This study analyzes the performance of fluorescent probes for in vitro applications. Firstly, the photophysical properties of Phosphatidylserine Sensor 794 were analyzed in various solvents systems. When compared to 100 % water solutions, there was an increase of at least three fold in the absorption and emission of the probe when 0.5 % of polar organic solvents were added to an aqueous solution. There was a fourfold increase in the emission of the probe when the pH was made acidic or the concentration of bovine serum albumin was increased. Comparative photobleaching and quenching studies of biotinylated squaraine rotaxanes demonstrated their higher photochemical stabilities when compared to fluorescein-4-biotin; likewise, streptavidin-conjugated squaraine rotaxanes displayed higher photostability when compared to commercially available Cy5-Streptavidin. Multivalent squaraine rotaxanes with ammonium and guanidinium surface groups exhibited similar affinity for bacterial cells but induced higher agglutination when compared to bis-DPA-squaraine rotaxane. Mammalian cells appeared to internalize the probes, and preliminary studies indicate the probes are highly phototoxic.