How surface plasmon microscopy differs from conventional optical microscopy in the focal region?

Objective-coupled surface plasmon microscopy (SPM) shares a similar configuration with conventional optical microscopy (OM), but critical differences in their focal mechanisms are often neglected. Although SPM and OM exhibit operational similarities, the unique interaction between the focused beam and the sensor chip, driven by surface plasmon (SP) propagation, remains poorly understood. This leads to widespread errors, such as misaligning SPM's focal plane with OM's, which degrades resolution and sensitivity. While prior studies recognized the “defocus” effect, ambiguous definitions hindered its practical resolution. Herein, we systematically clarify the distinction between SPM, OM, and focused SPs. By analyzing the origin of defocus, we demonstrate that SPM's focal plane results from a balance between optical focusing and SP self-interference. We define SPM's ‘real focal plane’ (optimized at 1–2 wavelengths of negative defocus) and ‘focal spot’ (smaller than OM's diffraction limit), supported by simulations consistent with experimental data. These definitions provide actionable criteria for positioning the sensor chip, maximizing resolution without sacrificing signal strength. This work resolves the ambiguities and establishes practical guidelines for the accurate implementation of SPM in bio-sensing and surface analysis.