We chemically immobilized live, motile Escherichia coli on micrometer-scale, photocatalytically patterned silicon surfaces via amine- and carboxylic acid-based chemistries. Immobilization facilitated (i) controlled positioning; (ii) high resolution cell wall imaging via atomic force microscopy (AFM); and (iii) chemical analysis with time-of-flight-secondary ion mass spectrometry (ToF-SIMS). Spinning motion of tethered bacteria, captured with fast-acquisition video, proved microbe viability. We expect our protocols to open new experimental doors for basic and applied studies of microorganisms, from host-pathogen relationships, to microbial forensics and drug discovery, to biosensors and biofuel cell optimization.