Optically Distortion‐Free Mica‐Glass Hybrid Substrates for Quantitative Correlative Atomic Force and Fluorescence Microscopy

Correlative atomic force microscopy (AFM) and fluorescence microscopy (FM) is a powerful technique that provides unique insights into individual nanomaterials by combining the strengths of both techniques. However, its broader application is constrained by the absence of suitable substrates. AFM requires atomically flat surfaces, commonly provided by mica, whereas FM demands optically transparent and non‐birefringent substrates such as glass. Although mica ensures superior surface flatness, its biaxial birefringence introduces significant fluorescence distortion. Here, we establish design criteria and validation metrics for mica‐glass hybrid substrates that achieve an atomically flat surface without optical distortion. By using glass substrates, we maintain optical transparency, while the atomically flat surface is obtained by attaching a thin mica layer to the glass. Optical distortion becomes negligible when the mica thickness is below 7 µm, as quantitatively confirmed using a method developed for precise thickness determination. The performance of these hybrid substrates is validated through quantitative correlative AFM‐FM imaging of diverse nanomaterials, including semiconductor quantum dots, boron nitride nanotube‐organic dye hybrid nanostructures, and organic J‐aggregate nanoparticles. This platform substantially enhances the reliability and versatility of correlative AFM‐FM, expanding its applicability to a wide range of nanomaterial systems and enabling more accurate structure‐property correlations at the nanoscale.