Investigation of the image contrast of tapping-mode atomic force microscopy using protein-modified cantilever tips

Abstract

In this work we have designed a simple system to investigate empirically the image contrast of tapping-mode atomic force microscopy (TMAFM). We modified the cantilever tips with protein molecules (bovine serum albumin or goat anti-biotin antibody) and used these protein-modified cantilevers to scan poly-L-lysine films and antibody layers deposited on mica in air under ambient conditions. We also investigated the effects of manipulating the setpoint voltage in this system. It was found that extra topographic features with a patchlike appearance were introduced into the TMAFM images of both the poly-L-lysine and antibody films when scanned with the protein-modified tips, even at initial preset setpoints, and were superimposed on the topography of the samples. The surface coverage of the patchlike features in the TMAFM images changes significantly with the setpoint voltage in a reversible and nonlinear manner. These are believed to arise from the surface indentation of the sample or from the structural deformation of the proteins at the tip induced in TMAFM imaging. Interestingly, it was observed in the experiment that no structural alteration or damage was discernible on the sample surface, even after continuous scanning with the protein-modified tips for a long period of time, with varying setpoint voltage. This study provides experimental evidence that cantilever tips modified with protein molecules or, under certain circumstances, even unmodified tips introduce extra topographical features (i.e., artifacts) and enhance the image contrast of TMAFM imaging of soft materials, which is dependent on their mechanical properties.

Topographical Pattern Dynamics in Passive Adhesion of Cell Membranes

“…9 D), using the same imaging conditions as above. This uniformity of the poly-L-lysine layer is in agreement with literature data (You and Yu, 1997;Leckband et al, 1993). Poly-L-lysine is known to adsorb electrostatically to clean glass or mica, both of which are negatively charged under physiological buffer.…”

“…Poly-L-lysine is known to adsorb electrostatically to clean glass or mica, both of which are negatively charged under physiological buffer. Atomic force microscopy of a dried poly-L-lysine monolayer on mica (N % 300 kDa, C % 0.25 mg/ml) has shown a uniform surface with only suboptical ;50-nm holes in it (You and Yu, 1997). AFM tapping mode imaging here of the poly-L-lysine-coated glass slides in PBS buffer (N % 3.8 kDa, bulk concentration of 10 mg/ml) also shows a uniform (nonreticulated) surface with an average roughness of 1.7 nm (Fig.…”

Two Dimensional Auto-Organized Nanostructure Formation of Hyaluronate on Bovine Serum Albumin Monolayer and Its Surface Tension

“…AFM has been employed to examine the topography of sample surfaces with atomic or molecular resolution. Tapping-mode AFM has the advantages of imaging soft biological molecules and live cells directly without using any complicated chemical method to immobilize samples onto the substrate. − AFM has been also a useful tool in studying surface interactions, and a great deal of work has been carried out on theoretical and experimental issues. , AFM is able to obtain force (>1 pN)−distance (lateral, >1 Å; vertical, >0.1 Å) curves from surfaces with high resolution . The BSA molecules adsorbed on the PMLG film and the surface structures of NaHA adsorbed on the BSA monolayer were imaged using AFM.…”

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“…However, the viscoelastic properties of the cell have not been fully taken advantage of, because the resonant frequency of the cantilever in liquid is relatively low, typically in the range of 8 to 35 kHz, resulting from the substantial liquid damping. On the other hand, the operation of tapping-mode AFM (TMAFM) in liquid appears to be more difficult than in the air [31] and the image contrast mechanism(s) of the TMAFM is unexpectedly complex and is not fully understood [32,33]. Nevertheless, it is expected that more AFM studies of cellular structures will be reported using this kind of imaging mode.…”