Auto-focus scanning surface plasmon resonance microscopy

Wide-field inspection, nano detection, and real-time observation are essential for investigating biomolecular interaction processes. Surface plasmon resonance microscopy (SPRM) is a label-free, real-time, and nano-imaging method that is widely employed for the dynamic detection of nanoscale biomolecules. The field of view (FOV) of SPRM is limited by the usage of high NA objectives, and a scanning SPRM is required to obtain a large FOV. However, during the scanning, the focus drift introduced by the mechanical vibrations blurs the imaging quality of SPRM, making the detection deviate from the true status. To this end, this paper presents the development of autofocus scanning SPRM (AFS-SPRM) that is capable of performing automated real-time focus drift correction during auto-scanning, thereby enabling high-quality SPRM imaging with large FOV. Only 80 ms is taken to process each defocusing event, and the ability to maintain focus has been improved by 30 times by comparison with SPRM. The AFS-SPRM was successfully employed to distinguish nanoparticles of different sizes and to observe the changes of macrophages in a culture medium containing nanoparticles. This investigation illustrates the superior imaging capabilities of AFS-SPRM and demonstrates its potential for observing interactions between biomolecules at the nanoscale.