Abstract

Optical super-resolution microscopy has become a powerful technique to help scientists to monitor the sample of interest at nanoscale. Fluorescence emission difference (FED) microscopy, a very facile super-resolution method, does not require high depleting laser intensity and is independent on the species of agents, which makes FED microscopy possess great potential. However, to date, the biomarkers applied in FED microscopy usually suffer from a photo-bleaching problem. In this work, by introducing Er3+ activated upconverting nanoparticles with red-color emission and non-photobleaching properties, we demonstrate nonbleaching super-resolution imaging with FED microscopy. The dopant neodymium ions (Nd3+) can work as highly efficient sensitizing ions and enable near infrared 808-nm CW laser excitation of relatively low power, which would potentially reduce high intensity/short-wavelength light induced tissue damage. Both simulations and experiments on monodispersed NaYF4:Nd3+/Yb3+/Er3+@NaYF4:Nd3+ UCNPs also indicate that the easy saturation of the multiphoton properties of these UCNPs is beneficial to resolution enhancement in FED microscopy.

© 2017 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

1. Introduction

Optical super-resolution microscopy has attracted a lot of interest in recent years because of its ability to observe living sample at nanoscale size. Several microscopic techniques were proposed to overcome the diffraction limit [1–7]. Among these techniques, the fluorescence emission difference (FED) microscopy [5, 8], also named Switching LAser Mode (SLAM) microscopy [9] provides an alternative approach to perform super-resolution microscopic imaging by means of subtracting two different patterns under the excitations of solid and doughnut focused lasers beam, which enables retrofitting in commercialized laser scanning microscopy system without the requirement of specific dyes and emission spectrum.

However, similar to other laser scanning microscopy schemes, the current FED employs conventional fluorescent dyes, which suffer from the troublesome effect of photobleaching and are not capable for long-time imaging and record. High illumination intensities required to achieve saturated excitation effect in the saturated FED would further make photobleaching more serious and also cause photo-damage problem to the samples [10]. As a new class of biomarkers, lanthanide-doped upconversion nanoparticles (UCNPs) has enormous potential for its photostability and can be excited by cost-effective continued wave (CW) near-infrared (NIR) laser and efficiently emit multiphoton fluorescence [11–13]. UCNPs has been demonstrated to be a powerful candidate for various optical bioimaging applications, including deep tissue imaging, diffuse optical tomography, and high-resolution microscopy [14–19]. Recently, it was found that the blue emission of Yb3+/Tm3+ doped UCNPs under 980-nm excitation can be efficiently depleted by laser light around 810-nm [20, 21] and 1550-nm [22] laser, making UCNPs based STED nanoscopy become feasible. However, there still exist several restricts. For example, in all these STED schemes, the power density of the employed depletion laser is much higher than that of excitation laser, and both 980-nm and 1550-nm light have very large absorption in bio-samples. Second, the blue emission (455 nm) in these studies is high-order multi-photon (four/five-photon) emission thus the relatively low efficiency is not good for high sensitivity nanoscopic imaging. Our previous work successfully depleted the two-photon green emission (545 nm) of Er3+ by 1140-nm with about 30% depletion efficiency [23]. Efficiently depleting longer wavelength emission still remains a challenge and requires solution for future applications.

In this work, we for the first time propose a red-color upconversion fluorescence emission difference (FED) microscopic imaging with an 808-nm CW laser excited the Nd3+/Yb3+/Er3+ co-doped UCNPs. A commercialized low-cost diode laser was used as the laser source for multiphoton scanning microscopic imaging. For the designed UCNPs, the nonlinear dependence of the emission intensity on the excitation laser intensity was measured. And the simulation of point spread function (PSF) of imaging was performed based on the tested response curve. In addition, imaging experiments on the UCNPs were performed to verify the feasibility. The experimental results show that FED can significantly improve the resolution of UCNPs assisted microscopic imaging. Furthermore, the excellent photo-stability, nonbleaching and nonblinking properties of Nd3+/Yb3+/Er3+-UCNPs was also demonstrated.

2. Characterization and simulation

2.1. Structure and emission spectrum of UCNPs

With the wavelength located in biological tissue optical imaging window, the economical 808-nm laser is preferred for excitation laser source in the imaging application. The nanocomposites and the core-shell structure shown in Fig. 1(a) for UCNPs are designed to ensure the efficiency of 808-nm cascading excitation and energy transfer [16, 24]. As proposed in Fig. 1(b), the sensitizing ions Nd3+ in the shell layer firstly absorb the energy of 808-nm pumping photons and then transfer the energy to the internal secondary sensitizing ions, Yb3+. Through the subsequent energy transfer process from Yb3+ to Er3+, which enables the green and red emissions. The nanoparticles NaYF4:Nd3+/Yb3+/Er3+@NaYF4:Nd3+ was synthesized using typical solvothermal method with some modifications [21, 25, 26]. TEM images [Fig. 1(c)] indicate the dimension and morphology of core-shell nanoparticles, about 38 nm in diameter. A shell layer of about 5 nm thick was successfully coated onto the core (right part of Fig. 1(c), about 28 nm). The emission spectrum of these UCNPs under 808-nm excitation was collected, as shown in Fig. 1(d), in good agreement with the proposed mechanism with two strong emission located at green and red bands, respectively. Red emission was collected in the experiment for FED microscopic imaging, which hold large potential for deep tissue super-resolution.

 

Fig. 1 Nanoparticles Synthesis and Characterization. (a) Schematic design of NaYF4: Nd3+/Yb3+/Er3+ (1/10/0.5 mol %) nanoparticle coated with NaYF4:Nd3+ (20 mol %) shell. (b) Proposed luminescent mechanism of NaYF4: Nd3+/Yb3+/Er3+ @NaYF4:Nd3+ UCNPs under 808 nm excitation. (c) TEM image of the synthesized core-shell UCNPs, size bar: 20 nm. (d) Normalized luminescence spectrum of UCNPs under 808-nm excitation.

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2.2. Simulations

A numerical simulation was performed to illuminate the process of UCNPs-assisted FED microscopic imaging in this study. The FED image was obtained using a simple image subtractive method with the obtained negative values set to zeros [5]:

IFED=Isolidr×Idoughnut
where Isolid, and Idoughnut denote the normalized intensity profile of FED image, solid laser scanning image, and doughnut laser scanning image, respectively. The factor r represents the subtractive factor. Some parameters were set according to our FED system, which was built based on our laser scanning microscopy system with simple modifications. Lanthanide dopants in UCNPs have complex ladder-like state levels and interionic interactions, and thus their nonlinear response to specific excitation laser intensity might vary with the doping composite concentration and excitation pathway [19, 27]. In our previous research [19], the NaYF4:1%Nd3+/30%Yb3+/1%Tm3+@NaYF4:20%Nd3+ UCNPs was demonstrated to easily become saturated under 730-nm excitation and then the high order nonlinear process reduced to lower order nonlinear one. Similarly, the Nd3+/Yb3+/Er3+ activated UCNPs would have similar saturation property under 808-nm excitation, which should be taken into account in the simulation, where the excitation wavelength is set to 808 nm, and numerical aperture (NA) of objective is 1.35 according to our experimental system. The nonlinear response to excitation intensity of Nd3+/Yb3+/Er3+-UCNPs was experimentally investigated and compared with the simulation results. For multi-photon fluorescence microscopy, the excitation PSF, hNPL is proportional to the Nth power of intensity: hNPL(x,y)(hex(x,y))N. For example, in the two-photon process it is hTPL(x,y)(hex(x,y))2. However, the relationship in upconversion process of UCNPs is not constant due to the power dependent nonlinearity, the effective PSF huc should be calculated as:
huc(x,y)f(hex(x,y))
To determine the function relationship (f), the excitation response curve of synthetic NaYF4:Nd3+/Yb3+/Er3+@NaYF4:Nd3+ nanoparticles was measured under the varied irradiation intensity. As shown in Fig. 2(a), the slope of exponential plot curve decreases gradually from 2.09 to 0.59 while the excitation power density increase from 0.25 kW/cm2 to 25 MW/cm2, indicating that the excitation of Nd3+/Yb3+/Er3+-UCNPs degrades from two-photon to one-photon process.

 

Fig. 2 The relationship between the red fluorescence emission intensity and excitation intensity in NaYF4: Nd3+/Yb3+/Er3+@NaYF4: Nd3+ nanoparticles. (a) Double logarithmic plot and linear fitting. (b) Normalized linear coordinate fitting for corresponding range in (a).

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With fitting analysis, the experimental data, ln(Iem), ln(Iex) in Fig. 2(a) can be well fitted to a rational function: f(x)=(ax+b)/(x2+cx+d). Thus, as shown in Fig. 2(b), the relationship between the normalized fluorescence intensity, Iem, and the normalized excitation intensity, Iex, can be appropriately fitted to:

Iem=exp(aln(Iex.)+b(ln(Iex))2+cln(Iex)+d)(Iex>0)

The solid and doughnut laser focus spots are generated according to the custom method and used as excitation PSF [Figs. 3(a, b)]. Figure. 3(c) shows the corresponding PSF of FED microscopy calculated using Eq. (1) with the value of r set to 0.6. The excitation response curve in Figs. 2(a, b) obeys the square law, suggesting that the red upconversion luminescence is a two-photon process when the excitation power controlled in a relatively low level (Iex < 1 kW/cm2). In this case of two-photon excitation, the simulated solid PSF [Fig. 3(e)] can be narrowed by 1/2, compared to the solid laser focus spot [Fig. 3(a)]. Due to the same effect, the central dark zone of the two-photon excited doughnut PSF would be broadened [Figs. 3(f, h)], compromising the intended resolution enhancement [Figs. 3(g, h)] after subtracting operation with the same subtract factor (r = 0.6). While excitation laser intensity increases to the saturation region (Iex > 1 MW/cm2), the PSF for saturated excited upconversion luminescence is calculated using Eq. (2) and (3). A little positive offset is set to Iex in the simulation because Eq. (3) is not appropriate when Iex approaches to zero. Figures. 3(i) and (j) show the saturated excited PSFs (Iex = 10 MW/cm2). It is obvious that the saturated solid PSF and outer-ring of saturated doughnut PSF are widened. Meanwhile, the dark center of the doughnut PSF significantly shrinks, which would result in spatial resolution improvement in FED [Figs. 3(k, l)] when control the negative values to −0.5 by setting r to 0.83. Note that the range of negative values is broadened because of the widening of out-ring of doughnut PSF. This problem can be suppressed by employing the widely used confocal microscope set up. In this respect, the dual emission bands of Nd3+/Yb3+/Er3+-UCNPs as shown in Fig. 1(d) make it possible to further engineer two PSFs in two separately detecting channels, for example, employing pinholes with different sizes, adjusting the irradiation laser power and setting different voltage to two photomultiplier tubes (PMTs).

 

Fig. 3 Simulated PSFs in different modes under 808-nm laser excitation. In sequence are solid, doughnut and FED PSF for (a, b, c): excitation laser focus spot, (e, f, g): two photon luminescence when excitation intensity less than 1 kW/cm2, and (i, j, k): saturated excited upconversion luminescence with an excitation intensity of 10 MW/cm2. (d, h, l) The profile plots of corresponding PSFs in each row.

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3. Fluorescence emission difference microscopy

3.1. Optical setup

A home-made optical system was built to perform the FED imaging experiments [Fig. 4]. The 808-nm laser beam was output from a single mode, fiber coupled CW laser. A half-wave plate (P1) combined with a polarizing beam splitter (PBS1) were used to spit the laser into two beams and adjust the splitting ratio of laser power. These two separated beams were spatially overlapped again through another polarizing beam splitter (PBS2) before entering into a laser scanning microscope system. Shutters (S1 and S2) were used to switch two laser beams. A vortex phase plate (VPP-1a, 2π phase depth for 808 nm, RPC Photonics) was insert into one of the laser path to convert it into doughnut shape. The second halve wave plate (P2) combine with a quarter wave plate (QWP) were used to change the doughnut beam into right-handed circular polarization, opposite to the solid beam. The spatially overlapped lasers beams were reflected by a 690-nm short pass dichroic mirror (DM) inside the microscope, and then focused onto the sample through an oil-immersed objective lens (OL, NA = 1.35, Olympus). The emission luminescence was collected by the same objective, and then detected by a photomultiplier tube (PMT) after filtered by a 694-nm short pass filter (F1) and a bandpass filter (F2, FF01-660/30m, Semrock) for collecting the red emission from Er3+. The as-prepared UCNPs solution were highly diluted and dispersed on a coverslip using spin-coating method for imaging experiment. An image of solid spots and an image of doughnut spots would be captured by switching the two laser beams.

 

Fig. 4 Schematical illustration of the optical imaging setup. CL: Collimating lens, SL: Scanning lens, TL: Tube lens, OL: 60X objective lens. S1, S2: Shutters, M1-M5: Silver reflection mirrors, GM: Galvanometer mirror, DM: 690 short-pass dichroic mirror. P1, P2: Halve wave plates, VPP: Vortex phase plate, QWP: Quarter wave plate. F1: 694-nm short-pass filter, F2: 645-675 nm band-pass filter. PMT: photomultiplier tube.

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3.2. FED microscopy of single UCNPs

To verify the simulation study of PSFs in the upconversion process, experimental imaging study of single nanoparticle under different excitation intensities was carried out, as shown in Fig. 5. Laser intensity was adjusted to 0.1 MW/cm2, 1 MW/cm2 and 10 MW/cm2 for respectively, and the corresponding images were arranged in different rows of Fig. 5. In the experiment, the signal-to-noise ratio of single nanoparticle image is relatively low when excitation intensity was as low as 0.1 MW/cm2. In this case, FED is still beneficial to improve the signal-to-noise ratio, although results in insignificant spatial resolution enhancement. When the employed excitation intensity increased to 1 MW/cm2 or 10 MW/cm2, the signal-to-noise ratio of image was very high, facilitating the imaging resolution quantification. Comparing the FWHM of solid PSFs in Fig. 5(b) and 5(c), it is obviously seen that PSF is widened because of the saturated excitation. As for doughnut PSFs, higher intensity results in narrower dark center [Fig. 5(e) and 5(f)]. After subtraction process, the obtained FED PSFs, as shown in Fig. 5(h) and 5(i), present significant resolution enhancement and it is certified that excitation saturation caused by high intensity can benefit for resolution improvement. These results are in good agreement with the simulation.

 

Fig. 5 Imaging of single UCNP at corresponding excitation intensities, image size 1.56 μm. (a, b, c) Solid laser excited PSFs at 0.1 MW/cm2, 1MW/cm2 and 10MW/cm2, respectively. (d, e, f) Doughnut laser excited PSFs at the corresponding exctiation intensity. (g, h, i) FED PSFs at corresponding excitation intensity with subtraction factors set to 0.83. (j, k, l) The intensity profile along the image center of solid and FED PSFs.

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In order to further confirm the feasibility of upconverting fluorescence emission difference microscopy, imaging experiment was also demonstrated for the sample of multiple UCNPs. These nanoparticles deposited on the glass slide were imaged by 808-nm CW laser in the home-built optical system [Fig. 4]. 808-nm laser of 10 MW/cm2 was employed to achieve saturated excitation. Switching the laser path between solid and doughnut beams, a solid image [Fig. 6(a)] and a doughnut image [Fig. 6(b)] were obtained (scanning speed: 100 µs/pixel). The processed FED image with subtractive factor r = 0.83 was shown in Fig. 6(c). The intensity profile of one single spot was measured and fitted to one-dimensional Gaussian function [Fig. 6(d)], the FWHM of a single particle was reduced from 430 nm to 174 nm. Apart from this, two adjacent points were also separately resolved [Fig. 6(c), (e)], demonstrating the superior resolving ability of FED is advantageous over the traditional two-photon images. Although these two points seem to be smaller than other single points (133 nm and 80 nm as shown in Fig. 6(e)), their poorer signal-to-noise ratio indicated that deformation happens due to the negative values after subtracting operation. This is a common issue in FED but it could be diminished by further PSF engineering [28, 29].

 

Fig. 6 Imaging of monodispersed UCNPs under 10 MW/cm2 irradiation, image size 5 μm. (a) Image scanned by solid laser, and (b) by doughnut laser. (c) Subtraction image of (a) and (b), r = 0.83. (d) Intensity profile along the white arrow and (e) along the dashed line in (a) and (c), showing prominent resolution enhancement using FED strategy and two ajacent UCNPs were separately resolved.

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3.3. The photostability of UCNPs

For the laser scanning microscopic imaging, the photostability of employed agents is very crucial and need to be considered in the practice. Most of organic dyes suffer from serious photobleaching side-effect. High-efficiency semiconductor quantum dots suffer from photoblinking, although they have non-bleaching advantage. These kinds of instability are harmful to real-time FED microscopy, which is basically based on fluorescence difference between two rounds of scanning imaging. The photostability property of the synthesized Nd3+/Yb3+/Er3+-UCNPs was explored under long-term laser exposure. After half-hour laser scanning, images of monodispersed UCNPs show no intensity decrease [Fig. 7(a)]. The long-term time trace of the upconversion luminescence intensity was also recorded under settled point excitation on a spin-coated film sample. No decrease was observed in Fig. 7 (b) after being irradiated for 30 minutes with 100 µs temporal resolution, and the occurrence of intensity was coincide with a Gaussian distribution function [Fig. 7(c)], suggesting they do not suffer from photobleaching. By speeding up the scanning speed to 2 µs/pixels, the non-photoblinking property of UCNPs was also demonstrated in Fig. 7(d) since no on/off blinking behavior was observed in one second monitoring time period and the corresponding occurrence in Fig. 7(e) also fit well with the Gaussian distribution.

 

Fig. 7 The photostability of UCNPs. (a) Comparison of two scaned images of monodispersed UCNPs before and after 30 minutes scanning. (b) The time trace of emission intensity from UCNPs under 10 MW/cm2 continuous laser illumination for 30 minutes with 100 μs dwell time, and (d) for 1 second with 2 μs dwell time. The stability of time traces and the single peak Gaussian distributions of corresponding histograms in (c) and (e), showing absolutely no photobleaching and on/off blinking behavior.

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4. Discussion and conclusion

In this work, we successfully demonstrated that red emission FED microscopy can be realized under 808-nm laser excitation and the photo-bleaching problem of traditional FED was completely overcome by introducing lanthanide-doped UCNPs. Significant lateral resolution enhancement and sub-diffraction imaging were achieved in differentiating the single nanoparticle on the glass slide. In addition, the excellent photo-stability of UCNPs under saturated excitation was successfully demonstrated. This provides a very simple option to realize upconversion nanoparticles assisted nonbleaching super-resolution microscopy employing only single NIR CW laser. The super-resolution performance can be further improved by optimizing the nanoparticles property as well as the imaging setup, such as reducing the saturation intensity threshold and employing further PSFs engineering of introducing different confocal pinholes in two green and red detection channels. Furthermore, it also holds potential in terms of realizing low power, deep tissue super-resolution.

Funding

National Natural Science Foundation of China (61675071 and 61405062); the Pearl River Nova Program of Guangzhou (201710010010); the Innovation Project of Graduate School of South China Normal University; the Guangdong Innovative Research Team Program (201001D00104799318, 2011D039); the Natural Science Foundation of Guangdong province (2014A030313445); the China Postdoctoral Science Foundation (2016M600659, XJ2015018); the Guangdong Provincial Key Laboratory of Optical Information Materials and Technology (Grant No. 2017B030301007); Joint International Research Laboratory of Optical Information and the Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province.

References and links

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2. M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(Pt 2), 82–87 (2000). [PubMed]  

3. M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3, 793 (2006). [PubMed]  

4. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994). [PubMed]  

5. C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013). [PubMed]  

6. R. Han, Z. Li, Y. Fan, and Y. Jiang, “Recent advances in super-resolution fluorescence imaging and its applications in biology,” J. Genet. Genomics 40(12), 583–595 (2013). [PubMed]  

7. Y. Fang, C. Kuang, Y. Ma, Y. Wang, and X. Liu, “Resolution and contrast enhancements of optical microscope based on point spread function engineering,” Front. Optoelectron. 8, 152–162 (2015).

8. S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 5708 (2013).

9. H. Dehez, M. Piché, and Y. De Koninck, “Resolution and contrast enhancement in laser scanning microscopy using dark beam imaging,” Opt. Express 21(13), 15912–15925 (2013). [PubMed]  

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References

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  1. W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
    [PubMed]
  2. M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(Pt 2), 82–87 (2000).
    [PubMed]
  3. M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3, 793 (2006).
    [PubMed]
  4. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
    [PubMed]
  5. C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
    [PubMed]
  6. R. Han, Z. Li, Y. Fan, and Y. Jiang, “Recent advances in super-resolution fluorescence imaging and its applications in biology,” J. Genet. Genomics 40(12), 583–595 (2013).
    [PubMed]
  7. Y. Fang, C. Kuang, Y. Ma, Y. Wang, and X. Liu, “Resolution and contrast enhancements of optical microscope based on point spread function engineering,” Front. Optoelectron. 8, 152–162 (2015).
  8. S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 5708 (2013).
  9. H. Dehez, M. Piché, and Y. De Koninck, “Resolution and contrast enhancement in laser scanning microscopy using dark beam imaging,” Opt. Express 21(13), 15912–15925 (2013).
    [PubMed]
  10. G. Zhao, C. Kuang, Z. Ding, and X. Liu, “Resolution enhancement of saturated fluorescence emission difference microscopy,” Opt. Express 24(20), 23596–23609 (2016).
    [PubMed]
  11. Y. I. Park, K. T. Lee, Y. D. Suh, and T. Hyeon, “Upconverting nanoparticles: a versatile platform for wide-field two-photon microscopy and multi-modal in vivo imaging,” Chem. Soc. Rev. 44(6), 1302–1317 (2015).
    [PubMed]
  12. J. Shen, G. Chen, A. M. Vu, W. Fan, O. S. Bilsel, C. C. Chang, and G. Han, “Engineering the Upconversion Nanoparticle Excitation Wavelength: Cascade Sensitization of Tri‐doped Upconversion Colloidal Nanoparticles at 800 nm,” Adv. Opt. Mater. 1, 644–650 (2013).
  13. S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen, and P. J. Schuck, “Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” Proc. Natl. Acad. Sci. U.S.A. 106(27), 10917–10921 (2009).
    [PubMed]
  14. Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang, and S. Andersson-Engels, “Using 915 nm laser excited Tm3+/Er3+/Ho3+- doped NaYbF4 upconversion nanoparticles for in vitro and deeper in vivo bioimaging without overheating irradiation,” ACS Nano 5(5), 3744–3757 (2011).
    [PubMed]
  15. J. Liu, R. Wu, N. Li, X. Zhang, Q. Zhan, and S. He, “Deep, high contrast microscopic cell imaging using three-photon luminescence of β-(NaYF4:Er3+/NaYF4) nanoprobe excited by 1480-nm CW laser of only 1.5-mW,” Biomed. Opt. Express 6(5), 1857–1866 (2015).
    [PubMed]
  16. Y. Zhao, Q. Zhan, J. Liu, and S. He, “Optically investigating Nd3+-Yb3+ cascade sensitized upconversion nanoparticles for high resolution, rapid scanning, deep and damage-free bio-imaging,” Biomed. Opt. Express 6(3), 838–848 (2015).
    [PubMed]
  17. C. T. Xu, P. Svenmarker, H. Liu, X. Wu, M. E. Messing, L. R. Wallenberg, and S. Andersson-Engels, “High-resolution fluorescence diffuse optical tomography developed with nonlinear upconverting nanoparticles,” ACS Nano 6(6), 4788–4795 (2012).
    [PubMed]
  18. Q. Zhan, X. Zhang, Y. Zhao, J. Liu, and S. He, “Tens of thousands‐fold upconversion luminescence enhancement induced by a single gold nanorod,” Laser Photonics Rev. 9, 479–487 (2015).
  19. B. Wang, Q. Zhan, Y. Zhao, R. Wu, J. Liu, and S. He, “Visible-to-visible four-photon ultrahigh resolution microscopic imaging with 730-nm diode laser excited nanocrystals,” Opt. Express 24(2), A302–A311 (2016).
    [PubMed]
  20. Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
    [PubMed]
  21. Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Ågren, and S. He, “Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles,” Nat. Commun. 8(1), 1058 (2017).
    [PubMed]
  22. H. Zhang, T. Jia, L. Chen, Y. Zhang, S. Zhang, D. Feng, Z. Sun, and J. Qiu, “Depleted upconversion luminescence in NaYF4:Yb3+,Tm3+ nanoparticles via simultaneous two-wavelength excitation,” Phys. Chem. Chem. Phys. 19(27), 17756–17764 (2017).
    [PubMed]
  23. R. Wu, Q. Zhan, H. Liu, X. Wen, B. Wang, and S. He, “Optical depletion mechanism of upconverting luminescence and its potential for multi-photon STED-like microscopy,” Opt. Express 23(25), 32401–32412 (2015).
    [PubMed]
  24. X. Huang, “Giant enhancement of upconversion emission in (NaYF4:Nd3+/Yb3+/Ho3+)/(NaYF4:Nd3+/Yb3+) core/shell nanoparticles excited at 808 nm,” Opt. Lett. 40(15), 3599–3602 (2015).
    [PubMed]
  25. F. Wang, R. Deng, and X. Liu, “Preparation of core-shell NaGdF4 nanoparticles doped with luminescent lanthanide ions to be used as upconversion-based probes,” Nat. Protoc. 9(7), 1634–1644 (2014).
    [PubMed]
  26. X. Xie, N. Gao, R. Deng, Q. Sun, Q.-H. Xu, and X. Liu, “Mechanistic investigation of photon upconversion in Nd3+-sensitized core-shell nanoparticles,” J. Am. Chem. Soc. 135(34), 12608–12611 (2013).
    [PubMed]
  27. J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
    [PubMed]
  28. S. You, C. Kuang, Z. Rong, and X. Liu, “Eliminating deformations in fluorescence emission difference microscopy,” Opt. Express 22(21), 26375–26385 (2014).
    [PubMed]
  29. S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 125708 (2013).

2017 (3)

Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
[PubMed]

Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Ågren, and S. He, “Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles,” Nat. Commun. 8(1), 1058 (2017).
[PubMed]

H. Zhang, T. Jia, L. Chen, Y. Zhang, S. Zhang, D. Feng, Z. Sun, and J. Qiu, “Depleted upconversion luminescence in NaYF4:Yb3+,Tm3+ nanoparticles via simultaneous two-wavelength excitation,” Phys. Chem. Chem. Phys. 19(27), 17756–17764 (2017).
[PubMed]

2016 (2)

2015 (7)

Y. I. Park, K. T. Lee, Y. D. Suh, and T. Hyeon, “Upconverting nanoparticles: a versatile platform for wide-field two-photon microscopy and multi-modal in vivo imaging,” Chem. Soc. Rev. 44(6), 1302–1317 (2015).
[PubMed]

J. Liu, R. Wu, N. Li, X. Zhang, Q. Zhan, and S. He, “Deep, high contrast microscopic cell imaging using three-photon luminescence of β-(NaYF4:Er3+/NaYF4) nanoprobe excited by 1480-nm CW laser of only 1.5-mW,” Biomed. Opt. Express 6(5), 1857–1866 (2015).
[PubMed]

Y. Zhao, Q. Zhan, J. Liu, and S. He, “Optically investigating Nd3+-Yb3+ cascade sensitized upconversion nanoparticles for high resolution, rapid scanning, deep and damage-free bio-imaging,” Biomed. Opt. Express 6(3), 838–848 (2015).
[PubMed]

Q. Zhan, X. Zhang, Y. Zhao, J. Liu, and S. He, “Tens of thousands‐fold upconversion luminescence enhancement induced by a single gold nanorod,” Laser Photonics Rev. 9, 479–487 (2015).

Y. Fang, C. Kuang, Y. Ma, Y. Wang, and X. Liu, “Resolution and contrast enhancements of optical microscope based on point spread function engineering,” Front. Optoelectron. 8, 152–162 (2015).

R. Wu, Q. Zhan, H. Liu, X. Wen, B. Wang, and S. He, “Optical depletion mechanism of upconverting luminescence and its potential for multi-photon STED-like microscopy,” Opt. Express 23(25), 32401–32412 (2015).
[PubMed]

X. Huang, “Giant enhancement of upconversion emission in (NaYF4:Nd3+/Yb3+/Ho3+)/(NaYF4:Nd3+/Yb3+) core/shell nanoparticles excited at 808 nm,” Opt. Lett. 40(15), 3599–3602 (2015).
[PubMed]

2014 (2)

F. Wang, R. Deng, and X. Liu, “Preparation of core-shell NaGdF4 nanoparticles doped with luminescent lanthanide ions to be used as upconversion-based probes,” Nat. Protoc. 9(7), 1634–1644 (2014).
[PubMed]

S. You, C. Kuang, Z. Rong, and X. Liu, “Eliminating deformations in fluorescence emission difference microscopy,” Opt. Express 22(21), 26375–26385 (2014).
[PubMed]

2013 (8)

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 125708 (2013).

X. Xie, N. Gao, R. Deng, Q. Sun, Q.-H. Xu, and X. Liu, “Mechanistic investigation of photon upconversion in Nd3+-sensitized core-shell nanoparticles,” J. Am. Chem. Soc. 135(34), 12608–12611 (2013).
[PubMed]

J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
[PubMed]

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 5708 (2013).

H. Dehez, M. Piché, and Y. De Koninck, “Resolution and contrast enhancement in laser scanning microscopy using dark beam imaging,” Opt. Express 21(13), 15912–15925 (2013).
[PubMed]

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[PubMed]

R. Han, Z. Li, Y. Fan, and Y. Jiang, “Recent advances in super-resolution fluorescence imaging and its applications in biology,” J. Genet. Genomics 40(12), 583–595 (2013).
[PubMed]

J. Shen, G. Chen, A. M. Vu, W. Fan, O. S. Bilsel, C. C. Chang, and G. Han, “Engineering the Upconversion Nanoparticle Excitation Wavelength: Cascade Sensitization of Tri‐doped Upconversion Colloidal Nanoparticles at 800 nm,” Adv. Opt. Mater. 1, 644–650 (2013).

2012 (1)

C. T. Xu, P. Svenmarker, H. Liu, X. Wu, M. E. Messing, L. R. Wallenberg, and S. Andersson-Engels, “High-resolution fluorescence diffuse optical tomography developed with nonlinear upconverting nanoparticles,” ACS Nano 6(6), 4788–4795 (2012).
[PubMed]

2011 (1)

Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang, and S. Andersson-Engels, “Using 915 nm laser excited Tm3+/Er3+/Ho3+- doped NaYbF4 upconversion nanoparticles for in vitro and deeper in vivo bioimaging without overheating irradiation,” ACS Nano 5(5), 3744–3757 (2011).
[PubMed]

2009 (1)

S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen, and P. J. Schuck, “Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” Proc. Natl. Acad. Sci. U.S.A. 106(27), 10917–10921 (2009).
[PubMed]

2006 (1)

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3, 793 (2006).
[PubMed]

2000 (1)

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(Pt 2), 82–87 (2000).
[PubMed]

1995 (1)

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[PubMed]

1994 (1)

Ågren, H.

Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Ågren, and S. He, “Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles,” Nat. Commun. 8(1), 1058 (2017).
[PubMed]

Aloni, S.

S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen, and P. J. Schuck, “Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” Proc. Natl. Acad. Sci. U.S.A. 106(27), 10917–10921 (2009).
[PubMed]

Altoe, V.

S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen, and P. J. Schuck, “Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” Proc. Natl. Acad. Sci. U.S.A. 106(27), 10917–10921 (2009).
[PubMed]

Andersson-Engels, S.

C. T. Xu, P. Svenmarker, H. Liu, X. Wu, M. E. Messing, L. R. Wallenberg, and S. Andersson-Engels, “High-resolution fluorescence diffuse optical tomography developed with nonlinear upconverting nanoparticles,” ACS Nano 6(6), 4788–4795 (2012).
[PubMed]

Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang, and S. Andersson-Engels, “Using 915 nm laser excited Tm3+/Er3+/Ho3+- doped NaYbF4 upconversion nanoparticles for in vitro and deeper in vivo bioimaging without overheating irradiation,” ACS Nano 5(5), 3744–3757 (2011).
[PubMed]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3, 793 (2006).
[PubMed]

Bilsel, O. S.

J. Shen, G. Chen, A. M. Vu, W. Fan, O. S. Bilsel, C. C. Chang, and G. Han, “Engineering the Upconversion Nanoparticle Excitation Wavelength: Cascade Sensitization of Tri‐doped Upconversion Colloidal Nanoparticles at 800 nm,” Adv. Opt. Mater. 1, 644–650 (2013).

Carrington, W. A.

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[PubMed]

Chang, C. C.

J. Shen, G. Chen, A. M. Vu, W. Fan, O. S. Bilsel, C. C. Chang, and G. Han, “Engineering the Upconversion Nanoparticle Excitation Wavelength: Cascade Sensitization of Tri‐doped Upconversion Colloidal Nanoparticles at 800 nm,” Adv. Opt. Mater. 1, 644–650 (2013).

Chen, G.

J. Shen, G. Chen, A. M. Vu, W. Fan, O. S. Bilsel, C. C. Chang, and G. Han, “Engineering the Upconversion Nanoparticle Excitation Wavelength: Cascade Sensitization of Tri‐doped Upconversion Colloidal Nanoparticles at 800 nm,” Adv. Opt. Mater. 1, 644–650 (2013).

Chen, L.

H. Zhang, T. Jia, L. Chen, Y. Zhang, S. Zhang, D. Feng, Z. Sun, and J. Qiu, “Depleted upconversion luminescence in NaYF4:Yb3+,Tm3+ nanoparticles via simultaneous two-wavelength excitation,” Phys. Chem. Chem. Phys. 19(27), 17756–17764 (2017).
[PubMed]

Cohen, B. E.

S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen, and P. J. Schuck, “Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” Proc. Natl. Acad. Sci. U.S.A. 106(27), 10917–10921 (2009).
[PubMed]

Dawes, J. M.

J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
[PubMed]

De Koninck, Y.

Dehez, H.

Deng, R.

F. Wang, R. Deng, and X. Liu, “Preparation of core-shell NaGdF4 nanoparticles doped with luminescent lanthanide ions to be used as upconversion-based probes,” Nat. Protoc. 9(7), 1634–1644 (2014).
[PubMed]

X. Xie, N. Gao, R. Deng, Q. Sun, Q.-H. Xu, and X. Liu, “Mechanistic investigation of photon upconversion in Nd3+-sensitized core-shell nanoparticles,” J. Am. Chem. Soc. 135(34), 12608–12611 (2013).
[PubMed]

Ding, Z.

Fan, W.

J. Shen, G. Chen, A. M. Vu, W. Fan, O. S. Bilsel, C. C. Chang, and G. Han, “Engineering the Upconversion Nanoparticle Excitation Wavelength: Cascade Sensitization of Tri‐doped Upconversion Colloidal Nanoparticles at 800 nm,” Adv. Opt. Mater. 1, 644–650 (2013).

Fan, Y.

R. Han, Z. Li, Y. Fan, and Y. Jiang, “Recent advances in super-resolution fluorescence imaging and its applications in biology,” J. Genet. Genomics 40(12), 583–595 (2013).
[PubMed]

Fang, Y.

Y. Fang, C. Kuang, Y. Ma, Y. Wang, and X. Liu, “Resolution and contrast enhancements of optical microscope based on point spread function engineering,” Front. Optoelectron. 8, 152–162 (2015).

Fay, F. S.

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[PubMed]

Feng, D.

H. Zhang, T. Jia, L. Chen, Y. Zhang, S. Zhang, D. Feng, Z. Sun, and J. Qiu, “Depleted upconversion luminescence in NaYF4:Yb3+,Tm3+ nanoparticles via simultaneous two-wavelength excitation,” Phys. Chem. Chem. Phys. 19(27), 17756–17764 (2017).
[PubMed]

Fogarty, K. E.

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[PubMed]

Gao, N.

X. Xie, N. Gao, R. Deng, Q. Sun, Q.-H. Xu, and X. Liu, “Mechanistic investigation of photon upconversion in Nd3+-sensitized core-shell nanoparticles,” J. Am. Chem. Soc. 135(34), 12608–12611 (2013).
[PubMed]

Ge, J.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[PubMed]

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 5708 (2013).

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 125708 (2013).

Goldys, E. M.

J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
[PubMed]

Gu, Z.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[PubMed]

Gustafsson, M. G. L.

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(Pt 2), 82–87 (2000).
[PubMed]

Han, G.

J. Shen, G. Chen, A. M. Vu, W. Fan, O. S. Bilsel, C. C. Chang, and G. Han, “Engineering the Upconversion Nanoparticle Excitation Wavelength: Cascade Sensitization of Tri‐doped Upconversion Colloidal Nanoparticles at 800 nm,” Adv. Opt. Mater. 1, 644–650 (2013).

S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen, and P. J. Schuck, “Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” Proc. Natl. Acad. Sci. U.S.A. 106(27), 10917–10921 (2009).
[PubMed]

Han, R.

R. Han, Z. Li, Y. Fan, and Y. Jiang, “Recent advances in super-resolution fluorescence imaging and its applications in biology,” J. Genet. Genomics 40(12), 583–595 (2013).
[PubMed]

Hao, X.

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 5708 (2013).

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
[PubMed]

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 125708 (2013).

He, S.

Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Ågren, and S. He, “Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles,” Nat. Commun. 8(1), 1058 (2017).
[PubMed]

B. Wang, Q. Zhan, Y. Zhao, R. Wu, J. Liu, and S. He, “Visible-to-visible four-photon ultrahigh resolution microscopic imaging with 730-nm diode laser excited nanocrystals,” Opt. Express 24(2), A302–A311 (2016).
[PubMed]

Q. Zhan, X. Zhang, Y. Zhao, J. Liu, and S. He, “Tens of thousands‐fold upconversion luminescence enhancement induced by a single gold nanorod,” Laser Photonics Rev. 9, 479–487 (2015).

J. Liu, R. Wu, N. Li, X. Zhang, Q. Zhan, and S. He, “Deep, high contrast microscopic cell imaging using three-photon luminescence of β-(NaYF4:Er3+/NaYF4) nanoprobe excited by 1480-nm CW laser of only 1.5-mW,” Biomed. Opt. Express 6(5), 1857–1866 (2015).
[PubMed]

Y. Zhao, Q. Zhan, J. Liu, and S. He, “Optically investigating Nd3+-Yb3+ cascade sensitized upconversion nanoparticles for high resolution, rapid scanning, deep and damage-free bio-imaging,” Biomed. Opt. Express 6(3), 838–848 (2015).
[PubMed]

R. Wu, Q. Zhan, H. Liu, X. Wen, B. Wang, and S. He, “Optical depletion mechanism of upconverting luminescence and its potential for multi-photon STED-like microscopy,” Opt. Express 23(25), 32401–32412 (2015).
[PubMed]

Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang, and S. Andersson-Engels, “Using 915 nm laser excited Tm3+/Er3+/Ho3+- doped NaYbF4 upconversion nanoparticles for in vitro and deeper in vivo bioimaging without overheating irradiation,” ACS Nano 5(5), 3744–3757 (2011).
[PubMed]

Hell, S. W.

Huang, B.

Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Ågren, and S. He, “Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles,” Nat. Commun. 8(1), 1058 (2017).
[PubMed]

Huang, X.

Hyeon, T.

Y. I. Park, K. T. Lee, Y. D. Suh, and T. Hyeon, “Upconverting nanoparticles: a versatile platform for wide-field two-photon microscopy and multi-modal in vivo imaging,” Chem. Soc. Rev. 44(6), 1302–1317 (2015).
[PubMed]

Isenberg, G.

W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
[PubMed]

Jia, T.

H. Zhang, T. Jia, L. Chen, Y. Zhang, S. Zhang, D. Feng, Z. Sun, and J. Qiu, “Depleted upconversion luminescence in NaYF4:Yb3+,Tm3+ nanoparticles via simultaneous two-wavelength excitation,” Phys. Chem. Chem. Phys. 19(27), 17756–17764 (2017).
[PubMed]

Jiang, Y.

R. Han, Z. Li, Y. Fan, and Y. Jiang, “Recent advances in super-resolution fluorescence imaging and its applications in biology,” J. Genet. Genomics 40(12), 583–595 (2013).
[PubMed]

Jin, D.

Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
[PubMed]

J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
[PubMed]

Kuang, C.

G. Zhao, C. Kuang, Z. Ding, and X. Liu, “Resolution enhancement of saturated fluorescence emission difference microscopy,” Opt. Express 24(20), 23596–23609 (2016).
[PubMed]

Y. Fang, C. Kuang, Y. Ma, Y. Wang, and X. Liu, “Resolution and contrast enhancements of optical microscope based on point spread function engineering,” Front. Optoelectron. 8, 152–162 (2015).

S. You, C. Kuang, Z. Rong, and X. Liu, “Eliminating deformations in fluorescence emission difference microscopy,” Opt. Express 22(21), 26375–26385 (2014).
[PubMed]

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 125708 (2013).

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 5708 (2013).

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Li, S.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
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Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
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Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Ågren, and S. He, “Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles,” Nat. Commun. 8(1), 1058 (2017).
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Liu, W.

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
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G. Zhao, C. Kuang, Z. Ding, and X. Liu, “Resolution enhancement of saturated fluorescence emission difference microscopy,” Opt. Express 24(20), 23596–23609 (2016).
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Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
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J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
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Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
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J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
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Y. Fang, C. Kuang, Y. Ma, Y. Wang, and X. Liu, “Resolution and contrast enhancements of optical microscope based on point spread function engineering,” Front. Optoelectron. 8, 152–162 (2015).

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C. T. Xu, P. Svenmarker, H. Liu, X. Wu, M. E. Messing, L. R. Wallenberg, and S. Andersson-Engels, “High-resolution fluorescence diffuse optical tomography developed with nonlinear upconverting nanoparticles,” ACS Nano 6(6), 4788–4795 (2012).
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S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen, and P. J. Schuck, “Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” Proc. Natl. Acad. Sci. U.S.A. 106(27), 10917–10921 (2009).
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J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
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W. A. Carrington, R. M. Lynch, E. D. Moore, G. Isenberg, K. E. Fogarty, and F. S. Fay, “Superresolution three-dimensional images of fluorescence in cells with minimal light exposure,” Science 268(5216), 1483–1487 (1995).
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Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Ågren, and S. He, “Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles,” Nat. Commun. 8(1), 1058 (2017).
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Piper, J. A.

Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
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J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
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Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Ågren, and S. He, “Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles,” Nat. Commun. 8(1), 1058 (2017).
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Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang, and S. Andersson-Engels, “Using 915 nm laser excited Tm3+/Er3+/Ho3+- doped NaYbF4 upconversion nanoparticles for in vitro and deeper in vivo bioimaging without overheating irradiation,” ACS Nano 5(5), 3744–3757 (2011).
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H. Zhang, T. Jia, L. Chen, Y. Zhang, S. Zhang, D. Feng, Z. Sun, and J. Qiu, “Depleted upconversion luminescence in NaYF4:Yb3+,Tm3+ nanoparticles via simultaneous two-wavelength excitation,” Phys. Chem. Chem. Phys. 19(27), 17756–17764 (2017).
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J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
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S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen, and P. J. Schuck, “Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” Proc. Natl. Acad. Sci. U.S.A. 106(27), 10917–10921 (2009).
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Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang, and S. Andersson-Engels, “Using 915 nm laser excited Tm3+/Er3+/Ho3+- doped NaYbF4 upconversion nanoparticles for in vitro and deeper in vivo bioimaging without overheating irradiation,” ACS Nano 5(5), 3744–3757 (2011).
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Y. I. Park, K. T. Lee, Y. D. Suh, and T. Hyeon, “Upconverting nanoparticles: a versatile platform for wide-field two-photon microscopy and multi-modal in vivo imaging,” Chem. Soc. Rev. 44(6), 1302–1317 (2015).
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H. Zhang, T. Jia, L. Chen, Y. Zhang, S. Zhang, D. Feng, Z. Sun, and J. Qiu, “Depleted upconversion luminescence in NaYF4:Yb3+,Tm3+ nanoparticles via simultaneous two-wavelength excitation,” Phys. Chem. Chem. Phys. 19(27), 17756–17764 (2017).
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C. T. Xu, P. Svenmarker, H. Liu, X. Wu, M. E. Messing, L. R. Wallenberg, and S. Andersson-Engels, “High-resolution fluorescence diffuse optical tomography developed with nonlinear upconverting nanoparticles,” ACS Nano 6(6), 4788–4795 (2012).
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S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen, and P. J. Schuck, “Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” Proc. Natl. Acad. Sci. U.S.A. 106(27), 10917–10921 (2009).
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Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
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J. Shen, G. Chen, A. M. Vu, W. Fan, O. S. Bilsel, C. C. Chang, and G. Han, “Engineering the Upconversion Nanoparticle Excitation Wavelength: Cascade Sensitization of Tri‐doped Upconversion Colloidal Nanoparticles at 800 nm,” Adv. Opt. Mater. 1, 644–650 (2013).

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C. T. Xu, P. Svenmarker, H. Liu, X. Wu, M. E. Messing, L. R. Wallenberg, and S. Andersson-Engels, “High-resolution fluorescence diffuse optical tomography developed with nonlinear upconverting nanoparticles,” ACS Nano 6(6), 4788–4795 (2012).
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Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang, and S. Andersson-Engels, “Using 915 nm laser excited Tm3+/Er3+/Ho3+- doped NaYbF4 upconversion nanoparticles for in vitro and deeper in vivo bioimaging without overheating irradiation,” ACS Nano 5(5), 3744–3757 (2011).
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Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
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F. Wang, R. Deng, and X. Liu, “Preparation of core-shell NaGdF4 nanoparticles doped with luminescent lanthanide ions to be used as upconversion-based probes,” Nat. Protoc. 9(7), 1634–1644 (2014).
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Y. Fang, C. Kuang, Y. Ma, Y. Wang, and X. Liu, “Resolution and contrast enhancements of optical microscope based on point spread function engineering,” Front. Optoelectron. 8, 152–162 (2015).

S. Li, C. Kuang, X. Hao, Y. Wang, J. Ge, and X. Liu, “Enhancing the performance of fluorescence emission difference microscopy using beam modulation,” J. Opt. 15, 5708 (2013).

C. Kuang, S. Li, W. Liu, X. Hao, Z. Gu, Y. Wang, J. Ge, H. Li, and X. Liu, “Breaking the diffraction barrier using fluorescence emission difference microscopy,” Sci. Rep. 3, 1441 (2013).
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Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
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Wu, S.

S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen, and P. J. Schuck, “Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals,” Proc. Natl. Acad. Sci. U.S.A. 106(27), 10917–10921 (2009).
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C. T. Xu, P. Svenmarker, H. Liu, X. Wu, M. E. Messing, L. R. Wallenberg, and S. Andersson-Engels, “High-resolution fluorescence diffuse optical tomography developed with nonlinear upconverting nanoparticles,” ACS Nano 6(6), 4788–4795 (2012).
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Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
[PubMed]

J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
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X. Xie, N. Gao, R. Deng, Q. Sun, Q.-H. Xu, and X. Liu, “Mechanistic investigation of photon upconversion in Nd3+-sensitized core-shell nanoparticles,” J. Am. Chem. Soc. 135(34), 12608–12611 (2013).
[PubMed]

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C. T. Xu, P. Svenmarker, H. Liu, X. Wu, M. E. Messing, L. R. Wallenberg, and S. Andersson-Engels, “High-resolution fluorescence diffuse optical tomography developed with nonlinear upconverting nanoparticles,” ACS Nano 6(6), 4788–4795 (2012).
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X. Xie, N. Gao, R. Deng, Q. Sun, Q.-H. Xu, and X. Liu, “Mechanistic investigation of photon upconversion in Nd3+-sensitized core-shell nanoparticles,” J. Am. Chem. Soc. 135(34), 12608–12611 (2013).
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Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
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Zhan, Q.

Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Ågren, and S. He, “Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles,” Nat. Commun. 8(1), 1058 (2017).
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J. Liu, R. Wu, N. Li, X. Zhang, Q. Zhan, and S. He, “Deep, high contrast microscopic cell imaging using three-photon luminescence of β-(NaYF4:Er3+/NaYF4) nanoprobe excited by 1480-nm CW laser of only 1.5-mW,” Biomed. Opt. Express 6(5), 1857–1866 (2015).
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Q. Zhan, X. Zhang, Y. Zhao, J. Liu, and S. He, “Tens of thousands‐fold upconversion luminescence enhancement induced by a single gold nanorod,” Laser Photonics Rev. 9, 479–487 (2015).

R. Wu, Q. Zhan, H. Liu, X. Wen, B. Wang, and S. He, “Optical depletion mechanism of upconverting luminescence and its potential for multi-photon STED-like microscopy,” Opt. Express 23(25), 32401–32412 (2015).
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Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang, and S. Andersson-Engels, “Using 915 nm laser excited Tm3+/Er3+/Ho3+- doped NaYbF4 upconversion nanoparticles for in vitro and deeper in vivo bioimaging without overheating irradiation,” ACS Nano 5(5), 3744–3757 (2011).
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H. Zhang, T. Jia, L. Chen, Y. Zhang, S. Zhang, D. Feng, Z. Sun, and J. Qiu, “Depleted upconversion luminescence in NaYF4:Yb3+,Tm3+ nanoparticles via simultaneous two-wavelength excitation,” Phys. Chem. Chem. Phys. 19(27), 17756–17764 (2017).
[PubMed]

Zhang, L.

J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
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Zhang, S.

H. Zhang, T. Jia, L. Chen, Y. Zhang, S. Zhang, D. Feng, Z. Sun, and J. Qiu, “Depleted upconversion luminescence in NaYF4:Yb3+,Tm3+ nanoparticles via simultaneous two-wavelength excitation,” Phys. Chem. Chem. Phys. 19(27), 17756–17764 (2017).
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Zhang, X.

Q. Zhan, X. Zhang, Y. Zhao, J. Liu, and S. He, “Tens of thousands‐fold upconversion luminescence enhancement induced by a single gold nanorod,” Laser Photonics Rev. 9, 479–487 (2015).

J. Liu, R. Wu, N. Li, X. Zhang, Q. Zhan, and S. He, “Deep, high contrast microscopic cell imaging using three-photon luminescence of β-(NaYF4:Er3+/NaYF4) nanoprobe excited by 1480-nm CW laser of only 1.5-mW,” Biomed. Opt. Express 6(5), 1857–1866 (2015).
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Zhang, Y.

H. Zhang, T. Jia, L. Chen, Y. Zhang, S. Zhang, D. Feng, Z. Sun, and J. Qiu, “Depleted upconversion luminescence in NaYF4:Yb3+,Tm3+ nanoparticles via simultaneous two-wavelength excitation,” Phys. Chem. Chem. Phys. 19(27), 17756–17764 (2017).
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Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang, and S. Andersson-Engels, “Using 915 nm laser excited Tm3+/Er3+/Ho3+- doped NaYbF4 upconversion nanoparticles for in vitro and deeper in vivo bioimaging without overheating irradiation,” ACS Nano 5(5), 3744–3757 (2011).
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Zhao, G.

Zhao, J.

Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
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J. Zhao, D. Jin, E. P. Schartner, Y. Lu, Y. Liu, A. V. Zvyagin, L. Zhang, J. M. Dawes, P. Xi, J. A. Piper, E. M. Goldys, and T. M. Monro, “Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence,” Nat. Nanotechnol. 8(10), 729–734 (2013).
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Zheng, X.

Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi, and D. Jin, “Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy,” Nature 543(7644), 229–233 (2017).
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Zhou, C.

Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Ågren, and S. He, “Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles,” Nat. Commun. 8(1), 1058 (2017).
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Figures (7)

Fig. 1
Fig. 1 Nanoparticles Synthesis and Characterization. (a) Schematic design of NaYF4: Nd3+/Yb3+/Er3+ (1/10/0.5 mol %) nanoparticle coated with NaYF4:Nd3+ (20 mol %) shell. (b) Proposed luminescent mechanism of NaYF4: Nd3+/Yb3+/Er3+ @NaYF4:Nd3+ UCNPs under 808 nm excitation. (c) TEM image of the synthesized core-shell UCNPs, size bar: 20 nm. (d) Normalized luminescence spectrum of UCNPs under 808-nm excitation.
Fig. 2
Fig. 2 The relationship between the red fluorescence emission intensity and excitation intensity in NaYF4: Nd3+/Yb3+/Er3+@NaYF4: Nd3+ nanoparticles. (a) Double logarithmic plot and linear fitting. (b) Normalized linear coordinate fitting for corresponding range in (a).
Fig. 3
Fig. 3 Simulated PSFs in different modes under 808-nm laser excitation. In sequence are solid, doughnut and FED PSF for (a, b, c): excitation laser focus spot, (e, f, g): two photon luminescence when excitation intensity less than 1 kW/cm2, and (i, j, k): saturated excited upconversion luminescence with an excitation intensity of 10 MW/cm2. (d, h, l) The profile plots of corresponding PSFs in each row.
Fig. 4
Fig. 4 Schematical illustration of the optical imaging setup. CL: Collimating lens, SL: Scanning lens, TL: Tube lens, OL: 60X objective lens. S1, S2: Shutters, M1-M5: Silver reflection mirrors, GM: Galvanometer mirror, DM: 690 short-pass dichroic mirror. P1, P2: Halve wave plates, VPP: Vortex phase plate, QWP: Quarter wave plate. F1: 694-nm short-pass filter, F2: 645-675 nm band-pass filter. PMT: photomultiplier tube.
Fig. 5
Fig. 5 Imaging of single UCNP at corresponding excitation intensities, image size 1.56 μm. (a, b, c) Solid laser excited PSFs at 0.1 MW/cm2, 1MW/cm2 and 10MW/cm2, respectively. (d, e, f) Doughnut laser excited PSFs at the corresponding exctiation intensity. (g, h, i) FED PSFs at corresponding excitation intensity with subtraction factors set to 0.83. (j, k, l) The intensity profile along the image center of solid and FED PSFs.
Fig. 6
Fig. 6 Imaging of monodispersed UCNPs under 10 MW/cm2 irradiation, image size 5 μm. (a) Image scanned by solid laser, and (b) by doughnut laser. (c) Subtraction image of (a) and (b), r = 0.83. (d) Intensity profile along the white arrow and (e) along the dashed line in (a) and (c), showing prominent resolution enhancement using FED strategy and two ajacent UCNPs were separately resolved.
Fig. 7
Fig. 7 The photostability of UCNPs. (a) Comparison of two scaned images of monodispersed UCNPs before and after 30 minutes scanning. (b) The time trace of emission intensity from UCNPs under 10 MW/cm2 continuous laser illumination for 30 minutes with 100 μs dwell time, and (d) for 1 second with 2 μs dwell time. The stability of time traces and the single peak Gaussian distributions of corresponding histograms in (c) and (e), showing absolutely no photobleaching and on/off blinking behavior.

Equations (3)

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I F E D = I s o l i d r × I d o u g h n u t
h u c ( x , y ) f ( h e x ( x , y ) )
I e m = e x p ( a l n ( I e x . ) + b ( l n ( I e x ) ) 2 + c l n ( I e x ) + d ) ( I e x > 0 )

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