Adapting the Quesant Nomad atomic force microscope for biology and patch-clamp atomic force microscopy.

The Quesant Nomad atomic force microscope (AFM) was modified to produce a reliable patch-clamp AFM for demanding biologic applications. The AFM's laser optics forms the basis of a condenser that allows simultaneous Köhler illumination and AFM imaging on an inverted optical microscope. The original AFM scan head was replaced with plastic and glass to make it biologically inert. A bevel cut in the new scan head permits clearance for patch clamp pipets. Cantilevers are attached to the scan head with a quick setting silicone rubber that is readily removable. Software was developed to (a) automate a gentle approach and set a specific feedback force, (b) provide a mouse-driven control of the X-Y position of the probe tip and recall of saved locations, and (c) measure force-distance curves over user defined paths. Additional modifications were made to minimize mechanical noise. The patch-clamp AFM achieves 600 fA (3 kHz bandwidth) and 1 A RMS noise levels (10 kHz bandwidth). The correlation of electrical and mechanical information allows signal averaging and measures sub-Angstrom, sub-millisecond electromotile responses from cells.

Ciliary activity in differentiating and reactivated human respiratory epithelial cells.

OBJECTIVE: Experimental studies of mucociliary clearance, especially those involving drug effects, suffer from the difficulty of determining whether drugs act directly on ciliary motility or whether their effects are indirect, acting via changes in cell metabolism, viscous load, or alternative mechanisms. The present study provides a solution to this problem by comparing the motile characteristics of ciliated cells that have differentiated in primary cell culture with those of demembranated cilia reactivated with MgATP. METHODS: Human respiratory epithelial cells (REC) were dissociated from trachea, bronchus, nasal polyps, or turbinates and then placed in a dissociated cell culture system. Thirty-three percent of the dissociated cells contain beating cilia. Following 1 week in culture, the REC dedifferentiated, but then redifferentiated within 96 hours after they were brought to an air interface. RESULTS: The cilia on such cells beat with planar waves consisting of power and recovery strokes. Beat frequencies at 20 degrees C were 15+/-2.3 Hz. Ciliary beating often was coordinated both within and between cells with a defined antilaeoplectic pattern of coordination. Either fresh or cultured cells could be demembranated with Triton X-100 and reactivated with MgATP. The activity of these reactivated models was equivalent to those observed in living cells. CONCLUSION: The authors have demonstrated that ciliary beat frequency of demembranated human respiratory epithelial cells can be modulated by MgATP and can be adjusted to the same level of activity as measured in living cells. This allows them to selectively test whether a drug's effects on mucociliary transport are the result of direct interactions with the ciliary apparatus or are produced through other indirect mechanisms.

A small chamber for making optical measurements on single living cells at elevated hydrostatic pressure.

A 100-microliters chamber has been developed to facilitate the use of optical techniques for the study of living cells under conditions of high hydrostatic pressure and other environmental manipulations. The chamber attaches to the mechanical stage of an inverted microscope and is capable of sustaining cells under physiologic conditions over a range of hydrostatic pressure from 1 to 150 atmospheres (1 atm = 101.325 kPa). Cells are located directly on the pressure supporting window that forms the bottom of the chamber. This window is made from coverslip glass which is bonded to the chamber using an epoxy adhesive and is thin enough to permit ultraviolet epi-illumination using high magnification, high numerical aperture objectives. Electrodes for stimulating excitable cells are also located on this window. In operation, the chamber is installed in a custom stage mount which provides X, Y, Z, and rotational alignment of chamber contents with respect to the microscope's optical axis. This allows precise registration of the cell specimen with the image sensing devices attached to the microscope. Mechanical and optical alignment can be performed at any pressure over the working range of the chamber. The separate cover that seals the chamber is also fitted with a window to allow for simultaneous transillumination of the specimen. A high pressure liquid chromatography pump is used to continuously perfuse the chamber with a solution that is controlled for temperature, pressure, gas tension, and ionic composition.

Atrial veins of the human heart.

Modern anatomical description divides the cardiac veins into two groups: tributaries of the greater cardiac vascular system (GCVS) and tributaries of the smaller cardiac vascular system (SCVS), consisting of the Thebesian vessels. Both systems intercommunicate extensively. With the exception of the oblique vein of the left atrium (Marshall's vein), veins draining the walls of both the left and right atrium have not been well illustrated or described in anatomical atlases and textbooks. Consequently, we do not know exactly to which of the two groups (GCVS or SCVS) the atrial veins belong. There are three groups of left atrial veins: (1) tributaries of the left coronary vein and the coronary sinus; (2) special veins draining the right-sided walls of the left atrium that terminate via intramural sinuses in the right atrium, which vessels occur in 92% of cases and belong to the GCVS; (3) in 81% of cases special veins drain the myocardium of the posterior and superior walls of the left atrium. In most cases they empty into the left atrium itself; in almost 40% of the cases they are connected with mediastinal veins. These veins, also belonging to tributaries of the GCVS, constitute a distinctly separate category of cardiac veins and should be designated proper veins of the left atrium. The veins draining the walls of the right atrium fall also into three groups: (1) In most cases there are short or large intramural tunnels or sinuses in the basic walls of the auricle and atrioventricular node area. The generally valveless openings of all the venous tunnels and sinuses are lined up on a circle just above the tricuspid valve and between the openings of both venae cavae. (2) There are also thin veins at the junction of the right atrium with both the superior and inferior vena cava. (3) In addition, there are numerous cardiac veins of the "smallest size" (real Thebesian veins).