Frits F. M. de Mul

Local variation in absolute water content of human and rabbit eye lenses measured by Raman microspectroscopy

Raman spectra were obtained from fresh, fixed and sliced rabbit lenses and from human lens slices. For all lenses and lens slices the ratio R, defined as the Raman intensity at 3390 cm-1 divided by the Raman intensity at 2935 cm-1, was measured at different locations along the visual and equatorial axis. The ratios R were transformed to absolute water mass percentages by measuring solutions with known protein concentrations. It was shown that fixation and slicing have very little effect on the absolute water content of the lenses. The values obtained for the absolute water content are comparable to values given in literature. It was also shown that the water content in rabbit and human lenses rapidly decreases from the immediate anterior and posterior subsurface region to the deep superficial cortex and is relatively constant in the nucleus. Raman microspectroscopy appears to be a reliable method for the measurement of the absolute water content of small volumes on defined positions in the lens. This can be very useful when analyzing the possible relation between local variations in water content and the occurrence of opacities in the lens.

Laser Doppler perfusion imaging with a complimentary metal oxide semiconductor image sensor

We utilized a complimentary metal oxide semiconductor video camera for fast flow imaging with the laser Doppler technique. A single sensor is used for both observation of the area of interest and measurements of the interference signal caused by dynamic light scattering from moving particles inside scattering objects. In particular, we demonstrate the possibility of imaging the distribution of the moving red blood cell concentration. This is a first step toward laser Doppler imaging without scanning parts, leading to a much faster imaging procedure than with existing mechanical laser Doppler perfusion imagers.

Photoacoustic determination of blood vessel diameter.

A double-ring sensor was applied in photoacoustic tomographic imaging of artificial blood vessels as well as blood vessels in a rabbit ear. The peak-to-peak time (tau(pp)) of the laser (1064 nm) induced pressure transient was used to estimate the axial vessel diameter. Comparison with the actual vessel diameter showed that the diameter could be approximated by 2ctau(pp), with c the speed of sound in blood. Using this relation, the lateral diameter could also precisely be determined. In vivo imaging and monitoring of changes in vessel diameters was feasible. Finally, acoustic time traces were recorded while flushing a vessel in the rabbit ear with saline, which proved that the main contribution to the laser-induced pressure transient is caused by blood inside the vessel and that the vessel wall gives only a minor contribution.

Photoacoustic imaging of blood vessels with a double-ring sensor featuring a narrow angular aperture

A photoacoustic double-ring sensor, featuring a narrow angular aperture, is developed for laser-induced photoacoustic imaging of blood vessels. An integrated optical fiber enables reflection-mode detection of ultrasonic waves. By using the cross-correlation between the signals detected by the two rings, the angular aperture of the sensor is reduced by a factor of 1.9, from 1.5 to 0.8 deg. Consequently, photoacoustic images could be obtained in a manner analogous to the ultrasound B-scan mode. Next, the cross section of artificial blood vessels is visualized by reconstruction of the absorbed energy distribution. Finally, in vivo imaging and the subsequent reconstruction of the absorbed energy distribution is demonstrated for superficial blood vessels in the human wrist.

Prediction of the photodetector signal generated by Doppler-induced speckle fluctuations: theory and some validations

A theoretical framework is proposed for describing the laser Doppler photodetector signal. The theory allows for predicting the power of the photocurrent fluctuations. It is valid for a detector of arbitrary size. The input data required for application of the theory are the angular distribution of the detected light, the fraction of Doppler-shifted photons, and the active detector size. The theory is based on the time-domain approach to the statistics of dynamic speckle patterns on the photodetector. An experiment was carried out to validate some aspects of our theory. The consequences of the speckle dynamics for the various modes of laser Doppler flowmetry are discussed.

Path-length distribution and path-length-resolved Doppler measurements of multiply scattered photons by use of low-coherence interferometry.

We report first results of measurements by low-coherence Doppler interferometry of the path-length distribution of photons undergoing multiple scattering in a highly turbid medium. We use a Mach-Zehnder interferometer with multimode graded-index fibers and a superluminescent diode as the light source. The path-length distribution is obtained by recording of the heterodyne fluctuations that arise from the Brownian motion of particles in an Intralipid suspension as a function of the optical path length. The experimental path-length distribution is in good agreement with predictions of Monte Carlo simulations. In the heterodyne spectrum, an increase of the mean Doppler frequency with path length is observed.

Raman microspectroscopy of fixed rabbit and human lenses and lens slices: New potentialities

Raman spectroscopy is a non-invasive, non-destructive technique for the study of the macromolecular composition of tissues. Raman spectra were obtained from intact fresh and paraformaldehyde fixed rabbit lenses and from thin slices prepared from these lenses. In addition the Raman spectrum of an intact 82-yr-old human lens was compared with a slice of the same lens. It appeared that fixation with paraformaldehyde had only a minor qualitative effect on the Raman spectra and that Raman spectra of intact lenses and lens slices were comparable. It was also shown that in the slice of the old human lens the fluorescence, due to chromophores, could be reduced so that a reliable Raman spectrum could be obtained. The use of slices improves the accuracy of the position at which Raman spectra are recorded and fixation extends the time available for Raman analysis which is particularly important for the study of human lenses. Moreover, slicing enables Raman analysis of old human lenses, which up to now was thought to be impossible due to the overwhelming fluorescence of the chromophores present in these lenses.

Analysis of three-dimensional photoacoustic imaging of a vascular tree in vitro

We used photoacoustics in order to image a vasculature cast, which was obtained from a Wistar rat. The main objective was to investigate the imaging quality and the performance of the beamforming algorithm. For these purposes, the specimen was measured in several Intralipid™ 10% solutions and it was scanned with scanning steps of 100, 200, and 300 μm. The measurements were performed with a disk-shaped detector, with PVDF as an active material and four fibers for the light delivery. From the recorded signals, three-dimensional images of the cast were constructed.

Self-mixing feedback in a laser diode for intra-arterial optical blood velocimetry

Intra-arterial measurements of the velocity and the average flow of red-blood cells were investigated by means of a fiber-coupled laser Doppler velocimeter based on the self-mixing effect. The velocity of the red cells was calculated from the frequency of the signal that occurs when light, scattered back from a moving object in front of a fiber into a laser-diode cavity, interferes with the laser cavitys proper mode. These fluctuations occur at the Doppler frequency. The signal was obtained from the photodiode that is present in the laser diodes housing. Temperature control and stabilization of the diode cavity were introduced to reduce the light-intensity fluctuation that is due to mode hopping of the diode. The velocimeter was calibrated with a rotating disk covered with white paper (nonlinearity of 2.6% for velocities up to 0.4 m/s) and tested in vitro as a fluid velocimeter. The velocimeter was used in in vivo tests on the iliac artery of a 35-kg pig and on the arteria pulmonaris of a healthy calf. The optical fiber was placed in the iliac artery by a basket catheter 4 cm proximal to the bifurcation of the femoral artery. The average arterial blood flow velocity of the red cells were measured upstream and downstream. A special cleaving procedure for the fiber tip in downstream measurement is reported. Blood-velocity measurement is compared with values generated by an ultrasound flowmeter, and a difference of less than 9% is found.

Glass-fiber self-mixing intra-arterial laser Doppler velocimetry: signal stability and feedback analysis

We have developed a blood velocimeter based on the principle of self-mixing in a semiconductor laser diode through an optical fiber. The intensity of the light is modulated by feedback from moving scattering particles that contain the Doppler-shift frequency. Upon feedback the characteristics of the laser diode change. The threshold current decreases, and an instable region may become present above the new threshold. The amplitude of the Doppler signal turns out to be related to the difference in intensity between situations with and without feedback. This amplitude is highest just above feedback. The suppression of reflection from the glass-fiber facets is of paramount importance in the obtaining of a higher signal-to-noise ratio. Using an optical stabilization of the feedback, we optimized the performance of the laser-fiber system and the Doppler modulation depth and clarified its behavior with a suitable physical model. We also investigated the effect of the finite coherence length of the laser. We tested the efficiency of the self-mixing velocimeter in vivo with the optical glass fiber inserted in the artery with endoscopic catheters, both in upstream and in downstream blood flow conditions. For the latter we used a special side-reflecting device solution for the fiber facet to allow downstream measurements.