Table 1.

Strengths and weaknesses of analytical tools for nanoscale cell-material characterization

	Technique	Sample type			Resolution	Imaging thickness	Quantitative	Live imaging	Label free	Limitations	Future applications	References
		Cells	Material	Cell–material
Diffraction limited fluorescence microscopy	Confocal microscopy	✔	✔	✔	200 nm (XY), 500 nm (Z)	200 μm	Semi (diffraction limited)	✔	✖	-Photo-damage to cells, particularly when high laser power is required in weakly labeled samples. -Poor light penetration can limit observation of cell–material responses particularly in dense, opaque, or thick biomaterial systems. Multi-photon and light-sheet microscopy have better optical sectioning capabilities than confocal microscopy.	-Fluorescence cross-correlation spectroscopy (FCCS) and image correlation spectroscopy (ICS) have been used to study the molecular dynamics of fluorescently labeled proteins in living cells. Future applications could apply these powerful approaches to develop further understanding of the molecular dynamics of these adhesion proteins and proteins associated with their assembly.	[40, 41, 115–121]
Super-resolution fluorescence microscopy	Single-molecule localization microscopy (SMLM)	✔	✔	✖	20–50 nm (XY), 100 nm (Z)	1–5 μm	✔	✔	✖	-Both STORM and PALM require specific fluorophores. Photo-switching tags for STORM (e.g., Alexa Fluor 647, Cy3, Cy5), while PALM requires photo-switchable or photo-activatable proteins (e.g., photo-activatable GFP). -STORM and PALM require extensive optimization, including high labeling density (to obey the Nyquist–Shannon theorem) and the use of oxygen scavenging buffers in STORM.	-SMLM approaches are yet to be optimized to evaluate adhesive-functionalized hydrogels and the interactions which exist at the cell–material interface.	[12, 114, 122–125]
	Structured illumination microscopy (SIM)	✔	✔	✔	100 nm (XY), 250 nm (Z)	< 15 μm	✔	✔	✖	-To exploit the Moiré effect for super-resolution imaging, at least three rotating wide-field images per image slice in the Z-stack. Dyes must therefore demonstrate good photostability throughout image acquisition in order for effective image reconstruction without artifacts.	-Thick or dense biomaterials scatter light extensively. These may present challenges to imaging. Newer techniques including instant two-photon SIM or light-sheet SIM are being reported, but they are yet to be applied specifically to study the cell–material interface.	[38, 126–129]
Electron microscopy	Scanning electron microscopy (SEM) and Focused ion beam (FIB)-SEM	✔	✔	✔	< 5 nm (XY), 50 nm (Z)	-	✖	✖	✖	-Very time- and labor-intensive sample preparation. Can be subject to artifacts due to sample dehydration, processing, and staining. -More difficult to investigate dynamic processes which may be possible to observe using live-cell imaging techniques that are available using fluorescence imaging or Raman spectroscopy.	-Correlative light and electron microscopy or correlation of EM techniques to nanoscale secondary ion mass spectrometry (NanoSIMS) imaging could reveal new insights when combined together in the context of the cell–material interface.	[130]
Physical	Atomic force microscopy (AFM)	✔	✔	✔	< 1 nm (XY)	< 20 nm	✔	✔	✔	-Single-scan images achievable are around 150 μm2, which is more restrictive than EM techniques. -Thermal drift can be problematic, particularly for long imaging times.	-Combining AFM with optical fiber nanospectroscopy (e.g., tip-enhanced Raman Spectroscopy) enables precise spectroscopic assessments of samples enabling important spatial and compositional information to be resolved.	[131–134]
Secondary ion mass spectrometry	Time of Flight (ToF)-SIMS	✔	✔	✔	200 nm (XY), < 1 nm (Z)	Static SIMS < 5 nm	✖	✖	✔	-Static SIMS is capable of analyzing the surface of biomaterials and therefore ideal for monitoring surface treatments. Depth profiling is possible with dynamic SIMS, but the depth of penetration is challenging to control. -Material analysis conducted under ultra-high vacuum.	-Further enhancements in the mass resolving power with the OrbiTrap mass analyzer in Orbi-SIMS offer new potential to study in detail the metabolomic influences that functionalized biomaterials may have on cell response and behavior.	[135–137]
	NanoSIMS	✔	✖	✖	50 nm (XY), < 1 nm (Z)	< 5 nm	✖	✖	✔	-Very limited number of instruments available globally. -Unlike ToF-SIMS, NanoSIMS is limited to the detection of elements or small fragments such as CN-. Isotope labeling of biomolecules is therefore key to detecting biomolecules of interest with NanoSIMS.	-NanoSIMS imaging could be combined with other SMLM optical approaches in order to validate the resolution of the technique. Significant need to expand NanoSIMS to characterize material functionalization.	[138]
Vibrational spectroscopy	Raman	✔	✔	✔	250 nm (XY)	50 μm	Semi	✔	✔	-Raman scattering is inefficient, requiring prolonged image acquisition times and excessive sample exposure to laser power which can induce photo-toxic effects in cells. Newer Raman spectroscopy approaches may reduce acquisition times including Coherent anti-Stokes Raman spectroscopy (CARS) or Surface-enhanced Raman spectroscopy (SERS) by boosting signal intensities.	-Given that Raman spectroscopy can be conducted on live-cell samples, there remains a need to characterize spatiotemporal interactions of cells with functionalized biomaterial systems using this technique.	[139, 140]