Table 6 Main advantages and limitations of techniques used to evaluate cytoskeleton in 2D and 3D cultures

Confocal Microscopy

High-resolution imaging of cytoskeletal proteins;

Eliminates out-of-focus light;

Suitable for 2D and 3D models.

Phototoxicity and photobleaching with long-term imaging;

Complex sample preparation may be required.

McKayed and Simpson (2013); Laissue et al. (2017)

Light Sheet Microscopy

High-resolution imaging;

Minimal photobleaching and phototoxicity;

Ideal for dense 3D models.

Requires specialized equipment and optical alignment;

Limited accessibility in some laboratory setups.

Reynaud et al. (2008); Olarte et al. (2018); Stelzer et al. (2021)

Flow Cytometry

Rapid and high-throughput analysis of cytoskeletal proteins;

Measures structural rearrangements at the single-cell level.

Limited spatial information;

Requires single-cell suspensions;

Predominantly applied to 2D models.

Shin et al. (2021)

Atomic Force Microscopy (AFM)

Nanoscale resolution;

Provides quantitative assessment of mechanical properties (stiffness, contractility, elasticity);

Suitable for 2D and 3D models.

Complex data interpretation;

Requires specialized expertise and instrumentation.

Jalili and Laxminarayana (2004)