Microfabricated solenoids and Helmholtz coils for NMR spectroscopy of mammalian cells.

NMR-microprobes based on solenoids and Helmholtz coils have been microfabricated and NMR-spectra of mammalian cells have successfully been taken. The microfabrication technology developed for these probes consists of three electroplated copper levels for low resistance coils and three SU-8 layers for the integration of microchannels. This technology allows fabricating solenoids, Helmholtz and planar coils on the same wafer. The coils have inner diameters in the range of 160 to 400 microm and detection volumes of 5 to 22 nL. The solenoid and Helmholtz coils show improved RF-field characteristics compared to a planar coil fabricated with the same process. The fabricated solenoid has a particularly low resistance of only 0.46 Omega at 300 MHz. Moreover, it is very sensitive and has a very uniform RF-field, but shows large line width. The Helmholtz coils are slightly less sensitive, but display a far narrower line width, and are therefore a good compromise. With a Helmholtz coil, a SNR of 620 has been measured after one scan on 9 nL pure water. An NMR-microprobe based on a Helmholtz coil has also been used to take spectra of CHO cells that have been concentrated in the sensitive region of the coil with a mechanical filter integrated into the channel.

NMR spectroscopy and perfusion of mammalian cells using surface microprobes.

NMR spectra of mammalian cells are taken using surface microprobes that are based on microfabricated planar coils. The surface microprobe resembles a miniaturized Petri dish commonly used in biological research. The diameter of the planar coils is 1 mm. Chinese Hamster Ovaries are immobilized in a uniform layer on the microprobe surface or patterned by an ink-jet printer in the centre of the microcoil, where the rf-field of the planar microcoil is most uniform. The acquired NMR spectra show the prevalent metabolites found in mammalian cells. The volumes of the detected samples range from 25 nL to 1 nL (or 50,000 to 1800 cells). With an extended set-up that provides fluid inlets and outlets to the microprobe, the cells can be perfused within the NMR-magnet while constantly taking NMR spectra. Perfusion of the cells opens the way to increased cell viability for long acquisitions or to analysis of the cells' response to environmental change.

Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array.

We present parallel single molecule detection (SMD) and fluorescence correlation spectroscopy (FCS) experiments with a fully integrated complementary metal oxide semiconductor (CMOS) single-photon 2x2 detector array. Multifocal excitation is achieved with a diffractive optical element (DOE). Special emphasis is placed on parallelization of the total system. The performance of the novel single-photon CMOS detector is investigated and compared to a state-of-the-art single-photon detecting module [having an actively quenched avalanche photodiode (APD)] by measurements on free diffusing molecules at different concentrations. Despite the order of magnitude lower detection efficiency of the CMOS detector compared to the state-of-the-art single-photon detecting module, we achieve single molecule sensitivity and reliably determine molecule concentrations. In addition, the CMOS detector performance for the determination of the fraction of slowly diffusing molecules in a primer solution (two-component analysis) is demonstrated. The potential of this new technique for high-throughput confocal-detection-based systems is discussed.

Assessment of Left Ventricular Area at Risk by Myocardial Contrast Two-Dimensional Echocardiography: An Evaluation of a New Animal Model.

BACKGROUND: New echocardiographic contrast agents are commonly tested in the dog model. However, this species has a number of drawbacks, including difficulties in experimentation, cost, and ethical considerations. The rabbit has a number of advantages due to its relative simple coronary circulation. The present study was designed to evaluate the rabbit model for determination of areas of risk (ARs) by contrast echocardiography. METHODS: Eight rabbits were intubated and mechanically ventilated after occlusion of the left coronary artery with a ligature. The transducer (operating at 7.5 MHz) was positioned on the right ventricle through a right thoracotomy. The images were obtained after intra-aortic injection of 1 ml of Albunex, followed by 3 ml of dye (Blue Uniperse) for histological analysis postmortem. The ARs were obtained after circumscription of the various echocardiographic and histological images. RESULTS: Excellent echocardiographic images were obtained, largely due to the hemodynamic stability of the rabbit to ischemia. Echocardiographic ventricular areas, absolute AR, and relative AR correlated closely with postmortem data (r = 0.86, 0.94, and 0.94, respectively). The measurements were highly reproducible with low variability. CONCLUSIONS: The rabbit model shows promise for study by contrast echocardiography of myocardium subjected to ischemia. This method for determination of ARs was validated against postmortem findings. The method also should be of value in the evaluation of reperfusion.