Schematic overview of basic characteristics of fluorescence confocal microscope and total internal reflection fluorescence microscope. (a) Fluorescence confocal microscope (FCM) with excitation beam waist ∼250–300 nm. (b) Total internal reflected fluorescence microscope (TIRFM) with wide excitation area, which is typically ∼104 times larger than confocal spot in objective-type configuration, and with vertical resolution ∼100 nm.
Schematic of TIRFM imaging-guided confocal single-molecule fluorescence spectroscopy. Laser 1: cw laser source; Laser 2: pulse laser source; L1–L5: lenses; M1: reflection mirror; DM1–DM3: dichroic mirror beam splitters; P1: side port prism for left/vis obs; TL: tube lens; Obj: Objective; XYSS: x-y scanning stage; M2: beam path switch for visual obs; F1–F2: emission filters; PH: excitation pin hole; P2: side port prism for left. TIRFM mode beam path: Laser 1-L1-L2-M1-DM1-P1-TL-Obj-TL-P1-M2-DM2-F1-EMCCD; confocal mode beam path: Laser 2-L3-PH-L4-DM3-Obj-DM3-TL-P2-DM1-L5-F2-SPAD.
Calibration of TIRFM imaging-guided confocal single-molecule fluorescence spectroscopy. (a) Fluorescence image of the microspheres in EMCCD coordinates by TIRFM mode. (b) Fluorescence image of the microspheres in scanning translation stage coordinates by confocal mode. According to the special pattern, the microspheres a, b, and c in (a) are the same ones a, b, and c in (b).
Feasibility of TIRFM imaging-guided confocal single-molecule fluorescence spectroscopy. (a) Image of Rhodamine 6G molecules in TIRFM mode. (b) The corresponding confocal single-molecule spectroscopy measurement on molecule a in (a). The fluorescence intensity decay from single-molecule photon stamping recording is fitted with exponential decay with fit residual; the fluorescence lifetime of the single Rhodamine 6G molecule is 3.1 ± 0.3 ns. Inset: The intensity trajectory of the single molecule.
Application of TIRFM imaging-guided confocal single-molecule fluorescence spectroscopy on spatially and temporally randomly distributed single-molecule enzymatic reaction, horseradish peroxidase-catalyzed oxidation of amplex red. Horseradish peroxidase uses amplex red as an electron donor in the reduction of hydrogen peroxide to water. The product of enzymatic reaction, resorufin, is a highly fluorescent compound. (a) The single-molecule photon time-stamping raw data of an individual turnover event of fluorescent product from one of the active tethered enzyme. Each dot corresponds to a photon stamped with the chronic arrival time (t) and the single-photon delay time (Δt) between the photon emission and femtosecond laser pulse excitation. (b) Fluorescence intensity decay of a newly formed single resorufin molecule confined in the enzyme. The fluorescence intensity decay from single-molecule photon time-stamping recording is fitted with exponential, with fit residual. The fluorescence lifetime of the confined single resorufin molecule is 3.9 ± 0.2 ns.
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