JP2012003898A - Apparatus and method for two-dimensional imaging - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a two-dimensional imaging apparatus by which can increase resolution in mass spectrometry and a signal-to-noise ratio to acquire an accurate two-dimensional image, even when biological tissue and chemicals, which include molecules with weak chemical bonds therein are set as measurement objects.SOLUTION: A two-dimensional imaging apparatus is provided with a laser source 11; a sample stand 13 whereon samples 90 including measurement objects are placed; an optical system 30 which guides an infrared laser beam from the laser source 11 to the sample 90; a movement mechanism which relatively displaces the optical system 30 and the sample stand 13; a mass spectrometry unit 14 which separates ions, which the sample 90 generates by receiving an infrared laser beam, on the basis of the mass electric charge ratio of the ions; and an ion detector 17 which detects ions. A mass spectrum is acquired per part of a plurality of parts in the sample 90 where a matrix is applied and is stored after being associated with coordinate positions for each of the parts. A concentration map is created in accordance with the peak intensity indicating mass of specific molecules included in the measurement object.

Description

  The present invention relates to a two-dimensional imaging apparatus and method. More specifically, the present invention relates to an imaging apparatus and method for performing mass spectrometry (IR-MALDI) using infrared rays on a plurality of parts of a sample and displaying the obtained result as a two-dimensional image.

  Conventionally, for example, as disclosed in Non-Patent Document 1 (“MALDI-TOFMS Imaging”, Internet <URL: http://www.an.shimadzu.co.jp/apl/imaging/invitro01001015.htm>) There is known an imaging apparatus that performs mass spectrometry (MALDI-TOFMS) on a plurality of parts of a sample having a flat surface and displays the obtained result as a two-dimensional image.

JP 2007-005410 A

Shimadzu Corporation, “MALDI-TOFMS Imaging”, [online], 2010, [Search June 7, 2010], Internet <URL: http://www.an.shimadzu.co.jp/apl/imaging /invitro01001015.htm> Shimadzu Corporation, “The Principles of MALDI / TOFMS”, [online], 2010, [Search June 7, 2010], Internet <URL: http://www.an.shimadzu.co.jp/tofms/ axima / princpl1.htm>

  However, in the above conventional imaging apparatus, Non-Patent Document 2 related to Non-Patent Document 1 ("MALDI / TOFMS Principle", Internet <URL: http://www.an.shimadzu.co.jp/tofms/axima /princpl1.htm>), the sample is irradiated with nitrogen laser light (wavelength = 337 nm), that is, ultraviolet light for mass analysis. For example, when a measurement target is a biological tissue having a weak chemical bond in a molecule using ultraviolet rays, the chemical bond may be broken and a by-product may be generated. In such a case, it is difficult to increase the resolution and S / N ratio (signal to noise ratio) of mass spectrometry, and as a result, there is a problem that an accurate two-dimensional image cannot be obtained.

  Therefore, the object of the present invention is to increase the resolution of mass spectrometry and the S / N ratio even when a biological tissue or drug containing a molecule having a weak chemical bond in the molecule is used as a measurement object. An object of the present invention is to provide a two-dimensional imaging apparatus and method capable of obtaining a simple two-dimensional image.

  If the present inventor uses an IR-MALDI apparatus (infrared light-matrix-assisted laser desorption / ionization apparatus), the resolution of mass spectrometry and the S / N ratio may be improved, and an accurate two-dimensional image may be obtained. Focused on the fact that there is. As is well known, the IR-MALDI apparatus irradiates a measurement object with infrared light (especially mid-infrared light), so that by-products are generated without breaking molecules having weak chemical bonds in the molecule. This is because ionization can be performed while suppressing the generation of phosphine (see Patent Document 1 (Japanese Patent Laid-Open No. 2007-005410)).

Therefore, in order to solve the above problems, the two-dimensional imaging apparatus of the present invention is
A laser light source for generating infrared laser light;
A sample stage on which a sample including a measurement object and at least a matrix attached to the surface of the measurement object is placed;
An optical system for guiding the infrared laser light from the laser light source to the sample on the sample stage;
A moving mechanism for relatively moving the optical system and the sample stage in order to irradiate the portion of the sample to which the matrix is attached with the infrared laser light;
A mass analyzer that separates ions generated by the sample in response to the infrared laser light in accordance with a mass-to-charge ratio of the ions;
An ion detector for detecting ions separated by the mass spectrometer;
Based on the output of the ion detector, a data acquisition unit that obtains a mass spectrum for each of the plurality of parts to which the matrix is attached within the surface of the sample;
A data storage unit that stores the mass spectrum of each part of the sample in association with the coordinate position of each part in the surface of the sample;
A concentration map creating unit that creates a concentration map according to the intensity of a peak representing the mass of a specific molecule contained in the measurement object based on the mass spectrum of each part of the sample. .

  In the two-dimensional imaging apparatus of the present invention, an infrared laser beam from a laser light source is guided to a sample by an optical system, and the infrared laser beam is irradiated to a portion of the sample to which the matrix is attached. The optical system and the sample stage are relatively moved. Accordingly, the measurement object is sequentially ionized with the help of the matrix into ions for a plurality of portions of the sample to which the matrix is attached.

  Ions generated sequentially at the plurality of sites of the sample are separated according to the mass-to-charge ratio of the ions by the mass analyzer, reach the ion detector, and are sequentially detected. The data acquisition unit obtains a mass spectrum for each of the plurality of parts of the sample based on the output of the ion detector (time-of-flight mass spectrometry (TOFMS)). The data storage unit stores the mass spectrum of each part of the sample in association with the coordinate position of each part in the surface of the sample. Then, the concentration map creation unit creates a concentration map corresponding to the intensity of the peak representing the mass of the specific molecule contained in the measurement object, based on the mass spectrum of each part of the sample.

  In this two-dimensional imaging device, the sample is irradiated with infrared laser light (especially mid-infrared light is desirable), so that the generation of by-products is suppressed without breaking molecules that have weak chemical bonds in the molecule. Thus, the sample can be ionized. Therefore, the resolution of mass spectrometry and the S / N ratio (signal-to-noise ratio) can be increased as compared with the case where ultraviolet light such as a nitrogen laser is used. As a result, an accurate two-dimensional image is obtained by the density map creation unit.

In the two-dimensional imaging apparatus of one embodiment,
A comparison reference storage unit that stores a mass spectrum obtained under the same measurement conditions as the measurement conditions for the measurement object, with respect to the reference object serving as a reference for comparison for the measurement object,
The concentration map creation unit compares the mass spectrum of the measurement object with the mass spectrum of the reference object, and creates the concentration map.

  In the two-dimensional imaging apparatus of this one embodiment, the concentration map creation unit compares the mass spectrum of the measurement object with the mass spectrum of the reference object, and creates the concentration map. A dimensional image is obtained.

The two-dimensional imaging method of the present invention
A sample including a measurement object and at least a matrix attached to the surface of the measurement object is placed on the sample table,
An infrared laser beam from a laser light source is guided to the sample by an optical system, and the infrared laser beam is irradiated to a portion of the sample to which the matrix is attached. Are moved relative to each other, and the measurement object is sequentially ionized with the help of the matrix for each of the plurality of parts to which the matrix of the sample is attached,
Ions generated sequentially in the plurality of parts of the sample are separated according to the mass-to-charge ratio of the ions by a mass analyzer and detected by reaching an ion detector,
Based on the output of the ion detector, the data acquisition unit obtains a mass spectrum for each of the plurality of parts of the sample,
In the data storage unit, the mass spectrum for each part of the sample is stored in association with the coordinate position of each part in the surface of the sample,
Based on the mass spectrum of each part of the sample, a concentration map corresponding to the intensity of the peak representing the mass of the specific molecule contained in the measurement object is created.

  In the two-dimensional imaging method of the present invention, the sample is irradiated with infrared laser light (especially mid-infrared light is desirable), so that by-products are generated without breaking molecules having weak chemical bonds in the molecule. The above sample can be ionized while suppressing. Therefore, the resolution of mass spectrometry and the S / N ratio (signal-to-noise ratio) can be increased as compared with the case where ultraviolet light such as a nitrogen laser is used. As a result, an accurate two-dimensional image is obtained.

  In the two-dimensional imaging method of one embodiment, the sample is produced by spraying the matrix on the surface of the measurement object.

  In the two-dimensional imaging method of this embodiment, it is possible to easily produce a sample in a form in which adjacent matrices are separated from each other on the surface of the measurement object. It is desirable that the adjacent matrices are separated from each other within the surface of the measurement object because the components of the measurement object do not move between the adjacent matrices.

  In one embodiment of the two-dimensional imaging method, the sample is produced by dropping the matrix at a predetermined pitch on the surface of the measurement object.

  In the two-dimensional imaging method of this embodiment, a sample in a mode in which matrices having a substantially constant diameter are arranged vertically and horizontally can be easily produced by adjusting the dropping pressure and time.

  As is clear from the above, according to the two-dimensional imaging apparatus and method of the present invention, even when a biological tissue or a drug containing a molecule having a weak chemical bond in the molecule is used as a measurement object, mass spectrometry is performed. The resolution and S / N ratio can be increased, and an accurate two-dimensional image can be obtained.

(A) is a figure which shows schematic structure of the two-dimensional imaging device of one Embodiment of this invention. (B) and (C) are diagrams each schematically showing a two-dimensional image created by the two-dimensional imaging apparatus. It is a figure which shows the sample stand of the said two-dimensional imaging device. It is a figure which shows the block configuration of the data analysis apparatus of the said two-dimensional imaging device. It is a figure which shows one structural example of the laser light source and optical system of the said two-dimensional imaging device. It is a figure which shows another structural example of the laser light source and optical system of the said two-dimensional imaging device. (A) is a figure which represents typically the aspect by which the matrix was sprayed on the sample slice, (B) is a figure which represents typically the aspect by which the matrix was dripped at the predetermined interval on the sample slice. It is a figure explaining the method of dripping a matrix on a sample slice. It is a figure which shows the mass spectrum of the site | part represented by the coordinate position (25,5) in the surface of a certain sample. It is a figure which shows the mass spectrum of the site | part represented by the coordinate position (35,5) in the surface of the said sample. It is a figure which shows the mass spectrum of the site | part represented by the coordinate position (25,10) in the surface of the said sample. It is a figure which shows typically the two-dimensional image obtained about the said sample slice.

  Hereinafter, the present invention will be described in detail with reference to embodiments shown in the drawings.

  FIG. 1A shows a schematic configuration of a two-dimensional imaging apparatus (the whole is denoted by reference numeral 10) according to an embodiment of the present invention. The two-dimensional imaging apparatus 10 includes an IR light source 11 as a laser light source, a vacuum vessel 12, a sample stage 13 provided in the vacuum vessel 12, a mass analyzer 14, an ion detector 17, and a beam collector. The optical unit 30, the data analysis device 50, the image display device 51, and a system control unit 20 that outputs a control signal and controls the entire device are provided.

  The IR light source 11 generates infrared laser light, in this example, mid-infrared light within a wavelength range of 5 μm to 14 μm (micrometer). As the laser light source that generates such mid-infrared light, for example, the light sources disclosed in Japanese Patent Application Laid-Open Nos. 2005-331599 and 2007-323021 may be used.

  The inside of the vacuum vessel 12 is evacuated by a vacuum pump (not shown) controlled by a control signal from the system control unit 20. In this example, the vacuum container 12 accommodates a sample stage 13, a mass analyzer 14, and an ion detector 17.

  In the present embodiment, the sample stage 13 is provided in the vacuum vessel 12 similarly to the mass analyzer 14, but is not limited to this configuration. For example, the sample stage 13 and the mass spectrometer 14 may be provided in a container at atmospheric pressure. Alternatively, the sample chamber in which the sample stage 13 is provided may be set to atmospheric pressure, and ions may be guided to a mass analysis unit that becomes a vacuum environment via a capillary or an orifice. Such an AP-MALDI apparatus (atmospheric pressure matrix-assisted laser desorption / ionization apparatus) is highly convenient because the sample can be easily exchanged.

  In this example, the sample stage 13 is disposed below the vacuum container 12. On the sample stage 13, a sample 90 composed of a measurement object having a flat surface and a matrix attached to the surface of the measurement object is placed. As shown in FIG. 2, this sample stage 13 is controlled by a control signal from the system control unit 20 so that a desired part of the sample 90 can be irradiated with infrared laser light from the IR light source 11. The mechanism 40 is movable in the X and Y directions perpendicular to each other in the horizontal plane.

  As shown in FIG. 1, the beam condensing unit 30 guides and irradiates the infrared laser light from the IR light source 11 to the sample 90 on the sample stage 13.

  The ion detector 17 is provided at a position facing the sample stage 13 in the vacuum vessel 12, in this example, at an upper portion in the vacuum vessel 12. Between the electrode 15 on the sample stage 13 side and the electrode 16 on the ion detector 17 side, ions generated by the sample 90 are ion-detected by a high voltage power source (not shown) controlled by a control signal from the system control unit 20. A voltage (20 kV in this example) for accelerating toward 17 is applied.

  The mass analyzer 14 includes an electrostatic electromagnetic lens and a multipolar high-frequency ion guide provided between the sample stage 13 and the ion detector 17. The mass analyzing unit 14 receives the infrared laser light from the IR light source 11 and separates a plurality of ions generated by the sample 90 according to the mass-to-charge ratio m / z of those ions to the ion detector 17. Lead.

  The data analysis device 50 analyzes the output of the ion detector 17 and displays the data analysis result on an image display device 51 such as an LCD (liquid crystal display element).

  FIG. 4 schematically shows one configuration example of the optical system from the IR light source 11 to the beam condensing unit 30. Infrared laser light, which is parallel light emitted from the IR light source 11, is collected by an off-axis parabolic mirror 35 and is incident on one end of an optical fiber (in this example, a hollow fiber) 36. Then, the infrared laser light is transmitted through the optical fiber 36 and enters the housing 31 of the beam condensing unit 30 through the other end of the optical fiber 36.

The housing 31 of the beam condensing unit 30 is provided with a collimator lens 32, a condensing lens 33, and a position adjusting mechanism 34 including a helicoid in order from the side where the other end of the optical fiber 36 is connected. The infrared laser light that has entered the housing 31 of the beam condensing unit 30 spreads radially, is converted into parallel rays by the collimating lens 32, and is condensed toward the surface of the sample 90 by the condensing lens 33. The infrared laser light is applied to the surface of the sample 90 through a window 12w made of ZnSe provided on the wall surface of the vacuum vessel 12. The spot diameter of the irradiated infrared laser light is about several hundred μm (minimum 120 μm). Here, the window material may be a window material that transmits mid-infrared light such as CaF ₂ or BaF _{2 in} addition to ZnSe. In AP-MALDI, it is not always necessary to configure a window.

  An observation camera 18 is provided in the vacuum container 12. The observation camera 18 can observe the surface of the sample 90 during irradiation with infrared laser light.

  When the wavelength of the infrared laser light generated by the IR light source 11 is set variably, the focus of the infrared laser light tends to shift on the sample 90. In this example, such a focus shift can be eliminated by adjusting the focus with the position adjusting mechanism 34 while observing the surface of the sample 90 with the observation camera 18 provided in the vacuum vessel 12.

  FIG. 5 schematically shows another configuration example of the optical system from the IR light source 11 to the beam condensing unit 30. In FIG. 5, the same components as those in FIG. 4 are denoted by the same reference numerals, and redundant description is omitted.

  In the configuration example of FIG. 5, the infrared laser light that is a parallel light beam emitted from the IR light source 11 is reflected by the flat mirrors 37 and 38 and enters the housing 31 of the beam condensing unit 30 ′.

  The housing 31 of the beam condensing unit 30 ′ is provided with a condensing lens 33 and a position adjusting mechanism 34 made of a helicoid. The infrared laser light that has entered the housing 31 of the beam condensing unit 30 ′ is condensed toward the surface of the sample 90 by the condenser lens 33. The infrared laser light is applied to the surface of the sample 90 through a window 12w made of ZnSe provided on the wall surface of the vacuum vessel 12.

  The configuration example of FIG. 5 has an advantage that the number of parts in the beam condensing unit 30 ′ can be reduced as compared with the configuration example of FIG. In order to condense the infrared laser light entering the housing 31 of the beam condensing unit 30 ′ toward the surface of the sample 90, instead of the condensing lens 33 in the beam condensing unit 30 ′, An off-axis parabolic mirror may be provided.

  FIG. 3 shows a block configuration of the data analysis apparatus 50 shown in FIG. The data analysis device 50 includes a memory 52 as a data storage unit capable of storing data, a data acquisition unit 53, a density map creation unit 54, an image creation unit 55, and an LCD (liquid crystal display element), for example. An image display device 51 (also shown in FIG. 1) is provided.

  The memory 52 can store calculation process data handled by the data acquisition unit 53 and the concentration map creation unit 54, data obtained as a result of the calculation, that is, data representing a mass spectrum.

  The data acquisition unit 53 performs a calculation based on the output of the ion detector 17 to obtain a mass spectrum of the sample 90. This mass spectrum is, for example, as shown in FIGS. 8, 9, and 10 to be described later.

  Based on the mass spectrum obtained by the data acquisition unit 53, the concentration map creation unit 54 creates a concentration map corresponding to the intensity of the peak representing the mass of the specific molecule contained in the measurement object contained in the sample 90. For example, as shown in FIG. 1B and FIG. 1C, the created concentration map shows the concentration of the specific group in the surface of the measurement object 90 (or 90 ′) having a flat surface. Represents the distribution.

  The image creating unit 55 shown in FIG. 3 creates two-dimensional image data for displaying the density map created by the density map creating unit 54 on the image display device 51. As a result, a two-dimensional image representing the density map is displayed on the display screen of the image display device 51.

  The data acquisition unit 53, the density map creation unit 54, and the image creation unit 55 in the data analysis apparatus 50 described above are configured by software (computer program).

  Next, a method for creating a two-dimensional image representing the concentration distribution of the measurement object by the two-dimensional imaging apparatus 10 will be described with a specific example.

  In this example, the object to be measured is a section of biological tissue containing ganglicides (this is referred to as “sample section”). Gangliside is a kind of glycosphingolipid in which one or more sialic acids (N-acetylneuraminic acid: abbreviation NANA, Neu5Ac) are bonded on a sugar chain. As this ganglyside, more than 40 types such as GD1a, GT1b, GQ1b have been discovered. Each of these types has a different number and position of N-acetylneuraminic acid. In the following example, a two-dimensional image representing a concentration map of gangliosides GD1a, GT1b, and GQ1b as specific molecules is created.

  A two-dimensional image is created according to the following flow.

  Step S1: Slice the biological tissue containing ganglyside to prepare a sample section of the biological tissue.

  Here, the sample slice is prepared by slicing along a plane in which the ganglyside concentration distribution is to be observed in the living tissue.

  Step S2: The sample section is placed on a rectangular plate-like sample plate 19 (shown in FIG. 2).

  Step S3: The matrix is sprayed on the sample section on the sample plate 19. This is designated as sample 90.

  As this matrix, for example, known sinapinic acid is used. The spraying operation is performed using a work airbrush or a glass sprayer.

  Here, the spraying time is made relatively short so that most of the matrices 92, 92,... Are isolated from each other within the surface of the sample 90, for example, as schematically shown in FIG. . If adjacent matrices 92 are separated from each other within the surface of the sample 90, it is desirable because the components of the sample 90 (particularly biological tissue) do not move between the adjacent matrices 92.

  In consideration of the spot diameter of the irradiated infrared laser light being several hundred μm, the diameter of the matrix 92 to be irradiated with the infrared laser light is preferably 1.0 mm or more. On the other hand, from the viewpoint of the resolution of the created two-dimensional image, it is desirable that the diameter of the matrix 92 is as small as, for example, 2.0 mm or less. Therefore, the diameter of the matrix 92 is desirably 1.0 mm or greater and 2.0 mm or less, typically 1.5 mm. In addition, the interval between the matrices 92 is desirably 200 μm or more from the viewpoint of preventing the components of the sample (measurement object) from moving through the fusion of the matrices 92. On the other hand, from the viewpoint of the resolution of the created two-dimensional image, it is desirable that the interval between the matrices 92 be as small as 500 μm or less, for example. Therefore, the interval between the matrices 92 is desirably 200 μm or more and 500 μm or less, typically 300 μm.

  Step S4: The sample 90 on the sample plate 19 is dried.

  Step S5; The sample plate 19 on which the sample 90 is mounted is attached on the sample stage 13 in the vacuum vessel 12.

  Step S6: The inspection apparatus 10 is operated to irradiate the sample 90 with infrared laser light (in this example, a pulse laser having a wavelength of 6 μm, a spot diameter of several hundred μm, and a time of 10 nsec (nanoseconds)) from the IR light source 11. .

  Specifically, the sample stage 13 is moved and positioned in the horizontal direction along the surface of the sample 90 by the moving mechanism 40 with respect to the optical system 30, and the infrared laser light is matrix 92 in the surface of the sample 90. Are applied to the portions 92-1, 92-2, 92-3,... (See FIG. 11). As a result, the sample 90 is ionized sequentially with the help of the matrix 92 for each of the plurality of portions 92-1, 92-2, 92-3,. In FIG. 11, only the three portions 92-1, 92-2, and 92-3 are shown, but actually, the infrared laser light is irradiated to many portions within the surface of the sample 90, and sequentially. Ionize. Then, the ions generated by the plurality of portions 92-1, 92-2, 92-3,... Of the sample 90 are accelerated by the voltage between the sample stage 13 and the ion detector 17, and the mass analyzer 14 is used. Is separated according to the mass-to-charge ratio of the ions and reaches the ion detector 17 for detection.

  Step S7; A mass spectrum is obtained for each part of the plurality of parts of the sample 90 by the data acquisition unit 53 of the data analysis device 50. The obtained mass spectrum for each part is stored in the memory 52 in association with the coordinate position of each part in the surface of the sample 90.

  Here, the obtained mass spectrum is as shown in FIGS. 8, 9, and 10, for example. In these figures, the horizontal axis represents the mass-to-charge ratio m / z. The vertical axis represents the relative intensity of ions, and is normalized so that the maximum peak is 100%.

  FIG. 8 shows a mass spectrum of the portion 92-1 represented by the coordinate position (25, 5) in the surface of the sample 90. FIG. 9 shows a mass spectrum of the portion 92-2 represented by the coordinate position (35, 5) in the surface of the sample 90. FIG. 10 shows a mass spectrum of the portion 92-3 represented by the coordinate position (25, 10) in the surface of the sample 90.

  In the region represented by the coordinate position (25, 5) in FIG. 8, only a peak (for example, a peak at m / z = 18633.6533) representing the mass of ions derived from ganglioside GD1a appears (before normalization). The intensity (count number) is 1094.0 described in the upper right axis.) In the region represented by the coordinate position (35, 5) in FIG. 9, in addition to the peak representing the mass of the ion derived from ganglioside GD1a, the peak representing the mass of the ion derived from ganglioside GT1b and the mass of the ion derived from ganglioside GQ1b. And a peak representing. In the region represented by the coordinate position (25, 10) in FIG. 10, in the same manner as in FIG. 9, in addition to the peak representing the mass of ions derived from ganglyside GD1a, the peak representing the mass of ions derived from ganglyside GT1b And a peak representing the mass of ions derived from ganglyside GQ1b.

  Step S8; Based on the mass spectrum of each part of the sample 90 by the concentration map creation unit 54 of the data analyzer 50, the mass of the specific molecule (ganglyside GD1a, GT1b, GQ1b in this example) contained in the measurement object is calculated. Create a density map according to the intensity of the peak to be represented.

  In the examples of FIGS. 8, 9, and 10 described above, density maps as shown in FIG. 11 are obtained. In FIG. 11, the innermost region A indicates a region in which only the ganglyside GD1a is substantially observed and the relative intensity of peaks derived from the ganglysides GT1b and GQ1b is weak (for example, less than 20%). A region B surrounding the outer periphery of the region A indicates a region where, in addition to the peak derived from the ganglyside GD1a, the peak derived from the ganglyside GT1b, GQ1b is observed at an intermediate intensity (for example, 20% or more and less than 80%). A region C surrounding the outer periphery of the region B indicates a region where peaks derived from the ganglysides GT1b and GQ1b are observed with high intensity (for example, 80% or more) in addition to the peak derived from the ganglyside GD1a.

  In the density map, the color and density on the display screen are designated for these areas A, B, and C, respectively. The color on the display screen is specified for each type of substance by a known method, and the concentration on the display screen is set to increase as the relative intensity of the peak in the mass spectrum increases. It is specified.

  For example, in the example of FIG. 11, ganglysides GT1b and GQ1b are combined and the change in concentration is represented by regions A, B, and C. However, different colors are designated for ganglyside GT1b and ganglyside GQ1b, respectively. A change in density may be displayed.

  Step S9: The image creation unit 55 of the data analysis device 50 creates two-dimensional image data for displaying the density map created by the density map creation unit 54 on the image display device 51. As a result, a two-dimensional image representing the density map is displayed on the display screen of the image display device 51.

  According to this imaging method, the sample 90 is irradiated with infrared laser light (especially mid-infrared light is desirable), so that by-products can be generated without breaking molecules having weak chemical bonds in the molecule. The sample 90 can be ionized while being suppressed. Therefore, the resolution of mass spectrometry and the S / N ratio (signal-to-noise ratio) can be increased as compared with the case where ultraviolet light such as a nitrogen laser is used. As a result, an accurate two-dimensional image can be obtained.

  In the above example, the matrix is sprayed on the sample section (step S3), but the method of attaching the matrix to the sample section is not limited to this.

  For example, as shown in FIG. 7, the matrix may be dropped onto the surface of the sample section using a capillary tube 99. In the example of FIG. 7, the sample stage 13 'can be moved in the X and Y directions perpendicular to each other in the horizontal plane by the moving mechanism 40'. A sample plate 19 on which a sample section is placed is placed on the sample table 13 ', and the sample section is moved together with the sample table 13' by a moving mechanism 40 'at a constant pitch in the X direction and the Y direction. The matrix 92 is sequentially dropped on the part. The amount of the matrix 92 dropped on each part is set to a constant amount by adjusting the pressure and time pushed out through the capillary tube 99. As a result, as shown in FIG. 6 (B), the sample 90 in a mode in which the matrix 92 having a substantially constant diameter d3 is arranged in a matrix with the vertical and horizontal equal intervals c1 and c2 (c1 = c2) spaced apart from each other is ensured. can get.

  When the matrix is dropped, the diameter of the matrix 92 is set to 1.0 mm or more and 2.0 mm or less, typically 1.5 mm. The interval between the matrices 92 is set to 200 μm or more and 500 μm or less, typically 300 μm.

  According to the sample 90 obtained by dropping the matrix 92 in this way, when the sample 90 is irradiated with infrared laser light (step S6), the sample 90 is moved together with the sample stage 13 by the moving mechanism 40 in the X direction and Y direction. If it is moved in the direction at a constant pitch that is the same as the dropping pitch, the target site can be reliably irradiated.

  The memory 52 in the data analysis apparatus 50 in FIG. 3 stores a mass spectrum obtained under the same measurement conditions as the measurement conditions for the measurement object with respect to the reference object serving as a comparison reference for the measurement object. It can be used as a reference storage unit.

  That is, prior to the determination of the measurement object, the mass obtained in the memory 52 under the same measurement conditions as the measurement conditions for the measurement object for the reference object serving as a reference for comparison for the measurement object. Store the spectrum. Then, the concentration map creation unit 54 creates a concentration map by comparing the mass spectrum of the measurement object with the mass spectrum of the reference object. In this case, a more accurate two-dimensional image can be obtained.

  In the above embodiment, as shown in FIGS. 4 and 5, the optical system from the light source 11 to the beam condensing units 30 and 30 ′ is provided outside the vacuum container 12. However, the present invention is not limited to this. The optical system from the light source 11 to the beam condensing unit 30 may be provided in the vacuum vessel 12. On the other hand, the observation camera 18 may be arranged outside the vacuum vessel 12 so as to observe the surface of the sample 90 through a window.

  Moreover, although the sample 90 was moved with the sample stand 13 with the moving mechanism 40 with respect to the infrared laser beam irradiated to the sample 90, it is not restricted to this. Conversely, the optical system 30 may be translated with respect to the sample stage 13. Alternatively, the irradiation position in the surface of the sample 90 (sample stage 13) may be changed by the optical system 30 changing the emission direction (angle) of the infrared laser light.

  In this embodiment, the measurement object is a living tissue containing ganglicids. Needless to say, the present invention is not limited to this, and the two-dimensional imaging apparatus and method of the present invention can be widely applied to biological tissues and drugs containing elements other than ganglicides.

DESCRIPTION OF SYMBOLS 10 Two-dimensional imaging apparatus 11 IR light source 12 Vacuum container 13,13 'Sample stand 30,30' Beam condensing unit 40,40 'Moving mechanism 50 Data analysis apparatus 51 Image display apparatus 90 Sample 99 Capillary tube

Claims (5)

A laser light source for generating infrared laser light;
A sample stage on which a sample including a measurement object and at least a matrix attached to the surface of the measurement object is placed;
An optical system for guiding the infrared laser light from the laser light source to the sample on the sample stage;
A moving mechanism for relatively moving the optical system and the sample stage in order to irradiate the portion of the sample to which the matrix is attached with the infrared laser light;
A mass analyzer that separates ions generated by the sample in response to the infrared laser light in accordance with a mass-to-charge ratio of the ions;
An ion detector for detecting ions separated by the mass spectrometer;
Based on the output of the ion detector, a data acquisition unit that obtains a mass spectrum for each of the plurality of parts to which the matrix is attached within the surface of the sample;
A data storage unit that stores the mass spectrum of each part of the sample in association with the coordinate position of each part in the surface of the sample;
A concentration map creating unit that creates a concentration map according to the intensity of a peak representing the mass of a specific molecule contained in the measurement object based on the mass spectrum of each part of the sample. Two-dimensional imaging device. The two-dimensional imaging apparatus according to claim 1,
A comparison reference storage unit that stores a mass spectrum obtained under the same measurement conditions as the measurement conditions for the measurement object, with respect to the reference object serving as a reference for comparison for the measurement object,
The concentration map creating unit creates the concentration map by comparing the mass spectrum of the measurement object with the mass spectrum of the reference object. A sample including a measurement object and at least a matrix attached to the surface of the measurement object is placed on the sample table,
An infrared laser beam from a laser light source is guided to the sample by an optical system, and the infrared laser beam is irradiated to a portion of the sample to which the matrix is attached. Are moved relative to each other, and the measurement object is sequentially ionized with the help of the matrix for each of the plurality of parts to which the matrix of the sample is attached,
Ions generated sequentially in the plurality of parts of the sample are separated according to the mass-to-charge ratio of the ions by a mass analyzer and detected by reaching an ion detector,
Based on the output of the ion detector, the data acquisition unit obtains a mass spectrum for each of the plurality of parts of the sample,
In the data storage unit, the mass spectrum for each part of the sample is stored in association with the coordinate position of each part in the surface of the sample,
A two-dimensional imaging method, wherein a concentration map corresponding to the intensity of a peak representing the mass of a specific molecule contained in the measurement object is created based on a mass spectrum for each part of the sample. The two-dimensional imaging method according to claim 3,
A two-dimensional imaging method, wherein the sample is prepared by spraying the matrix on the surface of the measurement object. The two-dimensional imaging method according to claim 3,
A two-dimensional imaging method, wherein the sample is prepared by dropping the matrix onto the surface of the measurement object at a predetermined pitch.