Impact of wavelength and spot size on laser depth of focus: Considerations for mass spectrometry imaging of non‐flat samples

Abstract

Biospecimens with nearly flat surfaces on a flat stage are typically required for laserbased mass spectrometry imaging (MSI) techniques. However, sampling stages are rarely perfectly level, and accounting for this and the need to accommodate non-flat samples requires a deeper understanding of the laser beam depth of focus. In ablation-based MSI methods, a laser is focused on top of the sample surface, ensuring that the sample is at the focal point or remains within depth of focus. In general, the depth of focus of a given laser is related to the beam quality (M 2 ) and the wavelength (λ). However, because laser is applied on a biological sample, other variables can also impact the depth of focus, which could affect the robustness of the MSI techniques for diverse sample types. When the height of a sample ranges outside of the depth of focus, ablated area and the corresponding ion abundances may vary as well, increasing the variability of results. In this tutorial, we examine the parameters and equations that describe the depth of focus of a Gaussian laser beam. Additionally, we describe several approaches that account for surface roughness exceeding the depth of focus of the laser.

A Comprehensive Comparison of Tissue Processing Methods for High-Quality MALDI Imaging of Lipids in Reconstructed Human Epidermis

“…A major advantage of MALDI MSI of biological tissues is that it preserves the integrity of the sample so that the spatial distribution and abundance of biomolecules can be determined in conditions close to the native state. 50 Recent investigations on the influence of hardware parameters, such as the impact of the wavelength on biological nonflat samples 51 or the impact of the laser spot size on lipid signals from brain sections, 52 have led to the improvement of the signal intensity and the ability to image at cellular and subcellular levels. 53 In addition to these aspects, it is also essential to optimize the sample preparation to increase the number of biomolecules detected and the ion signal intensity.…”

Oversampling for Enhanced Spatial Resolution of Zebrafish by Top-Hat IR-MALDESI-MSI

“…14 Utilizing a laser in these sources creates specific challenges when attempting high spatial resolution experiments, which include but are not limited to the operating laser wavelength and the beam quality. 15 MALDI approaches can work under ambient conditions (i.e., AP-MALDI) 16 or vacuum and have been documented to employ infrared (IR) 17 or ultraviolet (UV) 18 lasers, although the latter is more common. For UV-MALDI, the laser is complemented with an organic matrix co-crystallized with the sample, where energy is absorbed at the same or similar wavelength as the laser (e.g., 337 or 355 nm).…”

“…Two soft laser-based ionization sources include matrix-assisted laser desorption/ionization (MALDI) and infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) . Utilizing a laser in these sources creates specific challenges when attempting high spatial resolution experiments, which include but are not limited to the operating laser wavelength and the beam quality . MALDI approaches can work under ambient conditions (i.e., AP-MALDI) or vacuum and have been documented to employ infrared (IR) or ultraviolet (UV) lasers, although the latter is more common.…”

“…MALDI approaches can work under ambient conditions (i.e., AP-MALDI) or vacuum and have been documented to employ infrared (IR) or ultraviolet (UV) lasers, although the latter is more common. For UV-MALDI, the laser is complemented with an organic matrix co-crystallized with the sample, where energy is absorbed at the same or similar wavelength as the laser (e.g., 337 or 355 nm) . In the case of IR-MALDESI, ice and sucrose-embedding , have been utilized as the energy absorbing matrix, as the laser wavelength for this platform is 2970 nm.…”

Ambient mass spectrometry imaging of banana (Musa spp.) tissue with infrared laser-based selected reaction monitoring mass spectrometry imaging

“…An advantage of using an IR laser is its ability to effectively couple energy into the O − H stretching mode of hydrogen-bonded water, commonly present in hydrated biological materials. This facilitates greater depth of sampling, making it suitable for imaging various tissues including non-at samples (Joignant et al 2023).…”