Strategies for Minimizing Interference from Metastable Water Clusters in ToF-SIMS 3D Imaging of Frozen Hydrated Biological Samples

Cryogenic analysis of hydrated biological samples is required to retain their native three-dimensional structure during time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging. However, stable and metastable water cluster ions, originating from water ice, result in spectral interferences over a wide mass range. These interferences worsen the detection of analytes or can lead to misassignments of mass peaks. In this study, we have investigated the water ice spectrum in a gridless multistage reflectron ToF mass analyzer with two acceleration zones prior to the reflectron. The influence of analyzer settings on the spectrum of a cryogenic frozen hydrated organic solution has been investigated. Distinct metastable peaks associated with the loss of a single water molecule or multiple water molecules in different field-free drift zones of the mass analyzer have been identified. The influence of analyzer settings on the position of these metastable peaks has been investigated and used to identify the processes giving rise to these metastable peaks. Strategies for minimizing spectral interference from these metastable peaks have been identified. The results of this work demonstrate that most of the cryogenic frozen hydrated background signals originate from either pure water clusters ((H2O)nH+) or cationized water clusters ((H2O)nR+, where R is a small cation). In the mass range between 200 m/z and 415 m/z, the spectrum primarily contains peaks from the decay of metastable pure and cationized water clusters with the loss of a single H2O molecule during the ion's flight to the detector. Above 415 m/z, metastable peaks due to the loss of multiple H2O molecules increase.