SpheroRuler - Calibration beads for SMLM microscopy

The SpheroRuler beads are 1μm-diameter polymer particles surrounded by 647-fluorophores covalently anchored to their surface. Beads will be visible as hollow rings or spheres when reconstructed in 2D or 3D SMLM experiments respectively.

SpheroRuler beads are coated with 647-fluorophores giving a stable blinking in SMLM microscopy, and have been initially developed for dSTORM imaging. Since then, they have also been successfully used in SFFR, SEM, confocal, Airyscan confocal and SEM microscopy.

The spherical particles making up Spheroruler beads have been selected based on very good monodispersity properties. The accuracy and reproducibility of the bead diameter have been characterized by SEM on 25 independent microspheres and showed a standard deviation of
1 +/- 0.05μm.

The SpheroRuler kit contains a 50μL suspension of SpheroRuler beads. All you need to have on your side is some blinking buffer, coverslips and imaging vessels of your choice.

One kit contains a 50μL suspension of SpheroRuler beads, allowing for 10 experiments when using the recommended 5μL volume in 35mm glass-bottom dishes.

Yes, SpheroRuler beads are resuspended in PBS and can be loaded together with biological specimens (cells, tissue sections, etc).

The SpheroRuler suspension is stable for at least 7 months when stored at 4°C.

In 2D, you should retrieve a hollow circle of 1μm diameter. In 3D, you should retrieve a hollow sphere of 1μm diameter.

SpheroRuler beads are highly fluorescent beads coated with a high density of fluorophores. We recommend using a “multi-emitter / high density” rather than a “single-emitter / low density”, algorithm, to guarantee an efficient localization of individual blinking events. Although the available options will vary on each imaging system used, an example of algorithm that was successfully used is the "Account for overlap" function on the ZEN software (Zeiss).

Fluorophores are directly coated on the bead surface without linkers or antibodies, and their thickness is therefore negligible compared to the measured diameter. The apparent thickness of the fluorophore ring will depend on the resolution of your imaging system (i.e. around 160-200nm when measured in dSTORM). The external periphery of the beads should be taken into account when measuring diameter.

Although 2D reconstructions of SpheroRuler beads should be accurately circular, obtaining an elongated shape in z is a common artefact that will depend on the system used for imaging and for 3D reconstruction (biplane reconstruction, astigmatism, etc). The measured diameter in z and its distance from the actual 1μm can therefore be used as a robust indicator to evaluate the fidelity of the 3D reconstruction for a given system. As an example, measured z diameter was 1.3 μm when tested on a Vutara VXL system using biplane imaging.