Fungal infections in humans have significantly increased, primarily due to various Candida species such as Candida albicans, Candida tropicalis, and Candida auris, along with a rise in antifungal resistance. In response to these challenges, new strategies like antimicrobial photodynamic therapy (APDT) have been explored. However, understanding the specific cellular responses to these treatments remains a challenge. In this context, microscopic manipulation with optical tweezers (OT) emerges as a valuable tool for investigating these responses.
A novel method combining OT and APDT is presented to monitor the effects of treatments on individual fungal cells, focusing on C. tropicalis. Cells were placed in a 96-well microplate with Sabouraud dextrose broth to create biofilms and promote the formation of opaque cells by phenotypic switching. Methylene blue with a 10 μM concentration was added to the cells and irradiated with a lethal light dose of 60 J cm-². Subsequently, an intracellular lipid body was captured using an OT system and the stiffness of the trap over time was determined by analyzing its Brownian motion. The results revealed an increase in the intracellular viscosity during cell death processes activated by the application of APDT. This multifaceted methodology lays the foundation for formulating more effective therapeutic strategies against fungal infections.
SignificanceSpeckle contrast analysis is the basis of laser speckle imaging (LSI), a simple, inexpensive, noninvasive technique used in various fields of medicine and engineering. A common application of LSI is the measurement of tissue blood flow. Accurate measurement of speckle contrast is essential to correctly measure blood flow. Variables, such as speckle grain size and camera pixel size, affect the speckle pattern and thus the speckle contrast.AimWe studied the effects of spatial correlation among adjacent camera pixels on the resulting speckle contrast values.ApproachWe derived a model that accounts for the potential correlation of intensity values in the common experimental situation where the speckle grain size is larger than the camera pixel size. In vitro phantom experiments were performed to test the model.ResultsOur spatial correlation model predicts that speckle contrast first increases, then decreases as the speckle grain size increases relative to the pixel size. This decreasing trend opposes what is observed with a standard speckle contrast model that does not consider spatial correlation. Experimental data are in good agreement with the predictions of our spatial correlation model.ConclusionsWe present a spatial correlation model that provides a more accurate measurement of speckle contrast, which should lead to improved accuracy in tissue blood flow measurements. The associated correlation factors only need to be calculated once, and open-source software is provided to assist with the calculation.
We compared MB-PDT effect on two breast cancer cell lines: MDA-MB-231 from a triple negative adenocarcinoma, and T47D from a ductal cell tumor of woman. Methylene blue concentrations at 5, 10 and 20 μM and red light doses of 20, 40 and 60 J/cm2 were employed. Cell viability was evaluated with the MTT test, obtaining around 80% of inhibition with 20 μM and 60 J/cm2 of light dose.
Breast cancer implies a very important health problem worldwide since it represents the cancer with the highest incidence and mortality rate among women in 2018. Traditional treatments are not always totally efficient, causing severe systemic side effects, therefore the search for alternative treatments such as photodynamic therapy (PDT) is of vital importance. In the present work, we show the in vitro effects of rose bengal (RB) as a photosensitizer (PS) and green light (500-550 nm) to eliminate two breast cancer cell lines: MDA-MB-231, a triple negative line highly aggressive and invasive, and T47D, a luminal line of the infiltrating ductal type. RB concentrations of 0.5, 1 and 5 μM and radiation fluences of 2.5, 5 and 10 J/cm2 were evaluated. Cell viability was evaluated with the MTT test, obtaining the best effect with 5 μM concentration and 10 J/cm2 light dose.
Speckle contrast analysis had been used for multiples purposes, for instance, laser speckle contrast imaging (LSCI) has been used to estimate the relative blood flow speed in a non-invasive way. The speckle contrast can be calculated using a spatial or temporal algorithm or a combination of both. Our work focuses into the contrast temporal algorithm. A contrast image calculated with the temporal contrast algorithm requires a sequence of L equal-sized frames. The contrast images are performed pixel by pixel, however, the experimental contrast calculation does not match with the current temporal theoretical model especially when the exposure time T is smaller than the correlation time τc. In this work, we propose to correlate neighboring pixels along the temporal axis to improve the contrast calculation. The contrast measurements using our proposal provide a better agreement than the current models.
A trustworthy speckle contrast calculation is fundamental in many applications, such as “laser speckle contrast Imaging” (LSCI), which is a non-invasive technique commonly employed to estimate relative blood speed. In LSCI, the local contrast of a speckle image is calculated using spatial, temporal analysis or a combination of both. In this work, we focus on the spatial analysis. To calculate the local spatial contrast, typically, a sliding window of 5x5 pixels is used to calculate the standard deviation (σs) and the mean intensity (<I>s) of those 5x5 pixels and the calculated contrast KS=σs/(<I>s) is assigned to the central pixel of the sliding window. In this work, we show that this experimental procedure to calculate the local speckle contrast does not match the corresponding spatial theoretical model and we propose an alternative method that considers correlations of the central pixel with the other ones. We have found a better agreement of the contrast measurement with our numerical calculation.
The aim of this study was to compare the effectiveness of Rose Bengal (RB) and Methylene Blue (MB) as photosensitizers (PS) in Photodynamic Inactivation (PDI) on planktonic cultures of Candida albicans, a well-known opportunistic pathogen. RB and MB at concentrations ranging from 0.5 to 60 μM and fluences of 10, 30, 45 and 60 J/cm2 were tested. The light sources consist of an array of 12 led diodes with 30 mW of optical power each; 490-540 nm (green light) to activate RB and 600 -650 nm (red light) to activate MB. We first optimize the in vitro PDI technique using a single light dose and the optimum PS concentration. The novelty of our approach consist in reducing further the PS concentration than the optimum obtained with a single light exposure and using smaller light fluence doses by using repetitive light exposures (two to three times). MB and RB were tested for repetitive exposures at concentrations ranging from 0.1 to 10 μM, with fluences of 3 to 20 J/cm2, doses well below than those reported previously. All experiments were done in triplicate with the corresponding controls; cells without treatment, light control and dark toxicity control. RB-PDI and MB-PDI significantly reduced the number of CFU/mL when compared to the control groups. The results showed that RB was more effective than MB for C. albicans inactivation. Thus, we show that is possible to reduce significantly the amount of PS and light fluence requirements using repetitive light doses of PDI in vitro.
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