Traceable Correlative Microscopy - From Fiducials to Visualization

Chibane, L., et al., Nanomaterials, 2025, 15, 1861.^[1]

In their work on graphene oxide reduction, Chibane et al. propose a rigorous correlative metrology workflow combining AFM, SEM, Raman spectroscopy, SMM, and SThM on a single graphene oxide flake. The objective is not only to correlate morphology, chemistry, electrical, and thermal properties, but to do so without altering or damaging the sample during successive measurements.

This study highlights several critical constraints inherent to correlative microscopy at the nanoscale:

• Substrate choice strongly impacts electrical grounding and thermal dissipation
• Measurement sequence must be carefully defined to prevent irreversible sample modification
• Repeated localization of the same flake is mandatory to ensure meaningful correlation
   

To address these challenges, the authors rely on lithographically patterned silicon substrates, which provide precise positioning references. While effective, this approach introduces notable limitations:

• high fabrication cost,
• limited substrate flexibility,
• incompatibility with alternative substrates such as mica or polymers,
• reliance on manual repositioning steps.
   

This case study clearly demonstrates both the scientific value of correlative metrology and the need for more flexible, substrate-independent positioning solutions.

O. Acher & al., Measurement Science and Technology, 2021, 32, 045402.^[2]

Acher et al. directly address these limitations by introducing machine-readable, multiscale, multimodal fiducial nanoGPS navYX tags as a metrology-driven backbone for correlative microscopy workflows.

Unlike conventional visual markers, nanoGPS navYX tags encode absolute position and orientation information. A single image of the tag—captured by SEM, optical microscopy, or Raman imaging—is sufficient to determine:

• absolute coordinates,
• in-plane orientation,
• relocation uncertainty.
   

Key advantages demonstrated in this work include:

• Substrate independence: tags can be affixed to virtually any sample or holder
• Multimodal compatibility: the same tag is readable across SEM, optical, and Raman systems
• Quantified uncertainty: relocation errors are measured and dominated by stage accuracy rather than the tag itself
• Long-term reproducibility: ROIs can be reliably revisited over time and across laboratories
   

Relocation accuracies ranging from a few micrometers up to ~20 µm are achieved, enabling fast, unambiguous correlation without iterative manual alignment. This approach effectively transforms correlative microscopy from a qualitative alignment exercise into a traceable, quantitative, and reproducible analytical workflow.

L. Sotelo & al., Advanced Materials Technologies, 2023, 8, 2300126.^[3]

The importance of robust correlative workflows is further illustrated by Sotelo et al. in their study on laser-induced periodic surface structures (LIPSS) on TiAl6V4 alloys for biomedical applications.

This work investigates how initial surface roughness and laser fluence influence:

• wettability,
• surface chemistry,
• biological cell differentiation,
• bacterial adhesion.
   

To establish meaningful correlations, the authors combine SEM, AFM, Raman spectroscopy, and contact angle measurements on identical surface regions. Such studies critically depend on:

• precise relocation of the same ROIs across techniques,
• controlled measurement order to preserve surface integrity,
• reproducibility across multiple samples and experiments.
   

The study demonstrates that even subtle misalignment or sample alteration can compromise the interpretation of structure–function relationships. It therefore exemplifies the growing need for robust, repeatable, and traceable correlative positioning, especially in application-driven research such as biomedical implants.

Taken together, these studies reveal a clear evolution in correlative microscopy: Correlation is no longer sufficient, traceability, uncertainty control, and reproducibility are becoming essential.

As correlative workflows move toward:

• quantitative materials characterization,
• regulatory and industrial environments,
• multi-site and long-term studies,
   

they increasingly require metrology-grade positioning strategies that decouple correlation quality from operator skill, substrate constraints, and trial-and-error alignment.

By combining advanced multimodal instrumentation with traceable positioning technologies such as fiducial nanoGPS tags, correlative microscopy can evolve into a reliable, scalable, and quantitative analytical framework, unlocking deeper insights across nanomaterials, advanced materials, and life sciences.

Watch this webinar to discover practical workflows for traceable alignment, drift correction, and multimodal data fusion using nanoGPS navYX technology.

References

[1] Chibane, L., Delvallée, A., Fleurence, N., Douri, S., Morán-Meza, J., Ulysse, C., ... & Flahaut, E. (2025). Toward a Correlative Metrology Approach on the Same 2D Flake: Graphene Oxide Case Study—Sample Preparation and Stability Issues. Nanomaterials, 15(24), 1861.

[2] Acher, O., Nguyên, T. L., Podzorov, A., Leroy, M., Carles, P. A., & Legendre, S. (2021). An efficient solution for correlative microscopy and co-localized observations based on multiscale multimodal machine-readable nanoGPS tags. Measurement Science and Technology, 32(4), 045402.

[3] Sotelo, L., Fontanot, T., Vig, S., Herre, P., Yousefi, P., Fernandes, M. H., ... & Christiansen, S. (2023). Influence of Initial Surface Roughness on LIPSS Formation and Its Consecutive Impact on Cell/Bacteria Attachment for TiAl6V4 Surfaces (Adv. Mater. Technol. 12/2023). Advanced Materials Technologies, 8(12), 2370056.