Improved spatial speckle contrast model for tissue blood flow imaging: effects of spatial correlation among neighboring camera pixels.

Significance: Speckle 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. Aim: We studied the effects of spatial correlation among adjacent camera pixels on the resulting speckle contrast values. Approach: We 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. Results: Our 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. Conclusions: We 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.

Organic light emitting diode for in vitro antimicrobial photodynamic therapy of Candida strains.

Organic light emitting diodes (OLEDs) are very attractive light sources because they are large area emitters, and can in principle be deposited on flexible substrates. These features make them suitable for ambulatory photodynamic therapy (PDT). A few reports of in vitro or in vivo OLED based PDT studies for cancer or microbial inhibition are published but to our best knowledge, none against yeasts. Yeast infections are a significant health risk, especially in low income countries with limited medical facilities. In this work, OLED-based antimicrobial PDT (aPDT), using methylene blue (MB) as photosensitizer (PS), is studied to inactivate opportunistic yeast of four Candida strains of two species: Candida albicans and Candida tropicalis. Before aPDT experiments, fluconazole-resistance was evaluated for all strains, showing that both strains of C. tropicalis were resistant and both strains of C. albicans were sensitive to it. We found that 3 repetitive irradiations work better than a single dose while keeping the total fluence constant, and that this result applies whether or not the strains are resistant to fluconazole.

Visualization of in vitro deep blood vessels using principal component analysis based laser speckle imaging.

Visualization of blood vessels is a fundamental task in the evaluation of the health and biological integrity of tissue. Laser speckle contrast imaging (LSCI) is a non-invasive technique to determine the blood flow in superficial or exposed vasculature. However, the high scattering of biological tissue hinders the visualization of those structures. In this paper, we propose the use of principal component analysis (PCA) in combination with LSCI to improve the visualization of deep blood vessels by selecting the most significant principal components. This analysis was applied to in vitro samples, and our results demonstrate that this approach allows for the visualization and localization of blood vessels as deep as 1000 µm.

Use of kurtosis for locating deep blood vessels in raw speckle imaging using a homogeneity representation.

Visualization of deep blood vessels in speckle images is an important task as it is used to analyze the dynamics of the blood flow and the health status of biological tissue. Laser speckle imaging is a wide-field optical technique to measure relative blood flow speed based on the local speckle contrast analysis. However, it has been reported that this technique is limited to certain deep blood vessels (about ? = 300 ?? ? m ) because of the high scattering of the sample; beyond this depth, the quality of the vessel's image decreases. The use of a representation based on homogeneity values, computed from the co-occurrence matrix, is proposed as it provides an improved vessel definition and its corresponding diameter. Moreover, a methodology is proposed for automatic blood vessel location based on the kurtosis analysis. Results were obtained from the different skin phantoms, showing that it is possible to identify the vessel region for different morphologies, even up to 900 ?? ? m in depth.

Fiber-based laser speckle imaging for the detection of pulsatile flow.

BACKGROUND AND OBJECTIVE: In endodontics, a major diagnostic challenge is the accurate assessment of pulp status. In this study, we designed and characterized a fiber-based laser speckle imaging system to study pulsatile blood flow in the tooth. STUDY DESIGN/MATERIALS AND METHODS: To take transilluminated laser speckle images of the teeth, we built a custom fiber-based probe. To assess our ability to detect changes in pulsatile flow, we performed in vitro and preliminary in vivo tests on tissue-simulating phantoms and human teeth. We imaged flow of intralipid in a glass microchannel at simulated heart rates ranging from 40 beats/minute (bpm) to 120 bpm (0.67-2.00 Hz). We also collected in vivo data from the upper front incisors of healthy subjects. From the measured raw speckle data, we calculated temporal speckle contrast versus time. With frequency-domain analysis, we identified the frequency components of the contrast waveforms. RESULTS: With our approach, we observed in vitro the presence of pulsatile flow at different simulated heart rates. We characterized simulated heart rate with an accuracy of and >98%. In the in vivo proof-of-principle experiment, we measured heart rates of 69, 90, and 57 bpm, which agreed with measurements of subject heart rate taken with a wearable, commercial pulse oximeter. CONCLUSIONS: We designed, built, and tested the performance of a dental imaging probe. Data from in vitro and in -vivo tests strongly suggest that this probe can detect the presence of pulsatile flow. LSI may enable endodontists to noninvasively assess pulpal vitality via direct measurement of blood flow.

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.

Spatial versus temporal laser speckle contrast analyses in the presence of static optical scatterers.

Previously published data demonstrate that the temporal processing algorithm for laser speckle contrast imaging (LSCI) can improve the visibility of deep blood vessels and is less susceptible to static speckle artifacts when compared with the spatial algorithm. To the best of our knowledge, the extent to which the temporal algorithm can accurately predict the speckle contrast associated with flow in deep blood vessels has not been quantified. Here, we employed two phantom systems and imaging setups (epi-illumination and transillumination) to study the contrast predicted by the spatial and temporal algorithms in subsurface capillary tubes as a function of the camera exposure time and the actual flow speed. Our data with both imaging setups suggest that the contrast predicted by the temporal algorithm, and therefore the relative flow speed, is nearly independent of the degree of static optical scattering that contributes to the overall measured speckle pattern. Collectively, these results strongly suggest the potential of temporal LSCI at a single-exposure time to assess accurately the changes in blood flow even in the presence of substantial static optical scattering.

Photothermal laser speckle imaging.

The analysis of speckle contrast in a time-integrated speckle pattern enables visualization of superficial blood flow in exposed vasculature, a method we call laser speckle imaging (LSI). With current methods, LSI does not enable visualization of subsurface or small vasculature, because of optical scattering by stationary structures. In this work we propose a new technique called photothermal LSI to improve the visualization of blood vessels. A 595 nm laser pulse was used to excite blood in both in vitro and in vivo samples. The high absorption coefficient of blood at this wavelength results in efficient conversion of optical energy to thermal energy, resulting in an increase in the local temperature and hence increased scatterer motion, and thus a transient decrease in speckle contrast. As a result, we found that photothermal LSI was able to visualize blood vessels that were hidden when imaged with a conventional LSI system.

Impact of velocity distribution assumption on simplified laser speckle imaging equation.

Since blood flow is tightly coupled to the health status of biological tissue, several instruments have been developed to monitor blood flow and perfusion dynamics. One such instrument is laser speckle imaging. The goal of this study was to evaluate the use of two velocity distribution assumptions (Lorentzian- and Gaussian-based) to calculate speckle flow index (SFI) values. When the normalized autocorrelation function for the Lorentzian and Gaussian velocity distributions satisfy the same definition of correlation time, then the same velocity range is predicted for low speckle contrast (0 < C < 0.6) and predict different flow velocity range for high contrast. Our derived equations form the basis for simplified calculations of SFI values.

Linear response range characterization and in vivo application of laser speckle imaging of blood flow dynamics.

Noninvasive blood flow imaging can provide critical information on the state of biological tissue and the efficacy of approaches to treat disease. With laser speckle imaging (LSI), relative changes in blood flow are typically reported, with the assumption that the measured values are on a linear scale. A linear relationship between the measured and actual flow rate values has been suggested. The actual flow rate range, over which this linear relationship is valid, is unknown. Herein we report the linear response range and velocity dynamic range (VDR) of our LSI instrument at two relevant camera integration times. For integration times of 1 and 10 ms, the best case VDR was 80 and 60 dB, respectively, and the worst case VDR was 20 and 50 dB. The best case VDR values were similar to those reported in the literature for optical Doppler tomography. We also demonstrate the potential of LSI for monitoring blood flow dynamics in the rodent dorsal skinfold chamber model. These findings imply that LSI can provide accurate wide-field maps of microvascular blood flow rate dynamics and highlight heterogeneities in flow response to the application of exogenous agents.

Microvascular blood flow dynamics associated with photodynamic therapy, pulsed dye laser irradiation and combined regimens.

BACKGROUND AND OBJECTIVES: Previous in vitro studies demonstrated the potential utility of benzoporphyrin derivative monoacid ring A (BPD) photodynamic therapy (PDT) for vascular destruction. Moreover, the effects of PDT were enhanced when this intervention was followed immediately by pulsed dye laser (PDL) irradiation (PDT/PDL). We further evaluate vascular effects of PDT alone, PDL alone and PDT/PDL in an in vivo rodent dorsal skinfold model. STUDY DESIGN/MATERIALS AND METHODS: A dorsal skinfold window chamber was installed surgically on female Sprague-Dawley rats. One milligram per kilogram of BPD solution was administered intravenously via a jugular venous catheter. Evaluated interventions were: control (no BPD, no light), PDT alone (576 nm, 16 minutes exposure time, 15 minutes post-BPD injection, 10 mm spot), PDL alone at 7 J/cm2 (585 nm, 1.5 ms pulse duration, 7 mm spot), PDL alone at 10 J/cm2, PDT/PDL (PDL at 7 J/cm2), and PDT/PDL (PDL at 10 J/cm2). To assess changes in microvascular blood flow, laser speckle imaging was performed before, immediately after, and 18 hours post-intervention. RESULTS: Epidermal irradiation was accomplished without blistering, scabbing or ulceration. A reduction in perfusion was achieved in all intervention groups. PDT/PDL at 7 J/cm2 resulted in the greatest reduction in vascular perfusion (56%). CONCLUSIONS: BPD PDT can achieve safe and selective vascular flow reduction. PDT/PDL can enhance diminution of microvascular blood flow. Our results suggest that PDT and PDT/PDL should be evaluated as alternative therapeutic options for treatment of hypervascular skin lesions including port wine stain birthmarks.

Thermal responses of ex vivo human skin during multiple cryogen spurts and 1,450 nm laser pulses.

BACKGROUND AND OBJECTIVE: Although cryogen spray cooling (CSC) is used to minimize the risk of epidermal damage during laser dermatologic surgery, concern has been expressed that CSC may induce cryo-injury. The objective of this study is to measure temperature variations at the epidermal-dermal junction in ex vivo human skin during three clinically relevant multiple cryogen spurt-laser pulse sequences (MCS-LPS). STUDY DESIGN/MATERIALS AND METHODS: The epidermis of ex vivo human skin was separated from the dermis and a thin-foil thermocouple (13 microm thickness) was inserted between the two layers. Thermocouple depth and epidermal thickness were measured using optical coherence tomography (OCT). Skin specimens were preheated to 30 degrees C before the MCS-LPS were initiated. Three MCS-LPS patterns, with total cryogen spray times of 38, 30, and 25 milliseconds respectively, were applied to the specimens in combination with laser fluences of 10 and 14 J/cm(2), while the thermocouple recorded the temperature changes at the epidermal-dermal junction. RESULTS: The thermocouple effectively recorded fast temperature changes during three MCS-LPS patterns. The lowest temperatures measured corresponded to the sequences with longer pre-cooling cryogen spurts. No sub-zero temperatures were measured for any of the MCS-LPS patterns under study. CONCLUSIONS: The three clinically relevant MCS-LPS patterns evaluated in this study do not cause sub-zero temperatures in ex vivo human skin at the epidermal-dermal junction and, therefore, are unlikely to cause significant cryogen induced epidermal injury.

Effect of ambient humidity on light transmittance through skin phantoms during cryogen spray cooling.

Cryogen spray cooling (CSC) is a technique employed to reduce the risk of epidermal damage during dermatologic laser surgery. However, while CSC protects the epidermis from non-specific thermal damage, it might reduce the effective fluence reaching the target chromophore due to scattering of light by the spray droplets and subsequent water condensation/freezing on the skin surface. The objective of this work was to study the effect of ambient humidity (omega) on light transmittance during CSC. An integrating sphere was employed to measure the dynamics of light transmittance through a deformable agar phantom during CSC. The study included two representative CSC spurt patterns studied using four omega: 57, 40, 20 and 12%. Results show that during CSC, as omega increased, light transmittance decreased. For the highest humidity level (57%) studied, light transmittance reached a minimum of 55% approximately 30 ms after spurt termination. In a controlled environment with omega = 12%, light transmittance reached a minimum of 87% approximately 30 ms after spurt termination. The reduced light transmittance immediately after spurt termination was most likely because of scattering of light caused by condensation of water vapour due to aggressive cooling of ambient air in the wake of the cryogen spurt.

Description and analysis of treatments for port-wine stain birthmarks.

Port-wine stain (PWS) birthmarks are congenital, low-flow vascular malformations of the skin. Lasers are the modality of choice for the treatment of PWS birthmarks, and for most patients the pulsed-dye laser in conjunction with epidermal cooling offers the greatest efficacy and safety. Other light devices, including the 532-nm frequency-doubled Nd:YAG laser, intense pulsed light, 1064-nm Nd:YAG laser, and combined 1064/532-nm system, may be useful during a treatment course for resistant PWS. Laser treatment results in blanching of most lesions, although complete resolution may not occur and some resistant PWS birthmarks respond minimally, if at all. Factors limiting laser treatment include variable vascular geometry, inadequate damage of some vessels, and lesional posttreatment recurrence as a result of neovascularization. Alternative or adjunct treatment options that address these limitations should be explored, including noninvasive real-time imaging to optimize the selection of treatment settings, photodynamic therapy, and perioperative use of antiangiogenic compounds.

Evaluation of single versus multiple cryogen spray cooling spurts on in vitro model human skin.

Many commercially available dermatologic lasers utilize cryogen spray cooling for epidermal protection. A previous tissue culture study demonstrated that single cryogen spurts (SCS) of 80 ms or less were unlikely to cause cryo-injury in light-skinned individuals. More recently, multiple cryogen spurts (MCS) have been incorporated into commercial devices, but the effects of MCS have not been evaluated. The aim was to study an in vitro tissue culture model and the epidermal and dermal effects of SCS vs patterns of shorter MCS with the same preset total cryogen delivery time (Deltat(c)) and provide an explanation for noted differences. Four different spurt patterns were evaluated: SCS: one 40-ms cryogen spurt; MCS2: two 20-ms cryogen spurts; MCS4: four 10-ms cryogen spurts; MCS8: eight 5-ms cryogen spurts. Actual Deltat(c) and total cooling time (Deltat(Total)) were measured for each spurt pattern. RAFT tissue culture specimens were exposed to cryogen spurt patterns and biopsies were taken immediately and at days 3 and 7. Actual Deltat(c) was increased while Deltat(Total) remained relatively constant as the preset Deltat(c) of 40 ms was delivered as shorter MCS. Progressively more epidermal damage was noted with exposure to the MCS patterns. No dermal injury was noted with either SCS or MCS. For a constant preset Deltat(c) of 40 ms, delivering cryogen in patterns of shorter MCS increased the actual Deltat(c) and consequently the observed epidermal cryo-injury as compared to an SCS.

Skin model surface temperatures during single and multiple cryogen spurts used in laser dermatologic surgery.

BACKGROUND: Although cryogen spray cooling (CSC) is used to minimize the risk of epidermal damage during laser dermatologic surgery, concern has been expressed that CSC may induce cryo-injury. In order to address this concern, it is necessary to evaluate the effects of prolonged exposure of human skin phantoms (HSP) to CSC. OBJECTIVE: To measure the minimum surface temperature (T(min)) and the time at which it occurs (t(Tmin)) as well as determine the time the sprayed HSP surface remains below 0 degrees C (sub-zero time, Deltat(s)) and -26 degrees C (residence time, Deltat(r)) during the application of single (SCS) and multiple (MCS) cryogen spurts. Two initial HSP substrate temperatures were studied, T(i): 23 and 70 degrees C. STUDY DESIGN/MATERIALS AND METHODS: An epoxy-based HSP was constructed to measure T(min), t(Tmin), Deltat(s), and Deltat(r), for 17 spray patterns: 1 SCS with a total cryo-delivery time (Deltat(c)) of 40 milliseconds; 8 MCS patterns with identical Deltat(c), but with a total cooling time (Deltat(total)) varying from 50 to 280 milliseconds; and 8 SCS patterns that matched the Deltat(total) of the MCS patterns. RESULTS: For both T(i), our results show that it is possible to distinguish between two different cooling regimes. For Deltat(total)

Generation of partially coherent diffraction-free fields with tunable geometry.

The synthesis of diffraction-free fields with different profiles is described. The synthesis is done by illuminating a screen containing a circular slit with a cosine beam. The treatment is equivalent to the modulation of the slit transfer characteristics and makes possible a tunable interference interaction of Bessel beams with noncommon axes. These results are generalized, and it is shown that coherent diffraction-free fields with arbitrary profiles can be expressed as the superposition of shifted zero-order Bessel beams and temporary parametric representations of the shifted functions. Diffraction-free fields with partially coherent features can be obtained. Experimental results are shown for each case.