Hole-burning spectroscopy, a high-resolution spectroscopic technique, allows details of heterogeneous nano-environments in biological systems to be obtained from broad absorption bands. Recently, this technique has been applied to proteins, nucleic acids, cells, and substructures of water to probe the electrostatic conditions created by macromolecules and the surrounding solvent. Starting with the factors that obscure the homogeneous linewidth of a chromophore within an inhomogeneously broadened absorption or emission band, we describe properties and processes in biological systems that are reflected in the measured hole spectra. The technique also lends itself to the resolution of perturbation experiments, such as temperature cycling to elucidate energy landscape barriers, applied external electric fields (Stark effect) to measure net internal electric fields, and applied hydrostatic pressure to find the volume compressibility of proteins.

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