First results for a new atmospheric-pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging source operating at 213 nm laser wavelength are presented. The activation of analytes in the 213 nm MALDI process at atmospheric pressure was evaluated and compared to results for 337 nm MALDI and electrospray ionization using thermometer molecules. Different sample preparation techniques for nicotinic acid, the matrix with the highest ionization efficiency at 213 nm of all tested matrices, were evaluated and optimized to obtain small crystal sizes, homogenous matrix layer sample coverage, and high ion signal gains. Mass spectrometry imaging experiments of phospholipids in mouse tissue sections in positive- and negative-ion mode with different lateral resolutions and the corresponding pre-/post-mass spectrometry imaging workflows are presented. The use of custom-made objective lenses resulted in sample ablation spot diameters of on average 2.9 μm, allowing mass spectrometry imaging experiments to be performed with 3 μm pixel size without oversampling. The ion source was coupled to an orbital trapping mass spectrometer offering high mass resolution (>100.000), high mass accuracy (≤ ±2 ppm), and high sensitivity (single pixel on-tissue tandem MS from 6.6 μm2 ablation area). The newly developed 213 nm atmospheric-pressure MALDI source combines the high mass resolution and high mass accuracy performance characteristics of orbital trapping mass spectrometers with high lateral resolution (pixel size ∼3 μm) mass spectrometry imaging.

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Matrix-assisted laser desorption/ionization mass spectrometry imaging is a promising tool in the life sciences for obtaining spatial and chemical information from complex biological samples. State-of-the-art setups combine high mass resolution and high mass accuracy with high lateral resolution, offering untargeted insights into biochemical processes on the single-cell length scale. Despite recent technological breakthroughs, the sensitivity and acquisition speed of many setups are often in competition with achievable pixel resolutions below 25 μm. New measurement modes were developed by implementing a high-repetition-rate laser into an AP-SMALDI10 ion source, coupled to an orbital trapping mass spectrometer. These new MSI modes allow for a modular use of the new setup. We demonstrate that the system allows single cell features to be visualized in mouse brain tissue sections at a pixel resolution of 5 μm and an imaging speed of 18 pixels/s. Furthermore, the analytical sensitivity was improved in another measurement mode by applying multiple pulses of a highly focused laser beam over larger square pixels ≥25 μm edge length, increasing ion signal intensities up to 20-fold on tissue and decreasing the limit of detection by 1 order of magnitude.

In the mammalian olfactory bulb, the inhibitory axonless granule cells (GCs) feature reciprocal synapses that interconnect them with the principal neurons of the bulb, mitral, and tufted cells. These synapses are located within large excitable spines that can generate local action potentials (APs) upon synaptic input ("spine spike"). Moreover, GCs can fire global APs that propagate throughout the dendrite. Strikingly, local postsynaptic Ca 2ϩ entry summates mostly linearly with Ca 2ϩ entry due to coincident global APs generated by glomerular stimulation, although some underlying conductances should be inactivated. We investigated this phenomenon by constructing a compartmental GC model to simulate the pairing of local and global signals as a function of their temporal separation ⌬t. These simulations yield strongly sublinear summation of spine Ca 2ϩ entry for the case of perfect coincidence ⌬t ϭ 0 ms. Summation efficiency (SE) sharply rises for both positive and negative ⌬t. The SE reduction for coincident signals depends on the presence of voltage-gated Na ϩ channels in the spine head, while NMDARs are not essential. We experimentally validated the simulated SE in slices of juvenile rat brain (both sexes) by pairing two-photon uncaging of glutamate at spines and APs evoked by somatic current injection at various intervals ⌬t while imaging spine Ca 2ϩ signals. Finally, the latencies of synaptically evoked global APs and EPSPs were found to correspond to ⌬t Ϸ 10 ms, explaining the observed approximately linear summation of synaptic local and global signals. Our results provide additional evidence for the existence of the GC spine spike.Here we investigate the interaction of local synaptic inputs and global activation of a neuron by a backpropagating action potential within a dendritic spine with respect to local Ca 2ϩ signaling. Our system of interest, the reciprocal spine of the olfactory bulb granule cell, is known to feature a special processing mode, namely, a synaptically triggered action potential that is restricted to the spine head. Therefore, coincidence detection of local and global signals follows different rules than in more conventional synapses. We unravel these rules using both simulations and experiments and find that signals coincident within ϷϮ7 ms around 0 ms result in sublinear summation of Ca 2ϩ entry because of synaptic activation of voltage-gated Na ϩ channels within the spine.