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

Biospecimens with nearly flat surfaces on a flat stage are typically required for laser-based 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 (lambda). 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.

Automated summary

Biospecimens with flat surfaces are needed for laser-based mass spectrometry imaging. However, imperfections in the sampling stages and non-flat samples require a deeper understanding of the laser beam's depth of focus. This tutorial explores the parameters and equations that describe the depth of focus of a Gaussian laser beam and presents methods to address surface roughness exceeding the laser's depth of focus.