ABSTRACT
Optical absorption and fluorescence spectroscopies are by many considered to be primary research tools in biophysics and biochemistry. In this chapter, subsequent to defining the main concepts relevant to these methods, we focus on modern developments of the basic absorption and fluorescence methods, including polarization spectroscopy techniques, with proven success record in photosynthesis research. Especially fluorescence, although a mere by-product of photosynthesis, demonstrates expanding applications, due to exceptional sensitivity of current detectors suitable for single molecule recording. The techniques introduced in this chapter include spectrally selective hole burning, fluorescence line narrowing, and differential fluorescence line narrowing spectroscopy, single molecule spectroscopy, linear dichroism and polarized fluorescence emission, and isotropic and anisotropic circular dichroism, as implemented in differential polarization laser scanning microscopes. Rather than describing the instrumentation, we foremost explain the fundamental physics principles behind each technique. Examples that illustrate how the concepts are used in different applications are included, with special attention to the effects of the microenvironment of the pigment molecules, including the influence of the protein matrix and the nature of excitonic interactions between the chromophores on the migration of excitation energy in hierarchically organized light-harvesting antenna systems in different prokaryotic and eukaryotic organisms—both in their native and isolated forms, as well as in reconstituted and artificial systems. We also emphasize the importance of optical spectroscopic techniques for the characterization of extended arrays of light-harvesting antenna complexes, highly organized systems originating from different levels of the structural complexity, which play significant roles in regulatory mechanisms.
