Resolving surface details with reflection and fluorescence video-confocal profilometry.

Video-confocal profilometry has been exploited to characterize reflecting and non-reflecting surfaces in materials with tilted and corrugated areas. An alternative method based on fluorescence detection has been developed and tested to characterize metal surfaces modified by intense laser irradiation. Combined representations of surface topography have been obtained on the basis of both reflectance and fluorescence signals. A discussion of results and problems encountered in reflection and fluorescence based techniques is provided.

A chimeric saporin-transferrin conjugate compared to ricin toxin: role of the carrier in intracellular transport and toxicity.

Human transferrin (Tf) and saporin-6 (Sap), a ribosome inactivating protein from Saponaria officinalis, were chemically conjugated: the reaction generated two chimeras (called Tf-Sap) that proved to be cytotoxic to HepG2 cells. Electrophoretic and chromatographic analysis revealed that the two conjugates contained saporin and Tf in a 2:1 or 1:1 molar ratio (140 and 110 KDa, respectively). Free saporin is essentially nontoxic, whereas Tf-Sap efficiently kills HepG2 cells, although its ID50 (= 6 nM) is 1000-fold greater than that of ricin. Intracellular transport of these toxins was followed by in vivo fluorescence video microscopy, preparing the conjugates starting from rhodamine isothiocyanate-labeled saporin. Image analysis of living HepG2 cells exposed to fluorescent Tf-Sap revealed that the endocytotic pathway involving passage through secondary endosomes is dictated by Tf and is different from that of ricin (the dimeric toxin from Ricinus communis), which is delivered to the Golgi apparatus, the probable site of activation. We discuss whether differences in toxicity between ricin and Tf-Sap can be attributed to the different mechanisms of transport and activation.

Intracellular dynamics of ricin followed by fluorescence microscopy on living cells reveals a rapid accumulation of the dimeric toxin in the Golgi apparatus.

The intracellular dynamics of fluorescent conjugates of the toxic lectin ricin was followed by video fluorescence microscopy on living CHO cells, demonstrating that the ricin heterodimer and its isolated B chain, after binding to the plasma membrane receptors, migrate to and accumulate in the Golgi apparatus following internalization. A ricin derivative labelled with fluorescein on the A chain and rhodamine on the B chain did not display significant splitting of the A-B heterodimer during translocation of the toxin to the Golgi; this novel finding provides support for the hypothesis that further processing of ricin takes place in this cellular compartment.

Confocal-line microscopy.

The confocal-line (CL) technique combines some of the characteristics of confocal-scanning microscopy with those of conventional-imaging methods. It is based on the introduction of line-shaped illumination and linear image detection, as an alternative to the current confocal-point (CP) approach. Although confocal only in one dimension, the proposed solution offers performance and features adequate to a variety of biological and non-biological applications and is also adaptable to an increased number of microscopical observations and measurements. The absence of moving components in the optical path and the use of electronic linear imagers permits flexible and fast operation that appears particularly relevant in many fields of basic and applied research. For instance, transmission, reflection and emission images can simultaneously be collected from the same area of the specimen, with slight adjustments to the optical setup. Useful extensions of CL microscopy to the field of spectral imaging are obtained with the introduction of a slit, a polychromator and an area detector, substituting for the linear imager. Prototype instrumentation has been constructed working from the cited principles and some tests have been performed on selected applications.

Binary data acquisition from instrumentation with BCD-coded output: a hardware converter and an example of the relative software control.

Simple hardware for conversion from BCD into binary code is described. The device is of low cost, performs a 16-bit conversion in a maximum time of 400 nsec, and is able to output the converted number to either an 8-bit or a 16-bit data bus. An example of the software necessary to perform data acquisition from a digital voltmeter by using this converter is also presented.