Part I I wasraised in a redbrick Baltimore row housewhere summer was marked by the ti- honored ritual of firefly-chasing a backyard tradition that has endured the gene- tions.Amidtheexcitement,myfatheroftentoldthestoryofhow,whenhewasachild, researchers at the Johns Hopkins University had appealed for the systematic capture of live fireflies en masse. Science had engaged the Baltimore youth in an entrepreneurial quest to jar as many lightning bugs as the dwindling light of dusk would permit. The very next morning, each 100-count glass jar of glowing crawling insects could be exchanged at the University for exactly one crisp dollar bill. Unrecognized at the time by my father, his joyous endeavors had contributed in a profoundwaytotheadvancedmolecularbiologicaltechniquesthatserveasthebasisfor thistextbook.In1947,WilliamMcElroyusedextractsfromthoseveryfirefliestodefine the fundamental reaction underlying the mystical phenomenon of luminescence, and published The energy source for bioluminescence in an isolated system in the Proceedings of the National Academy of Sciences. Inthedecadessincethatsummer,thestudyandapplicationofbioluminescencehave allowed us to leverage the enduring power of natures elegance. We have painstakingly harnessedapowerfultoolthatenablesustoseekadeeperunderstandingofthecomplex mechanisms underpinning so many vital biologic systems. This second edition of Methods in Molecular Biologys Bioluminescence: Methods and Protocols serves as a re- ableandutilitariancompilationofthenewestandmostinnovativetechniquesthathave emerged in this rapidly expanding and progressively diverse field. We are indebted to the authors for their thoughtful contributions, inspired by their rigorous dedication to the science of bioluminescence, humbled by theunyielding supportofourcolleagues,andgratefulfortheopportunityprovidedusbyJohnandJan Walker.