Because bacteria are found widely spread in almost every habitat on earth and even in, or on, other organisms, they are vital for the global ecosystem and greatly impact human health. Pseudomonas aeruginosa is an opportunistic pathogen that can rapidly develop resistance during antibiotic treatment by genetic mutation utilizing a horizontal gene transfer mechanism. Moreover, the tendency of P. aeruginosa to form biofilms and its ability to utilize multiple mechanisms of motility make P. aeruginosa biofilms hundreds of times more resistant to antibiotics than the corresponding planktonic cells. The work described in this thesis uses multimodal chemical imaging – Raman microspectroscopy, mass spectrometric imaging and electrochemical measurements - together with micro- and nanofabricated structures of high spatial definition to investigate the mechanisms of P. aeruginosa biofilm growth by characterizing the spatial and temporal distributions of signaling molecules and other secreted metabolites.Using a combination of confocal Raman microscopy and mass spectrometry imaging, P. aeruginosa biofilm growth from both mucoid and nonmucoid strains was investigated on lithographically defined patterns, allowing differences in the spatial and temporal distributions of secreted molecules - including quinolones, rhamnolipids, and phenazines – to be characterized as a function of the surface environment. Results indicate that microbial attachment was accompanied by secretion of 2-alkyl-4-quinolones and rhamnolipids from both mucoid and nonmucoid strains, while pyocyanin was only detected from the mucoid strain. The mucoid strain was found to be sensitive to composition and patterning, while the nonmucoid strain was not; unpatterned surfaces were better than patterned surfaces in promoting community development in the mucoid strain; and mucin was also better than mercaptoundecanoic acid in this regard. Also, the mucoid strain was observed to secrete the virulence factor pyocyanin in a way that correlates with distress. Confocal Raman microscopy was also applied to investigate the response of P. aeruginosa biofilm grown on lithographically defined patterned or unpatterned mucin to antibiotic treatment and to characterize the spatial and temporal distribution of secreted metabolites from static biofilms on the substrate, from supernatant of the bacterial broth, and from pellicle biofilms at the air-liquid interface. The significant differences observed in metabolite secretion between P. aeruginosa mucoid and nonmucoid strains, static and pellicle biofilms, and patterned and unpatterned mucin surfaces suggests that tobramycin exposure and spatial patterning both strongly affected P. aeruginosa growth. Under tobramycin treatment, static biofilms of the mucoid strain favor unpatterned mucin surfaces, while static biofilms of the nonmucoid strain prefer patterned mucin surfaces as judged by their secreted Pseudomonas quinolone signals. Phenazines were only detected in pellicle biofilms and the supernatant in both strains, and the differences between phenazine species in supernatant showed the same preference of mucin surfaces as the static biofilms.A Hierarchically organized pH-responsive block co-polymer (BCP) membrane was combined with gold nanoparticle (AuNPs)-filled nanopore electrode arrays (NEAs) to separate and detect phenazine-1-carboxylic acid (PCA) from bacterial broth. By adjusting the pH of bacterial broth to 4.5 which is above the pKa of PCA but below the pKa of pyocyanin (PYO) and phenazine-1-carboxamide (PCN), negatively charged PCA was selectively captured. Because of the selectivity of BCP membrane at pH 4.5, only PCA was transported into the AuNPs-filled NEAs, while PYO and PCN were blocked. PCA secreted from P. aeruginosa with various incubation time was quantified by square-wave voltammetry and SERS on this electrochemical SERS sensor with high sensitivity and efficiency.Overall, this work illustrates the effectiveness of a multimodal approach combining Raman microspectroscopy, mass spectrometry imaging and electrochemistry to investigate biofilm growth, formation, and response to different stimuli in P. aeruginosa by characterizing the spatial and temporal distributions of signaling molecules and other metabolites. The potential application of multimodal techniques to future investigate other biologically relevant systems is also explored.