Laser induced degradation of bacterial spores during micro-Raman spectroscopy

Dmitry Malyshev,Rasmus Öberg,Tobias Dahlberg,Krister Wiklund,Lars Landström,Per Ola Andersson,Magnus Andersson

doi:10.1016/j.saa.2021.120381

Abstract

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores’ structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.

Highlights

Simultaneously analyze eukaryotic cells, bacteria, and spores [1]
Optical tweezers combined with micro-Raman spectroscopy are valuable tools for investigating bacterial spores
Spores are normally considered very resilient to environmental stress, but as we show in this work they can be affected when trapped in a NIR laser beam

Summary

Introduction

Simultaneously analyze eukaryotic cells, bacteria, and spores [1]. In general, trapping different objects has been considered noninvasive at low laser power [2]. The proposed mechanisms of action were absorbance of light by NADH and flavins for laser light in the 650–750 nm range, and absorbance by water at wavelengths 800–1064 nm [4,5], leading to DNA damage [5] In these studies, wavelengths in the region 800 nm and 1064 nm were found to be the least damaging to the cells. Similar effects have been observed on trapped bacterial cells where, for example, it was shown that a 1064 nm laser could inhibit the growth and division of E. coli cells at laser power as low as 3 mW [7,8], and dose ranging from 0.54 J to over 10 J[9]. When the intensity of this peak is reduced, we know that CaDPA is lost from the spore core, most likely by spore body disruption and diffusion to surrounding medium. To gain further insight in the mechanisms of the interaction between the laser light and the spore, we support the experimental data and conclusions with a multiphysics simulation model of a spore to estimate any photothermal contribution

Methods

Results

Conclusion