Chromophore-assisted light inactivation (CALI) was developed as a means of targeted inactivation of proteins to generate a protein loss of function at the time of light excitation [1]. In its original version, light energy is targeted to destroy a protein of interest by binding it with a specific antibody labeled with the chromophore, Malachite green. Short-lived free radicals are generated when the dye is excited by laser light at 620 nm, a wavelength not well absorbed by cellular components, resulting in photochemical damage to the bound antigen with minimal collateral damage to nearest neighbors. Since its inception, CALI has been useful in ascribing in situ function of a large number of proteins in cells and embryos and has spawned a family of related technologies that expand the power and diversity of application of light inactivation of protein function. Based on the twentieth anniversary of its invention, it is timely to review the development of CALI technology, its use to ascribe in situ function, and the future of its potential application. There have been other good comprehensive reviews on this technology and the reader is directed to these particularly for methods [2][3][4].
The development of CALI was derived from two ideas: (i) laser ablation of whole cells in vitro [5] and in simple organisms such as grasshopper [6] and (ii) the promise of monoclonal antibody technology to deliver highly specific inhibitors of protein function had not been realized. Combining these two ideas led the author to consider that laser energy might be targeted to damage specific proteins via antibodies using a dye that would absorb wavelengths of light not readily absorbed by cells or tissue. The excited chromophore would release the absorbed energy rapidly and in the form of heat, thus denaturing the bound antigen. Feasibility calculations were done with Allan Oseroff (then at Wellman Labs Massachusetts General Hospital) that suggested the possibility for successful denaturation of proteins by the proposed approach. In discussions with James Foley (then at the Rowland Institute) triarylmethanes were suggested and the author selected Malachite green as the first photosensitizer for CALI, because it absorbed 620 nm