Astigmatism-based focus stabilisation with universal objective lens compatibility, extended operating range and nanometre precision

Abstract

Focus stabilisation is vital for long-term fluorescence imaging, particularly in the case of high-resolution imaging techniques. Current stabilisation solutions either rely on fiducial markers that can be perturbative, or on beam reflection monitoring that is limited to high-numerical aperture objective lenses, making multimodal and large-scale imaging challenging. We introduce a beam-based method that relies on astigmatism, which offers advantages in terms of precision and the range over which focus stabilisation is effective. This approach is shown to be compatible with a wide range of objective lenses (10x-100x), typically achieving <10 nm precision with >10 um operating range. Notably, our technique is largely unaffected by pointing stability errors, which in combination with implementation through a standalone Raspberry Pi architecture, offers a versatile focus stabilisation unit that can be added onto most existing microscope setups.