PIT dependency was not clear. For biofilms of C. dubliniensis, the analysis of variance showed significant interaction of PIT and Cur concentration (p = 0.001) 17-AAG supplier in the P+L+ groups irradiated for 4 min. On the other hand, the interaction was not significant in the P+L+ groups irradiated for
8 min, with a significant effect of PIT (p < 0.001) and Cur concentration (p < 0.001). Tukey's test was applied to study the cases, and the results are presented in Fig. 5 and Fig. 6. The groups illuminated for 4 min were concentration-dependent for the extreme values (40 and 20 μM). No PIT dependency was clearly observed. Whereas, groups illuminated for 8 min were concentration and PIT-dependent. For all the microorganisms, CSLM was used to investigate Cur penetration into the deepness of the biofilms. Images of Candida spp. biofilms were captured by fluorescence mode ( Fig. 7 and Fig. 8) following incubation of the biofilms with Cur 40 μM for 5 min ( Fig. 7A, C and E) and 20 min (Figs. 7B, D, F and 8). In spite of the light green fluorescence observed after a 5-min incubation ( Fig. 7A, C and E), brighter fluorescence was observed following a 20-min incubation ( Fig. 7B, D and F). Fig. 8 presents cross sections and side views of C. albicans biofilms
after 5 and 20 min of incubation with Cur 40 μM ( Fig. 8B and C, respectively). Fig. 8A presents an image of the transmittance mode applied to C. albicans biofilm before the incubation with curcumin, due to the absence of fluorescence GSK1120212 signal from Candida biofilms without curcumin. On the side views of the same biofilm ( Fig. 8B and C), it is possible to determine the biofilm thickness (yellow lines) and curcumin penetration through the biofilm (red lines). Moreover, it is possible to observe the lack of sensitised cells in the deepest portions (yellow arrow) when compared to the outermost layers with a brighter fluorescence (red arrow). Among other factors, the effectiveness of antimicrobial PDT depends on the pre-irradiation time (PIT), which is the period required by the PS to remain in contact with the microorganisms before illumination. It seems that the PIT
sufficient to promote effective microbial killing depends on the properties of the PS. For example, the porphyrins, the phenothiazine and the aluminium phthalocyanine (AlPc)26, 34 and 35 require shorter PITs when compared with tetrasulfonated PDK4 aluminium phthalocyanine (AlPcS4).35 In contrast, other studies have stated that PIT had no significant importance on the effectiveness of PDT, and demonstrated that a longer PIT did not increase the reduction in cell viability.26, 33 and 34 In addition, the species of the microorganism studied is an important factor influencing PDT effectiveness.39, 45 and 51 Due to the vast diversity of microorganisms, a PS with distinct physicochemical properties may be required. For these reasons, different types of PS have been proposed for antimicrobial PDT.