A recording chamber for small volume slice electrophysiology.

Electrophysiological recordings from brain slices are typically performed in small recording chambers that allow for the superfusion of the tissue with artificial extracellular solution (ECS), while the chamber holding the tissue is mounted in the optical path of a microscope to image neurons in the tissue. ECS itself is inexpensive, and thus superfusion rates and volumes of ECS consumed during an experiment using standard ECS are not critical. However, some experiments require the addition of expensive pharmacological agents or other chemical compounds to the ECS, creating a need to build superfusion systems that operate on small volumes while still delivering appropriate amounts of oxygen and other nutrients to the tissue. We developed a closed circulation tissue chamber for slice recordings that operates with small volumes of bath solution in the range of 1.0 to 2.6 ml and a constant oxygen/carbon dioxide delivery to the solution in the bath. In our chamber, the ECS is oxygenated and recirculated directly in the recording chamber, eliminating the need for tubes and external bottles/containers to recirculate and bubble ECS and greatly reducing the total ECS volume required for superfusion. At the same time, the efficiency of tissue oxygenation and health of the section are comparable to standard superfusion methods. We also determined that the small volume of ECS contains a sufficient amount of nutrients to support the health of a standard brain slice for several hours without concern for either depletion of nutrients or accumulation of waste products.

Multi-step preparation technique to recover multiple metabolite compound classes for in-depth and informative metabolomic analysis.

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes. Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 µl.  Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.

Tandem mass spectrometry of bilin tetrapyrroles by electrospray ionization and collision-induced dissociation.

RATIONALE: Bilins are metabolic products of hosts and bacteria on porphyrins, and are markers of health state and human waste contamination. Although bilin tandem mass spectrometry reports exist, their fragmentation behavior as a function of structure has not been compared, nor has fragmentation been examined as a function of collision energy. METHODS: The fragmentation of bilins generated by positive ion mode electrospray ionization is examined by collision-induced dissociation (CID). CID on a quadrupole ion trap and on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer as a function of collision energy is compared. Methyl esterification was used to deduce which product ions contain the inner pyrrole rings. FT-ICR high mass accuracy measurements were used to determine the formulas of the resultant product ions. RESULTS: The central carbon's bonding to the inner pyrrole rings influences fragmentation. Bilirubin is unique because fragmentation adjacent to the central methylene group between innermost rings predominates, and loss of a terminal pyrrole is observed only with helium collision gas. The other bilins lose the terminal pyrroles first; as CID energy is increased, additional fragmentation due to neutral losses of small molecules such as H(2)O, CO, CO(2), and methanol occurs. CONCLUSIONS: Based on these observations, fragmentation schemes for the bilins are proposed that are strongly dependent on the molecular structure and collision energy; only bilirubin fragmentation is influenced significantly by the collision gas used. This report should have value in identification of this class of molecules for biomarker detection.

Ultra high-mass resolution paper spray by fourier transform ion cyclotron resonance mass spectrometry.

Paper Spray Ionization is an atmospheric pressure ionization technique that utilizes an offline electro-osmotic flow to generate ions off a paper medium. This technique can be performed on a Bruker SolariX Fourier transform ion cyclotron resonance mass spectrometer by modifying the existing nanospray source. High-resolution paper spray spectra were obtained for both organic and biological samples to demonstrate the benefit of linking the technique with a high-resolution mass analyzer. Error values in the range 0.23 to 2.14 ppm were obtained for calf lung surfactant extract with broadband mass resolving power (m/Δm(50%)) above 60,000 utilizing an external calibration standard.