【Nat.Photon.】复旦大学刘倩等联手上海交大李富友|连续闪烁数万帧不漂白！新一代超分辨显微镜实现10倍分辨率突破#

a, Schematic of the proposed photoblinking mechanism driven by a Yb3+ multiphoton process and defect-mediated energy trapping. ET, energy transfer; EM, energy migration; hν, photon energy. The energy gap between the highest and lowest yellow energy levels corresponds to the effective energy barrier of these traps, whereas the randomly distributed yellow levels illustrate the presence of multiple energetic states within the trap manifold. b, Representative luminescence time traces of individual nanoparticles under excitation at 976 nm, showing distinct on/off blinking behaviour with no photodegradation for 165 min (limited by memory; 99,000 frames at 10 frames per s (fps)). _τ_on, bright-state dwell time; _τ_off, dark-state dwell time. Scale bar, 1 μm. c,d, Correlative wide-field (c) and SEM (d) images, which confirm that blinking originates from individual nanoparticles. The correlative wide-field microscopy images show the UCL intensity, measured in photons per s per pixel (pps per px); the colour scale in c also applies to b. e, Bright-state (‘on’) dwell-time distribution fitted with a single-exponential decay (ExpDec1). f, Dark-state (‘off’) dwell-time distribution fitted with a double-exponential decay (ExpDec2). All single-particle imaging experiments were carried out under excitation at 976 nm (11.6 kW cm−2).#

a, Schematic of the multilayer nanoparticle architecture. b, UCL spectra under irradiation at 980 nm, showing emission variations with the Ho3+ content. c, Luminescence decay curves of the 541 nm green emission under pulsed 980 nm excitation at 15 W cm−2. The colours of the traces are the same as the x values in b. Fitted lifetimes are summarized in Supplementary Table 3. d, Representative single-particle luminescence traces under 976 nm excitation (11.6 kW cm−2), displaying the intensity fluctuations between bright and dark states (shown as the normalized UCL intensity; left). The intensity distributions for both states are shown on the right. Ho3+ doping concentration (top to bottom): x = 0.005, 0.01, 0.02, 0.04, 0.08, 0.16. e, Bright-state emission intensity of the nanoparticles as a function of Ho3+ concentration under 976 nm excitation (11.6 kW cm−2). f, Switching frequency (right axis) and duty cycle (left axis) versus Ho3+ concentration under 976 nm excitation (11.6 kW cm−2). g, Power-dependent photoblinking dynamics of Yb0.99/Ho0.01-codoped UCNPs under 976 nm excitation with the irradiance varied from 4.0 to 21.7 kW cm−2, revealing the tunable switching frequencies (right axis) and duty cycles (left axis). N > 5,000 events of bright states with over 200 particles across 3 different fields of view (FOVs) for each data point. In e, the data are presented as the mean ± standard error of the mean (s.e.m.).#

a, Schematic of the CSS architecture with tunable active shell (NaYb0.99Ho0.01F4) and inert shell (NaLuF4) thicknesses. b–f, Representative single-particle luminescence traces under 976 nm excitation (11.6 kW cm−2) for various shell dimensions (shown as the normalized UCL intensity): CS32nmS37nm (b), CS27nmS33nm (c), CS19nmS24nm (d), CS19nmS26nm (e) and CS19nmS28nm (f). Note that the trace in d is the same as in Extended Data Fig. 3c (y = 0.99), to make a more direct and intuitive comparison. g–i, Histograms of the upconversion intensity, corresponding to panels b (g), c (h) and d (i), fitted with multi-gaussian functions to resolve distinct emission states. j, Duty cycles under 976 nm excitation (4.0–21.7 kW cm−2) for UCNPs with various shell thickness: CS19nmS24nm (blue), CS19nmS26nm (cyan) and CS19nmS28nm (pink). k, Bright-state UCL intensity versus excitation power (4.0–11.6 kW cm−2) for UCNPs with various shell thickness as detailed in j. N > 5,000 events of bright states with over 200 particles across 3 different FOVs for each data point. In k, the data are presented as the mean ± s.e.m.#

a, Power-dependent switching frequency and bright-state intensity of CS19nmS24nm Yb0.99/Ho0.01-codoped UCNPs under 976 nm excitation (4.0–21.7 kW cm−2). b, Switching frequency versus bright-state intensity measured under 976 nm excitation (4.0–21.7 kW cm−2), showing a linear correlation (Pearson’s r = 0.99). c, Structural comparison of as-synthesized (pristine; left) and Lu3+-annealed (right) UCNPs. d, Duty cycles of pristine (cyan, equivalent to CS19nmS26nm in Fig. 3j) and Lu3+-annealed (yellow-green) UCNPs under 976 nm excitation (4.0–21.7 kW cm−2). e, Proposed mechanism of suppressed photoblinking in Lu3+-annealed nanoparticles. f, I–V curves for ITO interdigitated electrodes spin-coated with ~12-nm-diameter NaYb0.99Ho0.01F4 UCNPs. Initial measurements were performed under dark conditions (red). Subsequently, the devices were irradiated with a 976 nm laser for 3 min, after which immediate I–V measurements were taken (green). Finally, the devices were allowed to recover in the dark for 5 min (purple) and 10 min (orange), followed by additional I–V measurements. g, Representative luminescence time trace of a single CS19nmS23nm Yb3+/Tm3+-codoped nanoparticle under 976 nm excitation (6.6 kW cm−2). h, Comparison of the switching frequencies for CS19nmS24nm Yb3+/Ho3+-codoped UCNPs and CS19nmS23nm Yb3+/Tm3+-codoped UCNPs under 976 nm excitation (6.6 kW cm−2). i, Energy-transfer pathways in Yb3+/Er3+, Yb3+/Tm3+ and Yb3+/Ho3+-codoped nanoparticles (left, middle and right, respectively). The blue arrows represent ground-state absorption (GSA) in the activator ions. Δ_E_, energy mismatch. N > 5,000 events of bright states with over 200 particles across 3 different FOVs for each data point. In a and b, the data are presented as the mean ± s.e.m.#

基于这一近乎无限的荧光产额，研究团队将SPUM技术应用于极具挑战性的生物样本成像。在干燥盖玻片和水溶液体系中，单颗粒的定位精度达到了惊人的0.38纳米，在自组装的咖啡环纳米结构上实现了35纳米的超高空间分辨率，相比传统宽场成像提升了10倍以上。随后，研究人员利用该亲水性纳米探针对活体海拉细胞的质膜进行了超分辨成像，细胞毒性测试证实其具有极佳的生物相容性。更重要的是，团队成功对活细胞内的内吞体进行了长达数十分钟的实时示踪，空间分辨率稳定在30至60纳米，不仅完美还原了内吞体的纳米级运动轨迹，还首次捕获到了两个内吞体之间高度协同的偶联运输事件以及罕见的细胞间物质转移过程。

a,b, Maximum projection image (a) and super-resolution reconstructed image (b) of densely packed UCNPs over 6,000 frames at 10 fps under 976 nm irradiation (6.6 kW cm−2). c, Line profiles (of the areas highlighted by the dashed boxes in a and b) comparing the diffraction-limited (red) and super-resolved (green) images, showing enhanced FWHM values for the super-resolved image. d, Super-resolution image of a self-assembled UCNP ring structure formed via the coffee-ring effect under 976 nm irradiation (6.6 kW cm−2). Left: reconstructed super-resolved image with a region of interest (ROI) highlighted by the white dotted box. Right: overlay of the diffraction-limited maximum projection image. Inset: enlarged view of the highlighted ROI. e, Line profiles (corresponding to the yellow line in d and Supplementary Fig. 33c) comparing the super-resolved (red) and diffraction-limited (black) parts of the image. f, FRC analysis of the reconstructed ring structure. The FRC resolution cut-off (dashed purple line, 1/7 threshold) confirms a final resolution of 35 nm. g, Representative luminescence time trace of a single biotinylated UCNP in aqueous solution. h, Wide-field maximum projection (large image left) and super-resolution image (large image right) of UCNP-labelled HeLa cell membranes (inset: bright-field image of a HeLa cell), with enlarged views of the regions in the highlighted by the dashed boxes for the maximum projection (small image top) and super-resolution image (small image bottom).#

a, SPUM image of endosomes reconstructed from a 60-frame subset compared with maximum-intensity-projected conventional wide-field imaging in 3 living U2OS cells. b,c, Magnified view of the SPUM image (b) and the corresponding wide-field image (c) for a single endosome in a. d, Line profiles corresponding to the white lines in b and c, demonstrating the FWHM enhancement of SPUM (red) compared with wide-field imaging (black and grey). e, FRC analysis of the 100-frame reconstruction confirms a final FRC resolution of 61.4 nm; the dashed blue line indicates the FRC resolution cut-off. f,g, Histograms of the localization accuracy (f) and single-particle UCL intensity (g). ppf, photons per frame. h, Full super-resolution SPUM image of endosomes in 3 living U2OS cells reconstructed from all 6,000 frames. i,j, Long-term super-resolved tracking of endosomes in ROI1 (i) and ROI2 (j) from h. The pseudo-coloured reconstructions from sequential 60-frame subsets show the trajectories of the endosomes over time. For i, the tracking in ROI1 shows a synchronized, coupled transport event between two endosomes. For j, the tracking in ROI2 visualizes a rare intercellular transport event. The dashed white lines indicate the cell boundaries, and the white solid lines with arrows denote the trajectory and direction of motion. All experiments were performed with a 0.1 s exposure time per frame under 976 nm excitation (6.6 kW cm−2).#