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

Abstract

SummaryScanning (atomic) force microscopy (SFM) permits highresolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm, Lumbricus terrestris, were imaged in NH 4 Ac 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, h 1 and h 2 , respectively, were measured for each protein and ranged between 1 . 58 and 16 . 2 nm for h 1 and 1 . 23 and 13 . 6 nm for h 2 . The apparent diameter was measured and ranged from 44 . 9 to 86 . 6 nm (63 . 2 Ϯ 10 . 5 nm, n ¼ 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 h 1 and h 2 above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29 . 1 Ϯ 3 . 85 nm, which is close to the reported scanning transmission electron microscopy data 30 . 0 Ϯ 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.

Tip artifact in atomic force microscopy observations of InAs quantum dots grown in Stranski–Krastanow mode

“…The dependence of images on tip size does not deteriorate the value of the correction using the artifact. Some groups have determined a tip size using a standard object; 12,26,27 this procedure allows us to confirm the tip size. Combination of the procedure and the correction using the broadening assures reliability of the correction.…”

“…In AFM observations, it is widely known that the tip artifact prevents the determination of actual sizes 4,5,6,7,8,9,10,11,12,13,14,15,16,17 ( Fig. 1).…”

“…To suppress the artifact, some research groups have attached a carbonnanotube tip to a commercially available probe, 9,10 (A). Other groups have tried to reconstruct the actual shape of an object from its image by observing a sphere on a flat surface as a reference, 4,5,6,10,11,12,13 (B). A sphere is suitable as a reference for the following reason.…”

Nanosphere lithography using thermal evaporation of gold

“…Table 1 summarises the expected size and separation of individual nanostructures from both SEM and the geometric calculations and compares them to the obtained dimensions from AFM. The AFM dimensions obtained by taking an average of 19 measurements and the de-convoluted dimensions calculated using a modification of the method described by Xu et al [14].…”

Height measurement of dsDNA and antibodies adsorbed on solid substrates in air by vibrating mode scanning polarization force microscopy

“…24,25 Here, we select rigid colloidal gold particles as standards in height measurement. 24,25 Here, we select rigid colloidal gold particles as standards in height measurement.…”