Spectral phasor analysis of live bacteria in biofilms

Abstract

Biofilms have become a significant issue both clinically and industrially due to their ability to survive conventional treatment regimens. Subpopulations of bacteria with altered metabolic activity are a major contributor to the increased ability of biofilms to survive. Therefore, providing an insight into the metabolic activity of individual bacteria within biofilms is expect to lead to the development of effective treatment strategies. Real-time non-invasive characterisation of metabolic activity is achievable with fluorescent molecules as shifts in the emission spectrum of a fluorescent molecule can occur due to microenvironment changes. Spectral Phasor analysis enables the detection of these spectral shifts and their mapping within an image thereby providing an insight into cellular microenvironments. This thesis reports the application of Spectral Phasor in order to develop methodology which can characterise metabolic activity of live bacteria in biofilms. In this study, Spectral Phasor analysis was applied to green fluorescent protein (GFP) as well as nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H) in biofilm bacteria. Spectral shifts in GFP were detected in bacteria as a result of treatment with gentamicin. Wavelengths between 489.0 nm - 505.7 nm were primarily detected in bacteria prior to treatment with gentamicin, while wavelengths detected in bacteria treated with gentamicin for 45 minutes were predominantly between 506.8 nm - 523.5 nm. Following treatment with gentamicin for 60 minutes detected wavelengths were between 489.0 nm - 505.7 nm. The detection of spectral shifts in bacteria treated with gentamicin indicates that GFP can act as a bio indicator of microenvironments within bacteria. Furthermore, linked cursor analysis enabled the visualisation of bacteria with distinct emission wavelengths to other bacteria within a treatment condition. The advantages of application of Spectral Phasor analysis to GFP are that GFP is stable over a wide range of cellular condition and is minimally toxic. However, a limitation is that this approach requires bacteria expressing GFP. In contrast, NAD(P)H is found endogenously within bacteria; however, NAD(P)H is weakly fluorescent and the concentration of NAD(P)H present is affected by the state of bacteria. Due to this modifications in the acquisition parameters were required to enable the collection of data from NAD(P)H to increased the detected fluorescence from within bacteria and minimised background fluorescence. This research demonstrates that Spectral Phasor analysis can detect spectral shifts in the emission of fluorescent molecules in bacteria with altered cellular activity through treatment with an antibiotic. Further development of Spectral Phasor analysis for bacteria may provide an insight into the activity in individual bacteria within biofilms during treatment with antimicrobial agents in real-time.

Date of Award	2017
Original language	English