Scanning (atomic) force microscopy imaging of earthworm haemoglobin calibrated with spherical colloidal gold...

Scanning (atomic) force microscopy (SFM) permits high-resolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm. Lumbricus terrestris, were imaged in NH4Ac solution using calibrated SFM. Individual molecules and their top and side views were clearly identified and were comparable with the images of the same molecule obtained by scanning transmission electron microscopy (STEM). A central depression, the presumed mouth of the hole, was detected. We analysed 75 individual molecules for their lateral dimensions. Compression varied for different molecules, presumably because of the variation of the interaction between the SFM tip and the protein molecule. Two effective heights which correspond to the heights of the points of the haemoglobin molecules first and last touched by the tip, h1 and h2, respectively, were measured for each protein and ranged between 1.58 and 16.2 nm for h1 and 1.23 and 13.6 nm for h2. The apparent diameter was measured and ranged from 44.9 to 86.6 nm (63.2 +/- 10.5 nm, n = 75), which is about twice the diameter of the molecule reported by STEM for the top view orientation. The higher the measured effective heights, the worse was the tip convolution effect. In order to determine the tip parameters (semivertical angle, curvature of radius and the cut-off height) and to calibrate images of earthworm haemoglobin molecules, spherical gold particles were scanned as standards. The tip sectional radii at distances of h1 and h2 above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29.1 +/- 3.85 nm, which is close to the reported scanning transmission electron microscopy data 30.0 +/- 0.8 nm. The results presented here demonstrate that the calibration approach of imaging gold particles is practical and relatively accurate. Calibrated SFM imaging can be applied to the study of other biomacromolecules.