1. Field of the Invention
The present invention relates to an information acquisition method for acquiring information including a two-dimensional distribution state of a substance to be analyzed by an imaging mass spectrometry method using time-of-flight secondary ion mass spectrometry (hereinafter, referred to as TOF-SIMS), and more particularly, to an information acquisition method including subjecting an organic substance such as a lipid or a protein as a substance to be analyzed to high-sensitivity analysis.
2. Description of the Related Art
In the fields of biochemistry and medicine, there is a demand for acquisition of distribution information of a specific substance that constructs a biological tissue. As an example, in the field of medicine such as pathology, when distribution information of a specific antigen protein at a “cellular level” can be acquired in definitive diagnosis, a type of the disease can be determined and a therapy for the disease can be appropriately selected.
Conventionally acquisition of distribution information of a substance has been carried out by “immunostaining” including indirect observation of a substance to be detected using an antigen-antibody reaction of the substance. The immunostaining, however, has a problem of poor reproducibility due to the fact that an antibody is unstable and antigen-antibody reaction efficiency is difficult to control. Further, in the future, if the number of antigen proteins of interest providing evidence for definitive diagnosis becomes too large, e.g., when there arises a need of detection of more than hundreds of kinds of proteins, the current immunostaining cannot deal with the need.
Based on such backgrounds, there is an expectation for appearance of novel analysis techniques for exhaustively visualizing a substance to be detected. As one of the techniques, an imaging mass spectrometry method, which is an application of a mass spectrometry method, has been extensively developed in recent years.
The imaging mass spectrometry method is a technique for visualizing a two-dimensional distribution state of a specific substance in a sample by dividing any area in the sample of interest into smaller areas and analyzing each of the smaller areas by a mass spectrometry method. In the mass spectrometry method, relative intensity (mass spectrum) of each ion at each mass-to-charge ratio can be obtained by allowing an electric field or a magnetic field to act on a sample substance ionized in vacuum. A molecular weight of the detected substance can be determined from the mass-to-charge ratio of each ion peak in the resultant mass spectrum, and a mass of the substance can be determined from the height of the ion peak. Further, the mass can be divided by the molecular weight to determine an abundance of the substance. In addition, by subjecting each of the smaller areas to mass spectrometry and reconstructing an image using an ion peak of each substance included in the resultant mass spectrum, a two-dimensional distribution state of the specific substance can be imaged. Representative examples of the imaging mass spectrometry method include a secondary ion mass spectrometry method (SIMS) including irradiating each area with an ion as a primary probe (primary ion) and subjecting a secondary ion released by sputtering to mass spectrometry.
In order to allow measurement of a mass of a substance to be analyzed in a mass spectrometry method, it is necessary that the substance to be analyzed form an independent particulate state including, as a unit, an atom, a molecule, a cluster, or the like, and that the substance to be analyzed, in an independent particulate state, have a positive or negative charge. That is, it is necessary that, in mass spectrometry, desorption and ionization of a substance to be analyzed be achieved by some method.
Further, in order to assign a detected substance with high accuracy, it is preferred that both of inhibition of a degree of fragmentation of an ion to be analyzed, the so-called “soft ionization” and high ionization efficiency be achieved.
As a soft ionization method for proteins or lipids, or a variety of biologically-relevant molecules which are composite molecules of the proteins and lipids in SIMS analysis, there have been proposed, for example, a method including analyzing a mixed sample prepared by dispersing or dissolving a sample in a matrix substance (Analytical Chemistry 1996, 68, P. 873), a method including forming a metal thin layer on a sample by vapor deposition (Analytical Chemistry 2002, 74, P. 4955), and a method including laminating a nonvolatile liquid compound such as glycerol as a liquid matrix on a sample without destructing or dissolving the sample (Applied Surface Science 2008, 255, P. 929).
As a highly efficient ionization method for proteins or lipids, or a variety of biologically-relevant molecules which are composite molecules of the proteins and lipids, in SIMS analysis, there is known, for example, a method including carrying out cationization by protonation. The inventors of the present invention have hitherto proposed methods each of which achieves high ionization efficiency by detecting a trace amount of a biologically-relevant substance using an SIMS-specific sensitizing substance. For example, Japanese Patent Application Laid-Open No. 2009-264911 proposes a method including ionizing a substance to be analyzed over a long period of time or each measurement site uniformly with high efficiency by applying a fluorocarboxylic acid which has a strong proton-donating ability and is nonvolatile, to a sample.