Use of Tetra-ammonium Tetrakis(4-Sulphonato)Phenyl Porphyrin for Pseudomonas and Bacillus Cell Imaging

The use of tetraammonium tetrakis(4-sulphonato)phenyl porphyrin (TPPS), a water-soluble anionic compound, as a stain to analyse bacterial cells using fluorescent microscopy was investigated. TPPS was effectively used to analyse two different bacteria: Pseudomonas aeruginosa and Bacillus cereus. The variation in brightness with varying concentrations of TPPS was studied. The patterns of variations for these bacteria were found to be the same, but with consistently higher brightness for Bacillus cereus. 1. Introduction Fluorescent microscopy is a robust technique to study cells [1]. This technique requires tagging of cells with a fluorophore, which is a molecule that will fluoresce when light at appropriate wavelengths is incident on it. Fluorophores should have good photostability and high quantum yield, and when used to image live cells they should be noncytotoxic and stable in vivo [2, 3]. They should also have low susceptibility to photobleaching without losing their sensitivity to image the cells [4]. In order to overcome photon attenuation in living cells, fluorophores with long emission at the near-infrared (NIR) region are generally preferred [5]. At present, the most commonly used fluorophore to image bacterial cell is fluorescein, but owing to a variety of factors it is not a good choice [6]. Many other fluorophores are also in current use, and new fluorophores with high quantum yield and low cytotoxicity are also being continuously screened for imaging cells. Porphyrin and its derivatives are nontoxic organic compounds whose fluorescent properties have been widely studied. Amphiphilic derivatives of porphyrin have been studied for second-harmonic generation-imaging [7]. We have investigated the use of tetra ammonium tetrakis (4-sulphonato)phenyl porphyrin (TPPS) (Figure 1), a water-soluble anionic compound, with multiple excitation bands and wide excitation range, as a fluorescent dye for imaging bacterial cells. Figure 1: Tetrakis(4-sulphonato)phenyl porphyrin. In earlier studies, TPPS has been used as a photosensitising agent for photo-dynamic therapy. It was found to be moderately effective against Gram-positive bacteria but had no cytotoxic effect on Gram-negative bacteria, even after prolonged exposure to light at high frequency. Studies with tetra-cationic phthalocyanine as photosensitizer have also shown that the entry of photosensitizer into the cytoplasm increases brightness of cells, which was observed in images obtained from fluorescence microscope. The uptake of photosensitizer was further found to be an important step in affecting