1. Experimental procedure to study the correlation between the PDT effects and  pO 2 Delayed fluorescence lifetime measurements were performed during and at the end of the illumination of Chick chorioAllantoic Membranes (CAM) Samples preparation:  Fertilized eggs were incubated during 11 days (membrane development).  20 µl of ALA (0.15 M) were administered topically, 4h before PDT and the DF measurements PDT illumination conditions:  Light source: CW diode laser @ 405 nm; 2.5 mW/cm 2 ; up to 2.25 J/cm 2.  Circular spot produced by a frontal light distributor from Medlight SA: 8 mm diameter. CAM membrane at embryonic development day 11 1) Egg2) Laser3) PDT fiber 4) DF Spectrometer5) DF measurement fiber 6) DF measurement fiber holder 1) CAM2) Protective drop of aqueous serum 3) 0.19 mm cover-glass4) PDT fiber 5) DF measurement fiber (1 mm 2 probed area) 6) DF measurement fiber holder  - oxymetry For PDT Piffaretti et al., JBO, 2012

2. Experimental procedure (cont.) Vascular damage assessment 17 h after PDT the eggs were taken out of the incubator and vascular damage were examined with an epifluorescence microscope (Ex. 450 – 490 nm, Em. > 520 nm). 20 µl of a 150 µM solution of fluorescein isothiocyanate dextran (150 KD) were administered intravenously and 0.1 ml of india ink was injected under the CAM to reduce the background fluorescence. The diameter of the largest closed vessel was measured and used as an index of vascular damage. (method described in: Lange et al., IOVS 2001) 80μm 4X objective 10X objective 200 µm  - oxymetry For PDT Piffaretti et al., JBO, 2012

3. Delayed fluorescence lifetime measurement setup  - oxymetry For PDT Piffaretti et al., JBO, 2012

4. Tissular oxygen depletion during PDT The pO 2 decreases monotonically as the PDT illumination increase. An horizontal asymptotic value is reached after ~ 500 sec (~1.3 J/cm 2 ) Bars: SEM N = 17 16 Different symbols: different measurement series  - oxymetry For PDT Piffaretti et al., JBO, 2012

5. Vascular damage index observed after PDT versus the reciprocal lifetime differences (before/after PDT) An excellent correlation is observed between the vessel damages and the pO 2 reduction. Bars: SEM N = 20  - oxymetry For PDT Piffaretti et al., JBO, 2012

6. Setup for micro-imaging and photo-treatment - Assessment of the vascular damages based on the diameter of the largest closed vessel. - The index of vascular damage (Ivd) range assessment between “0” (no detectable photodamage) and “5” (total closure of all blood vessels in the irradiated area). FITC-dextran angiographies 24 h post irradiation  - oxymetry Exo. probe

7. - Phototoxic threshold: 1 mg/kg; 10 J/cm 2 @ 420 + 20 nm - Dark toxicity after iv injection of 10, 50 and 100 mg/kg: 100 % survival after 24 h (6 eggs/condition) 10 min after i.v. injection 1 min after i.v. injection  - oxymetry Exo. probe Phototoxicity and leakage of Ru(Phen) 3 2+ versus PdTCPP Lethal dose 4 eggs / condition ( PdTCPP ) Phototoxicity of Ru(Phen) 3 2+ << PdTCPP ( PdTCPP ) Huntosova et al., Metallomics, 2014 Huntosova et al., JBO, 2014

8. Light dose < 120 mJ/cm 2 Much smaller than the phototoxic threshold 10 J/cm 2 & 1 mg/kg CAM were subjected to different pO 2 conditions in a gas chamber. Detection was performed 10 min after 1mg/kg b.w. Ru(Phen) 3 2+ 20  l injection.  - oxymetry Exo. probe Luminescence lifetime measurement setup

9.  - oxymetry Exo. probe pO 2 dependence of the Ru(Phen) 3 2+ reciprocal lifetime Measurements performed 10 min after iv injection of a 20  l solution of Ru(Phen) 3 2+ (1 mg/kg of b.w.). CAM were subject to different environmental pO 2 in a gas controlled and temperature stabilized (25 °C) chamber. 6 eggs; 4 measurements / condition. Light dose @ 420 nm necessary to perform one pO 2 measurement: < 120 mJ/cm 2 Much smaller than the phototoxic threshold: 10 J/cm 2 (0.9% NaCl isotonic solution) Huntosova et al., Metallomics, 2014 Huntosova et al., JBO, 2014