High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies

• Published in: Biochimica et biophysica acta. General subjects Vol. 1864; no. 2; p. 129325
• Main Authors: Umakoshi, Takayuki, Fukuda, Shingo, Iino, Ryota, Uchihashi, Takayuki, Ando, Toshio
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2020
• Subjects: accuracy; atomic force microscopy; DNA; fluorescence microscopy; High-speed atomic force microscopy; image analysis; light microscopy; Plasmonics; proteins; Scanning near-field fluorescence microscopy; Single molecule analysis; Super-resolution optical microscopy; Scanning near-field fluorescence microscopy; HS-AFM; Single molecule analysis; TIRFM; HS-SNOM; High-speed atomic force microscopy; Super-resolution optical microscopy; Plasmonics; SNOM
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2019.03.011

High-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy. Scanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever. In tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated. This study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research. We achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution. •High-speed AFM is combined with tip-enhanced TIRFM/SNOM for biological research•Fluorescence SNOM images can be captured in synchrony with high-speed AFM imaging•Resolutions of 39 nm and 3 s/frame are achieved for fluorescence SNOM imaging•Improved correlation between fluorescence/AFM topography images is achieved