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laboratories continues to impede reproducibility and making it ideal for neuroscience and developmental
185
constrains the comparability of findings between studies. biology studies. ScaleSQ induces tissue expansion,
which facilitates higher-resolution imaging, although this
5.2. Optical clearing techniques and their expansion can complicate volumetric analyses. SeeDB
186
applications in high-resolution imaging of brain enhances resolution for thin samples but may alter cellular
organoids arrangements, complicating interpretations of cellular
187
Optical clearing techniques, combined with high- structures. The choice of clearing method depends on
resolution microscopy methods such as confocal and light study objectives, with each presenting unique strengths and
sheet microscopy, have emerged as powerful tools for 3D trade-offs.
visualization. These methods preserve tissue integrity Despite these advancements, challenges persist in high-
while enhancing imaging depth, enabling more accurate resolution imaging. Large datasets require substantial
morphological assessments (Table 5). Various clearing computational power for processing and analysis, and
techniques offer distinct advantages: ClearT2 minimizes chromatic aberrations can distort reconstructed 3D models.
distortion and is particularly suitable for deep tissue imaging, Segmentation of cellular structures is further complicated

Table 5. Comparative exploration of tissue clearing techniques
Method Key feature Advantages Limitations Applications Optimal tissue Use with References
type organoids
ClearT2 Preserves Ideal for deep May require Neuroscience, Brain, muscle, Suitable for 188,189
structural tissue imaging longer processing developmental and other solid imaging organoids
integrity without distortion times biology organs
ScaleSQ Induces tissue Enables Expansion can 3D imaging, Soft tissues, Applicable for 190
expansion higher-resolution complicate vascular studies including brain organoid studies
imaging imaging analysis and vascular
tissue
SeeDB Tissue shrinkage Enhances Shrinkage may Histology, Thin slices of Compatible with 187
resolution for thin alter cellular cellular various tissues thin organoid
samples arrangement morphology slices
analysis
Organic Effective deep Suitable for dense Potential toxicity Cancer research, Lipid-rich Possible, but 191
solvent- tissue clearing or lipid-rich and prolonged lipid studies tissues, requires careful
based tissues processing times including fat handling
and brain tissue
CUBIC Minimal High Time-consuming Imaging whole Various tissues, Effective for 192
distortion transparency and multiple steps organs and including brain organoid imaging
preservation of tissues and kidney
morphology
PARS Enzyme-assisted Preserves proteins Limited to specific Molecular Fixed or Limited use; 193
clearing and nucleic acids tissue types biology, protein frozen tissues mostly for specific
localization with complex types
studies structures
TDE Dehydration Allows for Requires careful Developmental Various types Highly suitable 194
followed by detailed imaging handling to studies, organoid of organoids for organoid
expansion at high resolutions prevent damage research and cultured applications
cells
ClearMap Combines Simultaneous Limited to In vivo imaging, Tissues Usable for 195
clearing and imaging and certain imaging dynamic studies amenable dynamic studies
imaging clearing modalities to dynamic with organoids
imaging
iDISCO Immunolabeling Retains Potential loss of Developmental Embryonic and Effective for 196,197
combined with antigenicity cellular detail biology, adult tissues immunolabeling
clearing while providing during processing immunological organoids
transparency studies
3DISCO 3D imaging Facilitates Complexity of Organ and large Whole organs Suitable for whole 198
capability visualization of protocol and cost tissue studies like brain, liver, organoid imaging
entire organs and heart
Abbreviation: TDE: Tissue dehydration and expansion.

Volume 1 Issue 3 (2025) 12 doi: 10.36922/OR025100010