The AFM pulls a tethered molecule through its cantilever. Deflection, multiplied by a calibrated cantilever spring constant, measures force; support motion must be corrected for that deflection and any linker compliance to obtain molecular extension. Initially folded structure can resist extension, then contacts break and more polypeptide polymer contour length becomes available. The readable trace rises with increasing extension, with fluctuations and a strongly rising large-extension part. It should not be interpreted as a direct image of individual amino acids or as establishing a unique microscopic unfolding pathway.
An ideal inextensible freely jointed chain with the stated segment count and length hasIts mean extension is strictly below at every finite force. Thus the quoted maximum does not establish ideal FJC behaviour: if both numbers are taken literally, it exceeds the model's polymer contour length by about . Finite segment stretch, inaccurate effective segment parameters, additional linkers or an extension-zero/calibration error can account for such a difference. If the numerical descriptions are approximate, the discrepancy may be within their uncertainty; even exact agreement with a polymer contour length would not prove freely jointed statistics. Low-force fluctuations and the full force–extension relation are separate tests.
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