MALDI-MS imaging of features smaller than the size of the laser beam

Abstract

The feasibility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging of features smaller than the laser beam size has been demonstrated. The method involves the complete ablation of the MALDI matrix coating the sample at each sample position and moving the sample target a distance less than the diameter of the laser beam before repeating the process. In the limit of complete sample ablation, acquiring signal from adjacent positions spaced by distances smaller than the sample probe enhances image resolution as the measured analyte signal only arises from the overlap of the laser beam size and the non-ablated sample surface. [3] has revolutionized the investigation of biological molecules by providing soft ionization methods linking biochemistry with the powerful analysis tools of mass spectrometry (MS). In the analysis of complex samples, such as biological tissue, MALDI is of particular interest because of its ability to desorb and ionize molecules of high molecular weight, such as proteins and peptides, providing excellent sensitivity while retaining considerable tolerance towards salts and other small molecules found at high concentration in tissue. It has been roughly 10 years since the first published applications in which MALDI-MS was used to create a chemical images of substrates [4,5]. The intervening decade has seen a considerable growth in techniques and instrumentation for MALDI-MS imaging, developed largely by Caprioli and coworkers [6 -12] with contributions to sampling techniques [13][14][15][16] and instrumentation [17,18] provided by others.It is generally accepted that the maximal spatial imaging or profiling resolution of microprobe imaging techniques is determined by a combination of the size of the microprobe and the precision of the sample or microprobe positioning device. MALDI mass spectrometers typically use lasers having relatively large beam sizes (about 100 m diameter) in the analysis of standard, dried-droplet preparations. Some effort has been put into decreasing the size of the laser beam sizes for MALDI-MS imaging and profiling of biological samples, particularly for samples containing a high proportion of peptidergic neurons or other secretory cells. Investigated biological samples of this nature include rat pituitary and rat pancreas [6], mouse brain and human brain tumor xenografts [8,14], rat brain and rat brain tumors [9,19], mouse epididymis [20], molluscan atrial gland [15], and molluscan peptidergic neurons [16].Ideally, the spatial resolution of MALDI-MS imaging of analyte-rich tissues would approach the size of a single mammalian cell, 5 to 20 m in diameter. Several strategies have been used or suggested to decrease the laser beam diameter in imaging applications, including the placement of a pinhole aperture between the outlet of the laser and the focusing optics of the mass spectrometer [6,13], decreasing the size of the fiber optic used to direct the laser into the MALDI source [9], and placing multiple lenses between the laser and the M...

Solvent-free matrix dry-coating for MALDI imaging of phospholipids

“…The dry-coated images were acquired at 100 ϫ 100-, 40 ϫ 50-, and 30 ϫ 35-m raster step sizes using the oversampling technique [17]. The spraycoated sections were imaged at a 100-m step size.…”

“…Common methods of depositing matrix for MALDI imaging include robotic spotting [16], electrospray deposition [17], and spray-coating with an airbrush [18]. Robotic spotting usually yields high signal quality and very reproducible results, but the image resolution is limited to the size and spacing of the matrix spots.…”

Vacuum compatible sample positioning device for matrix assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry imaging

“…One technique which allows circumvention of this shortcoming is "oversampling." 17 A typical oversampling MS imaging experiment consists of complete sample ablation at the initial position followed by moving the sample by a fraction of the laser spot size and collecting spectra (until sample exhaustion as well), hence acquiring sub-laser spot size information. To put it to a test, a simple oversampling experiment with a pixel size of 50 microns (whereas the laser spot diameter is 300 microns) was conducted on a single MALDI spot containing a 50:50 bradykinin, angiotensin mixture ( Fig.…”

“…While MALDI offers an unmatched molecular resolution and range (capable of ionizing whole macromolecules and their non-covalent complexes), its spatial resolution is limited by the laser spot diameter. Even though modification of the laser optics [12][13][14][15][16] and such experimental techniques as sample exhaustion 17 is capable of producing much smaller "pixels," the typical limit is >50 μm.…”

Preparation of Single Cells for Imaging Mass Spectrometry

“…Although more commonly used for tissue analysis, recent advances in matrixassisted laser desorption/ionization (MALDI) MS imaging have greatly enhanced the imaging resolution, opening the possibilities for imaging of single cells [8,9]. To date, however, very few reports of single-cell MALDI imaging have been published [10].…”