Innovative instrumentation for microarray scanning and analysis: application for characterization of oligonucleotide duplexes behavior.

Accuracy in microarray technology requires new approaches to microarray reader development. A microarray reader system (optical scanning array or OSA reader) based on automated microscopy with large field of view, high speed 3 axis scanning at multiple narrow-band spectra of excitation light has been developed. It allows fast capture of high-resolution, multi-fluorescence images and is characterized by a linear dynamic range and sensitivity comparable to commonly used photo-multiplier tube (PMT)-based laser scanner. Controlled by high performance software, the instrument can be used for scanning and quantitative analysis of any type of dry microarray. Studies implying temperature-controlled hybridization chamber containing a microarray can also be performed. This enables the registration of kinetics and melting curves. This feature is required in a wide range of on-chip chemical and enzymatic reactions including on-chip PCR amplification. We used the OSA reader for the characterization of hybridization and melting behaviour of oligonucleotide:oligonucleotide duplexes on three-dimensional Code Link slides.

A new image analysis system for biological dosimetry by fluorescent in situ hybridization. Step 1: metaphase finder and automatic metaphase acquisition validation.

Because of the large number of cells to be analyzed in cases of overexposure to ionizing radiation, an automated imaging system is desirable for scoring both translocations and dicentrics. This system should include three essential steps: automatic metaphase finding, automatic image capture at high magnification, and, finally, optimized data analysis for aberration interpretation. We evaluated a new image analysis system (CYTOGEN, IMSTAR, France) and found that its metaphase finder saved time, as much as quadrupling the speed of scoring chromosomal aberrations. Automatic metaphase selection did not appear to induce bias. We confirmed the equivalence of observing aberrations on a screen after automatic image capture and direct observation under a microscope. This work validated all of the steps necessary for obtaining images for automatic chromosomal aberration detection. The protocols for the detection of translocations may now be applied for biological dosimetry. This step will be validated in a future study.

A CpG-rich RNA and an RNA helicase tightly associated with the DNA demethylation complex are present mainly in dividing chick embryo cells.

In the developing chicken embryo, active DNA demethylation requires both RNA and proteins (Nucleic Acids Res. 25, 2375-2380, 1997; ibid. 25, 4545-4550, 1997, FEBS Lett. 449, 251-254, 1999a). In vitro assays indicate that in the 5- and 12-day-old embryos the highest specific activity of 5-methylcytosine DNA glycosylase is found in the brain, the eyes and the skin. In situ hybridization with antisense CpG-rich RNA tightly associated to the DNA demethylation complex shows a restricted expression pattern only in proliferating tissues such as the neuroepithelia of the brain in 5-day-old embryos. The RNA is absent in differentiated tissues like the skeletal and heart muscle, liver and the crystallin-producing cells in the lens. The CpG-rich RNA is transcribed in a developmental stage-specific rather than in a cell-specific manner. In contrast transcripts of DNA methyltransferase are found in dividing and quiescent cells. In situ hybridization with a probe of a RNA helicase which is also associated with the DNA demethylation complex shows a very similar localization in mitotically active tissues as the CpG-rich RNA. The content of 5-methylcytosine in individual cells was determined with a specific monoclonal antibody and cytometric analysis on tissue sections. The results indicate that proliferating cells have on the average 15% more methylated cytosines than non-dividing cells. This represents roughly 3x10(6) more methylation sites per haploid genome.

Computer-aided quantification of RNA levels detected by in situ hybridization of tissue sections.

A semi-automatic program, designed for non-computer scientists, was developed for quantification of RNA levels detected by in situ hybridization in heterogeneous tissues. A video camera was used to acquire microscopic images of autoradiographed tissue sections which are then digitized on a video monitor for semi-automated quantification of silver grains. We describe a data entry and analysis procedure for systematic quantification of RNA levels in which about 300 cells per tissue sample can be analysed within 10 min. When compared with visual counting, computer-aided quantification was found to be more objective and reliable, with the highest variation coefficient between individuals being 7.5% using computer-aided quantification, compared to 24% with manual counting of the same section areas. A comparative study of c-myc oncogene expression in 11 mammary adenocarcinomas from 3 independent experiments showed the good reproducibility of results using the computer-aided method, with an 18% maximum variation between experiments. The program, with its simple user-interface, reliability and rapidity, is convenient for measuring specific genetic expression levels in clinical studies requiring large numbers of specimens.

A technique for the correction of scattered radiation in a PET system using time-of-flight information.

The time-of-flight information has been used in the development of a correction technique for the effects of radiation scattering in a positron emission tomography system. A two-dimensional function describing the scattered radiation distribution is added to the response of the system. A deconvolution filter that depends on parameters that were experimentally determined from measurements of a linear source located in a scattering medium is derived. The method was tested on cold spot cylindrical phantoms. The relative intensity of events from scattered radiation is significantly reduced and the contrast is thus improved. This technique does not require any additional computing time and can be included in standard reconstruction procedure.

Brain tumor protein synthesis and histological grades: a study by positron emission tomography (PET) with C11-L-Methionine.

Brain protein synthesis may be evaluated in vivo by a PET three compartment methionine model. 14 human brain tumor patients were studied. Protein synthesis rate (PSR) was increased in any glial tumor even in low grades, but this increase was statistically more important in anaplastic tumor. Radiotherapy action was evaluated in two patients. Local tumoral PSR was reduced to normal brain PSR after treatment. No difference was seen in normal cortex contralateral to the lesion between pre and post radiotherapy examination. 11 C-L-Methionine incorporation measured by PET looks as a very sensitive method for studying tumor metabolism and treatment effects.

Tomographic mapping of brain intracellular pH and extracellular water space in stroke patients.

Functional images of regional intracellular pH (pHi) and of fractional volume of extracellular water (FVECW) were obtained in 10 patients with recent hemispheric infarction (between 10 and 19 days after onset of symptoms) using positron emission tomography (PET). The volume of extracellular water relative to that of total water was evaluated in each pixel of the PET scan 7-8 h after injection of 76Br. The pHi image was calculated from the data obtained after injection of [11C]5,5-dimethyl-2,4-oxazolidinedione and from the FVECW image. Regional CBF, oxygen extraction, and oxygen metabolism were also measured in the same patients. In normal hemisphere, mean +/- SD values for FVECW and pHi were 0.12 +/- 0.01 and 6.86 +/- 0.11, respectively. FVECW was increased in the infarcted area in most patients. pHi was increased in the infarct in seven patients and unchanged in three. The increase in pHi was not correlated with changes in FVECW, CBF, or CMRO2, but there was a significant correlation with the decrease in oxygen extraction fraction in the same region. Thus, the decreased H+ content in the infarcted area was correlated with the occurrence of perfusion in excess of metabolic demand. An alkaline shift in pHi enhances the glycolysis rate and could explain why the glucose metabolism is less affected than the oxygen metabolism in recent cerebral infarction. The pHi measured in the infarct could represent mainly the pHi of phagocytic cells that use aerobic glycolysis to synthesize hydrogen peroxide.

Kinetics and displacement of [11C]RO 15-1788, a benzodiazepine antagonist, studied in human brain in vivo by positron tomography.

The brain regional distribution and kinetics of RO 15-1788, a benzodiazepine (BZD) antagonist labeled with 11C was studied by time-of-flight positron tomography after intravenous injection in four normal human volunteers. In two control studies, there was a high uptake of [11C]RO 15-1788 in gray matter structures initially (brain/blood ratio approximately 3), and subsequent retention that was highest in cerebral cortex, a structure known to have a high density of BZD receptors in vitro. Variation in tissue kinetics of [11C]RO among different gray matter structures may, however, suggest regional differences in binding characteristics or environment of BZD receptors. In two displacement studies, unlabeled RO 15-1788 was injected ten minutes after the radioligand: there was an immediate and marked washout of [11C]brain radioactivity that reached 70% in the occipital cortex with a 0.05 mg/kg dose (indicating a high specific to non-specific binding ratio) but was less prominent with a 0.01 mg/kg dose. These data suggest that [11C]RO 15-1788 may be useful for in vivo mapping of human brain BZD receptors using positron tomography.

76Br-bromospiroperidol: a new tool for quantitative in-vivo imaging of neuroleptic receptors.

Bromine-76 labeled bromospiperone has been prepared with a very high specific activity (greater than 1 Ci/mumole). In-vivo studies in rat corroborated by PET studies in baboon have shown that the regional concentration of this radioligand parallels the morphologic distribution of dopamine receptors and that its binding in the striatum is saturable and displaceable. Tomographic images show a clear delineation of the striatal region 2.5 hours after administration of the radioligand.

Local interrelationships of cerebral oxygen consumption and glucose utilization in normal subjects and in ischemic stroke patients: a positron tomography study.

With the use of positron emission tomography (PET) and the 15O steady-state-[18F]fluorodeoxyglucose combined method, the local interrelationships between the cerebral metabolic rate for oxygen (CMRO2) and the cerebral metabolic rate for glucose ( CMRGlc ) were investigated in control subjects and in stroke patients. In addition to the classic in vivo autoradiographic approach, a kinetic method was used to measure CMRGlc because it was expected to be more reliable in cerebral ischemia. In control subjects local coupling between CBF, CMRO2, and CMRGlc was confirmed, and acceptable values for the CMRO2/ CMRGlc ratio were found; the latter, however, was lower in white matter than in gray. Uncoupling between CMRO2 and CMRGlc was observed in all stroke patients, suggesting that (1) enhanced anaerobic glycolysis occurred both in reperfused recent infarcts and in chronically ischemic tissue, and (2) substrates other than blood-borne glucose were being oxidized at the borders of recent infarcts. However, methodological uncertainties presently make such observations only tentative. Finally, a coupled depression of CMRO2 and CMRGlc was found in the contralateral cerebellum.

Coupling between regional blood flow and oxygen utilization in the normal human brain. A study with positron tomography and oxygen 15.

Using the oxygen 15 continuous-inhalation technique and positron emission tomography (PET), the regional cerebral blood flow (rCBF), oxygen extraction fraction (rOEF) and oxygen utilization (rCMRO2) were measured in 19 control subjects. The rCBF and rCMRO2 values, computed for small regions of interest (ROI), were within accepted normal ranges. Larger, more convenient ROI provided values for gray and white matter regions that were, respectively, lower and higher than those found using small ROI. The rOEF values were not affected by the ROI size, but seemed to slightly overestimate real OEF. These findings may be explained by (1) the partial volume effect, and (2) some limitations of the 15O steady state model. The 15O-PET technique therefore provides representative but clinically useful physiological indexes. In addition, our study demonstrated the normal coupling between local oxygen supply and demand.

[Water diffusion and measurement of cerebral blood flow by C15O2 inhalation]. / Diffusion de l'eau et mesure du debit sanguin cerebral par inhalation de C15O2.

Measurement of local Cerebral Blood Flow (CBF) using the C15O2 continuous inhalation technique coupled with Position Emission Tomography (PET) rests on the assumption that the in vivo labelled water (H215O) of blood diffuses freely within the brain water pool. This requirement however, may be disputed: in the rhesus monkey, Eichling et al (1974) showed a linear relationship between the single-pass extraction of water (E) and CBF after intra-carotid H215O injection, such that E decreases as CBF increases. Such a limitation in water transport across the blood brain barrier has also been suggested to exist in man (Paulson, 1977). In order to verify its possible effects on measured CBF in baboons, simple computer analysis was performed comparing the data obtained with CBF values reported in similar experiments, but using a freely diffusible inert gas (133Xe). This allowed calculation of a water extraction index (IE), which was shown to decline as CBF increased. This result may be considered as a presumption of the part played by the limited water diffusibility on the final CBF values, but other factors must also be considered, such as the inescapable admixture of grey and white matter (partial volume effect), a limitation of present day PET technology.

A Regularizing Method for Quantitative SPECT Reconstruction.

The problem of successive transverse plane reconstruction in single photon emission computerized tomography (SPECT) is modeled in its more general form, which implies the definition of emission tomographic operators (ETO's) for which an analytical solution can be derived. The properties of the attenuated tomographic operator (ATO) are described and discussed, including the attenuation which is distributed on the reconstruction domain. For this particular operator, a regularizing method (RIM) is proposed, for which it is demonstrated and tested with simulation studies that a filtered, accurate solution can be extracted for the tomographic images as obtained using a single photon emission tomograph based on a rotating gamma camera in clinical use.

Estimates of kidney volume by single photon emission tomography: a preliminary report.

Algorithms for the estimation of organ volumes have been developed which may provide whole organ volumes and which may or may not, at the operator's choice, take into account any voids which may be contained within the organ. These algorithms have been applied to the estimation of the volume of the kidneys, in normal subjects and in patients with renal disease. The dimensions of the kidneys were similar to those derived from ultrasound study measurements which were performed independently. Kidney volume should prove especially useful in the diagnosis of early graft rejection and in the diagnosis of glomerulonephritis.

Determination of organ volume by single-photon emission tomography.

A method for estimation of organ volume is proposed, based on analysis of individual slices obtained from SPET images. In a phantom simulating clinical circumstances, the data show that the level a threshold at 46% of the maximum activity predicts most closely the true volume over a wide range above one liter. The level at 45% predicted better volumes of less than one liter. For phantoms of 839 ml or less, the error was 6.3 ml (one standard error of estimation). This level seems to be independent of the plane or position of the phantom and also independent of the amount of scattering material around it. Nonradioactive voids ("holes") within a phantom may be included or excluded at will when their edges are not tangent to the edge of the phantom. In such cases, their edges are not distinguishable from the edge of the phantom and their volumes are excluded. Knowledge of organ volumes has both diagnostic and therapeutic importance and could lead to a more precisely quantitated total of the radioactivity contained in an organ or space.

An analytical approach to single photon emission computed tomography with the attenuation effect.

The problem of transverse plane reconstruction from an ensemble of projections is considered in its general formulation and an analytically exact solution to the attenuated tomographic operator is proposed. Such a technique, called the regularizing iterative method (RIM), allows the introduction of a priori knowledge on the size and shape of the activity distribution and in principle on the exact attenuation distribution. The relaxation factor used is so named because it provides noise filtering for a small number of iterations. The effectiveness of RIM was studied in the single photon emission computed tomography (SPECT) problem with the aim of correcting for attenuation before quantitative study. Its application involves the use of a rotating scintillation camera connected to a mini-computer system. Various mathematical and physical phantoms were studied, and a satisfactory attenuation correction was always obtained in the final image with an improvement in the contrast and signal-to-noise ratio. Preliminary clinical studies on liver transverse sections seems to indicate an improvement in deep lesion detectability, compared with images obtained by the filtered (Ramp) back projection technique.

Effects of oxygen breathing on regional distribution of ventilation and perfusion in hypoxemic patients with chronic lung disease.

Regional distribution of ventilation and perfusion were measured in 17 hypoxemic and 16 normoxic patients using 13N as tracer. The effect of 100% and 50% O2 breathing was tested. No variation was observed in normoxic patients. In hypoxemic patients, perfusion was not modified significantly during O2 breathing. Poorly-ventilated zones hypoventilated even more during O2 breathing. It is suggested that this effect may have been due to (1) a collapse of some alveoli, (2) a variation in the mechanical properties of the surrounding opened alveoli, or (3) a possible modification in tidal volumes and functional residual capacities during O2 breathing.

Rotating conventional gamma camera single-photon tomographic system: physical characterization.

A whole-body single-photon emission computed tomography system has been evaluated in terms of its physical performance. It consists of a standard GE 400 T maxi camera rotating on a ring stand, coupled to an Informatek Simis 3 computer system. In its standard mode of operation, 64 or 128 successive views of 64 X 64 or 128 X 64 matrices are collected at regular angular samples. The reconstruction of up to 64 possible adjacent transverse sections, sorted into coronal, sagittal, and oblique sections, is obtained by filtered backprojection. A conventional parallel-hole collimator is used. The uniformity of the camera field of view is corrected by the GE hardware module. The resolution in the transverse plane is typically of 15.5 mm in a 20 cm Lucite phantom, and independent of radial distance. The physical slice thickness is 19 mm (full width at half maximum of the longitudinal response using a point source) with only slight variations along the diameter of the image. The sensitivity is approximately 7,000 cps mCi-1 as measured using a 99m Tc thin source, enabling images of reasonable signal-to-noise ratio to be obtained in 10 min. The effect of the energy window, the number of angular samples, and the linear sampling modes on the transverse resolution and contrast is measured and discussed.

Noninvasive tomographic study of cerebral blood flow and oxygen metabolism in vivo. Potentials, limitations, and clinical applications in cerebral ischemic disorders.

The non-invasive continuous inhalation technique of C15O2 and 15O2 coupled with positron emission tomography (PET) provides brain images that are thought to represent local cerebral blood flow (CBF) and oxygen extraction fraction (OEF). Experimental studies in baboons have confirmed that C15O2 inhalation allows tomographic measurement of CBF. The numerous difficulties involved in PET absolute quantitation are stressed, as well as some limitations inherent to the 15O inhalation model. However, the values for local CBF, OEF and CMRO2 obtained in normal young subjects are satisfactory in view of the above-mentioned limitations. The clinical application to recent cerebral infarction has allowed two opposite types of flow-metabolism uncoupling to be identified, which appear to be often predictive if tissue prognosis. The time course of spontaneous changes in CBF and OEF within the infarct is also described. Our studies have, in addition, revealed the previously unknown phenomenon of "crossed cerebellar diaschisis" in supratentorial infarction. Lastly, a state of chronic watershed ischemia, potentially reversible by surgical revascularization, has been identified as presumably involved in the progression of watershed necrosis. The clinical potentials of this method appear considerable.