Mainly, lipidomics approaches have been demonstrated by the use of mass spectrometry to perform full characterization of lipid molecular species. Qualitative and quantitative knowledge of the lipid composition is the first step for lipidomics. The most widely used MS instrument is MS coupled with liquid chromatography (LCESI). LCESIMS can quantify the amounts of lipids as well as identify the structure of lipid molecules. Many reports have shown that shot-gun lipidomics analyses are the most suitable analytical method to investigate the full range of lipid molecules in cells or tissues or to find molecular-level indications of diseases by comparing the distribution of various lipid molecules in diseased tissue with that in control samples. However, this technique entails extraction and purification steps, leading to the loss of the lipid distribution over the biological tissue. Numerous techniques of chemical imaging enable the detection and the localization of lipids. Staining with Nile Red, osmium tetroxide is a common method to localize the lipid fraction on frozen sections, whereas few specific lipid antibodies are commercially available. These approaches target either the complete lipid fraction or only one specific family, not molecular species with a wide variety of fatty acid compositions. By contrast, imaging mass spectrometry is a relatively new imaging method based on MS. IMS is a two-dimensional MS technique used to visualize the spatial distribution of biomolecules. Several ionization methods, including (SIMS), (DESI), (MALDI), have been investigated as methods of IMS. In this review, we focus on lipid imaging by MALDI-MS because MALDI-IMS is the most common IMS method for lipid imaging. Indeed, the versatility of MALDI-IMS has opened a new frontier in several other fields, as lipidomics.
MALDI is a soft ionization technique triggered by a pulsed laser beam. The sample is mixed with a matrix or nanoparticles, which absorb light at the wavelength of the laser. The MALDI technique also has some disadvantages in terms of spatial resolution and sensitivity.
One requirement for performance of MALDI-IMS is the availability of an xy moving stage with electronic controls. TOF is the most widely-used technology. TOF analyzers allow the separation of ionized accelerated molecules according to their mass-to-charge ratio (m/z). TOF-MS offers suitable performance for MALDI-IMS, namely a good transmission ratio (50100%) sensitivity, and repetition rate. However, TOF-MS lacks the capability to perform effective tandem MS analyses for identification. This disadvantage of TOF-MS has been addressed with the introduction of hybrid analyzers pairing various technologies with TOF: a quadrupole mass analyzer and TOF, a quadrupole ion trap and TOF, an ion mobility spectrometer and TOF, and tandem TOF mass spectrometers. These combination systems have revolutionized the application of TOF-MS to structural analysis with tandem MS analyses. MALDI-IMS is a two dimensional laser scanning technology. The analyzing time depends on the number of spots, the frequency of the laser, the number of shots per spot, and the time required to moving the stage. The most relevant factor is the frequency of the laser. New MALDI instruments are equipped with 1000-Hz lasers. when users select the region of interest as a 11 cm area with a 10 ?m scan pitch, there are approximately 10,000 points to be analyzed. Using a 1000-Hz laser with common parameters, this will take only 1 h.
Spraying is the most frequently used method in MALDI-IMS. An entire tissue section can be coated with relatively small crystals homogeneously in a short time without special equipment. For its operation, several instruments including TLC sprayers and artistic airbrushes are available; we use a metal air-brush with a 0.2-mm nozzle
because of its simple and easy-to-handle design. Although this method seems to present few technical challenges, it nonetheless requires skillful operation because of the numerous parameters of the hand-operation of the air-brush. If there is an excess of matrix solution on the tissue, an inhomogeneous crystal can be formed with analytes that have migrated from their original location; on the other hand, if not enough matrix solution is sprayed and it evaporates without sufficiently moisturizing the tissue section, analytes cannot be adequately extracted from the tissue section. Beginners are recommended to practice spraying until homogeneous matrix spraying can be achieved reproducibly.
Sublimation is a new method for applying matrix to tissue sections. By using this technique, even an inexperienced researcher could easily apply a uniform coating of matrix over a large sample plate in a very short time without solvents. Additionally, previous reports demonstrated that this method increases the analyte signal and that the fine microcrystals formed from the condensed vapor reduce the limitation of image resolution caused by crystal size. Although it requires a special instrument, we believe that this method will be widespread in the near future.
To obtain a good spectrum in MALDI-IMS, the procedure is almost the same as that in traditional MALDI-MS; the mass range, detector gain, and laser power must be optimized. From the mechanical setting perspective, there are two differences between MALDI-MS and MALDI-IMS. One difference is that we have to set a two-dimensional region for analyses. In addition, the scan pitch, which decides the spatial resolution of the image, must be fixed because MALDI-IMS can ionize the molecules by laser pulse. The scan pitch, which means the distance between scans, depends on the laser size and mechanical movement control. At this moment, the commercially available instrument with the highest spatial resolution can analyze with a laser diameter of approximately 25 ?m.
Sometimes, we detected plural molecules at the same m/z value. In that case, a new imaging technique, MS/MS, was developed. By the use of this technique, we could separate each ion derived from their specific fragment ions. There are some reports which have shown MS/MS imaging to perform IMS of endogenous metabolites and an exogenous drug. In addition, the combination of ion-mobility separation with MALDI-IMS provides a unique separation dimension to further enhance the capabilities of IMS. It can be used to produce images without interference from background ions of similar mass, which can remove ambiguity from imaging experiments and lead to a more precise localization of the compound of interest.
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