Patent Publication Number: US-2012025068-A1

Title: Mass Spectrometry

Description:
The present invention relates to mass spectrometry and in particular, although not exclusively, to matrix-assisted laser deposition/ionisation mass spectrometry (MALDI-MS) in which a laser beam is delivered to a target by a multimode fiber optic feed. 
     Matrix-assisted laser desorption/ionisation (MALDI) is a highly adaptable soft ionisation technique for mass spectrometry (MS). It was developed in the late nineteen eighties and whilst MALDI-MS has found most application in proteomics, its versatility has been extended in recent years by the advent of protein profiling and imaging directly from the surface of thin biological tissue sections. 
     The use of mass spectrometry to obtain images started with the advent of secondary ion imaging mass spectrometry (SIMS). In imaging SIMS the surface of the sample is bombarded with high energy ions leading to the ejection (or sputtering) of neutral and charged species from the surface. The ejected species may include atoms, clusters of atoms and molecular fragments. In traditional SIMS it is only the positive ions that are mass-analysed. Since the technique utilises a beam of atomic ions (i.e. charged particles) as the probe, it is a relatively easy matter to focus the incident beam and then to scan it across the surface. The detector response for a selected mass at raster spot becomes a pixel in the image. The use of an ion beam results in sub-micron spatial resolution. 
     Imaging SIMS has been used in a range of pharmaceutical applications including monitoring drugs at the cellular and sub-cellular level. New developments apply SIMS to organic compounds and metabolites of low mass (&lt;500 u) in biological samples. However, a major limitation is the mass range that may be analysed by this technique. 
     The initial step in MALDI-MS imaging involves application of a thin layer of matrix to the sample. The chemistry of the sample is then imaged by moving the sample under a stationary laser and acquiring mass spectra from each point. Three-dimensional images may be obtained by plotting the spatial dimensions of x and y versus absolute ion abundance, which is considered to be proportional to analyte concentration. 
     A further development of MALDI-MS involves the shaping and delivery of the beam from the laser medium to the sample using a fiber optic feed. Typically, a single multimode fiber optic is used which generates multiple light paths by internal reflectance. The fiber optic serves to shape the profile into spatially modulated intensities distributed on the sample surface. Without the spatial shaping, the beam intensity on the sample, as with conventionally used solid state lasers, exhibits a Gaussian or near Gaussian distribution having a single maximum (intensity peak). 
     However, whilst the multimode fiber optic feed provides multiple intensity peaks on the sample, the sensitivity and speed of data acquisition is limited to the physical configuration of the fiber optic. 
     GB 2422954 discloses a MALDI based laser system configured to generate a pulsed laser beam that a spatially shaped such that the spatial intensity distribution on the sample exhibits more than one intensity peak. Optical or electro optical components are disclosed for spatially shaping the intensity of the laser beam and comprise a lens array, digital optical elements or masks that completely or partially absorb, reflect or scatter the laser beam at central points. The optical or electro optical components may be adjusted to create different spatial intensity distributions of the beam at the sample. 
     However, the laser system of GB 2422954 typically necessitates considerable data acquisition periods, of the order of four to ten hours, and importantly provides limited sensitivity. 
     What is required is MALDI-MS apparatus that provides increased sensitivity and a reduction in data acquisition time with possible improvements to resolution when implemented in imaging mass spectrometric analytical methods (IMS). 
     The present invention provides an analytical system utilising mass spectrometry providing enhanced sensitivity with a corresponding reduction in the data acquisition time over current mass spectrometry techniques. In particular, the present invention provides apparatus and method for use in MALDI-MS suitable for imaging a wide variety of non-biological and biological samples. According to specific implementations, an order of magnitude increase in sensitivity is observed over current MALDI imaging techniques. 
     The inventors have found that by vibrating a region of the optical fiber, used to deliver the laser beam to the sample/ion source, the intensity maxima are repeatedly displaced at the sample thereby increasing the degree of sample ionisation within a single pixel boundary. 
     The present invention utilises a multimode optical fiber, and in particular a single multimode fiber configured to spatially distribute the laser beam when delivered to the sample to generate a plurality of intensity maxima. By modulating the optical fiber using suitable vibration means the plurality of intensity maxima are effectively multiplied to increase the surface area of sample irradiation. 
     The present invention also comprises alternative means and method to generate a plurality of intensity maxima at the sample together with means to perturb the speckle generation so as to multiply the intensity maxima incident at the sample. 
     According to a first aspect of the present invention there is provided a mass spectrometer comprising a means for producing a laser beam, a multimode optical fiber to deliver the laser beam to an ion source and a vibration means configured to cause the optical fiber to vibrate such that the spatial intensity distribution of the laser beam at the ion source exhibits more than one intensity peak. 
     The present invention is suitable for use with a wide variety of different lasers adapted to provide a desired wavelength, typically of the order of 200 to 360 nm. According to one specific implementation, the laser is neodynium doped yttrium ortho vanadate Nd:YVO4 which is frequency tripled to give a wavelength of 355 nm. Alternative lasers include, by way example, neodymium doped yttrium aluminium garnet (Nd:YAG) In particular, and as will be appreciated by the skilled in the art, specific implementations of the present invention may comprise YAG, vanadate, yittruim lithium fluoride (YLF) with the active ion comprising neodymium, ytterbium or other host and active ion(s) combinations with or without various means of frequency conversion such as non linear crystal(s) designed to provide laser outputs at the appropriate wavelength(s). 
     The means by which the optical fiber is oscillated/vibrated may comprise any mechanical, electronic, sonic or air displacement based device being physically coupled or non-coupled with the optical fiber and designed to impart an oscillatory movement in the fiber optic in a direction transverse or perpendicular to its longitudinal axis. Example vibration means include an electric motor, a piezoelectric switch or speaker system designed to generate a tactile sonic pulse at the region of the fiber optic so as to induce movement. 
     As will be appreciated by those skilled in the art, the present mass spectrometer comprises three fundamental components, namely an ionisation source, an analyser and a detector. Preferably, the present system comprises a hybrid quadrupole type-of-flight analyser with a suitable detector system for use with a MALDI ionisation source, in particular an orthogonal MALDI (oMALDI) ion source. 
     Preferably, the vibration means is mounted at the mass spectrometer at a region towards one end of the optical fiber in close proximity to the ion source/sample chamber or sample support. In particular, it has been found advantageous to mount the vibration coupling approximately 1 to 5 cm from the region where the fiber optic is physically coupled towards the sample chamber. As will be appreciated by those skilled in the art, the vibration means may be positioned at any region along the length of the optical fiber so as to impart an oscillatory movement serving to physically move the intensity maximum at the MALDI ion source. 
    
    
     
       A specific implementation of the invention will now be described by way of example only, and with reference to the accompanying drawings in which: 
         FIG. 1  illustrates schematically a mass spectrometer comprising a vibration coupling positioned at the optical fiber to impart an oscillatory movement in a direction transverse to its longitudinal axis according to a specific implementation of the present invention; 
         FIG. 2  illustrates an ion chromatograph of intensity vs time with the mass spectrometer of  FIG. 1  operating in a dynamic modulating mode with the vibration means active and according to a second mode with the vibration means inactive to contrast the MALDI sample ionisation intensity; 
         FIG. 3  illustrates a mass spectrum acquired with the vibration means active to impart optical fiber modulation according to the first and highest intensity region of  FIG. 2 ; 
         FIG. 4  illustrates a mass spectrum acquired with the vibration coupling inactive according to the second and lower intensity region at  FIG. 2 . 
     
    
    
     The mass spectrometer comprises a laser  100  (based on a medium such as Nd:YVO4) coupled to an optical fiber  101  at a first end  105 . A second end  106  of fiber  101  is coupled to a sample housing  102  via a suitable screw thread type coupling  107 . The delivery end  106  of the optical fiber  101  is orientated so as to irradiate a region of a sample/MALDI ion source  104  mounted at a suitable sample support  103  within an internal chamber  108  of housing  102 . 
     A vibration coupling  109  is coupled to the optical fiber  101  towards beam delivery end  106  and approximately 1 to 5 cm from end  106 . Vibration means  109  may be supported and mounted within the spectrometer using suitable mountings (not shown) so as to be physically coupled to an exterior surface of the fiber  101 . According to further specific implementations of the present invention, vibration coupling  109  is not physically coupled to the external surface of optical fiber  101  but imparts an oscillating movement via a medium surrounding the external surface of the optical fiber  101  being a fluid, in particular air. In particular, vibration means  109  may comprise an air pump or speaker system designed to direct air pulses towards the external surface of fiber  101 . 
     In use, and with vibration coupling  109  active, fiber optic  101  is forced to oscillate back and forth along direction  110  aligned transverse, in particular perpendicular, to the longitudinal axis of optical fiber  101 . 
     Oscillating movement  110  at the region of vibration coupling  109  is transmitted along the length of optical fiber  101  to result in proportionally smaller movement oscillations at irradiation end  106 . This has the effect of physically moving the irradiation intensity maximum at sample surface  104 . Vibration coupling  109  is configured such that the movement modulation of optical fiber  101  at end  106  is sufficient to cause the intensity maxima to be displaced only within a single pixel of approximate dimensions 150×100 μm. Due to the enhanced sensitivity of the present mass spectrometer arrangement, the inventors provide a system capable of enhanced resolution with pixel dimensions of the order of 25×25 μm. 
     Investigation by MALDI Mass Spectrometry Imaging 
     A comparative investigation was undertaken to determine the effect on MALDI-MSI instrument sensitivity with vibration coupling  109  in an active and a non-active mode. The results are presented in  FIGS. 2 to 4 . 
     The mass spectrometric analysis was performed using an API ‘Q-Star’ Pulsar i hybrid quadrupole time-of-flight instrument from Applied Biosystems/MDS Sciex (Concord, Ontario, Canada), fitted with an orthogonal MALDI source and ‘o-MADLI Server 4.0’, ion imaging software. Image processing was carried out using BioMap imaging software (www.maldi-msi.org). 
     A neodynium doped yittruim ortho vanadate (Nd:YVO4) laser was used with a laser spot of approximate dimensions 150×100 μm. Images were acquired at 200 μm increments with an ablation time for each spot of approximately 2 s, using 30% laser power and a laser repetition rate of 1 kHz (although higher or lower frequencies could be used). A beta test version of the applied Biosystems/MDS Sciex ‘Dynamic Pixel’ MALDI MSI acquisition mode was used for all studies. 
       FIG. 2  illustrates the total ion chromatograph with vibration coupling  109  active to modulate the beam profile (region  200 ) and inactive without optical fiber  101  vibrated in direction  110  (region  202 ).  FIG. 2  illustrates the difference in intensity of the resultant sample ionisation due to the sample surface area irradiation as optical fiber end  106  moves back and forth whilst sample  104  is irradiated. As illustrated in  FIG. 2 , the intensity difference between region  200  and region  202  is approximately one order of magnitude. The sharp transition region  201  corresponds to the termination of power to the mechanical vibration coupling  109  resulting in a sharp decrease in intensity. 
       FIG. 3  illustrates in mass spectrum acquired with vibration coupling  109  inactive accordingly to region  202  of  FIG. 2 . 
       FIG. 4  illustrates a mass spectrum acquired with coupling  109  active according to region  200  of  FIG. 2  utilising the same MALDI ionisation source and instrument parameters as used in the investigation of  FIG. 3 . 
     Referring to  FIGS. 3 and 4  ionisation data appears only at regions  300  and  301  with vibration coupling  109  inactive. In contrast, the intensity profile is increased significantly with coupling  109  active to irradiate a greater sample surface area according to intensity regions  400  and  401 . In particular, due to the increased sensitivity of the present invention, data is acquired at region  402  with this data not being available with the arrangement of  FIG. 3 .