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Vital Microscopy and its use to Elucidate Photodynamic Effects by Tor Brustad1, Torhild Voss1, Pål Kristian Selbo1, Kaare Gärtner1, Trond Holmøy1, Jahn M. Nesland2, and Kristian Berg1. 1 Department of Biophysics, 2 Department of Experimental Pathology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello 0310 Oslo, Norway. # Abstract Vital microscopy (VM) deals with microscopic investigations of living cells and tissues. While useful samples of the former usually are easily obtainable from most types of cell cultures, sufficiently thin samples of living tissue are more demanding to attain. A transparent chamber technique is described, by which viable tissue layers of thickness down to 20 mm are generated. Some examples from the wealth of information inherent in the dynamic scenery associated with the in vivo processes are presented. This is achieved by combination of the video-based vital microscopy technique and the electronic information technology available at the present conference. The potency of this technology is demonstrated, with examples from studies related to photodynamic therapy (PDT), based on video recordings of chamber tissue as well as of cells from tissue culture. ### I) VM-studies of normal chamber tissues to elucidate response patterns of different types of vessels following PDT The effects were induced by light exposure of the tissue in the transparent chamber at specified times after intra-peritoneal (i.p.) or intra-venous (i.v.) injection of 25 mg kg-1 and 3.1 mg kg-1 body weight (b. w.) of tetra (4-sulfonatophenyl) porphine (TPPS4), respectively tetra (3-hydroxyphenyl) porphine (3-THPP). In both cases photochemical effects were demonstrated; in venules by showing growth of individual deposits at certain light-exposed sites in the vessel wall, until filling the entire lumen as permanent blood plugs. Alternatively the plugs are shed off and transported in the blood stream as emboli, until trapped when blocking up the blood stream in one arm of nearby vessel bifurcations downstream, in arterioles by showing a) that myriad's of emboli are formed and shed off in succession from given growth sites, and b) constriction of the vessel, which finally may give rise to permanent or transient interruption of the blood stream. ### II) VM-studies of location of photosensitizers in vivo This effect was demonstrated by fluorescence microscopic analysis of the i.p. injected photosensitizer TPPS2a (disulfonated tetraphenylporphine with the sulfonate groups in adjacent positions) in the viable granular normal tissue of the transparent chamber. In normal tissue, bright fluorescence from the photosensitizer was detected mainly in the microvascular network 24 hrs post administration, while the surrounding tissue was negative. A new method for quantitative analysis of photosensitizer distribution in living tissue was established, based on well founded methods for fluorescence quantification. ### III) PDT-effects on cells in tissue culture Two types of such effects are shown: a) The dynamics of intracellular release from lysosomes into the cytoplasm of photochemically activated disulfonated aluminium phthalocyanine with 2 sulfonate groups in adjacent positions, AlPcS2a. This effect was demonstrated by fluorescence video microscopy of NHIK 3025 cells treated with 10 mg/ml AlPcS2a for 18 hrs followed by continuos exposure to the excitation light of the microscope. b) The dynamics of PDT-induced formation and rupture of blebs on the plasma membrane. NHIK 3025 cells were treated with 5 mg/ml monosulfonated meso-tetraphenylporphine (TPPS1) for 18 hrs, followed by continuous exposure to the excitation light of the microscope. The treatment induced bleb formation on the plasma membrane, followed by release of the cellular cytoplasm into the extracellular medium. These results indicate that TPPS1 was partly located on the plasma membrane, and that the treatment killed the cells through a necrosis pathway.