Subclavian vein anatomic subtypes defined by digital cinefluroscopic venography prior to permanent pacemaker lead insertion.

Digital cinefluoroscopic venography of the subclavian vein was performed in 26 consecutive patients. The optimal stored image of the anticipated venipuncture site was magnified, road mapped, and used to compare with fluoroscopic-guided venipuncture. Two anatomic subtypes for both subclavian veins were observed. For the left subclavian vein, a gradual curve was seen most often (57%), while the remainder (43%) exhibited an "s"-shaped curve. For the right subclavian, a gradual curve was observed most frequently (60%) while an acute 90 degrees angle was noted in the remainder (40%). The "s"-shaped curve in the left subclavian vein necessitated redirection of the needle site both laterally and cranially. In three or 12% of patients venography showed either subclavian thrombosis or a persistent left superior vena cava and lead insertion was moved to the opposite side. Successful venipuncture and subsequent cannulation of the subclavian vein was achieved with the first or second passage of the needle in 22 or 85% of the 26 patients. Digital cinefluoroscopic venography appears to be both safe and rapid and may facilitate insertion of permanent pacemaker leads into the subclavian vein.

Endoluminal grafting for percutaneous aneurysm exclusion in an aortocoronary saphenous vein graft: the first clinical experience.

PURPOSE: Aneurysms develop only rarely in aortocoronary saphenous vein grafts (SVGs), and the usual treatment is surgical replacement of the diseased segment. However, in patients at appreciable risk for redo surgery, alternative therapies are desirable. We report the first compassionate use of a percutaneously delivered endoluminal graft (ELG) for internal exclusion of an SVG aneurysm. METHODS: A 47-year-old male with two coronary bypass procedures and SVG angioplasty presented with an 8-mm diameter aneurysm lying between 80% and 70% stenotic lesions in an SVG to the obtuse marginal branch. The risks of a third bypass operation were considerable, so the decision was made to attempt internal exclusion of the SVG aneurysm. RESULTS: An ELG composed of 2.0-mm diameter unexpanded PTFE graft material with Palmaz stents for fixation was delivered with a low-profile system, but a second ELG was necessary for complete exclusion of the aneurysmal sac. Both ELGs were dilated after initial deployment. The patient was discharged after 9 days without sequelae, and he remains asymptomatic with arteriographically documented ELG patency 5 months after treatment. CONCLUSIONS: In this patient with limited therapeutic options, percutaneous aneurysm exclusion in an SVG was effective in restoring a viable blood conduit. It remains to be seen if ELGs have a potential in aortocoronary SVGs.

Image intensification with color capability.

Image intensifiers applied to low light level phenomena provide outputs restricted to the color of the output phosphor. In many applications the ability to provide an image in color, or even color information, is desirable. A design of an intensifier system that can present images in color is described. A feasibility demonstration is presented, utilizing an intensified grating spectroscope.

Source and sinks for the calcium released during fertilization of single sea urchin eggs.

The source and sinks for the intracellular calcium released during fertilization were examined in single eggs from the sea urchin, Arbacia punctulata. Single eggs were microinjected with the calcium photoprotein, aequorin. The calcium-aequorin luminescence was measured with a microscope-photomultiplier or observed with a microscope-image intensifier-video system. In the normal egg a propagated release has been observed. The source of the calcium was investigated in the organelle-stratified centrifuged egg and by the use of mitochondrial uncouplers. In the organelle-stratified centrifuged egg, the calcium-aequorin luminescence was found to originate from the clear zone. The principal constituent of the clear zone is the endoplasmic reticulum. Other potential sources of calcium are the mitochondria. Their contribution to the calcium transient was investigated by exposure of aequorin-injected eggs to mitochondrial uncouplers either before or after fertilization. There was no calcium released from the mitochondria before fertilization. A very large calcium store was released from the mitochondria after fertilization. Interestingly, eggs fertilized in the presence of uncouplers showed no increase in the calcium-aequorin luminescence over untreated eggs. Apparently, in the absence of mitochondrial uptake, other sinks for calcium with affinity and capacity similar to the mitochondria exist, but their nature is unknown. We suggest that the endoplasmic reticulum is the source of the intracellular calcium released upon fertilization and that the mitochondria are the principal sink. The results are discussed with regard to the metabolic activation of the egg.

Temporal sequence and spatial distribution of early events of fertilization in single sea urchin eggs.

Measurements and observations of five early events of fertilization, singly and in pairs, from single sea urchin eggs have revealed the precise temporal sequence and spatial distribution of these events. In the Arbacia punctulata egg, a wave of surface contraction occurs coincident with membrane depolarization (t = 0). These two earliest events are followed by the onset of a rapid, propagated increase in cytoplasmic-free calcium at approximately 23 s as measured by calcium-aequorin luminescence. The luminescence reaches its peak value by 40 s after the membrane depolarization. The luminescence remains uniformly elevated for some time before its decay over several minutes. The onset of an increase in the pyridine nucleotide (NAD(P)H) fluorescence follows the membrane depolarization at approximately 51 s. The fertilization membrane begins its elevation in a wave-like fashion coincidentally with the increase in NAD(P)H fluorescence. Similar results are observed in the Lytechinus variegatus egg. The results suggest that while the increase in cytoplasmic-free calcium may be important for many changes occurring in the egg, the elevated-free calcium is not directly responsible for the propagated wave of cortical granule exocytosis.

Calcium transients during early development in single starfish (Asterias forbesi) oocytes.

Maturation and fertilization of the starfish oocyte are putative calcium-dependent events. We have investigated the spatial distribution and temporal dynamics of this calcium dependence in single oocytes of Asterias forbesi. We used the calcium photoprotein, aequorin, in conjunction with a microscope-photomultiplier and microscope-image intensifier. Surprisingly, in contrast to earlier work with Marasthenias glacialis, there is no detectable increase in intracellular-free calcium in the oocyte of A. forbesi in response to the maturation hormone 1-methyl adenine. During fertilization of the same, matured, A. forbesi oocyte there is a large increase in intracellular-free calcium. The calcium concentration increases to approximately 1 microM at the point of insemination and the region of elevated free calcium expands across the oocyte in approximately 20 s (17-19 degrees C). After the entire oocyte reaches an elevated concentration of free calcium, the concentration decreases uniformly throughout the oocyte over the next several minutes.

Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH.

An interface of our microspectrofluorometer with an image processing system performs microspectrofluorometric measurements in living cells by digital image processing. Fluorescence spectroscopic parameters can be measured by digital image processing directly from microscopic images of cells, and are automatically normalized for pathlength and accessible volume. Thus, an accurate cytoplasmic "map" of various spectroscopic parameters can be produced. The resting cytoplasmic pH of fibroblasts (3T3 cells) has been determined by measuring the ratio of fluorescein fluorescence exited by two successive wavelengths (489 and 452 nm). Fluorescein-labeled dextran microinjected into the cells is used as a pH indicator, since it is trapped in the cytoplasm but is excluded from the nucleus and other organelles. The average cytoplasmic pH is 6.83 (+/- 0.38). However, cytoplasmic pH exhibits a nonunimodal distribution, the lower mean pH being 6.74 (+/- 0.23). When 3T3 cells pinocytose medium containing fluorescein dextran, pinosomes peripheral to the nucleus exhibit a lower pH than those closer to the ruffling edge of the cell. The present image processing system is analyzed for linearity of detection, light scattering artifacts, signal to noise ratio, standard curves, and spatial resolution. The results obtained from digital image analysis are shown to be comparable to the results from standard microspectrofluorometry. We also discuss several other applications of this ratio imaging technique in cell biology.

X-ray diffraction analysis of wet isolated bovine rod outer segment disks. A dehydration study.

Sequences of X-ray diffraction patterns were obtained from dehydrating, artificially oriented multilayers of isolated, bovine rod outer segment disks. A direct-phase analysis was applied to highly hydrated specimens to determine sequences of low resolution (approx. 30 A) electron density profiles of the disks as dehydration proceeded. The profiles were found to evolve smoothly as the multilayer lattice simultaneously shrank and became increasingly ordered. The bilayer profiles were largely invariant under dehydration and the evolution of the diffraction consistent with simple decreases in fluid spacings. The specimens were observed to phase separate into characteristic primary and a secondary lattices when the multi-layer became too dehydrated. The small unit cell size of the secondary lattice was suggestive of a lipid phase. Large changes in the diffraction patterns from phase separated specimens were observed upon bleaching of the specimen. The changes were consistent with a reversible disordering of the primary lattice.

Applications of image intensification to low level fluorescence studies of living cells.

Microscopic observations of weak fluorescence from living cells can be achieved by using image intensification techniques in situations where conventional film recording is not feasible. A brief description is given of experimental arrangements that have been used, involving recording the intensifier output alternately on film, or TV vidicons. References are given to more detailed descriptions of particular systems, and an example is presented of the detection of Ca++ in Haemanthus by means of the fluorescence of chlorotetracycline.

High sensitivity image intensifier-TV detector for x-ray diffraction studies.

A sensitive, efficient image intensifier-TV x-ray detector is described that has been optimized for a large class of diffraction studies of biological structures. All of the major components are commercially available. The system is well suited to measuring the intensity of diffraction patterns that are weak, or changing with time. Because there is no count rate limitation, it is particularly well suited for studies utilizing the high fluxes of synchrotron sources.

A free calcium wave traverses the activating egg of the medaka, Oryzias latipes.

Aequorin-injected eggs of the medaka (a fresh water fish) show an explosive rise in free calcium during fertilization, which is followed by a slow return to the resting level. Image intensification techniques now show a spreading wave of high free calcium during fertilization. The wave starts at the animal pole (where the sperm enters) and then traverses the egg as a shallow, roughly 20 degrees-wide band which vanishes at the antipode some minutes later. The peak free calcium concentration within this moving band is estimated to be about 30 microM (perhaps 100-1,000 times the resting level). Eggs activated by ionophore A23187 may show multiple initiation sites. The resulting multiple waves never spread through each other; rather, they fuse upon meeting so as to form spreading waves of compound origin. The fertilization wave is nearly independent of extracellular calcium because it is only slightly slowed (by perhaps 15%) in a medium containing 5 mM ethylene glycol-bis[beta-aminoethyl ether]N,N'-tetraacetic acid (EGTA) and no deliberately added calcium. It is also independent of the large cortical vesicles, which may be centrifugally displaced. Normally, however, it distinctly precedes the well-known wave of cortical vesicle exocytosis. We conclude that the fertilization wave in the medaka egg is propagated by calcium-stimulated calcium release, primarily from some internal sources other than the large cortical vesicles. A comparison of the characteristics of the exocytotic wave in the medaka with that in other eggs, particularly in echinoderm eggs, suggests that such a propagated calcium wave is a general feature of egg activation.

The subcellular origin of bioluminescence in Noctiluca miliaris.

The light emitted by Noctiluca has its origin in 1 to 5 x 10(4) organelles ("microsources") which are scattered throughout the perivacuolar cytoplasm, and which appear to be the elementary functional units of bioluminescence. Microscopical techniques, image intensification, and microphotometry were employed in their investigation. Microsources are fluorescent, strongly phase-retarding, and range widely in diameter below 1.5 microns. The number of quanta emitted in a flash from a microsource ("microflash") is of the order of 10(5) photons. However, microflashes show a wide range of intensities, which are correlated with the size of the organelles from which they arise. Each organelle responds repetitively and with reproducible time course to a succession of invading triggering potentials. Reversible changes in the intensity of the flash emitted by the whole cell ("macroflash") occur because of graduations in intensity of microflashes rather than as a result of changes in the number of responsive organelles. The shape of the flash emitted by individual microsources resembles that of the macroflash except for slightly shorter rise and decay times. It is concluded that the macroflash results from somewhat asynchronous, but otherwise parallel summation of microflashes.

Sensitivity of an image intensifier film system.

Using calibrated light sources, the sensitivities of several commonly used films have been determined in terms of the number of photons incident required to develop one grain of emulsion. By incorporating an image intensifier in the system, the time required to obtain an image of a given quality or information content can be reduced significantly. Criteria are discussed whereby an estimate of the time advantage can be made for various experimental conditions. Noise, quantum fluctuations, spatial resolution, and over-all detection efficiency are discussed.