5:u.-~ II ELSEVIER 11 July 1997 Chemical Physics Letters 273 (1997) 37-41 CHEMICAL PHYSICS LETTERS Probing the microelastic properties of nanobiological particles with tapping mode atomic force microscopy L. Shao a, N.J. Tao b, R.M. Leblanc a a Department ofChemisto,, Universi~ of Miami, Coral Gables, FL 33124, USA b Department of Physics, Florida International UniL,ersity, Miami, FL 33199, USA Received 2 January 1997; in final form 17 April 1997 Abstract We have studied untreated photosystem II (PSII) membrane using tapping mode atomic force microscopy (AFM). The individual PSII particles distribute randomly in the membrane. Near the center of each particle, our AFM reveals an intramolecular cavity which confirms the previous electron microscopy of stained samples. The cavity can be reversibly enlarged from a few nm to as many as 40 nm in diameter by increasing the force on the AFM tip. A study of the particle's apparent height and cavity size under various forces provides unique information about the microelastic properties of single PSII particles. © 1997 Published by Elsevier Science B.V. I. Introduction Photosystem II (PS II) is a pigment-protein com- plex integrated into the thylakoid membrane of higher plants, cyanobacteria, red and green algae. Its major biological function is to split water to form molecu- lar oxygen, proton and electron by capturing solar energy which is vital for maintaining the present level of biomass on earth and for sustaining an oxygenic atmosphere. Despite many years of inten- sive studies [1-13], the exact structure of PSII is still not well understood. The techniques that have been predominantly used to probe PS II structure are X-ray crystallography [3,4] and electron microscopy (EM) [5-9]. While these techniques have made im- portant contributions to our understanding of PS II structure, the samples require crystallization, staining or metal coating. Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) have the capability of imaging biological molecules under conditions close to their native states [14]. However, since electrons cannot directly tunnel through large insulating molecules such as PSII, platinum repli- cates or metal coated PS II membranes were used in recent STM studies [10,11]. AFM works both for conductive and non-conductive samples therefore re- quires no metal coating of the samples, but the AFM tip can seriously distort and even damage soft bio- logical materials when operated in the contact mode. Furthermore, the large lateral tip force requires the samples to be strongly attached to a substrate, which results in a large sample-substrate interaction that may distort the natural structure of the sample. AFM has been recently used to image titanium coated PS II crystals [ 12] and Langmuir-Blodgett (LB) films of PS II membranes isolated from bacteria Rhodopseudomonas viridis deposited on glass sub- strates [13]. 0009-2614/97/$17.00 © 1997 Published by Elsevier Science B.V. All rights reserved. PII S0009-261 4(97)00585-X38 L. Shao et al. / Chemical Physics Letters 273 (1997) 37-41 In the present study, we report a tapping mode AFM [15,16] study of untreated PSII membranes from spinach deposited on an atomically flat mica substrate using the Langmuir-Blodgett method. Since the oscillating tip in the tapping mode AFM only briefly touches the sample during each cycle of oscillation, it drastically reduces tip-induced distor- tion to soft biological materials. The use of atomi- cally flat mica as substrate removes the complication in the image interpretation due to rough features on substrates such as glass. We have found that the PS II particles distribute in the membrane in a disor- dered fashion with a density of = 10-3/nm 2 in all the areas surveyed by AFM. Our AFM has revealed a cavity near the center of each PS II particle. In addition to structural studies, we have investigated the microelastic properties of the PS II by utilizing the unique advantage that the AFM tip can apply a local force onto each individual PS II particle. 2. Experiments The PS II membranes in this study were extracted from spinach using a procedure described in Ref. [17]. The sample was characterized by performing low-temperature LDS-PAGE and by measuring oxy- gen evolution using the Clark-type electrode. The oxygen evolution measurement demonstrated that the PS II membranes used in our experiments were ac- tive. The PS II membranes were spread at the inter- face of air and aqueous solution (2 mM CdC1 z, 2 mM sodium ascorbate, and 2 mM MES (pH 6.5)), compressed to a desired pressure and then deposited onto a freshly cleaved mica using the vertical method at a speed of 10 mm/min. The coverage of the PS II membranes on each substrate was examined with fluorescence spectroscopy (Fig. 1). The peak posi- tion and shape of the fluorescence spectrum of the membranes deposited on mica are nearly identical to those obtained in buffer solution. The peak height, as expected, increases as the pressure increases. The tapping mode AFM study was carried out on a MultiMode Nanoscope system in air at room tem- perature. Etched Si tips with a resonant frequency of = 319 kHz, force constant of = 50 nN/nm and nominal radius of curvature of 5-10 nm were used. The quality factor, Q, of the tip at the resonant j ¢- 600 800 I 700 Wavelength (nm) Fig. 1. Fluorescence spectra of PS II membrane LB films de- posited on mica at surface pressures of 10, 12.5 and 15 mN/m. frequency was determined to be = 500. The tip was driven to oscillate at a slightly lower frequency than the resonant frequency with an amplitude of = 30 nm. The images were obtained with various setting points which allows us to obtain information about the elastic properties of the sample. 3. Results and discussion Fig. 2A is a tapping mode AFM image of the PSII membrane prepared at 10 mN/m. The image reveals the PS II particles as blob-like features that appear to be randomly distributed on the surface. The height of the