F.F.M. de Mul

Three-dimensional photoacoustic imaging of blood vessels in tissue

We applied photoacoustics as a tissue tomography technique for the detection of blood concentrations, e.g., angiogenesis around tumors. We imaged blood vessels in highly scattering samples, using 532-nm light, to depths of ~1 cm . The samples were real tissue (chicken breast) or 10% dilutions of Intralipid-10%. The blood flowed through nylon capillaries. Polyvinylidene difluoride (PVdF) piezoelectric detectors were used in a surface-scanning mode. We demonstrate the sensitivity of the technique by photoacoustic detection of single red blood cells upon a glass plate. Lateral resolution is limited by the detector diameter (200 microm). The depth resolution is ~10 microm.

Laser irradiation and Raman spectroscopy of single living cells and chromosomes: Sample degradation occurs with 514.5 nm but not with 660 nm laser light

In Raman spectroscopic measurements of single cells (human lymphocytes) and chromosomes, using a newly developed confocal Raman microspectrometer and a laser excitation wavelength of 514.5 nm, degradation of the biological objects was observed. In the experiments high power microscope objectives were used, focusing the laser beam into a spot approximately 0.5 micron in diameter. At the position of the laser focus a paling of the samples became visible even when the laser power on the sample was reduced to less than 1 mW. This was accompanied by a gradual decrease in the intensity of the Raman signal. With 5 mW of laser power the events became noticeable after a period of time in the order of minutes. It is shown that a number of potential mechanisms, such as excessive sample heating due to absorption of laser light, multiple photon absorption, and substrate heating are unlikely to play a role. In experiments with DNA solutions and histone protein solutions no evidence of photo damage was found using laser powers up to 25 mW. No degradation of cells and chromosomes occurs when laser light of 660 nm is used. The most plausible explanation therefore seems to be that the sample degradation is the result of photochemical reactions initiated by laser excitation at 514.5 nm of as yet unidentified sensitizer molecules or complexes present in chromosomes and cells but not in purified DNA and histone protein samples.

Condensed Monte Carlo simulations for the description of light transport

A novel method, condensed Monte Carlo simulation, is presented that applies the results of a single Monte Carlo simulation for a given albedo micro(s)/(micro(alpha) & micro(s)) to obtaining results for other albedos; micro(s) and micro(alpha), are the scattering and absorption coefficients, respectively. The method requires only the storage of the number of interactions of each photon with the medium. The reflectance and transmittance of turbid slabs can thus be found from a limited number of condensed Monte Carlo simulations. We can use an inversion procedure to obtain the absorption and scattering coefficients from the total reflectance and total transmittance of slabs. Remitted photon densities from a semi-infinite medium as a function of the distance between the light source and the detector for all albedos can be found even from the results of a single condensed Monte Carlo simulation. The application of similarity rules may reduce further the number of Monte Carlo simulations that are needed to describe the influence of the distribution of scattering angles on the results.

Optical properties of human dermis in vitro and in vivo

Condensed Monte Carlo simulation results have been used for calculating absorption and reduced scattering coefficients from the literature data on the measured total transmittance and total reflectance of samples of the human skin in vitro. The results of several measuring methods have been compared. We have also estimated the range for absorption coefficients and reduced scattering coefficients at 660 and 940 nm from measured intensities at the skin surface as a function of the distance from the location where the light enters the skin by using condensed Monte Carlo simulations for a homogeneous semi-infinite medium. The in vivo values for the absorption coefficients and the reduced scattering coefficients appear to be much smaller than the values from the in vitro measurements, that have been assumed until now. The discrepancies have been discussed in detail. Our in vivo results are in agreement with other in vivo measurements that are available in the literature.

Raman microspectroscopic approach to the study of human granulocytes

A sensitive confocal Raman microspectrometer was employed to record spectra of nuclei and cytoplasmic regions of single living human granulocytes. Conditions were used that ensured cell viability and reproducibility of the spectra. Identical spectra were obtained from the nuclei of neutrophilic, eosinophilic, and basophilic granulocytes, which yield information about DNA and protein secondary structure and DNA-protein ratio. The cytoplasmic Raman spectra of the three cell types are very different. This was found to be mainly due to the abundant presence of peroxidases in the cytoplasmic granules of neutrophilic granulocytes (myeloperoxidase) and eosinophilic granulocytes (eosinophil peroxidase). Strong signal contributions of the active site heme group(s) of these enzymes were found. This paper illustrates the potentials and limitations for Raman spectroscopic analysis of cellular constituents and processes.

Age-related changes in local water and protein content of human eye lenses measured by Raman microspectroscopy

The Raman microspectroscopic method was used to determine the local water and protein content in human lenses. In 18 lenses of varying age position-defined water/protein content measurements were carried out along the visual and the equatorial axis. A main characteristic of the human lens is its constant and relatively low protein content. In addition this constant nuclear value is reached within a short distance from the capsular surface. For statistical analysis of age-related changes the data points in individual lenses were piecewise linearized. (1) The mean nuclear water content was calculated from the data points in the inner 80% of the visual axis. (2) The steep drop in water content was linearized using a least-squares linear regression approach. The distance between lenticular surface and the intersection of the regression line with the line representing the nuclear mean was denominated as surface layer width. It proved that: (i) the mean nuclear water content significantly increased with age, (ii) the width of the surface layer was age independent in the anterior and posterior poles of the visual axis, and (iii) in the equatorial axis the surface layer width significantly decreased with age. Seven human lenses with small opaque spots were also investigated. The opaque spots proved to have a normal-for-site water content and some of them were flanked at their capsular side by a zone with a high-for-site water content.(ABSTRACT TRUNCATED AT 250 WORDS)

Laser Doppler velocimetry and Monte Carlo simulations on models for blood perfusion in tissue

Laser Doppler flow measurements and Monte Carlo simulations on small blood perfusion flow models at 780 nm are presented and compared. The dimensions of the optical sample volume are investigated as functions of the distance of the laser to the detector and as functions of the angle of penetration of the laser into the sample. The effects of homodyne and heterodyne scattering are investigated.

Laser Doppler velocimeter based on the self-mixing effect in a fiber-coupled semiconductor laser : theory

A laser Doppler velocimeter that consists of a semiconductor laser coupled to a fiber and that uses the self-mixing effect is presented. The velocimeter can be used for solids and fluids. A theoretical model is developed to describe the self-mixing signals as a function of the amount of feedback into the laser and the distance from the laser to the moving object. Good agreement is found between this theory and measurements.

Small laser Doppler velocimeter based on the self-mixing effect in a diode laser

A very small and simple velocimeter is presented consisting of a diode laser with a gradient-index lens in front of it. The basis of the velocity measurement is the mixing that occurs when light, scattered back by the moving object into the laser cavity, interferes with light inside the laser. This mixing induces large fluctuations of the laser intensity with the Doppler frequency. These fluctuations can be detected either with a photodiode or by measuring the voltage across the diode laser. As an illustration of the performance of the velocimeter, velocity measurements of a rotating disk covered with white paper are described. The differences arising because of using a single-mode or a multilongitudinal mode laser were calculated and verified in experiments. The advantage of the use of a multimode laser is that differential measurements of the distance between laser and moving object are also possible.

Monte Carlo simulations of laser Doppler blood flow measurements in tissue.

Light propagation in a model for blood perfusion in tissue was simulated with Monte Carlo calculations to investigate the dependence of the output of laser Doppler perfusion meters on the configuration of the optical probe and on the multiple scattering of photons by moving particles in the tissue. Laser Doppler perfusion meters registrating the first moment ?v? and the first weighted moment ?v?(s) of the spectral power density S(v) of intensity fluctuations on a detector viewing tissue illuminated by a laser are considered. The model was scaled up about a factor of 10 compared with real tissue, to make experimental tests possible. From the simulations of the Doppler scattering, it will be shown that the location of the effective probe volume of the perfusion meter can be extended to deeper layers in tissue by increasing the distance between the illuminating light beam and the detector. This opens the possibility to measure perfusion in skin layers as a function of the distance to the surface. Other calculations show how the degree of multiple scattering of individual photons by moving cells determines which flow parameter is measured with the perfusion meter. If the degree is low, the output of the meter depends linearly on the mean velocity of cells. For high degrees, a dependence on the root mean square value of this distribution is found. At a high moving particle concentration, multiple scattering by moving particles also results in deviations from the linear dependence of ?v? on the concentration of moving particles and in deviations from the concentration independence of ?v?(s). Intensity distributions of light inside the tissue model were obtained from the simulations.