generator in the laser beam.
A beam-shaping element is located between the solid-state laser
system and the conversion crystals. The beam-shaping element
converts the circular laser beam with Gaussian profile into a beam
with approximately rectangular cross-section and an approximately
homogeneous energy density across the whole rectangular
cross-section.
[0016] An infrared solid-state laser system may be used. Economic
advantages can be realized, for example, by the fact that, firstly,
beam-shaping elements for infrared light cost significantly less
than beam-shaping elements for ultraviolet light; secondly, the
nonlinear conversion crystals can be utilized better and with a
higher conversion rate due to the rectangular cross-section and the
homogeneous energy density; thirdly, the lifetime of the conversion
crystals increases; and fourthly, the beam has a cross-section at
the exit of the conversion crystals which has a homogeneous energy
density in a square middle section containing more than 90 percent
of the beam energy. It is thus ideal for homogeneous illumination
of the pattern generator without substantial parts of this
higher-energy light having to be cut off and destroyed.
[0017] Thus, a mass spectrometer with a pulsed UV laser system is
proposed, with relatively low energy consumption, which produces a
spatially distributed spot pattern with peaks of uniform energy
density on the sample, increasing the degree of ionization for
analyte ions as compared to conventional spot patterns. The spot
pattern with peaks of uniform energy density can be produced by
homogeneous illumination of a pattern generator, for example a lens
array. The homogeneous illumination can be generated by a low-cost
beam-shaping element, which does not act on the UV beam but on the
original infrared beam, in conjunction with changes to the beam
cross-section and beam profile brought about by the nonlinear
conversion crystals. This beam shaping not only produces a beam
profile that illuminates the pattern generator homogeneously with
low losses, but at the same time increases the efficiency of the
frequency multiplication and the lifetime of the conversion
crystals so that significant cost savings are achieved because less
laser energy is required and the lifetime is increased.
[0018] The beam profile generated by the pattern generator (e.g.,
for example a structure with nine individual small-diameter spots
in a square arrangement) can then be imaged in reduced size onto
the sample via a telescope with downstream lens optics. In this
example, nine spots with diameters of around five micrometers and
identical energy densities can be generated.
[0019] The invention thus provides a mass spectrometer with a laser
system with a long lifetime that generates a spatially distributed
spot pattern on the sample with relatively low energy losses,
achieving a high degree of ionization for analyte ions.
[0020] These and other objects, features and advantages of the
present invention will become more apparent in light of the
following detailed description of preferred embodiments thereof, as
illustrated in the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWING
[0021] FIG. 1 shows, in the left column, the components 1 to 9
which are necessary for the generation of a structured UV beam,
their optical arrangement and the image planes A to F; next to this
are shown the intensity profiles of the infrared, green or
ultraviolet light in the image planes transverse to the respective
laser beam; and in the right column are the shapes of the beam
cross-sections, shown for heights of equal intensity. The beam
generator 1 contains pump diodes and resonator with laser crystal
and, if applicable, a Pockels cell; this is where the temporally
pulsed infrared beam 10 is generated. The beam-shaping optical
device 2 converts the circular beam with Gaussian profile 13, 14
into a rectangular beam 15 of around 5 by 6 square millimeters with
a profile 16 of constant energy density, which is formed in the
image plane B. Lens 3 images the image plane B into the image plane
C, reduced in size to around 500 by 600 square micrometers; the
reduction is not reproduced in FIG. 1 for the sake of clarity. The
distances here are not true to scale but greatly shortened for ease
of illustration; the rectangular cross-section of the beam of 500
by 600 square micrometers continues almost unchanged through the
two temperature-controlled conversion crystals 4 and 5. In the
nonlinear crystal 4, frequency doubling creates green light 11,
which is deflected laterally at a small angle (walk-off) by the
birefringence, and exits with an offset of around 100 micrometers.
Between the two conversion crystals, the green light has an energy
density profile 18, transverse to the beam, which is almost
trapezoidal, the sides of the trapezoid being slightly curved due
to the drop in the energy density of the infrared beam along the
conversion crystal 4. In the appropriately oriented nonlinear
crystal 5, the green beam is steered back to the infrared beam 10
and reacts with it to form the ultraviolet beam 12, which again has
a trapezoidal energy density profile 20, but here the sides have
less curvature. The infrared light 10 and the green light 11 are
masked in the filter 6 so that only the ultraviolet light 12 is
transmitted. Lens 7 images the image plane D in enlarged form into
the image plane E, which is formed by the surface of the beam
splitter 8; here an approximately rectangular section, measuring
around 4 by 4 square millimeters, of the cross-section 21 has a
homogeneously uniform intensity 22. The beam splitter 8, for
example a lens array with short focal length, is illuminated by the
homogeneous part of the cross-section, and forms the desired beam
pattern from it, in which the spots have the same intensity. A lens
9 then generates a parallel beam with cross-sectional pattern 23
and energy density profile 24 in the plane F. This beam is then
imaged onto the sample in reduced size via suitable optics, as can
be seen in FIG. 2.
[0022] FIG. 2 shows how the assembly 1-9 of all the optical
elements 1 to 9 from FIG. 1 is schematically embedded into a laser
system 43, which is connected to a MALDI time-of-flight mass
spectrometer 44. This is a laser system which controls the position
of the laser light pattern on the sample support plate 35 by a
rotatable mirror system 30. The parallelized UV laser beam with
structured profile from plane F can be slightly deflected in both
spatial directions in the rotatable mirror system 30 with two galvo
mirrors. The deflected laser beam is then expanded in a Kepler
telescope 31 and shifted in parallel in accordance with the angular
deflection. The mirror 32 directs the exiting laser beam centrally
into the objective lens 33 again, with reduced angular deflection.
Depending on the angular deflection, the beam passes through the
objective lens 33 centrally but at slightly different angles, thus
shifting the position of the spot pattern on the sample support
plate 35. The ions generated in the plasma clouds of the laser spot
pattern are accelerated by voltages applied to the diaphragms 36
and 37, and form an ion beam 40; this ion beam passes through the
two deflection capacitors 38 and 39, which are rotated by
90.degree., for a trajectory correction, and is focused in the
reflector 41 onto the detector 42.
[0023] FIG. 3 depicts different regular laser spot patterns with 4,
7, 9, 16 and 19 individual laser spots. The separations between the
spots here are just about as large as the spot diameters, but it is
also possible to generate patterns with other separations and spot
diameters. Pattern generators for square patterns with 4, 9, 16 or
also 25 spots can be illuminated with relatively low losses by
square forms of the infrared beam and the thus generated square,
flat-top profile of the UV beam.
DETAILED DESCRIPTION OF THE INVENTION
[0024] A mass spectrometer comprises a laser system with a pulsed
infrared solid-state laser system 1, two conversion crystals 4 and
5 for tripling the frequency, and a pattern generator 8 in the
ultraviolet laser beam. A low-cost beam-shaping optical device is
located between the pulsed solid-state laser system and the
conversion crystals 4 and 5. This beam-shaping optical device
converts the circular infrared beam with Gaussian profile 14 into a
beam 10 of approximately rectangular shape 15 with approximately
the same energy density 15 everywhere. The laser system preferably
generates an infrared beam, whose frequency is tripled to supply UV
light with a wavelength of between about 300 and 450 nanometers,
preferably between about 330 and 370 nanometers.
[0025] The laser light beam is converted into a beam with
homogeneous energy density, in order to allow homogeneous
illumination of the pattern generator. That is, one consciously
refrains here from generating a UV laser light beam with a Gaussian
profile in the conventional way. Beam-shaping optical devices for
UV beams must be manufactured from clean, UV-transmitting
materials, preferably silica glass, and they are extremely
expensive because the materials are difficult to machine.
[0026] Beam-shaping optical devices 2 for an infrared beam are less
costly, in contrast. There are various refractive and diffractive
beam-shaping optical devices on the market, which are all based on
distributing laser light from the center of the Gaussian
distribution into the periphery, while maintaining the parallel
beam with as high a quality as possible. This can be achieved using
refraction with specially shaped lenses. Diffractive beam-shaping
elements are slightly more expensive, but usually provide
qualitatively better beam shapes. The beam-shaping elements allow
the circular infrared beams with a diameter of for example, around
5 millimeters and Gaussian energy distribution to be converted into
rectangular beam cross-sections with selectable dimensions of about
5 by 6 square millimeters, for example, and homogeneous energy
density.
[0027] A preferred embodiment is illustrated in FIG. 1. The left
column of FIG. 1 shows the components 1 to 9 for generating a
structured UV beam. The image planes A to F are shown in addition
to the optical arrangement of the components. Next to this, in a
second column, the energy density profiles, i.e., the intensity
distributions transverse to the respective laser beam, are shown
for the image planes A to F. In the right-hand column of FIG. 1,
the beam cross-sections of the infrared, green and ultraviolet
light are shown with a freely chosen (i.e. not true to scale)
intensity. The beam generator 1 contains pump diodes, resonator
with laser crystal and, if required, a Pockels cell and a beam
attenuator also; this is where the pulsed infrared beam is
generated. The beam-shaping optical device 2 converts the beam with
circular cross-section 13 and Gaussian profile 14 into a
rectangular beam with cross-section 15 and the rectangular profile
16 of approximately the same intensity across the whole
cross-section, which is shown for the image plane B. Lens 3 images
the image plane B into the image plane C. The distances here are
not shown true to scale but greatly shortened for the sake of
clarity; the rectangular cross-section of the beam, reduced to
around 500 by 600 square micrometers, continues without any
significant changes through both conversion crystals 4 and 5. Green
light 11 is generated in the nonlinear crystal 4 by frequency
doubling, and this green light propagates with a small angle of
lateral deflection. This deflection is called "walk-off"; on
exiting the around 15 millimeter long conversion crystal 4, the
green beam is shifted in comparison to the infrared beam by around
100 micrometers. As is known in the prior art, this walk-off is
steered back in the appropriately cut and arranged nonlinear
crystal 5 with a walk-off compensation and reacts with the
remaining infrared beam 10 to fog n the ultraviolet beam 12. The
necessary phase matching for the deflections for the two nonlinear
conversion crystals are set by precise temperature control; the
temperature control ovens are not shown in FIG. 1. The remaining
infrared light 10 and the remaining green light 11 are masked in
the filter 6 so that only the ultraviolet light 12 is transmitted.
As is shown for image plane D, the UV beam formned has an
approximately trapezoidal intensity profile 20 with slightly curved
sides, while the base line of the trapezoid is larger than the top
line by an amount which corresponds to around twice the walk-off.
In the center part of the cross-section there is a roughly square
section of around 500 by 500 square micrometers with homogeneously
uniform energy density. The lens 7 images the image plane D in
enlarged form onto the surface of the pattern generator 8 (image
plane E), which is uniformly illuminated with the square section of
the homogeneous energy density. Only a small proportion of the UV
beam's energy of around 5 to 10 percent below the side lines of the
trapezoid is lost in the illumination. The pattern generator 8,
with its lenses of short focal length, shapes the desired beam
pattern, in which all the spots have approximately the same
intensity. A further lens 9 images the focal points of the lenses
of the pattern generator 8 into infinity. In the image plane F, the
parallel beam has the desired cross-sectional pattern 23 with the
profile 24 of the same energy densities in the spots of the
pattern. This beam is then imaged, as can be seen in FIG. 2, onto
the sample in reduced size via the appropriate optics.
[0028] Due to the exponential decrease of the energy density in the
infrared beam 10 within the conversion crystal 4, the cross-section
17 of the green light has a trapezoidal profile 18 with slightly
curved side lines; this shape, and the process of reverse
compensation in the conversion crystal 5, results in the
cross-section 19 of the UV beam also having a trapezoidal profile
20 with slightly curved side lines, although the curvature is less
pronounced.
[0029] The rectangular cross-section 15 and the homogeneous energy
density profile 16 of the infrared beam enable the two nonlinear
conversion crystals 4 and 5 to produce a better conversion; and in
the image plane D at the exit of the conversion crystals, the UV
beam has a cross-section 19 which has an approximately trapezoidal
profile 20 with a square center part of homogeneous energy density.
While theoretically a maximum of 43 percent of an infrared beam
with Gaussian profile can be converted into UV light, it is
possible to achieve a higher conversion rate with homogeneous
rectangular profiles. For a Gaussian profile, the energy density in
the maximum of the beam cross-section is just below the destruction
limit for the crystal, which reduces its lifetime. For the
rectangular profile, in contrast, the energy density is
significantly lower; thus increasing the lifetime of the nonlinear
conversion crystals. If the laser system is required to be operated
at ten kilohertz pulse frequency for around a year, the lifetime of
the laser system must be 10.sup.11 laser pulses. Since the lifetime
is essentially determined by the conversion crystals, the
rectangular infrared beam is beneficial in terms of cost here
also.
[0030] The beam pattern of the pattern generator 8, for example the
square structure 23 with nine individual partial beams of small
diameter, is then imaged via the lens 9 into infinity As can be
seen in FIG. 2, this beam pattern is expanded by a telescope 31 and
imaged onto the sample by lens optics 33, where nine spots, each
with a diameter of around five micrometers and the same energy
density, can be generated, for example. FIG. 2 also depicts how
positional control of the spot pattern on the MALDI sample can be
achieved with the aid of a double rotatable mirror system 30.
[0031] Instead of the nine spots, it is also possible to generate
other patterns of 4, 7, 9, 16 or 19 (or 25 or even more) spots, for
example, as shown in FIG. 3. The separations and diameters of the
spots on the sample can also be varied, although the type of
imaging with the optical lens system 33, which is necessarily far
removed from the sample, can only achieve, by theory, a minimal
diameter of around four to five micrometers. If a change to other
spot patterns is desirable, different pattern generators 8 can be
introduced into the laser beam with the aid of a mechanical system,
such as to be found in a slide projector. For a high degree of
ionization, the individual spots on the sample should always have
diameters which are smaller than 10 micrometers.
[0032] As has been explained in the introduction, although the aim
is to increase the degree of ionization for the analyte molecules
in order to increase the ion yield, as a rule the number of
metastable ions should be limited at the same time. For most
investigations, spontaneous fragmentations should be avoided.
Furthermore, one has to ensure that no more than around a thousand
analyte ions are generated per laser shot in order to avoid
saturation of the ion detector system. The prerequisites for the
simultaneous fulfillment of these differing requirements are not
completely known; there are indications, however, that a pattern of
nine spots, each about five micrometers in diameter, comes close to
an optimum for the most common methods of preparing the matrix
layers and for most analytical goals. For other types of
preparation or for other analytical goals, it is sometimes
necessary to select other patterns. The yield of analyte ions can
probably be increased, with the aid of suitable patterns, to around
ten percent of the analyte molecules, i.e., to around a thousand
times the yield of the conventional MALDI method. Analytical goals
deviating from the norm can require spontaneous fragmentations (for
in-source dissociation, ISD) or high proportions of metastable ions
(for daughter ion spectra with post-source dissociation, P SD), for
example.
[0033] The laser system of the mass spectrometer is not only
advantageous due to its energy savings and its high yield of
analyte ions, but it is also particularly advantageous because the
formation of the pattern with very small spots also suppresses the
splashing of liquefied matrix material during the desorption, which
saves sample material. Especially in the case of a large number of
samples per unit of time, as is possible with lasers of high pulse
frequency in MALDI-TOF spectrometers, the reduced contamination of
the ion optics is an enormous advantage. A further advantage is
also that the front of the adiabatically expanding plasma cloud of
the pattern accelerates the ions preferentially into the flight
direction of the time-of-flight mass spectrometer.
[0034] Different types of mass spectrometer may be used. The
analyte ions which are produced with the laser system can
preferably be detected and analyzed in a special MALDI
time-of-flight mass spectrometer with axial ion injection, as
depicted schematically in FIG. 2. It is also possible to feed the
analyte ions to different types of mass analyzer for the analysis,
such as time-of-flight mass spectrometers with orthogonal ion
injection (OTOF-MS), ion cyclotron resonance mass spectrometers
(ICR-MS), RF ion trap mass spectrometers (IT-MS) or electrostatic
ion trap mass spectrometers of the Kingdon type, for example.
[0035] The example embodiments cited do not represent a definitive
list. With knowledge of this invention, those skilled in the art
can design further advantageous embodiments of mass spectrometers
with laser systems which are to be covered by the scope of
protection of the patent claims.
[0036] Therefore, while the invention has been shown and described
with reference to a number of embodiments thereof, it will be
recognized by those skilled in the art that various changes in form
and detail may be made herein without departing from the spirit and
scope of the invention as defined by the appended claims.
[0037] Although the present invention has been illustrated and
described with respect to several preferred embodiments thereof,
various changes, omissions and additions to the form and detail
thereof, may be made therein, without departing from the spirit and
scope of the invention.
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