. 2021 Mar 5;14(5):1240. doi: 10.3390/ma14051240

Table 2.

Overview of the main micro- and nano-scale imaging technique.

Micro- and Nano-Scale Imaging Technique	Brief Description of the Technique	Invasiveness	Outcomes	Spatial Resolution	2D or 3D	Advantages	Disadvantages
Micro- and Nano-Scale Imaging Technique	Brief Description of the Technique	Invasiveness	Outcomes	Spatial Resolution	In Vitro/In Vivo Application	Advantages	Disadvantages
Stereomicroscopy Based on Histological Sections	Histology from the bone tissue is obtained and then the sample is properly treated (fixation, dehydration and clearing, embedding, sectioning, staining and mounting). The histological section is then observed by means of an optical microscope	Yes	Traditional technique for the visualization of bone microarchitecture	~1.6 µm [58]	2D	Bone remodeling assessment [59]	Destructive and invasive technique Limitations related to the bidimensional output images: the three-dimensional features are lost. High-resolution images (at least 1.4 µm or better) are required to identify and measure individual resorption cavities in the process of bone remodeling [59]
Stereomicroscopy Based on Histological Sections		Yes		~1.6 µm [58]	In vitro	Bone remodeling assessment [59]
Micro-Computed Tomography (Micro-CT) and Nano-Computed Tomography (Nano-CT)	Micro- and nano-CT scans use radiographs to generate cross-sections of bone, that are generally processed (image reconstruction) to generate a virtual 3D model without destroying the original bone sample	No Generally, the samples are obtained from surgical wastes that derive from prosthetic treatment	Microarchitectural 3D data for both the cortical and the trabecular sections (tissue volume, bone volume, bone surface, bone volume fraction, bone surface to tissue volume, trabecular/cortical thickness, degree of anisotropy, cortical porosity, etc.) [37]. Local and global parameters related to the lacunar network are obtained [36]	1.2 µm (micro-CT) ~50–150 nm (nano-CT)	3D	Large number of obtainable outputs (morphological parameters at different scales) Detailed finite element 3D models could be implemented by using micro-CT images	Static evaluation of micro-scale features Not suitable for in vivo human evaluation due to the high radiation dose No detection of the canalicular network (insufficient resolution for the micro-CT scans) Nano-CT
				1.2 µm (micro-CT) ~50–150 nm (nano-CT)	In vitro
Peripheral QCT (pQCT) and High-Resolution pQCT (HR-pQCT)	Dedicated CT scanners for the forearm (radius and ulna) and leg (tibia and fibula)	No Low radiation dose (≈0.003 mGy) [37]	Analysis of the trabecular and cortical sections (BMD, bone mineral content and bone geometrical parameter calculation). Acquisition of biomechanical parameters, such as the cross-sectional moment of inertia. Evaluation of the functional muscle–bone unit [60].	Isotropic voxel size of 82 μm with HR-pQCT	3D	High precision and accuracy Low radiation dose Applicable for the study of a large number of diseases, especially pediatric (useful in applications where trabecular and cortical sections are affected in a different way)	Evaluation restricted to the appendicular bone Only transversal data are available for fracture risk prediction Low spatial resolution
		No Low radiation dose (≈0.003 mGy) [37]		Isotropic voxel size of 82 μm with HR-pQCT	In vivo
Synchrotron Radiation Imaging (SR)	A high-intensity white beam travels around a fixed closed loop. It permits a high level of detail in bone visualization (ultra-structural porosity detection)	No Generally, the samples are obtained from surgical wastes that derive from prosthetic treatment	Morphological analysis of ultra-structural porosities	Voxel size of 0.9 μm for the white beam [61]	3D	Visualization of the lacunar and canalicular network Phase contrast permits the clear detection of micro-cracks	Reduced field of view
Synchrotron Radiation Imaging (SR)			Morphological analysis of ultra-structural porosities	Voxel size of 0.9 μm for the white beam [61]	In vitro		Reduced field of view
Micro-MRI and nano-MRI	The technique generates images by exploiting the nuclear magnetic behavior of different atoms in a sample tissue placed in a magnetic field	No	Structural parameters, such as trabecular bone thickness and mean bone volume fraction, associated with bone biomechanical properties and fracture resistance	Spatial resolution up to 25 µm (micro-MRI) and ~10 nm for the nano-MRI	3D	Non-destructive technique Good special resolution Good contrast resolution [62]	Long acquisition times High costs [62]
Micro-MRI and nano-MRI		No			In vivo		Long acquisition times High costs [62]
Laser Scanning Confocal Microscopy (LSCM)	LSCM employs lasers at proper wavelengths to excite fluorochromes that are used to stain bone sections	Yes	Correlation between micro-crack parameters and bone matrix toughness Comparison among damage morphologies [13]	180 nm laterally and 500 nm axially [63]	2D/3D images of consecutive planes can be reconstructed into a 3D image in vitro.	Evaluation of bone microdamage	Axial resolution in depth impaired by spherical aberration [63] High costs
Scanning Electon Microscopy (SEM)	SEM produces images of the bone sample by scanning the surface with a focused beam of electrons	Yes	Quantitative analysis of fracture surfaces Visualization of microdamage morphology, fiber bridging and interlamellar separation [13]	~1 nm	3D In vitro	Significant information related to sub-micro-scale damage	Destructive technique (sample surfaces should be conductive → bone needs to be coated with conductive materials)
Atomic Force Microscopy (AFM)	The deflections of a cantilever on the surface of the bone sample are transduced into electrical signals	Yes	Topographical parameters of fractured bone surfaces (mineral particle sizes) Identification of sacrificial bonding	Vertical resolution → up to 0.1 nm Lateral resolution → ~30 nm	3D In vitro	Versatile imaging technique for the visualization of fracture surfaces High accuracy Non-destructive technique [64]	Small dimensions of the single scan image size (150 × 150 µm, compared with mm for SEM) Slow scan time [64]