Silicon Intensified Target Camera Research Articles

Phospholipid order in gel- and fluid-phase cell-size liposomes measured by digitized video fluorescence polarization microscopy

Low-light digitized video fluorescence microscopy has been utilized to measure the steady-state polarized fluorescence from the membrane probe diphenylhexatriene (DPH) and its cationic and phosphatidylcholine derivatives 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 2-[3-(diphenylhexatrienyl)propanoyl]-3-palmitoyl-L-alpha-phosphati dylcholine (DPH-PC), respectively, in cell-size (10-70 microns) unilamellar vesicles composed of gel-or fluid-phase phospholipid. Using an inverted microscope with epi-illumination optics and an intensified silicon intensified target camera interfaced to a minicomputer, fluorescence images of single vesicles were obtained at emission polarizer orientations of 0 degrees, 45 degrees, 90 degrees, and 135 degrees relative to the excitation light polarization direction. Fluorescence intensity ratios F90 degrees/F0 degrees (= F perpendicular/F parallel) and F135 degrees/F45 degrees were calculated on a pixel-by-pixel basis from digitized image pairs. Theoretical expressions were derived for collected polarized fluorescence as a function of position on the membrane surface as well as the degree of lipid order, in terms of the fluorophore's maximum angular motional freedom in the bilayer (identical to theta max), using a modification of the method of D. Axelrod (1979. Biophys. J. 26:557-574) together with the "wobbling-in-a-cone" model of probe rotational diffusion. Comparison of experimental polarization ratios with theoretical ratios yielded the following results. In gel-phase dipalmitoyl-phosphatidylcholine, the data for all three probes correspond to a model in which the cone angle theta max = 17 +/- 2 degrees and there exists a collective tilt of the phospholipid acyl chains of 30 degrees relative to the bilayer normal. In addition, approximately 5% of DPH and TMA-DPH molecules are aligned parallel to the plane of the bilayer. In fluid-phase palmitoyloleoyl-phosphatidylcholine, the data are well fit by models in which theta max = 60 +/- 2 degrees for DPH and DPH-PC and 32 +/- 4 degrees for TMA-DPH, with approximately 20% of DPH molecules and 10% of TMA-DPH molecules aligned parallel to the bilayer plane, and a net phospholipid tilt at or near the headgroup region of approximately 30 degrees. The results demonstrate that lipid order can be measured with a spatial resolution of approximately 1 micron2 in cell-size vesicles even with high aperture observation through a microscope.

Imaging cytoplasmic free calcium in histamine stimulated endothelial cells and in fMet-Leu-Phe stimulated neutrophils

Intracellular free [Ca 2+] ([Ca 2+] i) was imaged in Fura-2 loaded human umbilical vein endothelial cells, using dual excitation light, an inverted epifluorescence microscope and a silicon intensified target camera. Oscillations of [Ca 2+] i were induced in these cells by 0.5 μM histamine. Because the image processor contained a large random access memory of 32 Mbyte, it was possible to follow these oscillations over a period of several minutes with a time resolution of approximately 2 s. Plots of the variation of average [Ca 2+] i measured from these images in neighbouring cells showed that not only were the oscillations asynchronous but also that they occurred at different frequencies. This indicates that there is no strong coupling of [Ca 2+] i in adjacent endothelial cells. The rise in [Ca 2+] i stimulated by 0.5 μM histamine was qualitatively imaged at 80 ms intervais by recording images at 380 nm only. [Ca 2+] i-independent variations in image intensity were removed by ratioing these images against a 380 nm image obtained before the stimulation by histamine. Such images revealed that [Ca 2+] i rises first locally and then propagates across the cell at approximately 50 μm.s −1. Neutrophils were imaged following stimulation by fMet-Leu-Phe, also with a time resolution of 2 s. Three responses were determined from the images: (a) the rise in [Ca 2+] i; (b) the cell spreading; and (c) the chemokinesis. Measuring these responses in single cells clearly showed the temporal relationships with the responses occurring in the order listed above.

Rapid Detection and Size Determination of PuO2 Particles Using a Charged-particle Imaging Video Monitor System

A charged-particle imaging video monitor system was constructed and applied to video imaging of the position profile of PuO2 particles on filter paper. The imaging video monitor system consists of a detector head, a silicon intensifier target (SIT) camera, a video frame memory, and a personal computer. The system can display the distribution image of PuO2 particles becoming gradually clearer on a video monitor. The integrated image that is transferred to the computer is analyzed quantitatively for the radioactivity of each PuO2 particle deposited on the filter paper. The system can not only rapidly distinguish PuO2 particle contamination from Rn daughter products, but can also determine the size distribution of the PuO2 particles.

B105 火花点火機関の燃焼撮影用低光量高速度撮影装置の開発

In order to analyze combustion characteristics of the spark ignition engine, a new photographic system was developed to photograph the high-speed and low-luminescent phenomenon of flame propagation.This system, comprised of an electronic framing high-speed camera, high-sensitivity SIT (Silicon intensifier Target) camera and digital image processing device, is capable of taking images of flames with a luminescence of 1, 000 cd/m2 at a speed of 10, 000 frames/second and displaying the images on a screen in real-time.

Viability of inner cell mass versus trophectodermal cells of frozen‐thawed horse embryos

SummaryFluorescein diacetate (FDA), a fluorogenic vital stain, was used in conjunction with a silicon‐intensified target (SIT) camera and computer‐based image analysis to estimate the viability of cells of the inner cell mass (ICM) and trophectoderm (TE) of freshly collected and frozen‐thawed horse embryos. Fluorescence intensities were greater (p<0.02) for fresh ICM (0.1500) and TE (0.1686) than for frozen‐thawed ICM (0.0323) and TE (0.0694). Fluorescence intensity ratios (ICM:TE) also differed (p<0.004) between fresh (0.966) and frozen‐thawed (0.482) embryos. These results indicate that 1) the use of FDA coupled with SIT camera image analysis is a repeatable, objective method for assessing embryo viability and 2) the freeze‐thaw process is more detrimental to ICM cells than to TE cells of horse embryos.

Enumeration and sizing of aquatic bacteria by use of a silicon-intensified target camera linked-image analysis system

Modern image analysis systems incoroporating video cameras and epifluorescence microscopy provide accurate cell counts and size measurements of natural populations of aquatic bacteria. This paper presents comparisons of counts and size measurements made manually and with an Image Technology Corporation Model 2000 image analysis system of bacteria from diverse aquatic environments of southwestern Florida, including the Alafia and Crystal Rivers, Tampa Bay, and the southeastern Gulf of Mexico. The modification of automated counting and sizing software and addition of a silicon-intensified target camera to the image-analysis system enabled collection of data statistically equivalent to manually derived data in all cases. Bacterial cells from 0.18 > 7.00 μm were measured at cell densities up to 1.14 × 10 7 cells·ml −1. Estimation of biomass based upon published values of g C·μm −3 and image analysis-derived cell abundances and volumes ranged between 2.79 × 10 −8 g C·ml −1 for the oligotrophic Crystal River and 1.27 × 10 −6g C·ml −1 for the eutrophic Alafia River.

Charged-particle imaging video monitor system

A charged-particle imaging video monitor system was constructed for video imaging of the distributions of alpha-emitting and low-energy beta-emitting nuclides. The imaging video monitor system consists of a detector head, a silicon intensifier target (SIT) camera, a video frame memory, and a personal computer. The system can display not only the scintillation image due to radiation on the video monitor, but also the integrated video image becoming gradually clearer on another video monitor. The integrated image is transferred to a personal computer for image processing. The distortion of the image is about 5%, and the spatial resolution is about 2 line pairs (lp) mm−1.

Isoproterenol and cyclic AMP increase intracellular free [Ca] in MDCK cells.

We examined the relationship between cyclic AMP and the intracellular free calcium concentration ([Ca]i) in MDCK cells, a hormonally responsive and chloride-secreting cell line. We measured [Ca]i using the calcium-sensitive dye fura-2, fluorescence microscopy, and a silicon intensifier target camera to amplify the signal. Isoproterenol, known to stimulate chloride transport via cyclic AMP, increased [Ca]i from 112 +/- 17 to 373 +/- 53 nM. The rise appeared due to an increase in cyclic AMP: isobutylmethylxanthine enhanced the effect of a submaximal dose of isoproterenol on [Ca]i, the cyclic AMP analogue 8-bromo-cyclic AMP caused [Ca]i to increase from 100 +/- 8 to 278 +/- 40 nM, and direct activation of adenylate cyclase with forskolin increased [Ca]i from 192 +/- 29 to 279 +/- 35 nM. The rise in [Ca]i after cyclic AMP may be due to an influx of calcium from the outside: the cyclic AMP-induced increase in [Ca]i was prevented either by lowering extracellular [Ca] or by addition of 1 mM lanthanum. The mechanism by which calcium enters the cell may not be a calcium channel because neither verapamil nor nitrendipine prevented cyclic AMP from increasing [Ca]i. Cyclic AMP also does not appear to act directly on sodium-calcium exchange. [Ca]i increased as well in low [Na] as in high [Na] following addition of the nucleotide. Thus, isoproterenol, acting through an increase in [cyclic AMP] causes an increase in [Ca]i in MDCK cells. The source and route of entry of calcium into the cytoplasm remain uncertain.

3D biological imaging using TSRLM confocal microscopy

Tandem scanning reflected light (TSRLM) confocal microscopy permits the imaging of optical sections through translucent material in real time. We are currently using a TSRLM microscope to examine the morphological components of biological tissue. Optical sections of either stained or unstained biological specimens can be rapidly obtained by a through focus series of a potentially dynamic specimen. Each optical section shown here is an average of 256 512X512x8 bit frames collected from a silicon intensified target camera at video rates.Unstained living frog muscle fibers were imaged with sufficient contrast to obtain 1μ optical sections traversing a total distance of approximately 80μ. Fig. 1 shows 4μ sections through a blood vessel. Frog red cells can be clearly seen in panels 1c-e. Frog muscle fibers were mounted and stretched to a defined length. The total fiber volume was easily imaged by progressive 1μ sections.

Voltage dependence of intracellular [Ca2+]i transients in guinea pig ventricular myocytes.

[Ca2+]i transients, elicited by voltage-clamp depolarization of single guinea pig cardiac ventricular cells, were observed through use of the fluorescent Ca2+ indicator, fura-2. Individual cells, loaded with fura-2 either by internal perfusion or by exposure to fura-2/AM, were studied with the use of an inverted microscope that was equipped with ultraviolet epifluorescence illumination, an intensified silicon intensifier target camera, and a photomultiplier tube. Variation of membrane voltage and exposure of cells to verapamil (a Ca2+ channel blocker) and ryanodine (which was assumed to abolish selectively the release of Ca2+ from the sarcoplasmic reticulum) were used to investigate the cellular processes that determine the [Ca2+]i transient. The principal results of the study are: When appropriate methods are used, the properties of cytosolic fura-2 inside guinea pig cells are similar to those of fura-2 in solution, irrespective of the method of loading. The amplitude (at 100 msec) of verapamil-sensitive fluorescence transients elicited by pulse depolarization (range -30 to 80 mV) has a bell-shaped dependence on membrane voltage (maximum at 10 mV). Rapid, ryanodine-sensitive and verapamil-sensitive "tail transients" are elicited on repolarization from membrane potentials greater than 30 mV; their amplitude increases as the amplitude of the preceding pulse increases. The amplitude of slow fluorescence transients that are insensitive to verapamil and ryanodine increases continuously with membrane potential throughout the range -20 to 80 mV. The voltage dependence and pharmacology of the rapid transients elicited by pulse depolarization or by repolarization are consistent with their having arisen from Ca2+ released from the sarcoplasmic reticulum, via Ca2+-induced Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of PaO2 on cerebral macro- and microcirculation as observed by light reflection: time course of changes.

A silicon intensified target camera was used to study cerebral cortical vessels of the cat through a skull window implant, and red cell content changes were measured by light reflectance. Red cell content changes were observed in cerebral arterioles, capillaries, and venules when the PaO2 was decreased by lowering the PiO2. The time course of change in the diameter of the arterioles and venules was measured by selecting a cross section of the vessel plus some surrounding tissue. From the averaged cross-sectional reflectance signal, the change in vessel diameter was followed as a function of time following the PiO2 change. All vessels of greater than 10 microns were observed in focus. Substantial areas where no vessels could be discriminated would contain only capillaries, and changes in light reflectance from such areas would indicate changes in capillary red cell content. The time course of these changes following a step decrease in PiO2 was recorded. Results show that the sequence of red cell content increase in cerebral microcirculation during hypoxia is capillary before venule and arteriole. The times of initial red cell content increase are 37.9 +/- 7 s, 59.7 +/- 7.9 s, and 60.8 +/- 9.1 s, respectively. These results suggest an increase in the capillary bed red cell content as the initial response to hypoxia, but venules and arterioles change only on longer exposure to hypoxia. The sequence of the increase in red cell content suggests the capillaries rather than the arterioles are the vessels which respond to the oxygen autoregulation signal.

Quantitative fluorescence image analysis.

So far quantitative fluorescence histochemistry has been carried out separately by two different methods, cytofluorometry for densitometry and image analysis for morphometry. We described here a method of quantitative fluorescence image analysis to realize simultaneous densitometric and morphometric measurements. This method gives complementary informations of quantity of substances of interest together with data on shape and pattern of the biological materials.DAPI staining for DNA is employed. The fluorescence images are successively taken from SIT (Silicon Intensifier Target) camera and sent to an intelligent color terminal. The data obtained from the image analyzer are compared with cytofluorometric data. Integration technique can remove random noise from the image.Brightness values of all pixels in the frame buffer or brightness of the pixels on nuclear area were summed up. Both parameters responded linearly and proportionally to fluorescence values determined by cytofluorometry within an adequate range. Nuclear DNA measurements revealed that rat hepatocytes are composed of diploid, tetraploid and octaploid subpopulations with proportional DNA contents. All cerebellar neurons showed DNA values strictly in diploid class.The conclusion is that both densitometric and morphometric informations can be obtained simultaneously by quantitative fluorescence image analysis within an adequate dynamic range. This will enable us to extend quantitative investigation on histochemical reaction and shape analysis in variety of biological systems.

Microscopic spectral imaging using a video camera.

A technique is described which permits the simultaneous acquisition of multiple fluorescent emission and/or absorption spectra from discrete regions of a specimen under microscopic observation. The instrument consists of a modified inverted microscope, an optical diffraction grating, a silicon intensified target (SIT) camera, and a digital video image processor. Observation of the zero diffraction order of the grating with the SIT camera permits an optical slice of the specimen to be selected by positioning the region of interest over the entrance slit of the grating housing. To obtain the spectral characteristics of this optical slice, the grating is rotated to impinge the first order diffraction on the camera. The video image of this first order diffraction maintains spatial integrity along the slit's long axis and provides spectral dispersion on the perpendicular axis. Thus, each of the horizontal video lines along the long axis of the slit represents a spectral analysis of the corresponding spatial location within the specimen. The spectral resolution (0.2 nm/channel) of each video line is determined by the resolution of the camera system in conjunction with the resolution of the grating. The image processing system acquires and processes all 500 spectra in 33 ms and permits the accurate localization of the source of each spectrum in the slice. This type of topological spectral analysis permits the determination of both spatial and spectral characteristics of intrinsic or extrinsic chromophores within the specimen. In addition, this technique permits the detection of and the possible correction for photobleaching, light scattering and image plane effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy-Filtered TEM With an External Spectrometer: Problems and Solutions

Some of the requirements for obtaining reliable compositional images with high resolution energy filtered electron microscopy (EFEM) were tested through EFEM of negatively stained (with uranyl acetate) catalase crystals. We present energy filtered inelastic images of the catalase crystals showing the distribution of both uranium (U) and carbon (C) to 3.4 nm resolution, and introduce a more rigorous approach of image analysis of inelastic CTEM images. This approach is intended to eliminate two artifacts commonly encountered by EFEM, i.e. (a) contrast arising from variations in the shape of energy-loss background as a function of position (b) the amplitude contrast modulation at all energies. We have performed EFEM with a spectrometer that is a homogeneous field magnetic sector corrected for second order energy aberrations and is mounted below the projection chamber of a Philips EM (FEG) 400. The energy filtered electron image was converted into a light image by a transmission phosphor screen, detected with a silicon intensifier target(SIT) camera and displayed on a TV monitor.

Improvements in Photomultiplier and TV Camera Tubes for Nuclear Medicine

Electro-optic devices such as photomultiplier tubes or TV camera tubes are used in nuclear medicine applications directly or indirectly as sensors of scintillations resulting from excitation of phosphor or scintillators by x- or ..gamma..-rays. Ideally, a detector would provide 100 percent quantum efficiency, have a read-out mechanism whose noise does not obscure the response from a single photon, and have a response time sufficiently short so as not to degrade the scintillation pulse. Photomultiplier tubes have been improved in quantum efficienctly and in response time. Photomultiplier tubes have been improved in quantum efficiency and in response time. Photocathodes, however, do not run better than 20 to 30 percent quantum efficiency. Response times can be less than 1 ns so that for the detection of ordinary scintillations, this is more than adequate. Vidicon camera tubes utilize photoconductive type sensors which can have essentially 100 percent quantum efficiency in parts of the spectrum. Unfortunately the read out mechanism introduces lag and noise so that the inherent photoconductor quantum efficiency cannot be fully realized. Recently developed silicon intensifier target (SIT) camera tubes provide a read out mechanism which overcomes much of the amplifier limitation inherent in vidicons, but again, the primary sensor ismore » a photocathode so that 100 percent quantum efficiency is not achieved. SIT tubes are capable, however, of detecting remarkably low level light images with a minimum of lag.« less