We report on the development of a Hyperbaric Atomic Force Microscopy (HAFM) imaging system. Initial system performance is provided and cantilever dynamics are investigated. HAFM results of human fibroblasts and rat hippocampal neurons under graded levels of hyperbaric gases (up to 6 atmospheres absolute; ATA) are shown. Hyperbaric AFM provides the capability to study the cellular and molecular mechanisms of pressure-dependent disorders.

Atomic Force Microscopy (AFM) has experienced tremendous growth in the variety of techniques and applications since its inception in 1986 [1]. UHV, ambient and fluid [2] environments have been used to image samples of interest from atoms [10] to living neural cells [8]. AFM can also be used to measure material properties [9] and pulling forces [7]. Tip-Sample interaction forces involved in these experiments continue to be a topic of investigation as the techniques of AFM mature [2]. In this report we describe the development of a hyperbaric imaging system which includes an AFM.

Currently, there exists no commercially available research AFMs designed to operate at hyperbaric pressures, although a custom-design AFM has been used to study materials at high pressure and temperature [6]. Our hyperbaric imaging system was designed to study living cells at physiologically relevant pressures during compression and decompression. There exists a need to understand how gases at increased atmospheric pressure affect the structure and function of living cells because the underlying mechanisms that govern pressure-dependent disorders such as oxygen toxicity, decompression sickness, and nitrogen narcosis are still largely unknown. The effects of hyperbaric pressure, especially hyperbaric oxygen (HBO₂), have been shown to disrupt normal cellular activity in vivo and in vitro [4]. More recently, an AFM study demonstrated that acute hyperoxia caused oxidative damage, observed as an increase in nanoscopic membrane blebs in human glioblastoma cells [3]. Using hyperbaric AFM we can study the fundamental understanding of cellular and molecular mechanisms that underly a wide range of disorders resulting from breathing gases at hyperbaric pressure, including CNS and pulmonary O₂ toxicity.

Description of Hyperbaric Imaging System

Features of the hyperbaric chamber (fig.

1) were designed to ensure that the chamber could house most commercially available hybrid imaging systems (AFM + fluorescence microscope), confocal attachments, and additional equipment for future applications (e.g. electrophysiology, amperometry). The chamber was designed and fabricated in collaboration with Riemers Systems Incorporated (RSI). The resulting design yielded a 3.25 ton (~3000 kg) hyperbaric chamber with a maximum working pressure of 100 psi, the pressure equivalent to ~196 feet of seawater. A total of 38 penetrations were made in the chamber for electric, gas and fluid connections. Following fabrication the chamber was hydrostatically pressure tested before being transported and installed into University of South Florida's Hyperbaric Biomedical Research Laboratory (HBRL).

Design characteristics that were considered for the hyperbaric imaging system include the ability to use live cell preparations; thermoregulation to counteract pressure effects on temperature; full control of existing microscope features at hyperbaric pressures; as well as the ease of use under ambient conditions. The maximum working pressure encompasses pressures most commonly experienced in conventional dive operations. Current limitations of the hyperbaric imaging system include thermal changes with rapid compression and decompression, especially with thermally conductive inert gases (e.g. helium.)

A Bioscope SZ (Veeco, Inc., Santa Barbara, CA) was mounted inside the hyperbaric chamber and consists of an inverted biological microscope (Nikon, TE2000) mated with the AFM head, specialized XY stage and control electronics. Standard AFM probes (DNP-20 or OTESPA) (Veeco, Inc.) were used for imaging in contact and tapping mode respectively. A 30 to 60 minute thermal equilibration period was done after positioning the scanning probe above the sample. In tapping mode the resonance frequency (7-9 Hz) was determined for maximum tip oscillation, and the drive amplitude of 200-800 mV was applied to obtain a free amplitude of 0.5 V RMS. AFM calibration scans were done using a standard calibration grid (200 nm depth, 10 µm pitch) supplied by the Veeco Instruments. AFM performance and system noise were not compromised under hyperbaric pressures (see D'Agostino, et al. to be published.)

In experiments with live cells, the lateral positioning of the AFM probe tip was done over the cell by visual guidance using the optics (10-40 objectives) of the Nikon inverted TE2000E. Vertical positioning of the probe tip within close proximity (200 µm) to the sample was performed just prior to engaging. Integral gain, proportional gain, deflection setpoint, scan rate and scan size were all optimized to maximize sensitivity and minimize lateral friction. AFM scanning was continuous and scan parameters were kept constant during compression and decompression.