Abstract:
A MALDI/LDI source is disclosed that includes an ion optical device and beam-focusing optics disposed on opposite sides of a sample support that is at least locally transparent in a region underlying the sample to allow transmission of a radiation beam therethrough. A laser or other radiation source, located adjacent a rear surface of the sample support, emits a beam of radiation that is focused by the beam focusing optics and traverses the transparent region of the sample support to impinge on the sample. Ions produced by irradiation of the sample are collected by an ion optical device located adjacent the front surface of the sample support. By locating the ion optical device and beam-focusing optics on opposite sides of the sample support, short focal length beam-focusing optics may be utilized, thereby facilitating smaller beam spot sizes. This may be particularly useful for mass spectral tissue imaging and other applications where high spatial resolution analysis of a differentiated sample is desirable.

Description:
BACKGROUND 
   1. Field of the Invention 
   The invention is in the field of mass spectrometry and more specifically in the field of ionization sources for mass spectrometry. 
   2. Related Art 
   Laser-based ionization techniques, which include laser desorption/ionization (LDI) and matrix-assisted laser desorption/ionization (MALDI), are useful tools for mass spectrometric analysis. These techniques involve irradiating a sample containing an analyte substance with a short pulse of radiation, typically emitted by a laser. The radiation is absorbed by the sample, resulting in the desorption and ionization of analyte molecules from the sample. In the MALDI process, the sample is prepared by associating the analyte substance with a matrix material, which is highly absorbent at the irradiation wavelength and which assists in the desorption and ionization of the analyte molecules. MALDI is a particularly useful technique for the analysis of large biological molecules, such as peptides or proteins, that may undergo fragmentation when subjected to alternative ionization methods. Furthermore, MALDI tends to produce singly-charged ions, thereby facilitating interpretation of the resultant mass spectra. The ions produced by the LDI or MALDI source (or product ions derived therefrom) may be analyzed using any one or combination of mass analyzers known in the art, including quadrupole mass filters, quadrupole ion traps, time-of-flight analyzers, Fourier transform ion cyclotron resonance cells, and electrostatic traps. 
   Recently, there has been growing interest in the use of LDI/MALDI mass spectrometry to generate spatially resolved maps of analyte concentrations in a biological material, such as a tissue sample. This process, which is often referred to as mass spectral tissue imaging, offers great promise as a tool for the study of drug absorption and excretion by selected tissues. Because analyte concentrations in a tissue sample may exhibit large spatial gradients, it is generally desirable to perform tissue imaging experiments at high spatial resolution in order to gain useful information regarding analyte concentration profiles at areas of interest within the sample. 
   The minimum spatial resolution that can be obtained using a MALDI or LDI source will be partially determined by the spot size, i.e., the area of the sample that is irradiated by the laser or other irradiation source. In most commercially available MALDI sources, the spot size has a diameter of around 100 μm, which is too large for some tissue imaging applications. The spot size may be reduced by more tightly focusing the radiation beam at the sample surface, e.g., by using a beam-focusing lens having a shorter focal length. However, the presence and positioning in the ionization source chamber of the ion guide or other optics, which transport the ions from the sample location to the mass analyzer, will often interfere with the placement of a short focal length lens, thereby making it difficult or impossible to focus the beam to the desired size. The placement of a short focal length lens may also be rendered more difficult by the presence of discrete viewing optics employed to acquire an image of the sample. 
   In view of the above discussion, there is a need in the art for an LDI or MALDI source that allows for reduction of the radiation spot size and facilitates tissue imaging or other applications that require high spatial resolution. 
   SUMMARY 
   According to embodiments of the present invention, an LDI or MALDI source is provided in which a sample is arranged on a front surface of a sample plate that is at least locally transparent at the irradiation wavelength. In various implementations, the transparency may be achieved by fabricating the sample support from a transparent material, or by fabricating the sample support from a non-transparent material and adapting the sample support with openings or transparent windows in the region or regions underlying the sample(s). An ion optical device, such as a multipole ion guide, is positioned adjacent the sample support front surface for transporting the ions emitted from the sample. Beam-focusing optics, which may include one or more short focal length lenses, are positioned adjacent the rear surface of the sample support. The radiation beam, focused by the beam-focusing optics, traverses the transparent sample plate and impinges upon the sample as a tightly-focused spot to desorbs and ionize the sample. 
   In some embodiments, viewing optics are disposed adjacent the rear surface of the sample support to enable viewing of an image of the sample by the operator (via, for example, a video camera or other imaging device). 
   By positioning the beam-focusing optics and/or the imaging optics on a different side of the sample support from the ion optical device, the design of the LDI/MALDI source is less constrained by the limited space around the sample, thereby permitting use of a short focal length beam-focusing lens that must be positioned at close proximity to the sample. Use of a short focal length lens produces a smaller beam spot than would be possible using prior art LDI/MALDI system architectures, which in turn allows for acquisition of mass spectral images at higher resolutions. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is an illustration of an LDI/MALDI source according to one embodiment of the invention, wherein beam-focusing optics and an ion optical device are disposed on opposite sides of the sample support. 
       FIG. 2  is an illustration of another embodiment of an LDI/MALDI source, wherein the viewing optics are disposed on the same side of the sample support as the beam-focusing optics. 
       FIGS. 3A and 3B  are illustrations of further embodiments of LDI/MALDI sources, wherein the ion optical device and beam-focusing optics are located on the same side of the sample support and the viewing optics are located on an opposite side of the sample support. 
       FIG. 4  is an illustration of a transparent sample support, according to various embodiments of the invention. 
       FIG. 5  illustrates a method of analyzing a sample using a mass spectrometer having an LDI/MALDI source, according to various embodiments of the invention. 
   

   DETAILED DESCRIPTION 
   In one aspect of the invention, a laser desorption/ionization source or matrix-assisted laser desorption/ionization source (referred to collectively as an LDI/MALDI source) is provided which accommodates a sample support configured to support one or more sample(s) on a front surface thereof. The sample support is at least locally transparent at the wavelength of the irradiation beam. Transparency may be provided by the modification of a non-transparent sample support with transparent windows or openings that underlie the sample(s); alternatively, the entire sample support may be constructed from a transparent material such as quartz. Beam focusing optics and/or viewing optics may be disposed adjacent a rear surface of the sample support for, respectively, focusing a beam of radiation onto the sample and acquiring an image of the sample. An ion optical device, such as a multipole ion guide, is disposed adjacent the front surface of the sample support and functions to collect and guide ions produced by irradiation of the sample. 
     FIGS. 1–3  illustrate different embodiments of an LDI/MALDI source having various arrangements of beam-focusing and viewing optics. In each of these embodiments, the beam-focusing optics optionally includes a short focal length lens that generates a compact beam spot on the sample. 
     FIG. 1  is an illustration of an LDI/MALDI source generally designated  100 . LDI/MALDI source  100  accommodates a sample support  110 , and includes beam-focusing optics  120 , viewing optics  130  and an ion optical device  140 . Sample support  110  includes a front surface  116 , on which one or more samples are deposited, and a rear surface  115 . Front surface  116  may be flat and featureless, or may optionally include a conductive coating for application of an offset voltage, one or more chemical reagents configured to react with the analyte, and/or indentations configured to receive and hold the sample. 
   As noted above, each embodiment of the invention makes use of a transparent sample support. As used herein, the terms “transparent” or “transparency” are not intended to require complete transparency; rather, any sample support may be utilized that allows substantial transmission therethrough of radiation having the wavelength(s) of interest. Furthermore, the sample support may be only locally transparent, i.e., may be transparent only at regions thereof that underlie the sample(s), and the remaining portions of the sample support may be opaque. 
   In some embodiments, sample support  110  is supported by a positioning stage  117  that is moved with respect to ion optical device  140  and beam-focusing optics  120 . A positioning stage driver  119  is configured to move (e.g., translate or rotate) positioning stage  117 . Positioning stage driver  119  may includes a stepper motor, piezoelectric device or mechanism known in the art that is capable of precise control of the sample support position. In some embodiments, positioning stage driver  119  is configured to move positioning stage  117  such that a selected one of a plurality of samples on sample support  110  is aligned with the radiation beam and the proximal end of ion optical device  140 . In various embodiments, positioning stage driver  119  is configured to move positioning stage  117  with lateral (i.e., in the X-Y plane defined by the sample support) resolutions of 10 micrometers, 5 micrometers, 3 micrometers, 1 micrometer, or less. 
   Beam-focusing optics  120  are disposed adjacent to rear surface  115  of sample support  110 . As used herein, the term “adjacent” does not require immediate adjacency, i.e., the beam-focusing optics should still be considered to be disposed adjacent to rear surface  115  even if one or more structures are interposed between the beam-focusing optics  120  and rear surface  115 , or if they are separated by a substantial distance. Rather, the beam-focusing optics should be considered adjacent to the rear surface  115  if they are located in a region that is closer to rear surface  115  than front surface  116 . Beam-focusing optics  120  will typically include at least one lens that focuses a beam of radiation  122 , which may be supplied by a radiation source, for example laser  124 , onto a sample disposed on or near sample support  110  front surface  116 . It is noted that beam-focusing optics  120  may, without limitation, consist of a single lens, as depicted in the figures. Laser  124  will typically take the form of a nitrogen or solid-state laser capable of emitting short pulses of radiation at a wavelength or wavelengths that are strongly absorbed by the sample and matrix. In various embodiments, beam-focusing optics  120  are configured to produce a beam spot (the area of the sample impinged by the radiation beam) having a diameter of 10 micrometers, 5 micrometers, 3 micrometers, 2 micrometers, 1 micrometer, or less. In various embodiments, beam-focusing optics  120  have a focal length of 15 millimeters, 12 millimeters, 10 millimeters, 8 millimeters, 5 millimeters, or less. Beam-focusing optics  120  are optionally positioned such that a major axis  125  is approximately parallel to surface front  116  and a center axis  126  is approximately perpendicular to front surface  116 . In some embodiments, a combination of laser pulse power and focal length may be selected to effect single-shot desorption/ionization of the irradiated region of the sample. That is, substantially the entire thickness of the sample can be desorbed and ionized at a predetermined location with a single shot of a laser. This could allow for more efficient use of limited sample volumes, enabling results to be attained from a relatively small amount of analyte, and for numerous results to be attained from a single small sample volume. 
   In some embodiments, laser  124  may operate in a selected one of two modes. In the first mode, the laser illuminates some, or all, of the sample for subsequent visual image acquisition via UV sensitive cameras, for example. In the second mode, the laser irradiates a target region of the sample for production of ions. Operation of the laser in the first mode may be employed, for example, to acquire and display an image that can be viewed by the instrument operator for use in selecting a portion of the sample to be analyzed. Typically, the illumination mode includes a lower beam flux than the ionization mode. 
   In some embodiments, beam-focusing optics  120  or a portion thereof are mechanically coupled to a lens manipulator  127  configured to move beam-focusing lens  120  relative to transparent sample support  110 . For example, in some embodiments lens manipulator  127  is configured to move beam-focusing optics  120  toward or away from front surface  116 . In some embodiments, lens manipulator  127  is configured to move beam-focusing optics  120  or other ionization optic parallel to first surface  116 . In these embodiments, lens manipulator  127  is optionally used to move the beam spot small distances between different target locations on the sample. Lens manipulator  127  may be operated in conjunction with positioning stage  117  to achieve highly precise control of the beam spot position; for example, movement of positioning stage  117  may provide gross control of the beam spot position, and movement of lens manipulator  127  may provide fine control of the beam spot position. In various embodiments, lens manipulator  127  is configured to move the focal point by 20 micrometers, 10 micrometers, 5 micrometers, 3 micrometers, 2 micrometers, 1 micrometer, or less than 1 micrometer. 
   Viewing optics  130  are configured for viewing (i.e., acquiring an image of) at least a portion of the sample disposed on sample support  110 . An image obtained using viewing optics  130  can be displayed to the operator and used to select a portion of interest of the sample (e.g., a region within a tissue sample) for mass spectral analysis. 
   Viewing optics  130  typically include at least a focusing element such as a lens  132 , reflector, or the like, and a viewing element such as an eye piece or CCD camera  134 . For example, in some embodiments, imaging optics  130  includes CCD camera  134 , lens  132  and a microscope aperture (not shown). In some embodiments, viewing optics  130  are configured to detect the incidence of laser beam  122  on the sample. Viewing optics  130  optionally include a visual distance indicator (not shown) configured to assist an operator in manipulating beam-focusing optics  120  using lens manipulator  127  to focus on a desired location within the sample. One or more illumination sources (not depicted in the figures) may be provided to illuminate the sample for viewing and/or image acquisition. 
   Ion optical device  140  is configured to collect ions desorbed from a MALDI sample disposed on front surface  116  of sample support  110 . Ion optical device  140  may comprise, for example, a multipole ion guide to which appropriate AC and DC voltages are applied in order to confine the ions and/or draw the ions along the longitudinal axis of the ion guide. In a typical mass spectrometer architecture, ion optical device  140  transports ions toward a mass analyzer, such as a quadrupole mass filter, ion trap, time-of-flight analyzer, or electrostatic trap, which separates ions according to their mass-to-charge ratios for subsequent detection and/or fragmentation. One or more intermediate chambers as well as various ion optics may be interposed in the ion path between ion optical device  140  and the mass analyzer. 
     FIG. 2  is an illustration of an LDI/MALDI source  200 , which is an alternative embodiment of LDI/MALDI source  100 . In this embodiment, both beam-focusing optics  120  and viewing optics  130  are disposed adjacent to rear surface  115  of sample support  110 . Viewing optics  130  are configured to acquire an image of a sample disposed on front surface  116  of sample support  110 . In this embodiment, beam-focusing optics  120  also functions to focus the sample image, in conjunction with partial reflector  210 . Partial reflector  210  is preferably highly reflective at the wavelength of laser  124  so as to direct the laser beam onto the sample and is at least partially transmissive at the wavelength range of visible light so as to enable viewing of the sample image therethrough by camera  134 . The wavelength-selective reflection/transmission of partial reflector  210  may be achieved, for example, by application of suitable dielectric layers to one or both surfaces of the reflector. In an alternative configuration, the relative positions of laser  124  and imaging optics  130  are exchanged relative to partial reflector  210 . 
     FIG. 3A  is an illustration of a MALDI source  300 , which is an alternative embodiment of MALDI source  100 . In MALDI source  300 , imaging optics  130  are disposed adjacent to rear surface  115  of sample support  110 , and ion optical device  140  and beam-focusing optics  120  are disposed adjacent to front surface  116  of sample support  110 . In this embodiment, ion optical device  140  optionally includes a skimmer configured to collect ions desorbed from a sample disposed on front surface  116 . Beam-focusing optics  120  is optionally configured to focus laser beam  122  onto front surface  116  at a perpendicular angle to front surface  116 . This orientation will typically produce the minimum spot size of laser beam  122  on the sample. However, in alternative embodiments, beam-focusing optics  120  are configured to focus laser beam  122  onto front surface  116  at other angles of incidence. One example of this arrangement is illustrated in  FIG. 3B . 
     FIG. 4  is a cross-sectional view of an exemplary implementation of sample support  110 , wherein local transparency is achieved by adapting a substrate  420  with openings  410  that underlie the samples  430 . Each opening  410  narrows upwardly to a reduced-diameter well  413  having a diameter indicated as  415 . A sample  430  may be deposited on sample support  110  by spotting a liquid solution containing the analyte material (and optionally a matrix substance) onto wells  413  and evaporating the solvent. The well diameter  415  should be sufficiently small to allow the liquid solution to be retained in the well by surface tension forces. In various embodiments, wells  413  have a diameter  415  of less than 50 micrometers, 25 micrometers, 10 micrometers or 8 micrometers. In some embodiments, wells  413  are each configured to hold a single cell. 
     FIG. 5  illustrates a method of analyzing a sample, according to various embodiments of the invention. In a Prepare MALDI Sample step  510  a MALDI sample is deposited on front surface  116  of sample support  110 , for example by adhering a thin tissue layer on the front surface and thereafter applying (e.g., by electrospraying) a matrix layer overlying the tissue. 
   In an optional View Sample step  520 , viewing optics  130  are used to view the sample prepared in Prepare Sample step  510 . The sample can either be viewed directly through a microscope aperture, viewed as an image captured using a digital camera, or the like. Typically, the sample is viewed in a magnified form. For example, in some embodiments the view may be in sufficient detail to identify areas of interest within the sample. 
   In an Ionize First Area step  530 , laser  124  is operated to desorb and ionize a part of the MALDI sample located at the focal point of beam-focusing optics  120 . Ionization may include simultaneous desorption and ionization or desorption followed by gas phase ionization. 
   In an Observe First Area step  540 , the location of the area of the sample ionized in Ionize First Area step  530  is observed using viewing optics  130 . This observation can occur either during the ionization process by imaging the ionization event or following the ionization process by imaging a change (e.g., loss of material) in the sample. 
   In a Change Locations step  550 , the location of the focal point of beam-focusing optics  120  on the sample is moved. This relative movement may be accomplished by moving positioning stage  117  using positioning stage driver  119  and/or by moving beam-focusing optics  120  using lens manipulator  127 . Change Locations step  550  is optionally performed while observing the sample through viewing optics  130  and/or using a distance measurement made using viewing optics  130 . 
   Change Locations step  550  is optionally performed while operating laser  124  in the illumination mode. For example, in one embodiment, Change Locations step  550  includes monitoring the position of the focal point of beam-focusing optics  120  by observing light of laser beam  122  striking the sample, while laser beam  122  is operated below a desorption/ionization threshold of the MALDI sample. During this observation, the focal point is optionally moved to a specific part of the MALDI sample to be analyzed. In various embodiments, the change in location of the focal point of beam-focusing lens, that occurs in Change Locations step  550 , is less than or equal to 15 micrometers, 10 micrometers, 8 micrometers, 5 micrometers, 3 micrometers or 2 micrometers. In some embodiments, Change Locations step  550  includes moving the focal point of beam-focusing optics  120  from one area of interest in a tissue sample to another. 
   In an Ionize Second Area step  560 , laser  124  is operated in the ionization mode to desorb and ionize a second area of the sample. This second area is that part of the MALDI sample to which the focal point of beam-focusing lens  120  was directed to in Change Relative Locations step  550 . 
   In a Determine M/Z step  570 , the mass-to-charge ratios of ions generated in Ionize Second Area step  560  is determined using a mass analyzer to which ions are transported by ion optical device  140  (or which is incorporated into ion optical device  140 ). These mass-to-charge ratios are optionally used to form a mass spectrum associated with the ionized part of the sample. By repeating Change Locations step  550  and Ionize Second Part step  560 , mass spectra associated with different areas of a tissue sample, or other sample, are generated. In alternative embodiments, an instance of Determine M/Z step  150  also follows Ionize First Part step  530 . 
   The embodiments discussed herein are illustrative of the present invention. As these embodiments of the present invention are described with reference to illustrations, various modifications or adaptations of the methods and or specific structures described may become apparent to those skilled in the art. All such modifications, adaptations, or variations that rely upon the teachings of the present invention, and through which these teachings have advanced the art, are considered to be within the spirit and scope of the present invention. Hence, these descriptions and drawings should not be considered in a limiting sense, as it is understood that the present invention is in no way limited to only the embodiments illustrated.