Sample preparation method and sample preparation device for MALDI including depositing matrix substance on sample substrate in two steps

Abstract

After a sample such as a biomedical tissue section is attached to an electrically-conductive slide glass (S1), the film layer of a matrix substance is appropriately formed by vapor deposition so as to cover the sample (S2). The crystal of the matrix substance in the film layer is very fine and uniform. Subsequently, the slide glass on which the matrix film layer is formed is placed in a vaporized solvent atmosphere, and the solvent infiltrates into the matrix film layer (S3). When the solvent sufficiently infiltrated is vaporized, a substance to be measured in the sample takes in the matrix and re-crystallized. Furthermore, the matrix film layer is formed again on the surface by the vapor deposition (S4). The added matrix film layer absorbs excessive energy of a laser beam during MALDI, which suppresses the denaturation of the substance to be measured and the like, so that high detection sensitivity can be achieved while high spatial resolution is maintained.

Claims

1. A sample preparation method for MALDI, the sample preparation method for preparing a sample for mass spectroscopy using a matrix assisted laser desorption ionization method and configured to execute steps, comprising: a) a matrix depositing step for vaporizing a matrix substance in vacuum and depositing the matrix substance to form a matrix layer on a surface of a sample substrate on which a sample to be measured is placed; b) a solvent introducing step for bringing a predetermined solvent in gaseous or liquid into contact with a surface of the matrix film layer formed on the sample substrate so as to infiltrate the solvent into the matrix film layer; and c) a matrix re-depositing step for vaporizing the matrix substance in vacuum and depositing the matrix substance again on the surface of the matrix film layer in a state where the solvent is infiltrated, or in a state where the infiltrated solvent is volatilized.

2. The sample preparation method for MALDI according to claim 1, wherein, in the solvent introducing step, the sample substrate on which the matrix film layer is formed is left in a container filled with a vaporized solvent for a predetermined period of time so as to infiltrate the solvent into the matrix film layer.

3. The sample preparation method for MALDI according to claim 1, wherein, in the solvent introducing step, the solvent is sprayed on the surface of the matrix film layer formed on the sample substrate so as to infiltrate the solvent into the matrix film layer.

4. A sample preparation method for MALDI, the sample preparation method for preparing a sample for mass spectroscopy using a matrix assisted laser desorption ionization method and configured to execute steps, comprising: a) a matrix depositing step for vaporizing a matrix substance in vacuum and depositing the matrix substance on a surface of a sample substrate on which a sample to be measured is placed; and b) a solution introducing step for spraying a matrix solution having a concentration of the matrix substance that is lower than a concentration of the matrix substance in the matrix depositing step a) on a surface of a matrix film layer formed on the sample substrate so as to infiltrate the solution into the matrix film layer.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a flowchart illustrating a procedure of processing in a sample preparation method for MALDI in a first embodiment of the present invention.

(2) FIG. 2 is a flowchart illustrating a procedure of processing in a sample preparation method for MALDI in a second embodiment of the present invention.

(3) FIG. 3 is a flowchart illustrating a procedure of processing in a sample preparation method for MALDI in a third embodiment of the present invention.

(4) FIGS. 4A to 4D are cross-sectional conceptual diagrams of a sample prepared in the sample preparation method for MALDI according to the present invention.

(5) FIG. 5 is a schematic configuration diagram of a sample preparation device to implement the sample preparation method for MALDI in the first embodiment.

(6) FIG. 6 is a photograph illustrating an analytical range in a sample to be measured that is used in a first experiment so as to verify the effects of the present invention.

(7) FIGS. 7A to 7C are mass spectra acquired by averaging mass spectra obtained at tall analytical points in an analytical range in the first experiment.

(8) FIGS. 8A and 8B are mass spectra acquired by averaging mass spectra obtained at all analytical points in the analytical range in the first experiment.

(9) FIGS. 9A to 9C are diagrams illustrating the comparison of mass spectroscopy imaging images obtained with an imaging mass spectrometer in the first experiment.

(10) FIGS. 10A and 10B are enlarged diagrams of a mass spectrum in a range of m/z 848.400 to 848.800 in the first experiment.

(11) FIGS. 11A and 11B are diagrams illustrating the mass spectroscopy imaging images in the vicinity of a mass-to-charge ratio range illustrated in FIGS. 10A and 10B.

(12) FIGS. 12A to 12C are diagrams in a case where only vapor deposition is conducted in a second experiment, and FIG. 12A illustrates a microscopic observation image of a sample surface after matrix application, and FIG. 12B illustrates a mass spectrum acquired by averaging mass spectra obtained at all analytical points in an analytical range, and FIG. 12C illustrates representative mass spectroscopy imaging images.

(13) FIGS. 13A to 13C are diagrams in a case where only a solvent is sprayed and applied after the vapor deposition in the second experiment, and FIG. 13A illustrates a microscopic observation image of a sample surface after matrix application, and FIG. 13B illustrates a mass spectrum acquired by averaging mass spectra obtained at all analytical points in an analytical range, and FIG. 13C illustrates representative mass spectroscopy imaging images.

(14) FIGS. 14A to 14C are diagrams in a case where a matrix solution having low concentration is sprayed and applied after the vapor deposition in the second experiment, and FIG. 14A illustrates a microscopic observation image of a sample surface after matrix application, and FIG. 14B illustrates a mass spectrum acquired by averaging mass spectra obtained at all analytical points in an analytical range, and FIG. 14C illustrates representative mass spectroscopy imaging images.

(15) FIGS. 15A to 15C are diagrams in a case where only the solvent is applied with the nebulizer after the vapor deposition in the second experiment, and FIG. 15A illustrates a microscopic observation image of a sample surface after matrix application, and FIG. 15B illustrates a mass spectrum acquired by averaging mass spectra obtained at all analytical points in an analytical range, and FIG. 15C illustrates representative mass spectroscopy imaging images.

(16) FIGS. 16A to 16C are diagrams in a case where the matrix solution having low concentration is applied with the nebulizer after the vapor deposition in the second experiment, and FIG. 16A illustrates a microscopic observation image of a sample surface after matrix application, and FIG. 16B illustrates a mass spectrum acquired by averaging mass spectra obtained at all analytical points in an analytical range, and FIG. 16C illustrates representative mass spectroscopy imaging images.

(17) FIGS. 17A to 17C are diagrams in which the results of the second experiment are compiled.

DESCRIPTION OF EMBODIMENTS

(18) Hereinafter, several embodiments of a sample preparation method for MALDI according to the present invention will be described. This embodiment represents the preparation of a sample of a case where a tissue section originating from a living organism is measured with an imaging mass spectrometer.

First Embodiment

(19) FIG. 1 is a flowchart illustrating a procedure of processing in a sample preparation method for MALDI according to a first embodiment in the present invention. FIGS. 4A to 4D are cross-sectional conceptual diagrams of a prepared sample.

(20) First, an operator places a thin-film sample 2 such as a tissue section, which is a target to be measured, on an electrically-conductive slide glass 1 that corresponds to a sample substrate in the present invention (Step S1). It is noted that a metallic plate such as stainless steel may be employed as the sample substrate, besides the electrically-conductive slide glass.

(21) Subsequently, a film layer of a predetermined matrix substance is formed by a vacuum vapor deposition method so as to cover the whole of the sample 2 placed on the electrically-conductive slide glass 1 (Step S2). As the matrix substance, substances generally used in a conventional sample preparation method for MALDI, for example, DHB, CHCA (α-cyano-4-hydroxycinnamic acid), 9-AA (9-aminoacridine), or various substances described above besides these can be used without processing. A matrix film layer 3 including crystals which are very fine and dense is formed on the sample 2 by the vacuum vapor deposition method (see FIG. 4A). The thickness of the matrix film layer 3 is adequate on the order of about 0.5 to 1.5 [μm].

(22) Subsequently, the electrically-conductive slide glass 1 on which the matrix film layer 3 is formed is placed in the atmosphere of the vaporized solvent, and the state is maintained in a predetermined period of time. As illustrated in FIG. 4B, this allows the solvent to gradually infiltrate into the matrix film layer 3 from the surface of the matrix film layer 3 being in contact with the vaporized solvent (Step S3). A solvent used in preparing the matrix solution by the conventional sample preparation method for MALDI, for example, methanol can be used as the solvent.

(23) When the solvent humidified in the matrix film layer 3 reaches the sample 2 and then vaporizes, a substance to be measured in the sample (for example, protein, or administered medicine) is taken in the matrix substance and re-crystallized, to form a cocrystal. The area of the cocrystal is illustrated by a reference number 4 in FIG. 4C. A film layer of the matrix substance is formed again by the vacuum vapor deposition method on the surface of the matrix film layer 3 on which the cocrystal area 4 is formed through humidification of the solvent (Step S4). As a result, as illustrated in FIG. 4D, the surface of the matrix film layer 3 on which the cocrystal area 4 is formed is covered with a matrix film layer 5. The thickness of the matrix film layer 5 is adequate on the order of about 0.5 to 1.5 [μm]. This completes preparation of the sample for MALDI (Step S5).

(24) The formation of the matrix film layers 3 and 5 in Steps S2 and S4 can be typically conducted with a vacuum vapor deposition device for forming a film on a targeted object by heating and vaporizing the matrix substance. The humectation of the solvent into the matrix film layer 3 in Step S3 can be conducted in the following manner. That is, the electrically-conductive slide glass 1 on which the matrix film layer 3 is formed, is placed in the interior of a hermetically-sealed container in which a predetermined amount of solvent is stored, and installed so as to bridge above a support body made of hydrophobic resin. The hydrophobic support body is provided to prevent the direct contact of the electrically-conductive slide glass 1 with the solvent that gradually oozes upward. The solvent generally has high volatility, but when a solvent that is relatively hard to volatilize, for example, water is used, vaporization may be facilitated by appropriately heating the solvent or vibrating the solvent with ultrasonic waves. As this fills the interior of the hermetically-sealed container with the vaporized solvent, the solvent can be humidified in the matrix film layer 3 by maintaining its atmosphere for a predetermined period of time.

(25) It is noted that, when the matrix film layer 5 is formed with the vacuum vapor deposition device, the matrix film layer 3 in which the solvent is humidified in the prior process needs not necessarily be dried. This is because when the electrically-conductive slide glass 1 is placed in the vacuum atmosphere to conduct the vacuum vapor deposition in Step S4, the solvent in the matrix film layer 3 vaporizes in a very short period of time and is removed.

(26) The mass spectroscopy is conducted for thus prepared sample with the imaging mass spectrometer, and the sample has the following characteristics in the analysis.

(27) As described above, the crystals of the matrix substance in the matrix film layers 3 and 5 formed by the vacuum vapor deposition are very fine and uniform. There occurs no needle-shaped crystallization, which causes the problem in the case where DHB and the like are applied to the sample surface by the spray method. When the laser beam having a microscopic diameter, which is narrowed for ionization, is irradiated to the sample, the crystals existed on the irradiated portion scatter, but the crystals do not scatter from the periphery of the irradiated portion because the crystals are fine, and therefore the substance to be measured is ionized in a state where the positional information on the sample 2 is secured. For this reason, as the irradiation diameter of the laser beam is reduced, the spatial resolution can be improved accordingly.

(28) Also, when the laser beam having a large amount of energy is used, the substance originating from a living organism, in particular, protein or the like is prone to suffer damage such as denaturation. This is one of factors in reduction of the ion generation amount from the target substance when the laser beam is repeatedly irradiated at plural times for signal integration. In contrast, in the prepared sample described above, the cocrystal area 4 in which the substance to be measured is distributed is covered with the matrix film layer 5, and therefore, when the laser beam is irradiated to the substance to be measured, the particles of the substance in the matrix film layer 5 appropriately absorb the laser beam and alleviate the energy applied to the substance to be measured. This suppresses denaturation of the substance to be measured, and the ion generation amount can be increased, compared with a case where there is no matrix film layer 5. As a result, the larger amount of ions contribute to the mass spectroscopy, and high detection sensitivity can be achieved.

Second Embodiment

(29) FIG. 2 is a flowchart illustrating a procedure of processing in a sample preparation method for MALDI according to a second embodiment in the present invention. Only Step S3 in the first embodiment is changed to Step S13, and each step except for Step S13 is the same with that of the first embodiment.

(30) In the sample preparation method for MALDI in the second embodiment, the solvent is directly sprayed with a spray device such as an airbrush on the surface of the matrix film layer 3 formed on the electrically-conductive slide glass 1. This attaches the minute droplets of the solvent to the surface of the matrix film layer 3 and infiltrates the solvent into the matrix film layer 3 (Step S13).

(31) In the sample preparation method according to the first embodiment, it takes a time, for example, the order of several hours, to cause the matrix film layer 3 to be humidified sufficiently, whereas in the sample preparation method according to the second embodiment, time required for it can be considerably shortened. However, when an operator sprays the solvent to the matrix film layer 3, a difference in finishing quality of the sample frequently arises depending on the skill of the operator.

Third Embodiment

(32) FIG. 3 is a flowchart illustrating a procedure of processing in a sample preparation method for MALDI according to a third embodiment in the present invention. Although Steps S1 and S2 are exactly identical to those in the sample preparation method in the first embodiment, the processes in Step S3 onward are different.

(33) In the sample preparation method for MALDI according to the third embodiment, after the matrix film layer 3 is formed on the electrically-conductive slide glass 1, the matrix solution having low concentration is directly sprayed with the spray device such as the airbrush on the surface of the matrix film layer 3 (Step S23), and subsequently the matrix film layer 3 is dried to remove the solvent (Step S24). This “low concentration” means the concentration lower than the concentration of the matrix solution used in a conventional general matrix application method, and specifically, the adequate concentration is about half to one fifth of the concentration of the saturation of the matrix solution.

(34) The matrix substance in the matrix solution applied on the surface of the matrix film layer 3 formed by the vacuum vapor deposition grows with crystals which are fine and uniform in the matrix film layer 3 as a core, and therefore, even when the matrix solution is applied with non-uniform, uniform crystals are easily generated. For this reason, the crystals of the matrix substance generated by the applied matrix solution are fine and uniform. Also, the solvent in the matrix solution infiltrates into the matrix film layer 3 to reach the sample 2, and forms cocrystals of the substance to be measured and the matrix substance in the sample, and a film layer of the matrix substance including the crystals in the matrix solution is formed such that the matrix film layer 3 is covered with the film layer. Accordingly, a sample having cross-sectional structure similar to that of the sample prepared in the sample preparation method in the first and second embodiments illustrated in FIG. 4D is completed. In this way, the sample prepared in the sample preparation method in the third embodiment has the effects and advantages similar to those of the sample prepared in the sample preparation methods in the first and second embodiments.

(35) Next, an embodiment of the sample preparation device for implementing the sample preparation method in the first embodiment will be described. FIG. 5 is a schematic configuration diagram of the sample preparation device in the present embodiment.

(36) The sample preparation device includes a base 10 and an openable/closable vacuum chamber 11, and a film forming chamber of which the interior can be maintained in a vacuum atmosphere is constituted by the base 10 and the vacuum chamber 11. A vacuum pump 13 and a vaporized solvent generating unit 15 are installed to the base 10 via a first valve 12 and a second valve 14, respectively, and further a vacuum gauge 16 for measuring a degree of vacuum in the film forming chamber and a leak valve 17 for reducing the degree of vacuum in the film forming chamber are installed to the base 10. A sample stage 18 on which the electrically-conductive slide glass (or a metal plate or the like) 1 is placed, a vapor deposition source 19 in which a matrix substance 20 is set, and a shutter 21 are installed in the film forming chamber.

(37) The vapor deposition source 19 heats the matrix substance 20 in the film forming chamber under vacuum atmosphere so as to scatter the matrix substance 20 in the form of particles in the space. The types of vapor deposition source 19 include a boat type, a basket type, a crucible type, and a wire type, which is appropriately selected in accordance with the form or amount of the matrix substance to be used, or the direction in which the evaporated particles are scattered. In the example of FIG. 5, the boat type is used. The sample stage 18 consists of a support plate 18b horizontally arranged and having an opening 18c formed approximately in the center thereof, and a support rod 18a holding the support plate 18b. The opening 18c is provided immediately above the matrix substance 20 of the vacuum vapor deposition source 19, and the electrically-conductive slide glass 1 is placed on the support plate 18b in a manner that the attached sample 2 faces downward, namely is opposed to the matrix substance 20. The shutter 21 consists of a support shaft 21a and a blocking plate 21b. The shutter 21 causes the blocking plate 21b to rotate about the support shaft 21a in a predetermined angle range so as to block or pass the particles of the matrix substance advancing upward, namely, toward the electrically-conductive slide glass 1 from the vacuum vapor deposition source 19.

(38) A control unit 30 that controls each unit for sample preparation in the sample preparation device includes functional blocks such as a heat control unit 31, a vacuum control unit 32, a gas supply control unit 33, and a shutter drive control unit 34. The control unit 30 can be embodied, for example, by a microcomputer including a CPU, a ROM, a RAM, a timer, and the like and can perform the control operation in the functional blocks, for example, in the process of executing control programs stored in the ROM or computational processing in accordance with control parameters by means of the CPU.

(39) The operations in the case of automatically preparing the sample in the sample preparation device in the present embodiment will be described in association with each step in FIG. 1.

(40) An operator puts the sample 2 on the electrically-conductive slide glass 1, and places the slide glass 1 on the support plate 18b of the sample stage 18 as illustrated in FIG. 5. Then, the operator puts an appropriate matrix substance such as DHB on the vapor deposition source 19, closes the vacuum chamber 11, and instructs the start using an operating unit not illustrated. Upon receiving the instruction, the vacuum control unit 32 of the control unit 30 closes the second valve 14 and the leak valve 17, activates the vacuum pump 13, and evacuates the film forming chamber through the first valve 12. After the start of the evacuation, the vacuum control unit 32 monitors gas pressure in the film forming chamber by means of the vacuum gauge 16, and when the actually-measured gas pressure reaches target gas pressure set in advance, the vacuum control unit 32 switches the operations of the vacuum pump 13 so as to maintain the actually-measured gas pressure in the vicinity of the target gas pressure.

(41) When the actually-measured gas pressure reaches the target gas pressure, as illustrated in FIG. 5, the heat control unit 31 starts heating of the vapor deposition source 19 in a state where the shutter 21 is closed (state where the blocking plate 21b is positioned above the vapor deposition source 19). A heating temperature can be controlled by adjusting a heating current fed to a vapor deposition board. When the heating temperature reaches a target temperature set in advance (sublimation temperature of the matrix substance 20, for example, about 130 degrees Celsius in DHB), the heating current is adjusted for keeping the heating temperature approximately constant.

(42) Upon elapse of a predetermined period of time after the heating temperature reaches the target temperature, the shutter drive control unit 34 opens the shutter 21. This causes the particles sublimated from the matrix substance 20 to reach the electrically-conductive slide glass 1, which starts the vapor deposition. For example, when the vapor deposition is conducted for a predetermined period of time so that the matrix film layer deposited on the electrically-conductive slide glass 1 has a predetermined thickness, the shutter 21 is closed, and the heating of the vapor deposition source 19 is stopped. It is noted that, preferably, the timing of stopping the vapor deposition is determined not by the time of the vapor deposition, but by a technique, for example, proposed in Patent Application No. 2012-159296 (see JP No. 2013-137294 A) by the applicant of the instant application in which the thickness of the matrix film layer is monitored, and the timing of stopping the vapor deposition is determined based on its monitoring result.

(43) When time has passed to the extent that the temperature of the vapor deposition source 19 is sufficiently lowered after stopping the vapor deposition, the vacuum control unit 32 stops the vacuum pump 13 and closes the first valve 12. Then the gas supply control unit 33 opens the second valve 14 and supplies the vaporized solvent generated in the vaporized solvent generating unit 15 into the film forming chamber. The vaporized solvent generating unit 15 appropriately heats the solvent or vibrates the accumulated solvent with supersonic to generate the vaporized solvent. This fills the interior of the film forming chamber with the vaporized solvent, and the electrically-conductive slide glass 1 on which the matrix film layer is placed under vaporized solvent atmosphere. The solvent infiltrates into the matrix film layer by maintaining this state for a predetermined period of time (normally for about several hours).

(44) When a predetermined period of time set in advance has passed, the gas supply control unit 33 closes the second valve 14 and stops supplying the vaporized solvent to the film forming chamber. Along with this, the vacuum control unit 32 activates the vacuum pump 13 again, opens the first valve 12, and evacuates the film forming chamber. Then, as is the same with the first formation of the matrix film layer, when the gas pressure in the film forming chamber reaches the target gas pressure, the heating of the vapor deposition source 19 is started, and when a predetermined period of time has passed after the heating temperature reaches the target temperature, the shutter 21 is opened, and the vapor deposition is executed.

(45) Then, when it is determined that the second matrix film layer has a predetermined thickness determined in advance, the shutter 21 is closed, and the heating of the vapor deposition source 19 and the vacuum vapor deposition are stopped, and the all processes complete.

(46) Naturally, the operator may manually perform a part or the whole of works or operations, instead of automatically conducting a series of works all, ranging from the initial vacuum vapor deposition to the completion of all processes. Specifically, a part or the whole of works such as the opening/closing of the valves 12, 14, 17, and the like, the activating and stopping of the vacuum pump 13, the heating and stopping of the vapor deposition source 19, the adjusting of the heating current, and the opening/closing of the shutter 21 may be carried out by instructions by the operator. Although these works takes time, the sample can be prepared without removing the electrically-conductive slide glass 1 on which the sample 2 is attached after it is stored in the film forming chamber. This sufficiently reduces the burdens imposed on the operator compared with a case where the solvent infiltration into the matrix film layer is conducted outside of the film forming chamber.

(47) Subsequently, the procedure and results of experiments implemented to verify the effects of the sample preparation method for MALDI according to the present invention will be described.

(48) [Procedure and Results of First Experiment]

(49) In this experiment, a sample to be measured is 10 [μm] section of a mouse cerebellum. FIG. 6 is a photograph illustrating an analytical range in the sample. The matrix substance is DHB, a used mass spectrometer is an imaging mass spectrometer manufactured by Shimadzu Corporation, the diameter of laser emitted from an MALDI ion source is 5 [μm], the pitch of a laser spot on the sample is 10 [μm], analytical points in the analytical range is 250×250, and the range of a mass-to-charge ratio is m/z 400 to 1200. Also, in the sample preparation method, three methods including the method in the third embodiment (referred to as “vapor deposition+spray method” in the description and drawings below), a conventional method with only the vapor deposition with no spray (referred to as “vapor deposition method” in the description and drawings below), and a conventional spray method (referred to as “spray method” in the description and drawings below) are examined. It is noted that a vapor deposition time in the vapor deposition+spray method is three minutes, and a vapor deposition time in the vapor deposition method is 12 minutes.

(50) FIGS. 7A, 7B and 7C are mass spectra acquired by averaging mass spectra obtained at all analytical points (250×250 points). FIGS. 8A and 8B are diagrams illustrating only the mass spectra in the vapor deposition+spray method and the vapor deposition method. It finds from these diagrams that the spray method has the largest number of detected peaks, and the vapor deposition+spray method has the second largest number of detected peaks, and the vapor deposition method has the least number of detected peaks. Also, it finds that the number of detected peaks is few only in the least method, but the number of detected peaks increases by combining a spray having a low concentration solvent with this.

(51) FIGS. 9A, 9B and 9C are diagrams illustrating the comparison of mass spectroscopy imaging images representing the two-dimensional distribution of a substance having a specific mass-to-charge ratio, which is obtained by the imaging mass spectrometer. In the case of the spray method, only unclear images are obtained at m/z 769.56, and images at m/z 760.58 is incapable of reflecting the boundary between tissues on the sample. That is, the number of detected peaks is large in the spray method, whereas the mass spectroscopy imaging image has low sharpness, which is not suitable for the imaging mass spectroscopy. On the other hand, in the vapor deposition method and the vapor deposition+spray method, very clear images are obtained compared with the spray method.

(52) FIGS. 10A and 10B are mass spectra in the narrow range of a mass-to-charge ratio of m/z 848.400 to 848.800. Attention needs to be paid in that the scale of a vertical axis (signal intensity axis) of FIG. 10A is ten times as much as that of FIG. 10B. For example, when peak intensity at m/z 848.648 is observed, the vapor deposition+spray method is four times as much as the spray method. That is, the vapor deposition+spray method represents high sensitivity, compared with the vapor deposition method. FIGS. 11A and 11B are mass spectroscopy imaging images in the vicinity of this mass-to-charge ratio range. As described above, the vapor deposition+spray method is higher in signal detection sensitivity than the vapor deposition method, and therefore the intensity value of a pixel in which the substance exists on the mass spectroscopy imaging image, increases, and as a result, a position in which the substance exists is clearly illustrated can be confirmed.

(53) Based on the results above, the vapor deposition+spray method which is one technique of the present invention is suitable, in particular, for the imaging mass spectroscopy, and the following advantages are confirmed: the number of detected peaks is large (that is, further many pieces of information on components is obtained) compared with the simple vapor deposition method, and a clear mass spectroscopy imaging image can be obtained, in particular, a clear mass spectroscopy imaging image for even a relatively small amount of components can be obtained, thanks to high sensitivity.

(54) [Procedure and Results of Second Experiment]

(55) In the second experiment, a 10 [μm] section of a liver of a normal mouse has been used as a sample to be measured. Also, in this experiment, the matrix substance is CHCA, a used mass spectrometer is an imaging mass spectrometer manufactured by Shimadzu Corporation, the diameter of laser emitted from the MALDI ion source is 20 [μm], the pitch of a laser spot on the sample is 25 [μm], analytical points in the analytical range is 70×52, and the range of a mass-to-charge ratio is m/z 100 to 670. A vapor deposition device manufactured by Shimadzu Corporation is used for the vapor deposition of the matrix substance on the surface of a sample placed on the electrically-conductive sample glass, and vacuum evaporation conditions are the following: gas pressure is 10 [Pa], a temperature of the vapor deposition source is 240 degrees Celsius, and a vapor deposition time is about four minutes. The gas pressure in this time is quite a low degree of vacuum as a general vapor deposition condition. It is noted that the vapor deposition time actually does not determine the stop timing of vapor deposition based on a time, but the vapor deposition is stopped at a time point when two interference fringes emerged on the surface of a deposited film layer become visible. As a result of this procedure, the vapor deposition time is about four minutes, and the thickness of the matrix film layer is about 0.6 [μm].

(56) For the sample preparation methods, the following four types of method are examined, in addition to “vapor deposition method” in the first experiment.

(57) (1) Only the solvent (75% ethanol, 25% water) is sprayed with the airbrush after the matrix substance is deposited (hereinafter referred to “vapor deposition+solvent spray method”).

(58) (2) A low-concentration matrix solution (CHCA having concentration of 10 [mg/mL] is dissolved into the solvent described above) is sprayed with the airbrush after the matrix substance is deposited (hereinafter referred to “vapor deposition+low-concentration solution spray method”).

(59) (3) Only the solvent (75% ethanol, 25% water) is sprayed with a nebulizer after the matrix substance is deposited (hereinafter referred to “vapor deposition+solvent nebulizer method”).

(60) (4) A low-concentration matrix solution similar to (2) is sprayed with the nebulizer after the matrix substance is deposited (hereinafter referred to “vapor deposition+low-concentration solution nebulizer method”).

(61) In (3) and (4), the spray with the nebulizer is repeated ten times for 10 seconds (the intervals are ten seconds or more) so as to carry out intermittent spray. In this way, by use of the nebulizer, considerably fine droplets are acquired from the sprayed solution compared with the spray with the airbrush.

(62) FIGS. 12A, 12B and 12C are diagrams in the case of executing the vapor deposition method. FIG. 12A illustrates a microscopic observation image of the sample surface after matrix application, FIG. 12B illustrates a mass spectrum acquired by averaging mass spectra obtained at all analytical points in the analytical range, and FIG. 12C illustrates representative mass spectroscopy imaging images.

(63) FIGS. 13A, 13B and 13C are diagrams in the case of executing vapor deposition+solvent spray method. FIG. 13A illustrates a microscopic observation image of the sample surface after matrix application, FIG. 13B illustrates a mass spectrum acquired by averaging mass spectra obtained at all analytical points in the analytical range, and FIG. 13C illustrates representative mass spectroscopy imaging images.

(64) FIGS. 14A, 14B and 14C are diagrams in the case of executing vapor deposition+low-concentration solution spray method. FIG. 14A illustrates a microscopic observation image of a sample surface after matrix application, FIG. 14B illustrates a mass spectrum acquired by averaging mass spectra obtained at all analytical points in an analytical range, and FIG. 14C illustrates representative mass spectroscopy imaging images.

(65) FIGS. 15A, 15B and 15C are diagrams in the case of executing vapor deposition+solvent nebulizer method. FIG. 15A illustrates a microscopic observation image of a sample surface after matrix application, FIG. 15B illustrates a mass spectrum acquired by averaging mass spectra obtained at all analytical points in an analytical range, and FIG. 15C illustrates representative mass spectroscopy imaging images.

(66) FIGS. 16A, 16B and 16C are diagrams in the case of executing vapor deposition+low-concentration solution nebulizer method. FIG. 16A illustrates a microscopic observation image of a sample surface after matrix application, FIG. 16B illustrates a mass spectrum acquired by averaging mass spectra obtained at all analytical points in an analytical range, and FIG. 16C illustrates representative mass spectroscopy imaging images.

(67) FIGS. 12B, 13B, 14B, 15B, and 16B show the mass spectrum acquired by averaging the mass spectra obtained at all analytical points (70×52 points). Also, FIGS. 12C, 13C, 14C, and 15C show the mass spectroscopy imaging images of three substances of spermidine, spermine, and CHCA (adduct ion) which is a matrix.

(68) It finds from these diagrams that general detection sensitivity is considerably low in the vapor deposition method in which the solvent or the low-concentration solution is not sprayed, and that the spermidine or the spermine assumed to be normally distributed over the whole of the sample on the mass spectroscopy imaging images, is hardly observed. In contrast, when the solution, in particular, the low-concentration solution is sprayed with the spray device or with the nebulizer, the detection sensitivity is generally improved, and the number of detected peaks increases. Also, the intensity value of a pixel corresponding to the spermidine or the spermine increases on the mass spectroscopy imaging images, and therefore it can be confirmed that the positions in which these substances exist are clearly shown. It is noted that the detection sensitivity in the solvent sprayed with the nebulizer is improved to the extent of that of the low-concentration solution spray, whereas the improvement of the detection sensitivity cannot be confirmed when the solvent is sprayed with the spray device. The reason is assumed that this is due not to the difference between the spray methods with the airbrush and the nebulizer but to the size of the droplet to be sprayed.

(69) FIGS. 17A, 17B and 17C are diagrams illustrating compiled experimental results of a peak area, an intensity ratio to a peak originating from the matrix, and the intensity ratio in the case of only the vapor deposition, with regard to peaks corresponding to spermidine, spermine, and CHCA, which emerge on the spectra illustrated in FIGS. 12B, 13B, 14B, 15B, and 16B. In view of FIG. 17B, it can be found that the spray implemented with the nebulizer in any of the solvent spray and the low-concentration solution spray increases the intensity ratio of the peak of the spermidine or the spermine. These substances are water-soluble polyamines, and as for these water-soluble substances, it can be concluded that when an organic solvent mixed with water is sprayed without spraying the matrix solution intentionally, the substantially great improved effects of the detection sensitivity are obtained.

(70) Also, as described above, when the low-concentration solution is sprayed, the detection sensitivity of the substance such as polyamines is enhanced, but as is evident from FIG. 17C, the increase in intensity of the peak originating from the matrix is conspicuous. In this way, when any of the solvent and the low-concentration solution is used, it can be said that the implementation of the spray of not large droplets but fine droplets is desirable.

(71) Also, the vapor deposition is carried out under sufficiently high degree of vacuum (gas pressure of the order of 10.sup.−3 [Pa]) in the first experiment, whereas the degree of vacuum in the case of vapor deposition of the matrix substance is considerably low in the second experiment. In this way, it finds that favorable analytical results can be obtained only by appropriately controlling the thickness of the matrix film layer even when the vapor deposition of the matrix substance is carried out under the condition with a low degree of vacuum.

(72) It is noted that any of the embodiments described above is a mere example of the present invention, and it is obvious that changes, additions, and modifications are appropriately included in the scope of the claims of the instant application within the scope of the gist of the present invention.

REFERENCE SIGNS LIST

(73) 1 . . . Electrically-conductive Slide Glass 2 . . . Sample 3, 5 . . . Matrix Film Layer 4 . . . Cocrystal Area 10 . . . Base 11 . . . Vacuum Chamber 12 . . . First Valve 13 . . . Vacuum Pump 14 . . . Second Valve 15 . . . Vaporized Solvent Generating Unit 16 . . . Vacuum Gauge 17 . . . Leak Valve 18 . . . Sample Stage 18a . . . Support Rod 18b . . . Support Plate 18c . . . Opening 19 . . . Vapor Deposition Source 20 . . . Matrix Substance 21 . . . Shutter 21a . . . Support Shaft 21b . . . Blocking Plate 30 . . . Control Unit 31 . . . Heat Control Unit 32 . . . Vacuum Control Unit 33 . . . Gas Supply Control Unit 34 . . . Shutter Drive Control Unit