Broadly speaking, there are two categories of imaging mass spectrometry for dealing with biological samples and industrial products and the like. The first is matrix-assisted laser desorption ionization (MALDI), and the second is secondary ion mass spectrometry (SIMS). These methods are described in the following literature, by way of example:
“Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections”, Current Opinion in Chemical Biology 2002, 6, 676-681, and “Direct molecular imaging of Lymnaea stagnalis nervous tissue at subcellular spatial resolution by mass spectrometry”, Anal. Chem. 2005, 77, 735-741.
To mention one example of a method of sample preparation by MALDI, a biological sample is cooled to −18° C. and a 15-μm section (piece) of the biological sample is produced as by using a stainless steel blade. The section is placed on an electrically conductive film and the sample is then dried. The sample surface is thinly coated with a matrix to thereby obtain a MALDI sample, the sample is inserted into a vacuum chamber and MALDI is carried out. There is also a method (laser capture microdissection) in which a biological sample is placed upon a polyethylene film and the macromolecular film is heated momentarily by irradiating it with a laser beam from the back side of the film, thereby transferring the cells at the contact interface to the film. Primarily, a 337-nm nitrogen laser is used in the desorption ionization of the sample ions.
It is difficult to reduce the beam diameter of the laser beam to less than several tens of microns with these methods, and since aberration extends over a wide area, the limit on spatial resolution is 50 μm. Further, by using a matrix, which is the most distinctive feature of MALDI, ion detection sensitivity increases markedly. On the other hand, however, spatial resolution is limited since the crystal size of the matrix applied to the sample exceeds 100 μm.
With the SIMS method, a metal ion source (Ga+, Au+, etc.) that approximates a point light source is employed and a spatial resolution of less than a micron is attained. However, the energy of the ions is large (10 to 20 keV), incident ions penetrate into the sample over a depth of several hundred angstroms and the sample sustains damage. Consequently, the yield of ions from a readily decomposed sample such as a biological sample declines rapidly with time. Since the sample desorbed is limited to the molecules in the proximity of the surface, the detection sensitivity of ions with respect to biologically related samples is low.
Cluster SIMS has been developed for the purpose of eliminating this drawback. It has become evident that the desorption efficiency of secondary ions increases sharply if gold cluster ions (Aun+) or C60+ ions, for example, are used as the incident ions.
However, a spatial resolution of less than a micron is difficult to obtain because the current of the primary ion beam is small and the ion beam diameter is greater than several microns. These SIMS methods are all difficult to apply to high-mass molecules such as biological macromolecules.
Imaging techniques using MALDI and SIMS of the kind described above are continuing to come into widespread use in the field of life science. With these methods, however, it is difficult to obtain a resolution of less than a micron owing to the fundamental limitations thereof. Accordingly, no matter what improvements are applied to the conventional MALDI and SIMS methods, it is difficult to realize a resolution of less than a micron so long as these are adopted as the basic techniques.
On the other hand, with conventional electrospray or nanolaser spray, a sample liquid forms a conical shape (referred to as a “Taylor cone”) at the tip of a capillary and a minute charged droplet is produced from the conical tip. Owing to the viscosity of the liquid, it is fundamentally impossible to impart this droplet with a size of less than a micrometer or submicrometer. The reason for this is that when the tip of the Taylor cone is torn off by the force of the electric field to produce the droplet, the diameter of the tip of the Taylor cone takes on the submicrometer size automatically owing to the viscosity of the liquid. Thus, droplet size capable of being produced by electrospray is decided in the manner of a natural occurrence and it is difficult to achieve a further reduction in size.
Further, with conventional electrospray, achieving the nanometer level (nanoelectrospray) is accompanied by the need to reduce capillary diameter. There are many limitations such as clogging. It is difficult to produce a spray and handling is troublesome. Furthermore, with conventional electrospray, an increase in salt concentration results in spraying difficulty and there is a sudden decline in desorption efficiency of ions into the gaseous phase. Accordingly, conventional electrospray cannot be applied to NaCl aqueous solutions on the order of 150 mM, such as physiological saline solution.