Necrosis response to photodynamic therapy using light pulses in the femtosecond regime

One of the clinical limitations of the photodynamic therapy (PDT) is the reduced light penetration into biological tissues. Pulsed lasers may present advantages concerning photodynamic response when compared to continuous wave (CW) lasers operating under the same average power conditions. The aim of this study was to investigate PDT-induced response when using femtosecond laser (FSL) and a first-generation photosensitizer (Photogem) to evaluate the induced depth of necrosis. The in vitro photodegradation of the sensitizer was monitored during illumination either with CWor an FSL as an indirect measurement of the PDT response. Healthy liver of Wistar rats was used to evaluate the tissue response. The photosensitizer was endovenously injected and 30 min after, an energy dose of 150 Jcm-2 was delivered to the liver surface. We observed that the photodegradation rate evaluated via fluorescence spectroscopy was higher for the FSL illumination. The FSL-PDT produced a necrosis nearly twice as deep when compared to the CW-PDT. An increase of the tissue temperature during the application was measured and was not higher than 2.5 °C for the CW laser and not higher than 4.5 °C for the pulsed laser. FSL should be considered as an alternative in PDT applications for improving the results in the treatment of bulky tumors where higher light penetration is required.

Evaluation of reference-based two-color methods for measurement of gene expression ratios using spotted cDNA microarrays

Abstract Background Spotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard. Results We performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable. Conclusion RefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.