ABSTRACT
Biological tissues can be eciently oxidized and damaged upon illumination with light at the appropriate wavelength when this light is absorbed by natural or synthetic photosensitizers (PSs). e photodynamic eect starts with electronic excitation of the PS to the singlet excited state, which is subsequently converted to a triplet excited state by intersystem crossing. Triplets are reactive species that live long enough to interact with nearby species, leading either to energy transfer to oxygen (type II process) or to direct reactions
9.1 Introduction 149
9.2Subcellular localization of PS 152
9.3 PS localization and cell death mechanisms 159
9.3.1Necrosis and apoptosis 160
9.3.2 Autophagy 163
9.4Conclusion and future directions 168
9.5 Experimental protocols 168
9.5.1 Subcellular localization 169
9.5.1.1 Intensity-based fluorescence microscopy 169
9.5.1.2 Fluorescence lifetime 169
9.5.2 Necrotic versus apoptotic cell death with annexin V/PI 169
9.5.2.1Identifying apoptotic nuclei 170
9.5.3 Detection of autophagy by fluorescence microscopy 170
9.5.3.1Staining acidic vacuoles with AO 170
9.5.3.2 Acidic vacuoles staining by MDC 170
9.5.3.3 Immunofluorescence to identify specific proteins present in autophagosomes170
9.5.3.4 Co-localization of organelles and autophagosomes 171
Acknowledgments 171
References 172
with biological substrates (type I process). In the former case, there is the formation of singlet oxygen (1 2O ), which is a highly electrophilic molecule. Type I processes are based on the reaction between excited states (usually triplets) and biomolecules that are in close proximity or contact, forming a variety of products, and usually starting radical chain reactions. Both type I and II processes cause oxidation of biomolecules, and depending on the extent of the damage, they also impair cell viability (Foote 1968; Halliwell 2009; Hamblin and Hasan 2014; Henderson and Dougherty 1992; Krinsky 1977). is phenomenon is the basis of the clinical treatment known as photodynamic therapy (PDT). In PDT, exogenous PSs are given to the patient, and the target of the treatment (tumors or diseases tissues sites with infected areas) is selectively irradiated.
