Table 2.

Imaging techniques for the assessment of the organization of bone ultrastructure.

technique	spatial resolutiona	feature imaged	sample format	FOV dimension	quantitative 3D orientation	main limitation(s)	additional information
CLSM	150 nm	fibril bundle	surface/section	cm	✗	cannot resolve single fibrils low depth penetration	macroscopic image of 3D tissue volume
SHG	100 nm	fibril bundle	surface/section	cm	✗	cannot resolve single fibrils low depth penetration expensive	macroscopic image of collagen in 3D tissue volume
SR-CT	20 nm	fibril bundle	volume	cm	✗	cannot resolve single fibrils (up to now) limited access to synchrotron facilities	mesoscopic 3D tissue image tissue mineral density trabecular architecture lacuno-canalicular network
phase-contrast CT	15 nm	fibril	volume	μm	✓	currently at the limit of resolving single fibrils time-consuming limited access to synchrotron facilities	microscopic 3D tissue image tissue mineral density lacuno-canalicular network
TEM	0.1 nm	fibril + HA platelet	section	μm	✗	very extensive sample preparation expensive limited FOV	nanoscopic 2D tissue image platelet shape and size mineral–collagen interface fibril diameter collagen D-period
SEM	1 nm	fibril	surface	μm	✗	extensive sample preparation only tissue surface expensive limited FOV	nanoscopic 2D tissue image
FIB SEM/SBF SEM	10 nm	fibril	volume	μm	✓	extensive sample preparation only tissue surface expensive limited FOV time-consuming	microscopic 3D tissue image lacuno-canalicular network
AFM	0.1 nm	fibril + HA platelets	surface	μm	✗	limited FOV irreproducible results due to possible probe tip damage	nanoscopic 2D tissue image platelet shape and size mineral–collagen interface fibril diameter collagen D-period mechanical properties

^aSpatial resolution values reflect the current resolution limits of each technique.