Effects of relative humidity and applied force on atomic force microscopy images of the filamentous phage fd

The filamentous phage fd was studied by both contact- and tapping-mode atomic force microscopy under conditions of controlled variations in relative humidity and changes in the applied tip force. By spin-coating freshly cleaved mica with phage containing solutions having very low salt content followed by rapid humidity control, stable and reliable sample preparation was achieved. The apparent height of the phage varied by about 10-fold with a quadratic dependence on the stabilized relative humidity, extrapolating to 73% of the accepted X-ray diffraction-based height at 0% relative humidity. The variation in measured height with relative humidity largely reconciles previous widely varying atomic force microscopy estimates of this dimension for the filamentous phage. Our finding that contact-mode images of phage are more difficult to analyze than those acquired in tapping mode are consistent with previously published results on other biological specimens such as DNA.