Tissue rotation chamber for electrophysiology, microinjection, and microdissection.

To generate complete 3-D maps of extracellular ion current patterns around insect ovarioles without removing the sample and chamber from the recording equipment, it was necessary to design a chamber permitting complete rotation of the tissue. While suction pipets have been used in previous studies to orient the tissue (1), their use can significantly disturb extracellular ionic gradients if an incomplete seal is obtained. Additionally, it was necessary to introduce a porous surface beneath the ovariole to cushion it and to permit free passage of ions so that current patterns would not be disturbed. Damage and stress to the ovariole during rotation and recording must be negligible, so a mechanism to lift the tissue off the surface was required. The recording chamber described here achieved these goals and may benefit studies of other tissues involving electrophysiology, microinjection, or microdissection where precise orientation of the sample is required. Figure 1A illustrates the completed chamber, which begins with a 35-mm plastic Petri dish modified as outlined in the figure legend. The rotating shaft and the roller can be manipulated from outside the dish, minimizing disturbance. To begin construction, a hot razor blade is used to remove a 25-mm section of the Petri dish wall. A piece of glass microscope slide the height of the dish (10–11 mm) is cut and siliconed to the opening, permitting sideview observation of the sample via a long focal distance video camera. After a hole 10–15 mm in diameter is cut in the dish floor using a Dremel tool, a 22-mm diameter glass coverslip is siliconed in place over the opening to improve optical clarity, a common practice. A 10-mm diameter plastic ring, made from a 1-mm slice of a 1.5-mL microcentrifuge tube, is then siliconed to the coverslip. This creates a shallow reservoir to be filled with agarose, providing a permeable, transparent tissue support. Construction of the mechanism for tissue orientation begins by making a hole in the side of the dish, approximately 2 mm above the dish floor, with a hot 16-gauge syringe needle. A 22gauge syringe needle is prepared by sealing the plastic base with silicone to prevent media leakage. The needle is inserted through a short piece (approximately 3 mm) of polyethylene tubing with an inner diameter that gives a snug fit, permitting easy rotation of the metal shaft without leaking media. The tubing and needle are then fitted into the hole in the Petri dish and the tubing is glued in place, oriented so that the syringe needle is parallel to the glass side and pointed toward the center of the plastic ring. Additionally, the needle tip is positioned so it is just below the top of the plastic ring (see Figure 1B). Proper positioning of the syringe is necessary to enable the roller, added subsequently, to adjust the tissue’s height relative to the agarose support. The plastic base of the syringe needle can be grasped to rotate the needle and the specimen. Next, a 15-mm length of thin (0.127 mm) tungsten wire (Alfa Products, Danvers, MA, USA) is bent to form a clip to grasp the tissue (Figure 1C) and inserted into the syringe needle tip. The wire must be long enough that the clip can be placed inside the plastic ring. A few slight bends are made in the wire to anchor it firmly in the syringe needle so Benchmarks