Surface analysis of bacterial systems using cryo‐X‐ray photoelectron spectroscopy

Abstract

Surface analysis of biological systems using XPS often requires dehydration of the sample for it to be compatible with the ultrahigh vacuum of the spectrometer. However, if samples are frozen to liquid-nitrogen temperature prior to and during analysis, water can be retained in the sample and the organization of the sample surface should be preserved to a higher degree than in desiccated samples. This article presents recent developments of cryo-X-ray photoelectron spectroscopy (cryo-XPS) for analyses of hydrated biological samples at liquid nitrogen temperature. We describe experiments on bacterial cells, bacterial biofilms, and bacterial outer membrane vesicles using a variety of bacterial species. Differences and similarities in surface chemistry are monitored depending on growth in liquid culture, on culture plates, as well as in biofilms, and are discussed. Two data treatment methods providing decomposition of the C 1s spectra into lipid, polysaccharide, and peptide/peptidoglycan content are used and compared.

Comparative Study of NAP-XPS and Cryo-XPS for the Investigation of Surface Chemistry of the Bacterial Cell-Envelope

“…In order to predict the contribution of peptides (proteins and peptidoglycans), lipids and polysaccharides in the C 1s spectra of the nine samples, "the Umeå method" was employed. It predicted that 66±4% of detected surface C atoms originated from proteins or peptidoglycans, 8±2% from lipids and 26±2 from polysaccharides (Supplementary Table S4), in line with what has previously been reported for bacterial cells (Hagberg et al, 2020;Ramstedt et al, 2011;Ramstedt et al, 2014).…”

“…Alternatively, the bacterial cells were washed with PBS (containing 8 g L −1 NaCl, 0.2 g L −1 KCl, 1.44 g L −1 Na 2 HPO 4 , 0.24 g L −1 KH 2 PO 4 at pH 7.4), centrifuged, the supernatant discarded, and approximately 20 µL of the pellet was transferred using an automatic pipette to the sample holder forming a dense droplet (this wash was performed only for some of the replica of cryo-XPS, and not for NAP-XPS). Thereafter, the bacterial biomass was fast-frozen in the sample introduction chamber under a gentle flow of dry nitrogen, the pressure reduced, the sample transferred into the analysis chamber, and analyzed by cryo-XPS in accordance with previously described procedures ( Ramstedt and Shchukarev, 2016 ; Hagberg et al, 2020 ). Previous studies have shown no significant differences in major peaks (C1s and N1s) between washed and non-washed P. fluorescens bacteria grown on LB agar culture plates ( Hagberg et al, 2020 ).…”

“…Thereafter, the bacterial biomass was fast-frozen in the sample introduction chamber under a gentle flow of dry nitrogen, the pressure reduced, the sample transferred into the analysis chamber, and analyzed by cryo-XPS in accordance with previously described procedures ( Ramstedt and Shchukarev, 2016 ; Hagberg et al, 2020 ). Previous studies have shown no significant differences in major peaks (C1s and N1s) between washed and non-washed P. fluorescens bacteria grown on LB agar culture plates ( Hagberg et al, 2020 ).…”

“…Today, applications of XPS exist that allow for analyses of hydrated samples without introducing a drying step ( Ramstedt et al, 2011 ; Kjaervik et al, 2018 ; Hagberg et al, 2020 ; Shchukarev et al, 2020 ). One alternative is cryo-XPS where the sample is fast frozen, a treatment that vitrifies the water in the cells conserving their spatial structure.…”

“…Comparisons between the “Umeå method” with the equation systems developed by Rouxhet et al have shown that they give comparable, albeit not identical, results. They both relate the XPS data to the composition of “ideal substances”, but the spectral fingerprints of these ideal substances are not identical ( Hagberg et al, 2020 ).…”