Table 1.

Comparison of Optical Imaging and Spectroscopy Systems for Engineered Tissue Characterization

Optical system	Resolution/size sensitivity	Penetration depth	Sources of contrast: information	Advantages	Limitations	Cost
Fluorescence spectroscopy	Probe and λ dependent (100s μm typical)	Probe and λ dependent (100s μm to few mm typical)	• Endogenous cellular fluorophores: cell biochemical, metabolic, differentiation status • Structural proteins: bulk matrix deposition, integrity, organization, remodeling • Exogenous labels: protein expression, cell lineage • Scaffolds, biomaterials: biomaterial status	• Simple implementation • Quantitative information on composition • Can be molecularly specific • Portable	• Low spatial resolution • Broad featureless spectra	Low
Elastic light scattering spectroscopy	Probe dependent (100s μm typical)	Probe and λ dependent (100s μm to few mm typical)	• Cellular organelles and membranes: cell structure, organelle packing • Collagen fibers: density/remodeling • Biomaterials, scaffolds: scaffold integrity	• Simple to implement • Portable	• Low spatial resolution • Not imaging based • Not molecularly specific	Low
Raman spectroscopy	Probe and system dependent (few μm to 100 μm)	Probe and λ dependent	Molecular bonds: biochemical composition, molecular structure	Molecular specificity	• Weak signal • Carefully designed fibers and/or detector required	Moderate
Confocal microscopy	Lateral: 0.5 μm or larger Axial: 1 μm or larger Both NA and λ dependent	10s μm to 300 μm	(Reflectance) • Collagen fibers, biomaterial scaffolds: detailed matrix morphology, organization, remodeling (Fluorescence) • Exogenous chromophores, FAD, collagen: cell presence, matrix morphology, blood flow/vascularization	• Depth-resolved imaging • Repeated monitoring possible	• Complex design • Limited depth resolution • Significant photobleaching • Cells usually visible with exogenous labels only	Moderate to high
MPM	Lateral: 0.5 μm or larger Axial: 1 μm or larger Both NA/λ dependent	100s μm to 1000 μm	• Endogenous cellular fluorophores: cell biochemistry, metabolism, organization • Elastin, collagen: matrix integrity, remodeling • Exogenous labels: molecular composition	• High resolution • Reduced out-of-focus photobleaching • Efficient signal detection (no pinhole) • Biochemical and morphological information	• High power density at focus • Not easily portable • Incompatible with traditional optical fiber delivery	High
SHG	Lateral: 0.5 μm or larger Axial: 1 μm or larger Both NA and λ dependent	100s μm to 1000 μm	Noncentrosymmetric structures: with polarization, alignment of structures. See Table 4.	• No photodamage • Efficient signal detection (no pinhole)	Highly directional	High
OCT	Axial: 3–15 μm (5–15 μm typical)Lateral: 1–15 μm (10–15 μm typical)	Up to 2–3 mm	• Changes in refractive index: matrix and scaffold remodeling • (Polarization mode) • birefringent molecules such as collagen: structure, orientation • (Doppler mode) moving/ flowing components: vascularization, perfusion	• Fast data acquisition • High depth penetration • Portable • Several commercial systems available	• Not ideal for visualizing cells (for systems with typical resolution) • Morphological but not biochemical information	Moderate