Effect of laser irradiation on cell function and its implications in Raman spectroscopy

Lasers 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 non-destructive 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 rate and lag time 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 a similar trend in response to laser irradiation dose. Laser irradiation could compromise 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 non-destructive 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 justify 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 detection of the 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 is that, unlike label-based fluorescence techniques, it provides a “fingerprint” that is specific to the identity and state of any (unlabelled) sample. Thus, it has emerged as a powerful method for studying living cells under physiological and environmental conditions. However, the laser9s 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 non-destructively measure the spectra of representative bacteria from several different groups.