Probing the Onset of the Bacterial Membrane Photodamage in Time using a Raman Optical Tweezer

Laser-induced photo damage of the optically confined microorganism is known to affect the bacterial membrane. We record the Raman spectra of a live, optically trapped Bacillus subtilis at different trapping time lapses to determine the changes in the bacterial membrane, which in turn impacts the flagellar rotation. A 1064 nm tightly focused laser traps a single de-flagellated bacterium and Surface Enhanced Raman Scattering (SERS) signals are recorded with silver nanoparticles (AgNPs) inserted into bacteria via the internal colloid method. The internal colloid method, albeit resulting in a modest signal increase, is employed in preference here to prevent undesirable cell aggregation or instabilities in trapping or compromised cell integrity resulting from the conventional nanoparticle dressing of the cell membrane. The second derivative of the Raman spectrum reveals subtle changes in the molecular structure of the bacterial membrane manifested by shifts in the phospholipid peak (1462 cm-1), amide III peak (1245 cm-1) and cytosine peak (792 cm-1), with increased trapping duration. This is followed by a Principal Component Analysis (PCA) to examine the changes occurring in the Raman spectral range (600 cm- 1-1800 cm-1). By comparing the spectral shifts at specific time lapses from the moment of trapping, with the diminishing frequency of rotation of the body and flagella of the trapped flagellated counterpart bacterium at these same time lapses, we establish a direct correlation between the changes in membrane structure and compromised rotation of the bacterium during photodamage. Our results confirm that the subtle changes that occur at the biomolecular level in a cell when subjected to photodamage can be identified with good sensitivity, and moreover, that the changes occurring at the biomembrane have a role to play in reduced rotation of the trapped bacterium during exposure to the laser.