Table 1.

Commonly used imaging technologies used for imaging organoids.

Imaging technology	Resolution	Sample preparation	Advantages	Disadvantages	Applications	Refs.
Electron microscopy	~1 nm	Requires fixation, dehydration, and thin sectioning of the sample	High resolution with detailed structural imaging.	Time-consuming preparation; not suitable for live imaging; limited to thin samples; high cost	Ultrastructural studies	[154–157]
Bright-field microscopy	~200 nm (xy); poor axial (z) resolution for large targets	Sample preparation is easy, often using a slide and a cover slip; thin or thick samples	May not need staining; large field of view; high speed; low cost.	Low contrast; low resolution	General cell morphologies; tissue structure	[158–160, 162]
Wide-field fluorescence microscopy	~200 nm (xy); poor axial (z) resolution for large targets	Requires fluorescent labeling; thin or thick samples	Easy to use; low cost; large field of view and low phototoxicity; high speed; live cell imaging	Lack of optical sectioning and can image only thin samples	General cell morphologies	[168, 170]
Laser-scanning confocal microscopy	~200 nm (xy); ~500 nm (z)	Requires fluorescent labeling	High axial resolution;3D imaging;	Limited depth of penetration; long imaging time; high photobleaching and phototoxicity	Detailed tissue architectures; intracellular structures	[173, 174]
Multiphoton microscopy	~200 nm (xy); ~500 nm (z)	Requires fluorescent labeling with limited fluorophores	Deeper penetration depth; less out-of-focus photobleaching	Complex, expensive equipment; long imaging time; phototoxicity with high peak excitation intensity	Deep tissue imaging; dynamic processes in live cells	[177, 178, 181]
Fluorescence lifetime imaging	~200 nm (xy); poor axial (z) resolution for large targets	Requires fluorescent labeling	Measures fluorescence decay time; provides functional information; less sensitive to photobleaching	Additional signal analysis; expensive setup with photon counting	Biochemical changes; protein interactions	[148, 182, 184, 185]
Light-sheet microscopy	~300 nm (xy); ~600 nm (z)	Requires fluorescent labeling; sample is often semitransparent and embedded in clear gel	High-speed imaging; low photobleaching	Complex setup; limited sample thickness	Long-term imaging of living samples	[188–192]
Super-resolution fluorescence microscopy	~30 nm (xy); ~100 nm (z)	Requires specific fluorescent dyes and sophisticated sample preparation.	Sub-diffraction resolution	may cause serious photobleaching; low imaging speed	Molecular interactions; subcellular structures	[206, 207, 209, 210]
Optical coherence tomography	~10 μm (xy), <10 μm (z)	Minimal preparation	Non-contact; deep penetration	Low resolution; lack of functional and molecular sensitivity	Tissue morphologies; developmental biology	[213–218]
Photoacoustic tomography	Scalable from a few micrometers to hundreds of micrometers (xy), tens of micrometers to hundreds of micrometers (z)	Minimal preparation; may use contrast agents labeling the molecules.	Deep penetration depth; high optical absorption contrast; inherent depth sectioning; can work with both fluorescent or non-fluorescent samples	Relatively low sensitivity; low imaging speed	Vascular imaging; tissue oxygenation studies	[219–222]