Ruben Ramos-Garcia

Time-resolved analysis of cavitation induced by CW lasers in absorbing liquids

We present novel results on thermocavitation using a CW medium-power near infrared laser (lambda=975 nm) focused into a saturated copper nitrate saline solution. Due to the large absorption coefficient at the laser wavelength, the solution can be heated to its superheat limit (T(sh) approximately 270-300 degrees C). Superheated water undergoes explosive phase transition around T(sh) producing approximately half-hemispheric bubbles (gamma approximately 0.5) in close contact with the substrate. We report the temporal dynamic of the cavitation bubble, which is much shorter than previously reported under similar conditions. It was found that the bubble radius and pressure wave amplitude emitted on bubble collapse decreases exponentially with the power laser. Thermocavitation can be a useful tool for the generation of ultrasonic waves and controlled ablation for use in high-resolution lithography.

Self-calibrating common-path interferometry

A quantitative phase measuring technique is presented that estimates the object phase from a series of phase shifted interferograms that are obtained in a common-path configuration with unknown phase shifts. The derived random phase shifting algorithm for common-path interferometers is based on the Generalized Phase Contrast theory [pl. Opt.40(2), 268 (2001)10.1063/1.1404846], which accounts for the particular image formation and includes effects that are not present in two-beam interferometry. It is shown experimentally that this technique can be used within common-path configurations employing nonlinear liquid crystal materials as self-induced phase filters for quantitative phase imaging without the need of phase shift calibrations. The advantages of such liquid crystal elements compared to spatial light modulator based solutions are given by the cost-effectiveness, self-alignment, and the generation of diminutive dimensions of the phase filter size, giving unique performance advantages.

Towards the enhancement of transdermal drug delivery through thermocavitation

Abstract Background and objective: Although for some highly lipophillic drugs the principal barrier to permeate the human skin may reside in the essentially viable epidermal membrane, for most molecules, the stratum corneum (SC) is the rate-limiting barrier to drug delivery. Today, several techniques have been developed to enhance transdermal drug delivery (TDD) by increasing the effective permeability of the SC (e.g., iontophoresis, electroporation, micro-needle, ultrasound, radio frequency and laser radiation). The goal of this study is to investigate the extent to which thermocavitation may be used as a novel alternative method to selectively pierce the SC and thus enhance TDD. Thermocavitation for this purpose is generated by a continuous wave (CW), low power laser beam focused on a highly-absorbing solution topically applied on the skin surface. The absorbed light creates a superheated volume in a tightly localized region followed by explosive phase transition and the formation of vapor-gas bubbles, which expand and later collapse very rapidly emitting intense acoustic shockwaves that disrupt the surface underneath. Materials and methods: Thermocavitation bubbles were induced close to the surface of skin models (agar gels) and ex-vivo porcine skin samples using a 975 nm CW laser, focused on a thin (100–300 μm) topical layer of copper nitrate (CuNO4). The damage induced by thermocavitation on the surface of agar tissue phantoms was analyzed by optical microscopy and the penetration depth of a fluorescent drug surrogate (FITC dextran, molecular weight=4 kDa), applied topically to the surface of ex-vivo porcine skin samples following thermocavitation. The corresponding histological structure was analyzed by fluorescent microscopy and hematoxylin and eosin (H&E) staining, respectively. Results: The damage observed on agar gel and porcine skin appears to be congruent with the relationship between laser power, focal point, cavitation frequency and extent of damage observed in previous studies. In particular, the greatest damage induced to the agar phantoms was produced with the lowest laser power (∼153 mW) and thinnest solution layer (∼100 μm) used. Similar laser and solution layer settings led to porcine skin damage of ∼80–100 μm in diameter, which was sufficiently large to break the SC and allow the penetration of 4 kDa, FITC-dextran to depths of ∼40–60 μm. Conclusion: This novel approach to achieve cavitation is attractive and seems promising because it can be generated with inexpensive, low power CW lasers, capable of selectively disrupting the SC and allowing the penetration of large, hydrophilic drugs topically applied to the skin. Zusammenfassung Hintergrund und Zielsetzung: Das Eindringen einiger lipophiler Wirkstoffe in die menschliche Haut wird durch die epidermale Membran begrenzt. Für die meisten Moleküle stellt aber das Stratum corneum (SC) die Barriere für die Wirkstoffverabreichung dar. Es sind verschiedene Techniken zur Wirkstoffverabreichung entwickelt worden, um die effektive Permeabilität des SC zu erhöhen (z.B. durch Iontophorese, Elektroporation, Mikronadeln, Ultraschall, Hochfrequenz, Laserstrahlung). Ziel der vorliegenden Studie war es zu untersuchen, ob das SC mittels Thermokavitation gezielt perforiert werden kann, um so die Durchlässigkeit für die Wirkstoffverabreichung zu verbessern. Thermokavitation kann erreicht werden, indem der Strahl eines kontinuierlich abstrahlenden (continuous wave, cw) Low-Power-Lasers auf die Hautoberfläche fokussiert wird, auf die zuvor eine stark absorbierende Lösung topisch aufgetragen wurde. Das absorbierte Licht erzeugt in einem begrenzten Volumenbereich eine überhitzung gefolgt von einem explosiven Phasenübergang und der Formierung von Dampfgasblasen, die expandieren und schließlich extrem schnell kollabieren. Die dabei erzeugten akustischen Schockwellen zerreißen die Gewebeoberfläche. Material und Methoden: Thermokavitationsblasen wurden dicht an der Oberfläche von Hautphantomen (Agargel) und Ex-Vivo-Hautproben (Schwein) mittels eines 975 nm, cw Lasers erzeugt, indem der Laserstrahl auf dünne Schichten (100–300 μm) von Kupfernitrat (CuNO4) fokussiert wurde. Die dadurch erzeugten oberflächlichen Schädigungen wurden bei den Agarphantomen mittels optischer Mikroskopie evaluiert. Von den Ex-Vivo-Hautproben wurden zusätzlich HE-Schnitte angefertigt und bewertet sowie die Eindringtiefe eines fluoreszierenden Wirkstoffsurrogates (FITC-Dextran, Molekulargewicht=4 kDa) mittels Fluoreszenzmikroskopie bestimmt. Ergebnisse: Die beobachteten Schädigungen an Agargel und Schweinehaut stimmen mit früheren Studien überein und spiegeln den Zusammenhang zwischen Laserleistung, Fokus, Kavitationsfrequenz und Schädigungsausmaß. Die größte Schädigung in Agargel wurde mit der geringsten Laserleistung (∼153 mW) und der dünnsten Schichtdicke (∼100 μm) erzeugt. Mit den gleichen Parametern konnten bei der Schweinehaut Schäden mit einem Durchmesser von ca. 80–100 μm erzeugt werden, was ausreichend war um das SC aufzubrechen, so dass das FITC-Dextran ca. 40–60 μm tief eindringen konnte. Schlussfolgerung: Das vorgestellte Verfahren zur Erzeugung von Thermokavitation erscheint vielversprechend, zumal preiswerte cw Low-Power-Laser eingesetzt werden können. Es ist geeignet, um das SC selektiv zu perforieren, so dass große, hydrophile Wirkstoffe, die zuvor topisch auf die Haut aufgetragen wurden, eindringen können.

In-vitro effect of antimicrobial photodynamic therapy with methylene blue in two different genera of dermatophyte fungi

Abstract Background and objectives: Antimicrobial photodynamic therapy (aPDT) is a technique that combines the photoactivation properties of an innocuous chromophore or photosensitizer (PS) and light, producing reactive oxygen molecules that trigger cell death processes. In this study the in-vitro application of aPDT to fight fungal infections was investigated using methylene blue (MB) as the PS. Materials and methods: The antimicrobial PDT process was carried out with MB and red laser light (λ=633 nm) to activate the PS. Testing was performed with suspensions of various species of dermatophyte fungi (Trichophyton mentagrophytes, Microsporum canis and Microsporum gypseum), including a fungus, which to our knowledge, has not been previously studied using this dye (Trichophyton tonsurans). For T. tonsurans further optimization tests were carried out. Results and discussion: The fungicidal effect of MB-aPDT was evident. Microsporum strains were slightly more sensitivity to the treatment than Trichophyton strains. The response of T. tonsurans to aPDT was less than to the other fungi tested under the same conditions, or even with higher fluence. However, repetitive aPDT treatment with very low doses of light can achieve a good effectiveness with this strain effecting total growth inhibition. Light may even disturb fungi growth in some circumstances, especially in strain such as T. tonsurans. Conclusion: This study with Trichophyton and Microsporum strains showed that MB was an effective PS to inhibit fungal growth through aPDT, reaching a total inhibition in most of the fungi tested. It was found that repeated exposure with low-power light within the framework of aPDT treatment can achieve better results than a single exposure at higher power.

Visualization of deep blood vessels in speckle imaging using homogeneity measurement of the co-occurrence matrix

The blood flow velocity optical monitoring techniques, such as laser speckle velocimetry, are attractive due its noninvasive character, but they are limited to superficial blood vessels. The visualization of deep blood vessel is difficult because of the highly scattering coefficient of the biological tissue. There are some techniques that allow the visualization of deep blood vessels, for example: optical clearing, Magnetomotive Laser Speckle Imaging and Pulsed Photo-Thermal Radiometry, unfortunately these techniques use an external agent to improve the visualization of blood vessels. In this work we improve the visualization and location of in-vitro deep blood vessels by speckle image processing without an external agents. The proposed methodology is based in a homogeneity measurement of the co-occurrence matrix by the direct processing of the speckle image. Our technique is able to determine the edges of a deep blood vessel and therefore improves its visualization.

Sorting of microparticles by optical landscapes generated with a spatial light modulator

The use of spatial light modulators to generate arbitrary optical field distributions has been extensively used to trap and manipulate dynamically a large number of particles. Here we show that by using phase computer generated holograms displayed on a spatial light modulator (SLM) sorting of microparticles can be achieved at relatively low power. The algorithm used for the generation of the PCGH is based on iterative Fourier transform algorithm which generate a spots array in the Fourier plane, then controlling some parameters as: the spot separation, the direction and velocity of the pattern displacement, optical sorting of micron-sized particles can be achieved.

Manipulation of photothermally generated microbubbles

Generation and 3D manipulation of microbubbles by means of temperature gradients induced by low power laser radiation is presented. Photodeposited silver nanoparticles on the distal end of two optical fibers act as thermal sources after light absorption. The temperature rises above liquid evaporation temperature generating a microbubble at the optical fibers end in non-absorbent liquids. Alternatively, switching the thermal gradients between the fibers, it is possible to generate forces in opposite directions, causing the migration of microbubbles from one fiber optic tip to another. Marangoni force induced by surface tension gradients in the bubble wall is the driving force behind the manipulation of microbubbles

Efficient in vitro photodynamic inactivation of Candida albicans by repetitive light doses

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.

Contrast temporal analysis using correlation between frames

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.

Speckle contrast calculation based on pixels correlation: spatial analysis

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 (s) of those 5x5 pixels and the calculated contrast KS=σs/(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.