Matija Milanič

Estimation of skin optical parameters for real-time hyperspectral imaging applications

Abstract. Hyperspectral imaging combines high spectral and spatial resolution in one modality. This imaging technique is a promising tool for objective medical diagnostics. However, to be attractive in a clinical setting, the technique needs to be fast and accurate. Hyperspectral imaging can be used to analyze tissue properties using spectroscopic methods, and is thus useful as a general purpose diagnostic tool. We combine an analytic diffusion model for photon transport with real-time analysis of the hyperspectral images. This is achieved by parallelizing the inverse photon transport model on a graphics processing unit to yield optical parameters from diffuse reflectance spectra. The validity of this approach was verified by Monte Carlo simulations. Hyperspectral images of human skin in the wavelength range 400–1000 nm, with a spectral resolution of 3.6 nm and 1600 pixels across the field of view (Hyspex VNIR-1600), were used to develop the presented approach. The implemented algorithm was found to output optical properties at a speed of 3.5 ms per line of image data. The presented method is thus capable of meeting the defined real-time requirement, which was 30 ms per line of data.The algorithm is a proof of principle, which will be further developed.

Numerical optimization of sequential cryogen spray cooling and laser irradiation for improved therapy of port wine stain

Despite application of cryogen spray (CS) precooling, customary treatment of port wine stain (PWS) birthmarks with a single laser pulse does not result in complete lesion blanching for a majority of patients. One obvious reason is nonselective absorption by epidermal melanin, which limits the maximal safe radiant exposure. Another possible reason for treatment failure is screening of laser light within large PWS vessels, which prevents uniform heating of the entire vessel lumen. Our aim is to identify the parameters of sequential CS cooling and laser irradiation that will allow optimal photocoagulation of various PWS blood vessels with minimal risk of epidermal thermal damage.

A spectrally composite reconstruction approach for improved resolution of pulsed photothermal temperature profiling in water-based samples

We report on the first experimental evaluation of pulsed photothermal radiometry (PPTR) using a spectrally composite kernel matrix in signal analysis. Numerical studies have indicated that this approach could enable PPTR temperature profiling in watery tissues with better accuracy and stability as compared to the customary monochromatic approximation. By using an optimized experimental set-up and image reconstruction code (involving a projected nu-method and adaptive regularization), we demonstrate accurate localization of thin absorbing layers in agar tissue phantoms with pronounced spectral variation of a mid-infrared absorption coefficient. Moreover, the widths of reconstructed temperature peaks reach 14-17% of their depth, significantly less than in earlier reports on PPTR depth profiling in watery tissues. Experimental results are replicated by a detailed numerical simulation, which enables analysis of the broadening effect as a function of temperature profile amplitude and depth.

Effective infrared absorption coefficient for photothermal radiometric measurements in biological tissues

Although photothermal radiometric (PTR) measurements commonly employ broad-band signal acquisition to increase the signal-to-noise ratio, all reported studies apply a fixed infrared (IR) absorption coefficient to simplify the involved signal analysis. In samples with large spectral variation of micro(lambda) in mid-IR, which includes most biological tissues, the selection of the effective IR absorption coefficient value (micro(eff)) can strongly affect the accuracy of the result. We present a novel analytical approach for the determination of optimal micro(eff) from spectral properties of the sample and radiation detector. In extensive numerical simulations of pulsed PTR temperature profiling in human skin using three common IR radiation detectors and several acquisition spectral bands, we demonstrate that our approach produces viable values micro(eff). Two previously used analytical estimations perform much worse in the same comparison.

Objective characterization of bruise evolution using photothermal depth profiling and Monte Carlo modeling

Abstract. Pulsed photothermal radiometry (PPTR) allows noninvasive determination of laser-induced temperature depth profiles in optically scattering layered structures. The obtained profiles provide information on spatial distribution of selected chromophores such as melanin and hemoglobin in human skin. We apply the described approach to study time evolution of incidental bruises (hematomas) in human subjects. By combining numerical simulations of laser energy deposition in bruised skin with objective fitting of the predicted and measured PPTR signals, we can quantitatively characterize the key processes involved in bruise evolution (i.e., hemoglobin mass diffusion and biochemical decomposition). Simultaneous analysis of PPTR signals obtained at various times post injury provides an insight into the variations of these parameters during the bruise healing process. The presented methodology and results advance our understanding of the bruise evolution and represent an important step toward development of an objective technique for age determination of traumatic bruises in forensic medicine.

Applicability of diffusion approximation in analysis of diffuse reflectance spectra from healthy human skin

Measurement of diffuse reflectance spectra (DRS) is a common experimental approach for non-invasive determination of tissue optical properties, as well as objective monitoring of various tissue malformations. Propagation of light in scattering media is often treated in diffusion approximation (DA). The major advantage of this approach is that it leads to enclosed analytical solutions for tissues with layered structure, which includes human skin. Despite the fact that DA solutions were shown to be inaccurate near tissue boundaries, the practicality of this approach makes it quite popular, especially when attempting extraction of specific chromophore concentrations from measured DRS. In this study we analyze the discrepancies between DRS spectra as obtained by using the DA solutions for three-layer skin model and more accurate predictions from Monte Carlo (MC) modeling. Next, we analyze the artifacts which result from the above discrepancies when extracting the parameters of skin structure and composition by fitting the DA solutions to the MC spectra. The reliability and usefulness of such a fit is then tested also on measurements of seasonal changes in otherwise healthy human skin.

Monte Carlo simulation of radiation transport in human skin with rigorous treatment of curved tissue boundaries

Abstract. In three-dimensional (3-D) modeling of light transport in heterogeneous biological structures using the Monte Carlo (MC) approach, space is commonly discretized into optically homogeneous voxels by a rectangular spatial grid. Any round or oblique boundaries between neighboring tissues thus become serrated, which raises legitimate concerns about the realism of modeling results with regard to reflection and refraction of light on such boundaries. We analyze the related effects by systematic comparison with an augmented 3-D MC code, in which analytically defined tissue boundaries are treated in a rigorous manner. At specific locations within our test geometries, energy deposition predicted by the two models can vary by 10%. Even highly relevant integral quantities, such as linear density of the energy absorbed by modeled blood vessels, differ by up to 30%. Most notably, the values predicted by the customary model vary strongly and quite erratically with the spatial discretization step and upon minor repositioning of the computational grid. Meanwhile, the augmented model shows no such unphysical behavior. Artifacts of the former approach do not converge toward zero with ever finer spatial discretization, confirming that it suffers from inherent deficiencies due to inaccurate treatment of reflection and refraction at round tissue boundaries.

Re-evaluation of pulsed photothermal radiometric profiling in samples with spectrally varied infrared absorption coefficient

Spectral variation of the sample absorption coefficient in mid-infrared (muIR) demands caution in photothermal radiometric measurements, because a constant muIR is regularly assumed in inverse analysis of the acquired signals. Adverse effects of such approximation were recently demonstrated in numerical simulations of pulsed photothermal radiometric (PPTR) temperature profiling in soft biological tissues, utilizing a general-purpose optimization code in the reconstruction process. We present here an original reconstruction code, which combines a conjugate gradient minimization algorithm with non-negativity constraint to the sought temperature vector. For the same test examples as in the former report (hyper-Gaussian temperature profiles, InSb detector with 3-5 microm acquisition band, signal-to-noise ratio SNR=300) we obtain markedly improved reconstruction results, both when using a constant value mueff and when the spectral variation muIR(lambda) is accounted for in the analysis. By comparing the results, we find that the former approach introduces observable artefacts, especially in the superficial part of the profile (z<100 microm). However, the artefacts are much less severe than previously reported and are almost absent in the case of a deeper, single-lobed test profile. We demonstrate that the observed artefacts do not result from sub-optimal selection of mueff, and that they vary with specific realizations of white noise added to the simulated signals. The same holds also for a two-lobed test profile.

Kinetically governed polymorphism of d(G4T4G3) quadruplexes in K+ solutions

It has been generally recognized that understanding the molecular basis of some important cellular processes is hampered by the lack of knowledge of forces that drive spontaneous formation/disruption of G-quadruplex structures in guanine-rich DNA sequences. According to numerous biophysical and structural studies G-quadruplexes may occur in the presence of K+ and Na+ ions as polymorphic structures formed in kinetically governed processes. The reported kinetic models suggested to describe this polymorphism should be considered inappropriate since, as a rule, they include bimolecular single-step associations characterized by negative activation energies. In contrast, our approach in studying polymorphic behavior of G-quadruplexes is based on model mechanisms that involve only elementary folding/unfolding transitions and structural conversion steps that are characterized by positive activation energies. Here, we are investigating a complex polymorphism of d(G4T4G3) quadruplexes in K+ solutions. On the basis of DSC, circular dichroism and UV spectroscopy and polyacrylamide gel electrophoresis experiments we propose a kinetic model that successfully describes the observed thermally induced conformational transitions of d(G4T4G3) quadruplexes in terms of single-step reactions that involve besides single strands also one tetramolecular and three bimolecular quadruplex structures.

Monte Carlo simulation of radiation transfer in human skin with geometrically correct treatment of boundaries between different tissues

In customary implementation of three-dimensional (3D) Monte Carlo (MC) numerical model of light transport in heterogeneous biological structures, the volume of interest is divided into voxels by a rectangular spatial grid. Each voxel is assumed to have homogeneous optical properties and curved boundaries between neighboring tissues inevitably become serrated. This raises some concerns over realism of the modeling results, especially with regard to reflection and refraction on such boundaries. In order to investigate the above concern, we have implemented an augmented 3D MC code, where tissue boundaries (e.g., blood vessel walls) are defined by analytical functions and thus maintain their shape regardless of grid discretization. Results of the customary and augmented model are compared for a few characteristic test geometries, mimicking a cutaneous blood vessel irradiated with a 532 nm laser beam of finite diameter. Our analysis shows that at specific locations inside the vessel, the amount of deposited laser energy can vary between the two models by up to 10%. Even physically relevant integral quantities, such as linear density of the energy absorbed by the vessel, can differ by as much as 30%. Moreover, the values obtained with the customary model vary strongly with discretization step and don’t disappear with ever finer discretization. Meanwhile, our augmented model shows no such behavior, indicating that the customary approach suffers from inherent inaccuracies arising from physically flawed treatment of tissue boundaries.