Construction of picoliter-nanoliter self-filling volumetric pipettes.

A technique is described whereby-volume, self-filling micropipettes can be constructed with minimal time and effort. The delivery volumes of such pipettes may range from several picoliters to more than 100 nl. The delivery of a given pipette of even the smallest volume is precise, generally yielding standard deviations much less than 1% of the mean volume as measured by liquid-scintillation counting of isotope dilutions. The technique offers considerable advantage to previously described techniques for constructing small constant-volume pipettes.     

The release of drugs from iontophoretic pipettes

A theoretical account is given of the relationship between applied current and release of drug ions from an iontophoretic pipette. The Nernst-Planck equation predicts a non-linear relationship except in the region of large ejecting currents. No finite value of retaining current can completely suppress spontaneous diffusional leakage. These conclusions apply to both conical and cylindrical tip geometries. An expression is derived giving release as a function of pulsed charge applied to a conical pipette. This relation is also non-linear. Dose-response curves constructed using charge as a measure of drug ejection should be interpreted with some caution.     

Phospholipid bilayers made from monolayers on patch-clamp pipettes.

Phospholipid bilayers were made from phospholipid monolayers at the air/water interface on patch-clamp pipettes. Lipid bilayers were characterized using the K+ carrier nonactin and the channel formers gramicidin and alamethicin. Bilayers were also formed from monolayers spontaneously assembled in a suspension of native vesicles from cardiac sarcolemma and lobster axonal membranes and an excess of lipids. In these types of bilayers we observed several different channels including one contained in the axonal membrane that shows delayed rectifier behavior. This technique permits the study of reconstituted channels on a time scale and noise comparable to cellular patch-clamp standards.     

Errors inherent in the use of piston activated pipettes

The accuracy and reproducibility of the volume of solution delivered from a number of piston-activated pipettes of different makes and sizes has been tested under a variety of experimental conditions. The results indicate that these devices deliver the nominal volume only when both the solution to be sampled, and the whole pipette are at the same temperature. When a temperature differential exists, the absolute accuracy is affected, and unless particular care is taken, the reproducibility is relatively poor. Thus it is only under well-controlled conditions that the behaviour of piston-activated pipettes falls within the specifications quoted by the manufacturers.     

Effect of mercury column on the microdynamics of the piezo-driven pipettes

This study is on an interesting phenomenon concerning cellular microinjection procedures which are used for various biomedical applications, and in particular intracytoplasmic sperm injection. Recent years have brought considerable practical improvements in these operations. One of them suggests aspirating a very small quantity of mercury in the injection pipettes prior to piercing into cells. This process is proven to enhance the rate of success considerably. We present a unique study in determining the influence of mercury on the microdynamics of the pipette. The effort contains both numerical simulations and corresponding experimental verification. Ultimately we offer two critical results: (1) The mercury column increases the mass loading and expectedly decreases the natural frequencies of the pipette and (2) The lateral oscillations, which play a destructive role in piercing, are subdued in amplitude due to the mass loading of mercury. Simulation results are presented, which are also verified experimentally using high-speed digital imaging. As a consequence of these findings we also propose some alternative design directions for future microinjection devices.     

'Crook' pipettes: embryonic emigrations from agriculture to reproductive biomedicine

While cloning, stem cells, and regenerative medicine are often imagined in a futurial idiom--as expectations, hype, hope and promises--this article approaches the remaking of genealogy in such contexts from a historical route. Through a series of somewhat disparate historical connections linking Australian sheep to the development of clinical IVF and the cloning of Dolly at the Roslin Institute in Scotland in 1996, this article explores the linkages through which agriculture, embryology, and reproductive biomedicine are thickly intertwined. Key to this examination is not only the history of experimental sheep breeding, and its somewhat unexpectedly genealogical connections to (Australian) national identity ('wool in the veins'), but also the re-emergence of a distinctive frontier ethos in the context of assisted conception, and later human embryonic stem cell derivation. I have set this scene of genealogical interconnection against the criss-crossing traffic between Britain and Australia, and the wool trade, to emphasise the importance of global, as well as local, connections in the bloodlines of animals such as Dolly. In sum, this article examines the idea of the 'biological frontier' by exploring its histories as a means to offset the assumption that this frequently encountered idiom describes a future that is, or must be, by definition, unknown and unknowable.     

Targeted axon-attached recording with fluorescent patch-clamp pipettes in brain slices

Understanding the physiology of axons in the central nervous system requires experimental access to intact axons. This protocol describes how to perform cell-attached recordings from narrow axon fibers (? <1 μm) in acute and cultured brain slice preparations (with a success rate of ～50%). By using fluorophore-coated glass pipettes and Nipkow disk confocal microscopy, fluorescently labeled axons can be visually targeted under online optical control. In the cell-attached configuration, axonal action potentials are extracellularly recorded as unit-like, sharp negative currents. The axon morphology labeling and cell-attached recordings of axons can be completed within 1-2 h. The recordings are stable for at least 30 min.     

Potassium channel "inactivation" induced by soft-glass patch pipettes

We have studied the potassium currents of rat pituitary pars intermedia cells kept in primary culture using whole-cell recording with patch pipettes. The potassium current recorded with hard-glass pipettes is mainly carried by voltage-dependent channels that show slow inactivation in the presence of 0.5 mM internal EGTA. Fast "inactivation" of the potassium current is seen with patch pipettes fabricated from soft glass (soda glass or potash lead glass), and is probably caused by block of the potassium channels by di- or multivalent cations released from the glass.     

The beginning of a new era in tissue expansion: self-filling osmotic tissue expander--four-year clinical experience

The osmotic tissue expander is a new device made of a hydrogel expanding skin that does not require external fillings. Once implanted, it absorbs body fluids, which leads to a gradual swelling of the device. The swelling phase is completed in 6 to 8 weeks and results in skin gain. Different shapes and sizes are available, and the devices can be used in almost every area of the body. Over a 4-year period, the osmotic tissue expander was used in 58 patients in different areas of the body. A round osmotic tissue expander was mainly used in breast reconstruction, and a rectangular expander was used for defect coverage after excision (i.e., of scars and tumors). The mean age of the patients was 49.34 years (range, 4 to 76 years). During the expansion phase, the patients noted only a little discomfort and pain for the first few days. Without a silicone membrane in the first-generation expander, the rate of successful explantation and good final result was 81.5 percent. In a few cases, rapid swelling of the device led to the introduction of a silicone membrane that encloses the expander and leads to a slower, more gradual, and consistent swelling. After introduction of the silicone envelope, the success rate improved to 91 percent. The expander is now used with a silicone membrane in every case. The osmotic tissue expander has many advantages compared with the conventional expander: there is no need for painful external fillings and the risk of external infections is avoided. The expander is 10 percent of its final volume and only requires a short incision and a small pocket. An operation can easily be performed under local anesthesia, with minimal tissue mobilization in older children and compliant patients.     

The use of quartz patch pipettes for low noise single channel recording.

Quartz has a dissipation factor of approximately 10(-4), which is an order of magnitude less than that of the best glasses previously used to fabricate patch pipettes; it's dielectric constant of 3.8 is also lower than that of other glasses. On the basis of these electrical characteristics it is expected that patch pipettes pulled from quartz tubing will produce significantly less noise than pipettes made from other glasses. Our work confirms these expectations and we describe theoretical and practical aspects of the use of quartz pipettes for single channel patch voltage clamp measurements. Methods for pulling quartz pipettes with a laser-based puller and coating them with low-loss elastomers are discussed, as are precautions that are necessary to achieve low noise recordings. We have shown that quartz pipettes can be pulled from tubing with outer diameter to inner diameter ratios as large as 3 and a method of applying heavy elastomer coatings all the way to the tip of pipettes is presented. Noise sources arising from the pipette and its holder are described theoretically, and it is shown that measured noise is in good agreement with such predictions. With low noise capacitive feedback electronics, small geometry holders, and thick-walled quartz pipettes coated with low-loss elastomers we have been routinely able to achieve noise of 100 fA rms or less in a 5-kHz bandwidth with real cell patches and a pipette immersion depth of approximately 2 mm. On occasion we have achieved noise as low as 60 fA rms in this bandwidth.     

Skeletonization of volumetric angiograms for display

The display of three-dimensional angiograms can benefit from the knowledge of quantitative shape features such as tangent and curvature of the centerline of vessels. These can be obtained from a curve-like skeleton representation. If connectivity and topology are preserved, and if geometrical constraints such as smoothness and centeredness are satisfied, it is possible to estimate length, orientation, curvature, and torsion. It is also required that no part of the original object be left unrepresented. An efficient method for the identification of such shape components is developed. First, a suitable representation is obtained using a voxel coding approach to yield connected and labeled unit-thick paths. The desired features are estimated from a smoothed version of the skeleton produced by a moving average filter. The computational cost is linear, of the order of N(object), the total number of object voxels contained in the binary volumetric data. The method is also shown to be robust to boundary noise. Examples are discussed.