Histology by mass spectrometry: label-free tissue characterization obtained from high-accuracy bioanalytical imaging.

Histological examination of biological and medical specimens has gained its universality and undisputed significance through distinct staining techniques and microscopical evaluation. Discrimination of tissue types after specific staining or labeling is an essential prerequisite for histopathological investigation, for example in accurate diagnosis of cancer. Histochemical staining techniques can only be used in a targeted manner for known compounds, and only a limited number of such targets can be visualized from a given sample at the same time. Another limitation of classical histology lies in the fact that a considerable amount of experience is required and that even well-trained pathologists often interpret histologically stained sections differently. Mass spectrometry (MS), on the other hand, offers complex but objective and reproducible information on biological material. Imaging of biological samples by MS gained interest after development of matrix-assisted laser desorption/ionization (MALDI) as a method to desorb and ionize biomolecules, such as peptides, proteins, glycans, or lipids, with a limit of detection in the attomole range. The first proof-of-principle of imaging by MALDI was presented in 1994, and was followed by numerous applications during the last decade. An extensive overview of instrumental developments and methodological approaches in MS imaging has been published recently. MS imaging allows the distribution of analytes to be investigated and displayed across a sample in a semi-quantitative manner and without the need to predefine or label selected substances prior to analysis. MALDI imaging is typically used with spatial resolutions of between 50 and 200 mm. Increasing the resolution into the lowmicrometer range has been demonstrated, but requires a very low limit of detection of the employed mass spectrometer, as the available amount of material per imaged spot is reduced quadratically with reduction of the spot diameter. Identification of molecules during MS imaging experiments is often limited if mass spectrometers with a rather low mass resolving power and accuracy are used. Additional offline bulk analyses of tissue material are typically used to back up imaging results. Imaging selectivity, that is, mass bin width for allocation to image signals, is typically set to onemass unit. Employing MS imaging for obtaining valid histological information requires a number of improvements: 1. The usable spatial resolution has to be high enough to resolve cellular features. 2. Analytical sensitivity has to be high enough to visualize the majority of interesting substances in high-lateralresolution experiments. 3. Mass resolving power and mass accuracy have to be as high as possible when complex biological samples are under investigation. To unequivocally assign a mass signal to an image and to identify substances by accurate mass, signals have to be stable and correct in detected mass values; that is, mass accuracy should be in the low-ppm range. 4. Image assignment to mass signals has to be both highly selective and flexible. To distinguish neighboring mass signals in biological tissue samples, the coding mass bin width must typically be smaller than 0.1 mass units. 5. To clearly identify imaged substances in complex samples, MS data from fragmentation of precursor ions has to be obtainable directly from individual imaged sample spots. 6. Ambient pressure conditions are often necessary, rather than high-vacuum conditions, for example when working under physiological conditions, imaging volatile substances such as drug metabolites, or using volatile matrices. 7. Sample handling and preparation have to be fast and robust. 8. Results have to be achievable in a reasonable timeframe.