Method for the Calibration of a Microscope Setup

Imaging and Microscopy

Ref.-No.: 1201-5661-BC

Errors in super-resolution microscopy (SRM) can be identified with pairs of emitters that have fixed and precisely known relative positions. Such reference and calibration structures are exceedingly rare on the nanoscale. Here, Nitrogen-vacancy centers in diamond (NVs) offer a formidable solution. These fluorescent defects are photo-stable and due to the excellent properties of their brilliant host, do not diffuse and are resilient against temperature or chemical environment changes.

On the nanoscale, the relative position of two NVs can be determined by their magnetic coupling, independent of optical microscopy. So, they offer a reference for the investigation and calibration of super-resolution microscopy.

Figure 1: The optical colocalization of emitters is often subject to inaccuracies on the nanometer range. The magnetic coupling allows for an independent verification of the distance.


Super-resolution microscopy, NV centers, defect centers, colocalization, localization, quantum registers, nanoscale reference, calibration of microscopes, accuracy in microscopy


The invention of super-resolution microscopy opened avenues into the previously hidden realm of nanoscopic optical structures. Techniques such as STED, STORM and MINFLUX achieve high spatial resolution, down to single nanometers. On such length scales, the optically determined position is prone to systematic errors, leading to a discrepancy between real and measured positions. Such inaccuracies are hard to identify without a reliable reference structure.


Figure 2: The relative position of two NVs can be determined through their magnetic dipolar coupling (left, red), which can be measured with electron spin resonance techniques. The determination via super-resolution microscopy (right, blue) is prone to systematic errors, leading to a wrong localization despite a good resolution. So, the known distance of the reference allows the calibration and verification of the microscopy method.

NVs have a fixed position within the diamond lattice and are exceptionally photo-stable. A plethora of SRM methods are established for NVs. Pairs of NVs can couple via the magnetic moments of their electron spins. This coupling can be measured through electron spin resonance using the defect centers themselves. The exact relative position in all three dimensions can then be determined down to a single lattice site, achieving Angstrom precision. The measurement of the magnetic coupling is a frequency measurement which is independent of the optical system and allows to trace the relative distance to the SI second.

The relative distance is fixed for millenia and hence, once determined, can be indefinitely used as a reference. A pair of NV centers within a macroscopic diamond can easily be transported around the world and thus allows for the direct comparison between an endless number of microscope setups, no matter the spatial or temporal distance.

Hence, NV centers are a versatile tool for the calibration and investigation of super-resolution techniques. While NVs are the best-known and best-researched solid state color centers, similar procedures are also possible with other defect centers, for example spins in silicon carbide.


  • NVs are persistent, photostable emitters, robust against nearly all environmental influences, allowing a high reproducibility for measurements. This enables a comparison between microscopes.
  • The relative position of close-by NV centers can be accurately determined by their magnetic dipolar coupling, independent of the optical resolution.
  • The optical transition dipole orientations are fixed and easily known. This allows the investigation of optical vector fields.
  • The reference is versatile: it also can be used to determine the rotation of the sample or the collection efficiency of the microscope.
  • The measurement of the magnetic dipolar coupling makes the relative position traceable to the SI second. The relative position does not change, so it only needs to be measured once and no recalibration down the road is necessary.

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