Feasibility of multiphoton microscopy-based quantification of antibiotic uptake into neutrophil granulocytes.

Antibiotic levels in livestock are usually evaluated through destructive analysis. Taking advantage of the fluorescent properties of marbofloxacin (MBX) and trovafloxacin (TVX), multiphoton microscopy (MPM) was evaluated as a minimally invasive and nondestructive method to determine the penetration of TVX and MBX into sheep neutrophils. Standard curves were measured with drug-only solutions and suggested that MBX was more suited for this type of analysis. The intracellular concentration of both TVX and MBX was higher than the extracellular concentration after incubating neutrophils for 30 min at concentrations ranging from 0.1 to 100  μg/ml for both the drugs. The intracellular concentration of TVX increased with the extracellular concentration but was always greater than the extracellular concentration, suggesting active internalization. On the other hand, intracellular/extracellular ratio (I/E) peaked at 1.6-fold I/E for 1  μg/ml and then gradually decreased with increased concentration to 1.2-fold I/E at 100  μg/ml. For the first time, this study showed the use of MPM to quantify antibiotic uptake by sheep neutrophils and observed that both antibiotics were taken up by sheep neutrophils beyond extracellular levels.