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Modern microscopes and profilometers such as the coherence scanning interferometer (CSI) approach sub-nm precision in height measurements. Transfer standards at all measured size scales are needed to guarantee traceability at any scale and utilize the full potential of these instruments, but transfer standards with similar characteristics upon reflection to those of the measured samples are preferred. This is currently not the case for samples featuring dimensions of less than 10 nm and for biosamples with different optical charasteristics to silicon, silica or metals. To address the need for 3D images of biosamples with traceable dimensions, we introduce a transfer standard with dimensions guaranteed by natural self-assembly and a material that is optically similar to that in typical biosamples. We test the functionality of these transfer standards by first calibrating them using an atomic force microscope (AFM) and then using them to calibrate a CSI. We investigate whether a good enough calibration accuracy can be reached to enable a useful calibration of the CSI system. The result is that the calibration uncertainty is only marginally increased due to the sample.

Imaging Confocal Microscopes (ICM) are highly used for the assessment of three-dimensional measurement of technical surfaces. The benefit of an ICM in comparison to an interferometer is the use of high numerical aperture microscope objectives, which allows retrieving signal from high slope regions of a surface. When measuring a flat sample, such as a high-quality mirror, all ICM's show a complex shape of low frequencies instead of a uniform flat result. Such shape, obtained from a λ/10, Sa < 0.5 nm calibration mirror is used as a reference for being subtracted from all the measurements, according to ISO 25178-607. This is true and valid only for those surfaces that have small slopes. When measuring surfaces with varying local slopes or tilted with respect to the calibration, the flatness error calibration is no longer valid, leaving what is called the residual flatness error. In this paper we show that the residual flatness error on a reference sphere measured with a 10X can make the measurement of the radius to have up to 10% error. We analyzed the sources that generate this effect and proposed a method to correct it: we measured a tilted mirror with several angles and characterized the flatness error as a function of the distance to the optical axis, and the tilt angle. New measurements take into account such characterization by assessing the local slopes. We tested the method on calibrated reference spheres and proved to provide correct measurements. We also analyzed this behavior in Laser Scan as well on Microdisplay Scan confocal microscopes.

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