A coded aperture microscope for X-ray fluorescence full-field imaging.

The design and construction of an instrument for full-field imaging of the X-ray fluorescence emitted by a fully illuminated sample are presented. The aim is to produce an X-ray microscope with a few micrometers spatial resolution, which does not need to scan the sample. Since the fluorescence from a spatially inhomogeneous sample may contain many fluorescence lines, the optic which will provide the magnification of the emissions must be achromatic, i.e. its optical properties must be energy-independent. The only optics which fulfill this requirement in the X-ray regime are mirrors and pinholes. The throughput of a simple pinhole is very low, so the concept of coded apertures is an attractive extension which improves the throughput by having many pinholes, and retains the achromatic property. Modified uniformly redundant arrays (MURAs) with 10 µm openings and 50% open area have been fabricated using gold in a lithographic technique, fabricated on a 1 µm-thick silicon nitride membrane. The gold is 25 µm thick, offering good contrast up to 20 keV. The silicon nitride is transparent down into the soft X-ray region. MURAs with various orders, from 19 up to 73, as well as their respective negative (a mask where open and closed positions are inversed compared with the original mask), have been made. Having both signs of mask will reduce near-field artifacts and make it possible to correct for any lack of contrast.

Electron microscope radioautographic evidence of in vivo androgen-binding protein internalization in the rat epididymis principal cells.

The androgen-binding protein (ABP) has been purified from rat testes with a yield of 14% using four steps of HPLC and was subsequently iodinated to a specific activity of 0.1 mCi/mg protein. Using a micromanipulator, [125I] iodo-ABP-dihydrotestosterone was injected intraluminally into the proximal caput of the rat epididymis. Epididymides were sampled from 3 to 120 min after the injection of the tracer and processed for transmission electron microscopy autoradiography. Our results showed the accumulation of detectable radioactive sources in the apical cytoplasm of only one of the epithelial cell type lining the ductus, the principal cells. In the interval from 3 to 120 min, the iodinated ABP was mainly present in the supranuclear region and was especially concentrated over coated structures, endosomes, multivesicular bodies, and over the Golgi apparatus. The same pattern was obtained using [3H]dihydrotestosterone-ABP complex instead of iodinated ABP. In addition, there was a negative correlation between the log time and the distribution of the silver grains in the luminal border and in the compartment of the apical vesicles. On the contrary, there was a positive correlation between the log time and the distribution of the silver grains in the Golgi apparatus. These results provide, for the first time, direct histological evidence of the in vivo ABP internalization by the principal cells. Since horseradish peroxidase, a fluid-phase endocytosis marker, when injected under the same conditions was internalized in both apical and principal cells, since labeled radioactive ABP appeared to be bound to the membrane of the endocytic apparatus rather than to its content, and since this binding and uptake could be prevented in the presence of an excess of unlabeled ABP, it is concluded that the internalization of ABP could not be a nonspecific fluid-phase endocytosis but should be dependent on its interaction with the apical plasma membrane of the principal cell. It still remains to be determined if these mechanisms involve the binding of ABP to a specific membrane receptor.

Purification of androgen-binding protein from rat testis using high-performance liquid chromatography and physicochemical properties of the iodinated molecule.

The androgen-binding protein (ABP) has been purified 87,500-fold from rat testis using 4 steps of HPLC, with a yield of 14%. The molecule was 99% pure with a specific activity estimated to 16,600 pmol/mg protein. The iodinated molecule was eluted in 2 peaks in Sephacryl S300 gel filtration with a molecular mass estimated to be 92,600 +/- 3300 and 50,300 +/- 4000 Da. The column isoelectrofocusing of 125I-ABP demonstrated 3 isoproteins isoelectric at pH 4.7, 4.9 and 5.3 and the sedimentation coefficient was estimated to be 4.7 S in sucrose gradient ultracentrifugation. The 125I-ABP had similar physiochemical properties to the non-labelled ABP of epididymis.