Abstract

ABSTRACTLasers are instrumental in advanced bioimaging and Raman spectroscopy. However, they are also well known for their destructive effects on living organisms, leading to concerns about the adverse effects of laser technologies. To implement Raman spectroscopy for cell analysis and manipulation, such as Raman-activated cell sorting, it is crucial to identify nondestructive conditions for living cells. Here, we evaluated quantitatively the effect of 532-nm laser irradiation on bacterial cell fate and growth at the single-cell level. Using a purpose-built microfluidic platform, we were able to quantify the growth characteristics, i.e., specific growth rates and lag times of individual cells, as well as the survival rate of a population in conjunction with Raman spectroscopy. Representative Gram-negative and Gram-positive species show similar trends in response to a laser irradiation dose. Laser irradiation could compromise the physiological function of cells, and the degree of destruction is both dose and strain dependent, ranging from reduced cell growth to a complete loss of cell metabolic activity and finally to physical disintegration. Gram-positive bacterial cells are more susceptible than Gram-negative bacterial strains to irradiation-induced damage. By directly correlating Raman acquisition with single-cell growth characteristics, we provide evidence of nondestructive characteristics of Raman spectroscopy on individual bacterial cells. However, while strong Raman signals can be obtained without causing cell death, the variety of responses from different strains and from individual cells justifies careful evaluation of Raman acquisition conditions if cell viability is critical.IMPORTANCE In Raman spectroscopy, the use of powerful monochromatic light in laser-based systems facilitates the detection of inherently weak signals. This allows environmentally and clinically relevant microorganisms to be measured at the single-cell level. The significance of being able to perform Raman measurement is that, unlike label-based fluorescence techniques, it provides a “fingerprint” that is specific to the identity and state of any (unlabeled) sample. Thus, it has emerged as a powerful method for studying living cells under physiological and environmental conditions. However, the laser's high power also has the potential to kill bacteria, which leads to concerns. The research presented here is a quantitative evaluation that provides a generic platform and methodology to evaluate the effects of laser irradiation on individual bacterial cells. Furthermore, it illustrates this by determining the conditions required to nondestructively measure the spectra of representative bacteria from several different groups.

Highlights

  • Lasers are instrumental in advanced bioimaging and Raman spectroscopy

  • The advent of lasers in the 1960s meant that Raman spectroscopy became a practical and affordable technology that has been rapidly implemented in many fields [2,3,4]

  • We exploit single-cell microfluidics for the quantitative evaluation of laser irradiation on bacterial cell growth and fate

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Summary

Introduction

Lasers are instrumental in advanced bioimaging and Raman spectroscopy. they are well known for their destructive effects on living organisms, leading to concerns about the adverse effects of laser technologies. The significance of being able to perform Raman measurement is that, unlike label-based fluorescence techniques, it provides a “fingerprint” that is specific to the identity and state of any (unlabeled) sample It has emerged as a powerful method for studying living cells under physiological and environmental conditions. The research presented here is a quantitative evaluation that provides a generic platform and methodology to evaluate the effects of laser irradiation on individual bacterial cells It illustrates this by determining the conditions required to nondestructively measure the spectra of representative bacteria from several different groups. In contrast to fluorescence techniques that detect known fluorescent labels, Raman spectroscopy does not require external labeling of samples or any a priori knowledge Instead, it provides a full spectrum of Raman “fingerprints” specific to the intrinsic chemical composition of a sample and has emerged as a powerful label-free method for studying living cells directly under their physiological conditions [5]. Considering the requirement to maintain cell function throughout Raman spectroscopic measurements, the growth characteristics of individual cells exposed to a wide range of irradiation conditions were quantified and correlated with their Raman spectra

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