A simulation method for the study of laser transillumination of biological tissues

The Monte-Carlo method is employed to simulate the illumination of a blood slab by a continuous laser. It is assumed that the geometry of the medium is bidimensional and that scattering or absorption takes place only when a photon strikes a red blood cell. The parameters involved in the calculations concern the photons free path lengths between two collisions, the scattering angles and the absorption probability at collision. These parameters are assessed according to experimental or literature data. Fortran programs allow the computation of diffuse and collimated transmittances (T_d and T_c, respectively), of transmittance measured with an optic fiber T_f and of reflectance R. The variations of T_c and T_f with blood thickness are in accordance with established laws. Moreover, measured and computed reflectances change with hematocrit ratio in a similar way. This work demonstrates that the Monte-Carlo method is a simple reliable tool which can be used to provide a realistic model of laser penetration in complex biological structures. Moreover, this method will permit investigations in laser tomospectrometry by providing a useful simulation of the interaction of ultrashort light pulses with biological media.     

Laser fluorescence bronchoscope for localization of occult lung tumors

A system for imaging occult bronchogenic carcinoma by the fluorescence of previously-injected, tumor-specific compound hematoporphyrin-derivative has been assembled and successfully used to locate a tumor 1 mm thick. The violet excitation source is a krypton ion laser coupled to fused quartz fiber light conductor. An electrostatic image intensifier attached to a standard flexible fiberoptic bronchoscope provides a bright image even at relatively low irradiance. A red secondary filter rejects most reflected background and autofluorescence. Sensitivity and contrast capability of the system should permit detection of a tumor less than 0.1 mm thick.     

Optimal laser parameters for port wine stain therapy: a theoretical approach

The optimal parameters for laser therapy of port wine stains (PWS) have been deduced from temperature calculations on two models: (i) the four-layer model; and (ii) the tube model in which two plane parallel layers, representing the epidermis and dermis, and a dermal rectangular blood vessel are considered. The calculations were performed with a vessel of average cross section 0.06 mm X 0.08 mm located in the centre of the laser beam. The numerical calculations were performed by an alternate direction-implicit finite difference method. The optimal parameters were: wavelengths lambda = 415, 577 and 540 nm; pulse time, 1 ms less than t1 less than or equal to 10 ms; and beam radius W1 greater than 0.1 mm. The energy densities E1 (for t1 = 1 ms) required to coagulate blood vessels down to a depth of 0.65 mm in order to establish bleaching of the PWS were 0.5, 1.6 and 2 J cm-2 for the different lambda respectively. The value for the argon laser (lambda = 488 and 514.5 nm) was E1 = 6.5 J cm-2 (t1 = 1 ms). Because, for this pulse time, heat summation effects at the boundary of the laser beam cause no drastic increase in local temperature at optimal wavelengths, the stripe technique was again considered and compared with the separated spot technique. The Nd-YAG and CO2 lasers prove even less selective than the argon laser. The influence of cooling the skin with water shows that only for lambda = 577 nm and t1 = 0.1 s is there an increase in E1 from 2.5 to 6 J cm-2 for which dermal damage occurs.     

Laser tomography of heterogeneous scattering media using spatial and temporal resolution

Time-resolved tomography is performed in transillumination by using 527 nm picosecond pulses from a passively mode-locked doubled Nd/glass laser and a streak camera to select photons according to their flight time. This work reports on the increase in contrast of a time-resolved profile of a 2 mm radius opaque object embedded in a scattering medium, constituted of diluted milk in a 30 mm thick cell. For spatial analysis, the emerging photons are detected through a 6 mm slit at the outlet face of the cell. Transmission profiles obtained as a function of time show that the contrast is enhanced for the shortest flight times, while the ‘shadow’ of the object is no longer detected after about 100 ps. Moreover, improvements in contrast are studied for different configurations of the model, to analyse separately the role of collimated and scattered photons. It is expected that such a tomographic method based on time-resolved absorption could be applied to imaging for more complex biological structures in the red and near-infra-red range.     

Monte Carlo fluorescence microtomography

Fluorescence microscopy allows real-time monitoring of optical molecular probes for disease characterization, drug development, and tissue regeneration. However, when a biological sample is thicker than 1 mm, intense scattering of light would significantly degrade the spatial resolution of fluorescence microscopy. In this paper, we develop a fluorescence microtomography technique that utilizes the Monte Carlo method to image fluorescence reporters in thick biological samples. This approach is based on an l(0)-regularized tomography model and provides an excellent solution. Our studies on biomimetic tissue scaffolds have demonstrated that the proposed approach is capable of localizing and quantifying the distribution of optical molecular probe accurately and reliably.     

Impact of simulated light scatter on the quantitative, noninvasive assessment of retinal arteriolar hemodynamics

We determine the impact of artificial light scatter on quantitative, noninvasive assessment of retinal arteriolar hemodynamics. One eye from each of 10 healthy young subjects between the ages of 18 and 30 (23.6+/-3.4) is randomly selected. To simulate light scatter, cells comprising a plastic collar and two plano lenses are filled with solutions of differing concentration of polystyrene microspheres (Polysciences Inc., USA). We prepare 0.002, 0.004, 0.006, and 0.008% microsphere concentrations as well as distilled water only. The Canon laser blood flowmeter (CLBF) is used to noninvasively assess retinal arteriolar blood flow. After a preliminary screening to confirm subject eligibility, seven arteriolar blood flow measurements are taken by randomly placing the cells between the instrument objective lens and the subjects' cornea. To achieve a baseline, subjects are first imaged with no cell in place. Both low- and high-intensity CLBF laser settings are assessed. Our light scatter model results in an artifactual increase of retinal arteriolar diameter (p<0.0001) and thereby increased retinal blood flow (p<0.0001). The 0.006 and 0.008% microsphere concentrations produce significantly higher diameter and flow values than baseline. Centerline blood velocity, however, is not affected by light scatter. Retinal arteriolar diameter values are significantly less with the high-intensity laser than with the low-intensity laser (p=0.0007). Densitometry assessment of vessel diameter is increasingly impacted as the magnitude of artificial light scatter increases; this effect can be partially negated by increasing laser intensity. A cataract is an inevitable consequence of aging and, therefore, care must be exercised in the interpretation of studies of retinal vessel diameter that use similar densitometry techniques.     

The use of near-infrared light for safe and effective visualization of subsurface blood vessels to facilitate blood withdrawal in children

Obtaining access to blood vessels can be difficult, especially in children. Visualization of subsurface blood vessels might be a solution. Ultrasound and visible light have been used to this purpose, but have some drawbacks. Near-infrared light might be a better option since subsurface blood vessels can be visualized in high contrast due to less absorption and scattering in tissue as compared to visible light. Our findings with a multispectral imaging system support this theory.A device, the VascuLuminator, was developed, based on transillumination of the puncture site with near-infrared light. The VascuLuminator was designed to meet the requirements of compact and safe use. A phantom study showed that the maximum depth of visibility (5.5 mm for a 3.6 mm blood vessel) is sufficient to visualize blood vessels in typical locations for peripheral venous and arterial access. A quantitative comparison of the VascuLuminator and to two other vessel imaging devices, using reflection of near-infrared light instead of transillumination, was conducted. The VascuLuminator is able to decrease failure at first attempt in blood withdrawal in pediatric patients from 10/80 (13%) to 1/45 (2%; P = .05).     

Using flow information to support 3D vessel reconstruction from rotational angiography

For the assessment of cerebrovascular diseases, it is beneficial to obtain three-dimensional (3D) morphologic and hemodynamic information about the vessel system. Rotational angiography is routinely used to image the 3D vascular geometry and we have shown previously that rotational subtraction angiography has the potential to also give quantitative information about blood flow. Flow information can be determined when the angiographic sequence shows inflow and possibly outflow of contrast agent. However, a standard volume reconstruction assumes that the vessel tree is uniformly filled with contrast agent during the whole acquisition. If this is not the case, the reconstruction exhibits artifacts. Here, we show how flow information can be used to support the reconstruction of the 3D vessel centerline and radii in this case. Our method uses the fast marching algorithm to determine the order in which voxels are analyzed. For every voxel, the rotational time intensity curve (R-TIC) is determined from the image intensities at the projection points of the current voxel. Next, the bolus arrival time of the contrast agent at the voxel is estimated from the R-TIC. Then, a measure of the intensity and duration of the enhancement is determined, from which a speed value is calculated that steers the propagation of the fast marching algorithm. The results of the fast marching algorithm are used to determine the 3D centerline by backtracking. The 3D radius is reconstructed from 2D radius estimates on the projection images. The proposed method was tested on computer simulated rotational angiography sequences with systematically varied x-ray acquisition, blood flow, and contrast agent injection parameters and on datasets from an experimental setup using an anthropomorphic cerebrovascular phantom. For the computer simulation, the mean absolute error of the 3D centerline and 3D radius estimation was 0.42 and 0.25 mm, respectively. For the experimental datasets, the mean absolute error of the 3D centerline was 0.45 mm. Under pulsatile and nonpulsatile conditions, flow information can be used to enable a 3D vessel reconstruction from rotational angiography with inflow and possibly outflow of contrast agent. We found that the most important parameter for the quality of the reconstruction of centerline and radii is the range through which the x-ray system rotates in the time span of the injection. Good results were obtained if this range was at least 135 degrees. As a standard c-arm can rotate 205 degrees, typically one third of the acquisition can show inflow or outflow of contrast agent, which is required for the quantification of blood flow from rotational angiography.     

Prospects for the diagnosis of breast cancer by noninvasive probing of calcifications using transmission Raman spectroscopy

Breast calcifications can be found in both benign and malignant lesions, and the composition of these calcifications can indicate the possible disease state. As current practices such as mammography and histopathology examine the morphology of the specimen, they cannot reliably distinguish between the two types of calcification, which frequently are the only mammographic features that indicate the presence of a cancerous lesion. Raman spectroscopy is an optical technique capable of obtaining biochemical information of a sample in situ. We demonstrate for the first time the noninvasive recovery of Raman spectra of calcified materials buried within a chicken breast tissue slab 16 mm thick, achieved using transmission Raman spectroscopy. The spectra of both calcium hydroxyapatite (HAP) and calcium oxalate monohydrate (COM) are obtained and chemically identified. The experimental geometry and gross insensitivity of the Raman signal to the depth of the calcified lesion makes the concept potentially well suited for probing human female breasts, in conjunction with existing mammography or ultrasound, to provide complementary data in the early diagnosis of breast cancer.     

利用图像相关法测量骨拉伸弹性模量随试件厚度的变化规律

本文采用在拉伸试件上选取双视场的方法。提高图像相关方法的应变测量精度。利用此方法测试牛骨拉伸弹性模量随试件厚度的变化。测试发现拉伸弹性模量随试件厚度的减小而减小，但非单调减小，分析认为这种尺度依赖性决定于骨的微观结构和成分。     

Scientific basis of breast diaphanography

Diaphanography, also known as transillumination, is a breast diagnostic technique based on differences in the diffuse transmittance of visible or near-infrared radiation. Previous papers by the authors reported on investigations of the effect of tumor size, depth at which the tumor is located, the thickness of the breast, and the effect of using photons of different wavelengths. The results from the study reported here indicate that absorption of light in hemoglobin is the basis for the luminance contrast, and shift in the infrared to red transmission ratio, in the diaphanographic image. Evidence is based on known extinction coefficients for oxyhemoglobin and deoxyhemoglobin as a function of wavelength, measurements of the transmitted spectrum in specimens, in vivo dual wavelength transillumination imaging of lesions containing different amounts of blood (bloody and clear cysts, hematomas, veins, fibroadenomas, and carcinomas), and comparison of preoperative diaphanographic images to blood vessel volumes measured by microscopic analysis of surgical specimens. Oxygenation affects the relative proportions of infrared and red light transmitted, but does not influence the diagnosis based on luminance contrast.     

An ultrasonic measurement for in vitro depth-dependent equilibrium strains of articular cartilage in compression

The equilibrium depth-dependent biomechanical properties of articular cartilage were measured using an ultrasound-compression method. Ten cylindrical bovine patella cartilage-bone specimens were tested in compression followed by a period of force-relaxation. A 50 MHz focused ultrasound beam was transmitted into the cartilage specimen through a remaining bone layer and a small hole at the centre of a specimen platform. The ultrasound echoes reflected or scattered within the articularcartilage were collected using the same transducer. The displacements of the tissues at different depths of the articular cartilage were derived from the ultrasound echo signals recorded during the compression and the subsequent force-relaxation. For two steps of 0.1 mm compression, the average strain at the superficial 0.2 mm thick layer (0.35 +/- 0.09) was significantly (p < 0.05) larger than that at the subsequent 0.2 mm thick layer (0.05 +/- 0.07) and that at deeper layers (0.01 +/- 0.02). It was demonstrated that the compressive biomechanical properties of cartilage were highly depth-dependent. The results suggested that the ultrasound-compression method could be a useful tool for the study of the depth-dependent biomechanical properties of articular cartilage.     

Lack of lymphangiogenesis in human primary cutaneous melanoma. Consequences for the mechanism of lymphatic dissemination.

Cutaneous melanoma has an initial preference for lymphatic spread. Remarkably, melanoma progression toward this metastasizing phenotype is accompanied by intense blood vessel angiogenesis (hemangiogenesis), but lymphangiogenesis, the formation of new lymph vessels in the tumor, has never been reported. To investigate how primary melanoma cells interact with the existing lymphatic microvasculature, and whether lymphangiogenesis occurs, an immunostaining was developed that differentially decorates blood and lymph vessels in frozen tissue sections. The density and distribution of both these vessel types in and around thin (< or = 1.5 mm) and thick (> or = 1.5 mm) primary melanoma lesions and in normal and uninvolved skin were determined. Although especially in thick melanoma lesions a significant increase in blood vessel density was observed, lymphatic density remained unaltered, showing that lymphangiogenesis did not occur. Morphological analysis indicated, however, that melanoma progression is accompanied by a sequence of events that involves hemangiogenesis supporting tumor expansion, especially in the vertical growth phase. Often, stromal sepia are formed around the blood capillaries in the tumor neovasculature protecting them from invasion. Lymph vessels inside the tumor were infrequently observed. However, subepidermal lymph vessels often seemed to be entrapped and penetrated by the expanding tumor mass. In this way, hemangiogenesis, as the driving force behind tumor expansion, might indirectly increase the chance of lymphatic invasion in the absence of lymphangiogenesis. This model explains the paradox that, although melanoma metastasis seems to require angiogenesis, a consistent relation of prognosis with blood capillary density in primary cutaneous melanoma is lacking.     

On the use of R1 and R2* for measurement of contrast agent concentration in isolated perfused rat liver

We present experimental MRI protocols at 4.7 T for quantitative determination of the Dotarem distribution volume in isolated perfused rat liver. The procedures involved either constant contrast agent (CA) concentration or bolus administration conditions. R1 and R2* effects of the CA in liver and perfusate were measured using gradient echo fast imaging (GEFI) experiments by varying either the excitation angle or the echo time. CA concentrations in liver and perfusate were also measured after MRI by inductively coupled plasma atomic emission spectroscopy, in order to determine in situ relaxivities in the perfusate (r1=4.2 +/- 0.1 s(-1) mm(-1), r2*=17 +/- 2 s(-1) mm(-1)) and in the liver (r1=7.2 +/- 0.2 s(-1) mm(-1), r2*=99 +/- 5 s(-1) mm(-1)). When CA concentrations were estimated from R1 measurements and r1, the CA distribution volume estimations in liver resulting from bolus (0.31 +/- 0.01) and stationary (0.32 +/- 0.05) experiments were not significantly different. In contrast, after a bolus, CA concentrations derived from R2* and r2* were overestimated in liver and even more in perfusate. However, with R1 and R2* being measured before CA bolus administration, zero echo time signal intensities computed from multiple TE measurements during multiple boli yielded good estimations of R1 and thus correct CA concentrations in liver and in perfusate. Under these conditions, a single multi-echo GEFI acquisition should be sufficient to determine the concentration-time curves. Consequently, this protocol should be appropriate to rapidly estimate the distribution volume in vivo when multiple boli have to be avoided.     

Principles of laser-induced separation and transport of living cells

Separation and transport of defined populations of living cells grown on a thin membrane can be achieved by laser microdissection (LMD) of the sample of interest, followed by a laser-induced forward transport process [laser pressure "catapulting" (LPC)] of the dissected cell cluster. We investigate the dynamics of LMD and LPC with focused and defocused UV-A laser pulses by means of time-resolved photography. Catapulting is driven by plasma formation when tightly focused pulses are used, and by confined thermal ablation at the bottom of the sample for defocused catapulting. With both modalities, the initial specimen velocity amounts to about 50 to 60 ms. Time-resolved photography of live cell catapulting reveals that in defocused catapulting, strong shear forces arise when the sample is accelerated out of the culture medium covering the cells. By contrast, pulses focused at the periphery of the specimen cause a fast rotational movement that minimizes the flow of culture medium parallel to the sample surface, and thus the resulting shear stresses. Therefore, the recultivation rate of catapulted cells is much higher when focused pulses are used. Compared to collateral damage by mechanical forces, side effects by heat and UV exposure of the cells play only a minor role.     

Comparison of human skin opto-thermal response to near-infrared and visible laser irradiations: a theoretical investigation

Near-infrared wavelengths are absorbed less by epidermal melanin, and penetrate deeper into human skin dermis and blood than visible wavelengths. Therefore, laser irradiation using near-infrared wavelengths may improve the therapeutic outcome of cutaneous hyper-vascular malformations in moderately to heavily pigmented skin patients and those with large-sized blood vessels or blood vessels extending deeply into the skin. A mathematical model composed of a Monte Carlo algorithm to estimate the distribution of absorbed light, numerical solution of a bio-heat diffusion equation to calculate the transient temperature distribution, and a damage integral based on an empirical Arrhenius relationship to quantify the tissue damage was utilized to investigate the optothermal response of human skin to near-infrared and visible laser irradiations in conjunction with cryogen spray cooling. In addition, the thermal effects of a single continuous laser pulse and micropulse-composed laser pulse profiles were compared. Simulation results indicated that a 940 nm wavelength induces improved therapeutic outcome compared with a 585 and 595 nm wavelengths for the treatment of patients with large-sized blood vessels and moderately to heavily pigmented skin. On the other hand, a 585 nm wavelength shows the best efficacy in treating small-sized blood vessels, as characterized by the largest laser-induced blood vessel damage depth compared with 595 and 940 nm wavelengths. Dermal blood content has a considerable effect on the threshold incident dosage for epidermal damage, while the effect of blood vessel size is minimal. For the same macropulse duration and incident dosage, a micropulse-composed pulse profile results in higher peak temperature at the basal layer of skin epidermis than an ideal single continuous pulse profile.     

A morphological study of the blood vessels associated with periodontal probing depth in human gingival tissue

Gingival tissues in human cadavers were examined the blood vessel diameter in the depths of the gingival pockets such as three groups: gingiva adjacent to a sulcus of 2 mm (Group 1); gingiva adjacent to a 2-4-mm sulcus (Group 2); and gingiva adjacent to a sulcus of > 4 mm (Group 3). A meaningful significant difference was seen observed in gingival pocket side, intermediate and outer layer side regions of the gingiva. A meaningful significant difference was seen found in intermediate part and the outer layer of the gingiva in Group 3. Other gingival biopsies were performed on a human body donation specimen to examine CD-31 positive endothelial cells of blood vessels by an immnohistochemical method. Our results suggest that the periodontal probing depth reflect the blood vessel organization of human gingival tissue.     

Tumor angiogenesis as a prognostic factor in thick cutaneous malignant melanoma

The prognostic value of the extent of neovascularization in cutaneous melanoma is a highly controversial issue. The aim of the current study was to evaluate whether the morphometric analysis of tumor vascularity may be helpful in predicting the clinical outcome of patients with thick cutaneous melanomas. A series of 15 patients with melanoma (>3 mm in thickness) who did not experience disease progression after long-term follow-up (10 years) and 30 matched controls who underwent recurrence and/or metastases were selected for the study. Microvessels were immunohistochemically stained with anti-CD31 antibody. Several parameters, including vessel number, vascular density, vessel area, equivalent circle diameter, perimeter, shape factor, compactness, and the number of vascular ramifications per 100 vessel sections, were quantitatively assessed by a computer-aided semi-automatic image analysis system. Mean vessel area was 341.69 microm2 in cases without progression and 512.55 microm2 in the progressed melanomas (P=0.008, Mann-Whitney U test). The mean equivalent circle diameter was 18.95 microm in non-progressed melanomas and 22.57 microm in progressed melanomas (P=0.009). The mean number of ramifications was 0.8 in cases without progression and 1.9 in the controls (P=0.03). Microvessel count and vascular density were higher in progressed cases (17.37 vs. 11.73 and 28.94/mm2 vs. 19.55/mm2, respectively), but the difference did not reach statistical significance (P=0.06). Our results suggest that neovascularization is a critical event in the progression of thick melanoma. Its prognostic significance is better assessed by quantification of vessel area, equivalent circle diameter, and microvessel branching, whereas microvessel count and vascular density do not provide significant prognostic information.     

Innovation for reducing blood culture contamination: initial specimen diversion technique

We hypothesized that diversion of the first milliliter of venipuncture blood-the initial specimen diversion technique (ISDT)-would eliminate incompletely sterilized fragments of skin from the culture specimen and significantly reduce our blood culture contamination rate (R). We studied our hypothesis prospectively beginning with our control culture (C) definition: one venipuncture with two sequentially obtained specimens, 10 ml each, the first specimen (M1) for aerobic and the second (M2) for anaerobic media. The test ISDT culture (D) was identical, with the exception that each was preceded by diverting a 1-ml sample (DS) from the same venipuncture. During the first of two sequential 9-month periods, we captured D versus C data (n=3,733), where DMXR and CMXR are R for D and C specimens. Our hypothesis predicted DS would divert soiled skin fragments from DM1, and therefore, CM1R would be significantly greater than DM1R. This was confirmed by CM1R (30/1,061 [2.8%]) less DM1R (37/2,672 [1.4%]; P=0.005), which equals 1.4%. For the second 9-month follow-up period, data were compiled for all cultures (n=4,143), where ADMXR is R for all (A) diversion specimens, enabling comparison to test ISDT. Our hypothesis predicted no significant differences for test ISDT versus all ISDT. This was confirmed by DM1R (37/2,672 [1.4%]) versus ADM1R (42/4,143 [1.0%]; P=0.17) and DM2R (21/2,672 [0.80%]) versus ADM2R (39/4,143 [0.94%]; P=0.50). We conclude that our hypothesis is valid: venipuncture needles soil blood culture specimens with unsterilized skin fragments and increase R, and ISDT significantly reduces R from venipuncture-obtained blood culture specimens.     

Discrimination of capillary and arterio-venular blood flow in skin by laser Doppler flowmetry

The scattering and absorption of light by tissue and blood is wavelength dependent; the tissue penetration of green light (λ=543·5nm) is about 60 per cent of that of red light (λ=632·8 nm) but the absorption of green light by blood is about 20 times greater than for red light. The effect of this difference has been studied by observing the responses of skin blood flow to heat and weal, measured by laser Doppler flowmetry at the two wavelengths. By using time autocorrelation function analysis (ACF) of the scattered light measured, low and high frequency components have been associated with capillary and larger vessel flow, respectively. The comparison of ACF from scattered green and red light has shown that measurements cannot be interpreted by only considering light penetration depth through a homogeneous tissue. Light absorption and multiple scattering by blood at the individual microvessel level, blood rheology and vessel morphology are parameters which are considered for greater attention.