Photodynamic damage study of HeLa cell line using ALA

The present study evaluates the photodynamic damage with 5-aminolevulinic acid (5-ALA) using HeLa as experimental model. HeLa cell line was irradiated with red light (He-Ne laser, λ = 632.8 CW nm). The influence of different incubation times and concentrations of 5-ALA, different irradiation doses and various combinations of photosensitizer and light doses on the cellular viability of HeLa cells were studied. The optimal uptake of photosensitizer ALA in HeLa cells was investigated by means of PpIX fluorescence intensity by exciting the HeLa cell suspension at 450 nm and a detection wavelength set at 690 nm. Cells viability was determined by means of trypan blue solution. The spectrometric measurements showed that the maximal cellular uptake of 5-ALA occurred after 4 h in vitro incubation. We found that the combination with 5-ALA and laser irradiation leads to time/concentration-dependent increase of cells death and also energy doses-dependent enlarge the cells death. The fluorescence intensity after PDD of carcinoma cells reduce when compared with the control group. The fluorescence emission spectral profiles after PDD of carcinoma cells showed a dip around 425–525 nm when compared with the control group. This may be due to the damage of mitochondria component of cells. The percentage of HeLa cells after PDD shows that the percentage of cells survival rate as function of laser dose (power). Hence it is clear that at 200 μg/ml ALA and 20 mW laser irradiation, more than 70% of HeLa cells were dead after 15 min.     

Fluorescence spectra of cultured normal and malignant lung cells

Fluorescence Spectroscopy has emerged as a new modality to characterize physicochemical properties of biomolecules. The biomolecules have certain photophysical properties based on their molecular structure and these properties have been considered as useful parameters to monitor alterations in the functional, morphological and micro environmental changes in the cells and tissues. In this study the fluorescence emission spectra of normal and malignant lung cells were recorded for different excitation wavelengths: 230, 300, 340, and 450 nm, corresponding to the absorption of tyrosine, tryptophan, collagen or elastin, Nicotinamide adenine dinucleotide (NADH) and flavin adeno dinucleotide (FAD). Similarly excitation spectra were also recorded at 340 nm. The emission profiles showed considerable difference between the malignant and normal cells with the malignant cells having more fluorescence intensity than that of normal cells keeping emission at 340 nm. Our study had shown the discriminating features between normal and carcinoma cells lines because of higher concentration of tryptophan (1.5 times), NADH (3 times), and flavin (4 times) in carcinoma cell lines.     

Photodynamic damage (PDD) study using stimulated raman scattering

In the current study, photodynamic damage in different cell cultures was examined using a pulsed laser light. Two different experiments were performed to analyse the photodynamic damage. For the first one, a stimulated Raman scattering laser has been obtained by exciting DMSO liquid with Nd-YAG laser, second harmonic generation, 532 nm. The resulted SRS wavelength is pulsed 630 nm. 1 ml ALA (200 μg/ml) was added to cell suspension and keep it for incubated for 4 h then irradiate the suspension with SRS wavelength 630 nm at different light dose 8, 12, 17, 25, 30, 40, 60, 100, 150, 200 μJ for 10 pluses and obtain the cell degradation. We repeat the step above but for 30 pluses. Finally for the second experiment, 1 ml ALA (200 μg/ml) was added to cell suspension and was incubated for 4 h and then irradiated with Nd-YAG Laser at wavelength 532 nm. Different doses range between 8 up to 200 μJ for 10 pluses only and the cell degradation rate was measured.