Table 1.

Comparison of 2D osteocyte/lacuna imaging methods

Method	Light microscopy	SEM w/ acid etching	BEI/qBEI	TEM	AFM	Light sheet microscopy	Confocal laser scanning microscopy
2D/3D	2D	2D	2D	2D	2D	3D	2D/3D
ROI/VOI	large	large	large	small	intermediate	large but limited depth	large but limited depth
Min. resolution	200 nm	1 nm	> 1 nm	0.2 nm	5 nm	6.5 μm	200 × 200 × 300 nm
Common preparation	fixation, embedding, sectioning (4–25 μm)	fixation, embedding, acid etching, gold coating	fixation, embedding, polishing, carbon/gold coating	fixation, (staining), embedding, ultra-thin sectioning (100 nm), possibly decalcification	fixation, embedding or dried	fixation, clearing, commonly fluorescent dye	commonly fluorescent dye, fixation, (embedding), sectioning, decalcification for 3D
Visualization of canaliculi?	yes, with staining	yes	yes	yes	yes	no	yes, with staining
Direct/indirect assessment of osteocyte?	direct	indirect	indirect	direct	indirect	depending on dye	depending on dye
Pros	functional imaging, soft tissue contrast, wet mounting possible, simple, wide-spread availability	simple preparation	imaging of mineralized lacunae contrast based on composition	imaging of subcellular structures, resolution high enough for canaliculi, crystallization information in dark field mode	mechanical and topographical information about tissue	fast acquisition time, functional imaging through fluorescent labeling	functional imaging/labeling of subcellular structures using dyes, soft tissue contrast, wet mounting possible,
Cons	2D, limited analysis of lacunae morphology	2D representation of 3D network - quantification with care	calibration necessary, if mineral to be quantified (qBEI)	complicated preparation	calibration necessary to recognize tip wear	resolution not sufficient to assess osteocyte morphology, long preparation	anisotropic voxel size, blurred edges due to dye diffusion, overestimation of canaliculi
Destructive	no	yes	no	no	no	no	no
Artifacts	sectioning, dye inhomogeneity, cracks and folds from cutting	incomplete infiltration may prevent full imaging of canaliculi, potential shrinkage	insufficient coating, charging, electron beam damage, cracking in vacuum, shrinkage	sectioning, insuff. coating, charging, electron beam damage, cracking in vacuum, shrinkage	probe wear/ contamination, scanner artifacts (creep, nonlinearity)	sectioning, dye inhomogeneity	sectioning + dye inhomogeneity, (depth-dependent) artifacts through signal distortion & attenuation
Reference	[21, 41]	[7, 20]	[7, 29]	[24, 35]	[30]	[60]	[9, 16]