Probing the Relationship Between Detected Ion Intensity, Laser Fluence, and Beam Profile in Thin Film and Tissue in MALDI MSI

Abstract

Abstract. Matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) is increasingly widely used to provide information regarding molecular location within tissue samples. The nature of the photon distribution within the irradiated region, the laser beam profile, and fluence, will significantly affect the form and abundance of the detected ions. Previous studies into these phenomena have focused on circular-core optic fibers or Gaussian beam profiles irradiating dried droplet preparations, where peptides were employed as the analyte of interest. Within this work, we use both round and novel square core optic fibers of 100 and 50 μm diameter to deliver the laser photons to the sample. The laser beam profiles were recorded and analyzed to quantify aspects of the photon distributions and their relation to the spectral data obtained with each optic fiber. Beam profiles with a relatively small number of large beam profile features were found to give rise to the lowest threshold fluence. The detected ion intensity versus fluence relationship was investigated, for the first time, in both thin films of α-cyano-4-hydroxycinnamic acid (CHCA) with phosphatidylcholine (PC) 34:1 lipid standard and in CHCA coated murine tissue sections for both the square and round optic fibers in continuous raster imaging mode. The fluence threshold of ion detection was found to occur at between~14 and~64 J/m 2 higher in tissue compared with thin film for the same lipid, depending upon the optic fiber employed. The image quality is also observed to depend upon the fluence employed during image acquisition.

Influence of the Laser Spot Size, Focal Beam Profile, and Tissue Type on the Lipid Signals Obtained by MALDI-MS Imaging in Oversampling Mode

“…For the larger spot sizes, these findings are in line with MALDI results obtained previously with drieddroplet sample preparations [23,27,28]. Essentially, they also reproduce the recent results obtained by Steven et al [29] with both coated tissue sections and thin-layer sample preparations The smallest dimensions produced in that work were about 50 × 120 μm 2 and might just have been above the transition point toward a different signal intensity-fluence behavior. Moreover, a different MALDI matrix (HCCA) was used as well as a more structured focal beam profile containing an array of hot spots (speckles).…”

“…Compared with the frequently used fiber-based approaches [23,24,27,29], the FBS is uncritical concerning the laser fluence (i.e., damage thresholds) under MALDI-typical conditions and also concerning speckle effects. However, a main prerequisite for obtaining the desired beam patterns with this modulating element is the use of a laser with high beam quality (M 2 ≤ 1.2).…”

“…Numerous fundamental studies were conducted to investigate the influence of relevant Binput parameters^on the MALDI ion yields, like the type and physicochemical properties of the MALDI matrix [21], the analyte-to-matrix ratio in the sample [22], or those of the laser irradiation parameters like wavelength [23][24][25], fluence [23,24,[26][27][28][29], and pulse width [30,31].…”

“…The effect of the laser spot size has been investigated roughly between 1.5 and 300 μm for both Gaussian [28,32] and flat-top beam profiles [23,24,26,27,29]. These studies generally revealed a strong increase of the ion detection threshold (and optimal fluence for maximizing the ion yields) with decreasing spot size that was larger than the reduction in irradiated area.…”

“…To address this additional complexity further, researchers have recently designed fundamental experiments in which matrix-coated tissue sections were used as Breal-life^test systems. For example, Steven et al performed experiments with mouse brain sections and investigated the phospholipid ion signals in dependence of beam profile, laser spot size, and laser fluence [29]. Notably, a fiber-based setup was used in this work that enabled production of square spot sizes.…”