72 Science Research Writing Th e IR camera that we used in the setup that is shown in Fig. 1 is a Raytheon Radiance HS that contains a 256×256 InSb focal plane array, and operates in the 3–5 µm spectral region. It is sensitive (with a 1 ms integration time) to surface temperature changes of ~0.03°C, and can be operated at full frame rates up to 140 Hz with that sensitivity. We have also observed the eff ects reported here with a considerably less expensive, uncooled, microbolometer focal plane array camera, operating in the long wavelength (7–10 µm) of the IR. Th e height of biomolecules measured with the atomic force microscope depends on electrostatic interactions MATERIALS AND METHODS Biological samples Aquaporin-1 (AQP1) from human erythrocyte solubilized in octyl-f3-glucopyranoside was reconstituted in the presence of Escherichia coli phospholipids to form two-dimensional (2D) crystalline sheets (Walz et al., 1994). Th e 2D crystals were prepared at a concentration of -0.5 mg protein/ml and 0.25 mg/ml lipid in 0.25 M NaCl, 20 mM MgCl2, 20 mM 2-(N-morpholino) ethanesulfonic acid (MES) (pH 6). Hexagonally packed intermediate (HPI) layer from Deinococcus radiodurans, a kind gift of Dr. W. Baumeister, was extracted from whole cells (strain SARK) with lithium dodecyl sulfate, and purifi ed on a Percoll density gradient (Baumeister et al., 1982). A stock solution (1 mg/ml protein) was stored in distilled water at 4°C. Purple membranes of Halobacterium salinarium strain ET1001 were isolated as described by Oesterhelt and Stoeckenius (1974). Th e membranes were frozen and stored at −70°C. Aft er thawing, stock solutions (10 mg protein/ml) were kept in distilled water at 4°C. Porin OmpF trimers from E. coli strain BZ 1 10/PMY222 (Hoenger et al., 1993) solubilized in octyl-polyoxyethylene were mixed with solubilised dimyristoyl phosphatidylcholine (99% purity;
Methodology — Writing Task 73 Sigma Chemical Co., St. Louis, MO) at a lipid-to-protein ratio (w/w) of 0.2 and a protein concentration of 1 mg/ml. Th e mixture was reconstituted as previously described (Hoenger et al., 1993) in a temperature-controlled dialysis device (Jap et al., 1992). Th e dialysis buff er was 20 mM HEPES, pH 7.4, 100 mM NaCl, 20 mM MgCl2, 0.2 mM dithiothreitol, 3 mM azide. 1,2-Dipalmitoyl-phosphatidylethanolamine (DPPE) from Sigma was solubilized in chloroform:hexane (1:1) to a concentration of 1 mg/ml. Th e resulting solution was diluted in buff er solution (150 mM KCl, 10 mM Tris, pH 8.4) to a concentration of 100 µg/ml. Layered crystals MoTe2, a layered crystal of the family of transition metal dichalcogenides (Wilson and Yoff e, 1969), was employed to calibrate the piezo scanner of the AFM. It was prepared by chemical vapor transport (CVT), with chlorine or bromine as carrier gases in a temperature gradient of 100°C across the quartz ampule (Jungblut et al., 1992), and was a kind gift of Y. Tomm. Muscovite mica (Mica New York Corp., New York) was used as the solid support for all samples. Mica minerals are characterized by their layered crystal structure, and show a perfect basal cleavage that provides atomically fl at surfaces over several hundreds of square microns. Th eir hydrophilicity and relative chemical inertness (Bailey, 1984) make them suitable for the adsorption of biological macromolecules. Atomic force microscopy A commercial AFM (Nanoscope III; Digital Instruments, Santa Barbara, CA), equipped with a 120-µm scanner (j-scanner) and a liquid cell, was used. Before use, the liquid cell was cleaned with normal dish cleaner, gently rinsed with ultrapure water, sonicated in ethanol (50 kHz), and sonicated in ultrapure water (50 kHz). Mica was punched to a diameter of −5 mm and glued with water- insoluble epoxy glue (Araldit; Ciba Geigy AG, Basel, Switzerland) onto a Tefl on disc. Its diameter of 25 mm was slightly larger than the diameter of the supporting steel disc. Th e steel disc was required to magnetically mount the sample on to the piezoelectric scanner.