[The demonstration of blood flow in focal breast lesions by power-Doppler sonography. A new approach to assessment?]. / Blutflussdarstellung in Herdbefunden der Mamma mittels der Power-Doppler-Sonographie. Ein neuer Ansatz zur Dignitätsbeurteilung?

PURPOSE: Power-Doppler sonography is regarded as a very sensitive method for detecting low-velocity and low-volume blood flows. The purpose of our study was to investigate whether increased vascularity in breast carcinoma can be visualized by power-Doppler sonography and whether new criteria for differentiating benign and malign lesions can be found. METHOD: 315 patients were examined with a 13-MHz high-resolution linear transducer. If a suspicious lesion was found, it was evaluated further by power-Doppler sonography. Compared to normal breast parenchyma (reference structure), a focal increase in blood flow signals was registered using a 3-step grading system with a 4th step for no flow increase. RESULTS: In 97 cases the sonographic findings were correlated with histology (n = 95) or cytology (n = 2). There were 50 benign lesions, 42 cases of invasive and 5 cases of in-situ carcinoma. 73.5% benign lesions showed no or just minimal increases in flow signal. 81% of invasive cancer presented middle- or high-flow increases compared to normal breast parenchyma. The extend of flow increase was linked to tumor size in invasive cancer. In stage T1b to T4, 94.3% of invasive carcinoma presented middle or high flow increases. CONCLUSION: Power-Doppler sonography is able to visualize vascularization in breast tumors. According to first clinical results PD sonography is a promising additional diagnostic tool which seems to offer new criteria for differential diagnosis in breast tumors.

Three-dimensional imaging of rhodamine 123 fluorescence distribution in human melanoma cells by means of confocal laser scanning microscopy.

Three-dimensional (3D) imaging of intracellular rhodamine 123 fluorescence distribution was performed by means of confocal laser scanning microscopy (CLSM). Human IGR melanoma cells grown in monolayer or multicellular spheroid culture were studied for elucidating mitochondrial membrane potential characteristics, and cell and nucleus volume dimensions. Microspheres 6 microns in diameter loaded with rhodamine B were used to calibrate our instruments for performing 3D imaging of optical sections as obtained by CLSM. Accurate optical slicing is only possible taking into consideration the physical characteristics of the objectives used like chromatic and spherical aberrations, depth discrimination or cover slip correction and the temperature dependence of the immersion medium. While 3D imaging of optical slices can be carried out showing the original shape of the object being tested without physical distortion, 3D images of microspheres show well-reproducible structures of rhodamine B fluorescence. These can be explained by a superposition of two effects, namely scattering of the fluorescence light and a gradient of the electromagnetic field strength of the laser beam due to the shape of the object. 3D imaging of optical slices of IGR cells in monolayer or multicellular spheroid culture, which have been loaded with rhodamine 123, show the location of the dye predominantly within the cytoplasm of the cells with a remarkable heterogeneity of fluorescence intensity within and between single cells, indicating differences in the mitochondrial membrane potential and thus in the metabolic activity. Due to the heterogeneity of the cell shape the cell nucleus occupies between 4 and 14% of the total cell volume. These data reveal calibrated 3D imaging as a valuable noninvasive tool to visualize the heterogeneity of cell parameters under different cell culture conditions.

Evidence for redistribution-associated intracellular pK shifts of the pH-sensitive fluoroprobe carboxy-SNARF-1.

Properties and peculiarities of the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1), in view of pHi measurements in single cells, were evaluated using confocal laser scanning microscopy. It was found that in human malignant glioma cells (U 118 MG) grown in multicellular spheroid culture, intracellular calibration curves (nigericin method) varied from one cell to another despite emission ratioing of the fluorescence signals. In addition, considerable deviations between indicator calibration in cell-free solution and intracellular calibration were observed. Microspectrofluorometric measurements revealed that these deviations are attributable to intracellular pK shifts of the indicator rather than to spectral changes of the fluorescence emission. The observed pK shifts are probably due to intracellular redistribution of the indicator between cytosol and lipophilic cell compartemants, e.g. plasma membrane, since the indicator can even be loaded efficiently into the cells via its active acid form (instead of the acetoxymethyl ester form). An approximate theoretical derivation of a cellular calibration curve confirms that a reversible, pH-dependent intracellular redistribution of the protonated indicator component results in an apparent pK shift of delta pK = log(1 + epsilon.P), with P the partition coefficient and epsilon a factor that depends on the different mean layer thicknesses of the cytosol and plasma membrane. Since the apparent pK shift amounts to about 1 pH unit in tumour cells of spheroids, the intracellular pH measuring range of carboxy-SNARF-1 is almost restricted to alkaline pH values. Further consequences of the redistribution phenomenon are discussed with special respect to intracellular ion imaging.

Electrochromic dyes, enzyme reactions and hormone-protein interactions in fluorescence optic sensor (optode) technology.

The analytical potential of fluorescence-based optochemical sensors (optodes) has been expanded by use of (1) electrochromic dyes incorporated in thin polymeric multilayers by means of Langmuir-Blodgett film techniques, (2) enzyme-catalysed biochemical reactions and (3) antibody-linked immunological reactions. Fluorescence optical biosensors have been developed for the determination of electrical potentials (e.g., those produced by ion-selective membranes) and of hormones (e.g., thyroxine) and metabolites (e.g., lactate, glucose, xanthine and ethanol).

Theory and development of fluorescence-based optochemical oxygen sensors: oxygen optodes.

As the preceding considerations concerning the physical and technical features of oxygen optodes have demonstrated, fluorescence-based optochemical oxygen sensors possess certain advantages and peculiarities compared to conventionally applied electrochemical sensors such as polarographic oxygen electrodes. First, in contrast to oxygen electrodes, oxygen measurements with oxygen optodes do not suffer from distortions caused by the reference electrodes. In addition, because of the polarographic process, platinum electrodes continuously consume oxygen, which falsifies the results, especially when small sample volumes or long-term measurements, or both, are involved, whereas the sensor layer of oxygen optodes must only be equilibrated. Moreover, the surface of the platinum wire has to be catalytically clean in order to obtain a plateau of the polarogram and, consequently, to achieve a low rest current at zero PO2. Unfortunately, the demand for catalytically clean platinum surfaces turns out to be rather critical, since surface contamination occurs even with membranized electrodes, resulting in the well-known phenomenon of "electrode poisoning." The question of the specificity of oxygen electrodes also must be considered. In this context, CO2 and halothane may interfere with oxygen measurements, whereas fluorescence quenching is unaffected by CO2 and halothane affects the measurements only slightly, depending on the special indicator used. Furthermore, because of the flow dependence, oxygen measurements with the oxygen electrode show a distinct "stirring effect" caused by the turbulence in front of the electrode, which disturbs the diffusion field. Because of the completely different physical principle of fluorescence optical sensors, such influences are not observed with oxygen optodes. In addition, isolation and shielding of electrical circuits found in electrodes are not necessary for optodes. Furthermore, the sensitivity of oxygen optodes can be tuned to the desired range of PO2 values, resulting in a higher resolution. Use of suitable polymer alloys as indicator matrices can even enhance oxygen sensitivity; therefore, the application of optodes for trace analysis of oxygen might be possible, especially with regard to the application of highly oxygen-sensitive phosphorescent indicators. Finally, owing to the reversibility of fluorescence quenching, monitoring of oxygen by fluorescence optical sensors allows a continuous and remote control of biomedical parameters as well as regulation of biotechnological processes.(ABSTRACT TRUNCATED AT 400 WORDS)