Free-running Er:YAG lasers are used for precise tissue ablation in various clinical applications. The ablated material is ejected into the direction perpendicular to the tissue surface. We investigated the influence of shielding by the ablation plume on the energy deposition into an irradiated sample because it influences the ablation dynamics and the amount of material ablated. The investigations were performed using an Er:YAG laser with a pulse duration of 200 µs for the ablation of gelatin with different water contents, skin, and water. Laser flash photography combined with a dark field Schlieren technique was used to visualize gaseous and particulate ablation products, and to measure the distance traveled by the ablating laser beam through the ablation plume at various times after the beginning of the laser pulse. The temporal evolution of the transmission through the ablation plume was probed using a second free running Er:YAG laser beam directed parallel to the sample’s surface. The ablation dynamics was found to consist of a vaporization phase followed by material ejection. The observation of droplet ejection during water ablation provided evidence that a phase explosion is the driving mechanism for material ejection. The laser light transmission was only slightly reduced by the vapor plume, but decreased by 25%–50% when the ejected material passed the probe beam. At radiant exposures ?10 times above the ablation threshold, the laser energy deposited into the sample amounted to only 61% of the incident energy for gelatin samples with 90% water content and to 86% for skin samples. For free-running Er:YAG laser pulses shielding must therefore be considered in modeling the ablation dynamics and determining the dosage for clinical applications.

A system for robotically assisted retinal surgery has been developed to rapidly and safely place lesions on the retina for photocoagulation therapy. This system provides real-time, motion stabilized lesion placement for typical irradiation times of 100 ms. The system consists of three main subsystems: a digital-based global tracking subsystem; a fast, analog local tracking subsystem; and a confocal reflectance subsystem to control lesion parameters dynamically. We have reported previously on these individual subsystems. This paper concentrates on the development of a second hybrid system prototype. Considerable progress has been made toward reducing the footprint of the optical system, simplifying the user interface, fully characterizing the analog tracking system, using measurable lesion reflectance parameters to develop a noninvasive method to infer lesion depth, and integrating the subsystems into a seamless hybrid system. These system improvements and progress toward a clinically significant system are covered in detail within this paper. The tracking algorithms and concepts developed for this project have considerable potential for application in many other areas of biomedical engineering.

To quantify dye leakage in ocular fluorescein angiography, the arterial concentration of sodium fluorescein has to be determined. We investigated whether the nonlinear relationship between the fluorescein concentration and the fluorescence intensity obtained by in vitro measurements corresponds with that measured in vivo in a retinal artery. The time series of fluorescence in a retinal artery were recorded using an in-house-designed and -built confocal scanning laser ophthalmoscope in 11 healthy volunteers. Three different doses of sodium fluorescein were injected successively. About 10 min after the last injection a venous blood sample was drawn. The three in vivo peak intensities were fitted by least squares on the in vitro calibration curve using the first peak concentration and an intensity scaling factor as the two unknown parameters. The fit showed that the saturation of the three in vivo peak intensities corresponded well with the in vitro data. Calculation of the intensity scaling factor from the blood sampling data confirmed the result of the fit. The fitted concentration was verified by showing that the cardiac output necessary to obtain this concentration was within the physiological range. The fluorescence measured in our in vitro experimental setup corresponded well with the in vivo measurements. Therefore, the results from in vitro measurements can be applied in the analysis of fluorescein angiograms.

Scanning laser polarimetry (SLP) assesses the retinal nerve fiber layer (RNFL) for glaucoma diagnosis by detecting the birefringence of the peripapillary RNFL. A detailed understanding of SLP requires an accurate value for RNFL birefringence in order to relate measured retardance to RNFL thickness, but current knowledge of this value is limited. A multispectral imaging micropolarimeter of PSC'A type was used to measure the retardance in transmission of the RNFL of isolated rat retina before (living) and after (fixed) 20 min of glutaraldehyde fixation. The thickness of the nerve fiber bundles measured was then determined histologically. As previously known from reflectance measurements, in transmission the RNFL behaved as a linear retarder. The retardance of the RNFL was constant at wavelengths from 440 to 830 nm and persisted after tissue fixation. In 37 nerve fiber bundles of 8 retinas, the average RNFL birefringence was 0.23 nm/µm before and 0.19 nm/µm after fixation, with an uncertainty of 0.01 nm/µm. The wavelength independence is consistent with a mechanism of form birefringence from thin cylindrical organelles. These results allow extrapolation of previous visible wavelength measurements to the near-infrared wavelengths used by SLP and validate the use of fixed tissue for RNFL research.

Using scanning confocal microscopy, we measure the backscattered second harmonic signal generated by a 100 fs laser in rat-tail tendon collagen. Damage to the sample is avoided by using a continuous scanning technique, rather than measuring the signal at discrete points. The second harmonic signal varies by about a factor of 2 across a single cross section of the rat-tail tendon fascicle. The signal intensity depends both on the collagen organization and the backscattering efficiency. This implies that we cannot use intensity measurements alone to characterize collagen structure. However, we can infer structural information from the polarization dependence of the second harmonic signal. Axial and transverse scans for different linear polarization angles of the input beam show that second harmonic generation (SHG) in the rat-tail tendon depends strongly on the polarization of the input laser beam. We develop an analytical model for the SHG as a function of the polarization angle in the rat-tail tendon. We apply this model in determining the orientation of collagen fibrils in the fascicle and the ratio ? between the two independent elements of the second-order nonlinear susceptibility tensor. There is a good fit between our model and the measured data.

A phase zero evaluation of a new fluorescence imaging technique for diagnosing cervical intraepithelial neoplasia (CIN) was performed. The fluorescence imaging prototype performed quantitative imaging of Protoporphyrin induced by a topically applied aminolevulinic acid using double ratio (DR) fluorescence imaging technique developed by our group. A total of 38 patients were in the protocol, with 16 colposcopically selected for biopsy. Fluorescence images of these 16 patients were taken, 19 sites were biopsied, and the disease was staged histopathologically. DR fluorescence imaging of the cervix using our general purpose prototype appeared to be cumbersome but feasible. In four cases strongly localized fluorescent hotspots were observed at the location where the disease was colposcopically visible. In the other cases the fluorescence showed a more diffuse multifocal image. The value of the DR determined at the site of biopsy correlated in a statistically significant way with the histopathologically determined stage of the disease [Spearman rank correlation, r=0.881, p<0.001 (confidence interval 0.7044–0.9552)]. This suggests that noninvasive staging of CIN using this technique is feasible. We believe that the results of this study justify the development of a dedicated device that combines regular white light colposcopy with DR fluorescence imaging.

Three methods by which to determine absolute total cerebral hemoglobin concentration (tHb in µmol/L) by near-infrared spectrophotometry (NIRS) have evolved: (1) tHbo, requiring oxygenation changes and arterial oxygen saturation measurements as a reference using a relative NIRS algorithm, (2) tHbg, using a geometrical multidistance principle and (3) tHbgo, a combination of both. The aim of this study was to compare the three methods quantitatively. Sixteen clinically stable preterm infants with a mean gestational age of 29.6 (range of 25.1–36.4) weeks, birthweight of 1386 (680–2820) g and a postnatal age of 2.5 (0.5–6) days, who needed supplemental oxygen, were enrolled. The mean6standard deviation tHbg was 150.2 ±41.8 µmol/L (range of 61.6–228.9 µmol/L), the tHbo was 62.1 ±27.2 µmol/L (26.0–110.8 µmol/L) and the tHbgo was 89.3 ±45.6 µmol/L (26.5–195.9 µmol/L). The correlation coefficient among the three methods were tHbg and tHbgo r=0.736; tHbo and tHbgo r=0.938; tHbg and tHbo r=0.598. A multiple regression with variable selection by Mellow’s C(p) showed, that tHbg was correlated to the birthweight, the postnatal age, the heart rate and the pCO2 (r2=0.588), tHbo and tHbgo were associated with the hemoglobin concentration in the blood, the mean arterial blood pressure and the pCO2 (r2=0.493 and 0.406, respectively). The three methods (tHbg, tHbo, and tHbgo) give systematically different tHb readings and large intersubject variability.

A general physiological model for the hemodynamic response during altered blood flow, oxygenation, and metabolism is presented. Calculations of oxy-, deoxy-, and total hemoglobin changes during stimulation are given. It is shown that by using a global hyperoxic or mild hypoxic challenge it is possible to normalize the activation response in terms of the fractional changes in the cerebral blood volume, tissue oxygenation index, and oxygen extraction ratio, which are independent of the optical pathlength. Using a dual wavelength spectrometer, the method is validated by measuring pathlength-independent hemodynamic responses during mild hypercarbia in a rat model. Phantom experiments showed that the changes in optical pathlength were small as the hemoglobin concentration was varied over a wide range. The determination of quantitative parameters facilitates the use of continuous-wave transcranial methods by providing a means by which to characterize activation response across subjects.

Microcirculatory blood flow can be measured using a laser Doppler flowmetry (LDF) probe. However, the readings are affected by the tissue’s optical properties (absorption and scattering coefficients, µa and µs ) and probe geometry. In this study the influence of optical properties [µa?(0.053,0.23) mm-1,µs?(14.7,45.7) mm-1] on LDF perfusion and LDF sampling depth was evaluated for different fiber separations. In vitro measurements were made on a sophisticated tissue phantom with known optical properties that mimicked blood flow at different depths. Monte Carlo simulations were carried out to extend the geometry of the tissue phantom. A good correlation between measured and simulated data was found. The simulations showed that, for fixed flow at a discrete depth, the influence of µs or µa on LDF perfusion increased with an increase in flow depth and decreased with an increase in fiber separation. For a homogeneous flow distribution, however, the perfusion varied 40% due to variations in the optical properties, almost independent of the fiber separation (0.23–1.61 mm). Therefore, the effect in real tissue is likely to vary due to the unknown heterogeneous blood flow distribution. Further, the LDF sampling depth increased with a decrease in µs or µa and an increase in fiber separation. For fiber separation of 0.46 mm, the e-1 sampling depth ranged from 0.21 to 0.39 mm.

Since hyperglycaemia changes the erythrocyte cell membrane fluidity and impairs cell deformity, our goal was to characterize hemoglobin and red blood cell (RBC) light refractive property changes in diabetic patients. Microscopic investigation was carried out on intact and fixed RBCs. To determine the refractive index (RI): smears of peripheral blood were air dried and fixed for 3 min in methanol. Mixtures of polyvinylpyrolidine and buffer of different pH (1:1) were used as embedding media. Intact RBCs were mixed with a buffered embedding medium, placed on a slide and overlaid with a coverslip. Interference microscopy was used for RI measurements at 18 different pH (pH=2 – 13). The results showed that curves of the RI of diabetic patients and of a control group were of similar configuration, with one branch in the acidic portion of the pH scale, a maximum and two minima in the neutral (middle) portion, and one branch in the alkaline portion. The curves of the individuals from the control group overlapped each other. To the contrary, the curves of the diabetic patients were not uniform in the neutral portion and the alkaline portion. The curves of the diabetic patients in the neutral zone were shifted towards the alkaline end of the pH scale, and the RBC RI curves were lower in comparison to the control curves. The center maximum of the curves of diabetic patients corresponded to pH=6.6 whereas the central maximum of the control group curves was at pH=6.2– 6.8. Contrary to in the diabetic group, intact RBC RI curves in the control group revealed only one significantly different minimum at pH of 7.2 in the neutral zone. Using this method it is possible to show phenotypic differences between uniform type intact and fixed cells, erythrocytes of diabetic patients and of healthy donors.

Colon cancer is the third leading class of cancer causing increased mortality in developed countries. A polyp is one type of lesion observed in a majority of colon cancer patients. Here, we report a microscopic Fourier transform infrared (FTIR) study of normal, adenomatous polyp and malignant cells from biopsies of 24 patients. The goal of our study was to differentiate an adenomatous polyp from a malignant cell using FTIR microspectroscopy and artificial neural network (ANN) analysis. FTIR spectra and biological markers such as phosphate, RNA/DNA derived from spectra, were useful in identifying normal cells from abnormal ones that consisted of adenomatous polyp and malignant cells. However, the biological markers failed to differentiate between adenomatous polyp and malignant cases. By employing a combination of wavelet features and an ANN based classifier, we were able to classify the different cells as normal, adenomatous polyp and cancerous in a given tissue sample. The percentage of success of classification was 89%, 81%, and 83% for normal, adenomatous polyp, and malignant cells, respectively. A comparison of the method proposed with the pathological method is also discussed.

The effectiveness and limitations of medical image processing using analog and digital methods are studied. Several types of errors introduced during the image processing are analyzed. For the analog optical Fourier transform, errors are introduced by the vignetting effect and lens aberration. For the digital Fourier transform, errors are introduced by the aliasing effect and the band limit. To compare the results obtained by the two techniques, a set of x-ray images was processed both optically and digitally. The former was achieved by an optical system containing a large Fourier telephoto lens and the latter by a personal computer using a Fourier transform algorithm. The veracity of both the optical and digital Fourier spectra is analyzed. Our results indicate that the optical method has high speed due to parallel processing. High veracity can be achieved in high frequency regions by using an optimal optical system. In comparison, the digital method has the advantages of high processing precision and programmability, but has low processing speed. The comparison of the two different techniques presented in this article can provide a basis for selection of the processing method in different clinical settings. Even with today’s fast computers, the optical method is still suitable for many clinical applications. The best choice lies in an analog–digital combination.

An investigation of the stress distribution patterns in post– core restored teeth and the behavior of dentin material to fracture propagation was conducted using experimental techniques such as digital photoelasticity (on photoelastic models), mechanical testing and scanning electron microscopy (SEM) (on extracted teeth). Digital photoelastic experiments showed that endodontic post–core restoration resulted in regions of high tensile stress and of stress concentrations in the remaining dentin structure. It was observed from mechanical testing that the fracture resistance in post–core restored teeth is significantly lower (p<0.0001) than that in intact tooth. There was a significant correspondence between the plane of stress concentrations identified in the photoelastic models and in those of the plane of fracture exhibited by the rehabilitated tooth specimens. While the fracture of post–core rehabilitated teeth was consistent, that of control teeth was not as distinct. The SEM highlighted varying dentin response to fracture propagation at the inner core and the outer regions. The fractographs showed brittle and ductile response to fracture propagation in the outer and inner core dentin, respectively. These photomechanical studies highlighted that the stress concentrations, high tensile stress and loss of inner ductile dentin associated with post endodontic rehabilitation diminished their resistance to fracture.