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Abstract
We demonstrate a new optical source in the form of a thin scintillating film with subdiffraction resolution for bioimaging. Atomically smooth, uniform and highly luminescent scintillating membranes have been successfully fabricated by radio frequency magnetron sputtering for application as a nanoscale imaging probe. This cathodoluminescence-activated optical transducer converts electron beam energy to near-field, visible radiation simultaneously satisfying the requirements for high-resolution, dye-free imaging. We discovered high-yield emission for YAlO3:Gd3+ perovskite thin films with enhanced scintillating properties by providing experimental evidence for quantum cutting in the ultraviolet-visible region. A subwavelength spatial resolution of ∼100 nm with a high signal-to-noise ratio (SNR = 20) under the atmospheric condition is confirmed by using a luminescent optical probe. The simple manufacturing process of such scintillating membranes broadens their application toward super-resolution microscopy, fulfilling prerequisites for biomedical imaging where noninvasive nanoprobe is required.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
In bioimaging, visualizing subcellular structures and molecular dynamics is crucial for understanding cellular function and protein behavior. To overcome the diffraction limit encountered in the classical microscopy of biological specimens, a variety of imaging methods with subdiffraction resolution have been recently developed for optical microscopy. Novel fluorescent imaging techniques such as structured illumination microscopy (SIM) [1], photoactivated localization microscopy (PALM) [2], stochastic optical reconstruction microscopy (STORM) [3], single-molecule localization microscopy (SMLM) [4] and stimulated emission depletion (STED) [5] have been successfully applied to the in-cell tracking of tagged biological specimens. Although such techniques transcend the diffraction limit with spatial resolution down to ∼20 nm, dye-free imaging of living cells in aqueous solution without dye labels remains challenging for these methods. However, in vivo imaging of complex cells, their structures, and dynamics down to the molecular scale requires high resolution as well as a noninvasive and tag-free approach.
In addition to fluorescent imaging [6], liquid scanning transmission electron microscopy (STEM) [7], and other super-resolution microscopy techniques [8], we have already developed the electron beam excitation-assisted (EXA) optical microscopy [9]. In the present study, we use a highly luminescent thin film as a near-field optical probe with salient features of the EXA microscopy for super-resolution, dye-free imaging. This film suppresses the invasive nature of electron beam irradiation. An electron beam focused onto a few-nanometer diameter spot on the luminescent film causes noninvasive nanoscale optical excitation (Fig. 1(a)), resulting in a high spatial resolution that is dependent on the material properties and local scattering of electrons on the luminescent film. Thin film scintillators aim to completely stop the electron beam and convert its energy into visible radiation via cathodoluminescence (CL). By offering the inherent advantages of conventional light microscopy, optical imaging assisted by electron excitation with the luminescent probe has no diffraction limit on the spatial resolution. An appropriate combination of film thickness and accelerating voltage is essential for high-resolution imaging with bright optical emission without transmitted electrons. Such a scintillating material must be radiation-resistant, robust against electron damage, and biocompatible. It must also have a high luminescence quantum yield with a narrow spectral bandwidth. Previously employed zinc oxide (ZnO) thin films offer most of these features but may be toxic to a few biological samples and have low luminosity with a reduced spatial resolution. Therefore, in this work, we propose the EXA microscope with perovskite rare-earth (RE)-doped thin films. RE-doped thin films are preferable for their unique scintillating properties and biocompatibility [10]. In contrast to luminescent NPs [11] or potentially toxic quantum dots (QDs) [12], we use flat ultrathin Gd3+-doped yttrium aluminum perovskite (YAlO3:Gd3+, YAP) thin film scintillators. The advantages of semiconductor technology facilitate the use of micromachined electron-transparent membranes [13] combined with RE-doped thin films. Thus, we elaborate the imaging platform, which consists of a tightly focused electron beam incident onto a luminescent material from one side, generating optical excitation beneath the sample on the opposite side for noninvasive imaging with a nanoscale optical probe (Fig. 1(a) inset). RE-doped materials are well-known phosphors and are widely used as high-yield optical scintillators for detecting high-energy particles and gamma rays in medical diagnostic imaging [14]. Numerous wide-band-gap phosphors have been commercialized for field-emission displays (FEDs) [15] and plasma display panels (PDPs) [16] due to their thermal properties, radiation resistance and photonic excitation in the vacuum-ultraviolet (VUV) region.
Fig. 1. (a) Schematic of an EXA microscope. The inset shows the principle of near-field optical imaging with YAP thin film as a nanoscale optical probe. (b) Energy level diagram of Gd3+ ions with an illustration of the QC processes. (c) Monte Carlo simulation of the energy distribution in a YAP thin film. (d) Simulated CL depth profiles for YAP thin films using an accelerating voltage of 1.5 kV (blue), 3 kV (black), and 5 kV (green). Experimental CL intensities for 30-, 35-, 40-, and 50-nm thin films are indicated in red.
Furthermore, quantum cutting (QC) from RE ions is highly promising due to a bright, narrow emission with the capability to excite autofluorescence [17,18] without any fluorescent dyes. Activated with Gd3+ trivalent lanthanide ions, YAP thin films show excellent luminescent properties attributed to abundant energy levels in the 4f7 configuration. Typical energy transfer from the VUV to ultraviolet-visible (UV-Vis) region with improved emission is made possible by highly efficient conversion due to QC. High energy from the electron beam generates a VUV photon, which excites two photons of lower energy, resulting in photon cascade luminescence. Figure 1(b) presents the energy level diagram for Gd3+ ions with the possible QC processes. A transition from the ground state 8S7/2 to the excited state 6Gj for the Gd3+ ion involves subsequent 6Gj$\; \to \; $6Pj, 6Pj$\; \to \;$8S7/2 radiative transitions. These transitions are expected to occur in the UV-Vis region [19].
We present the fabrication, material characterization, and optical properties of YAP thin films and demonstrate super-resolution EXA images for 100-nm gold (Au) NPs with a high signal-to-noise ratio (SNR). Sputter deposition, the de facto standard in the semiconductor industry, can potentially replace other difficult and costly fabrication methods for manufacturing high-quality scintillators at a low cost. Such a combination enables on-chip cell culturing and bioimaging with high spatial resolution. We anticipate the future integration of YAP thin films with micro/nanoelectromechanical systems and silicon integrated photonics for biological applications, radiation detectors, solar cells and display panels.
2. Experimental section
2.1 Fabrication
High-quality Gd3+-doped YAlO3 perovskite thin films were fabricated by radio-frequency (RF) magnetron sputtering (Sanyu-Electron, SVC-700RF II). Stoichiometric (Y0.97Gd0.03)AlO3 targets of 99.99% purity (Kojundo Co., Ltd) were used to deposit thin films in a pure argon and mixed oxygen/argon atmosphere. All films were deposited at room temperature with an RF power of 30, 50 and 60 W. The initial pressure of the system was 10−4 Pa. After an Ar/O2 reactive gas mixture was introduced, the pressure in the vacuum chamber was maintained at ∼5 Pa during deposition. The argon and oxygen flow rates were 20 sccm and 1 sccm, respectively, and the deposition times were 30, 60, 120, and 180 min. Silicon frames of 5.0 mm × 5.0 mm containing 35-, 50-, and 100-nm-thick silicon nitride (Si3N4) membranes (Silson Corp.) and 35-nm-thick single silicon (Si) membranes (EMJapan Co., Ltd.) were used as substrates. The dimensions of the Si3N4 and Si membrane windows were 50 µm × 50 µm, 100 µm × 100 µm, and 100 µm × 250 µm, respectively. After deposition, the thin films were annealed in a 3-zone quartz tube furnace at 1000° C for 0.5-1 hour in a nitrogen atmosphere. The nitrogen flow rate was 0.5 L/min. Heating rates of 4 °C/min and 8 °C/min were applied during annealing and cooling, respectively.
2.2 Characterization
The crystal structure was investigated by X-ray diffractometry (XRD) using an X-ray diffractometer (Rigaku, Rint Ultima 3) with CuKα radiation (λ = 1.542 Å). An atomic force microscopy (AFM) image of the YAP surface over an area of 1 µm × 1 µm was collected (Hitachi, SPI3800) in DFM and contact modes.
CL images were obtained using a field emission scanning electron microscope (Jeol, JSM-7001F) in combination with a parabolic mirror (Horiba, LS-100-EM-TYPE2), a monochromator (Princeton Instruments, Acton SP-2300i) and a highly sensitive photomultiplier tube (PMT) (H11461-P11, Hamamatsu Photonics) in the photon-counting mode. Lifetime measurements were performed using a PL quantum yield spectrometer (Hamamatsu Photonics, Quantaurus-QY C11347-11) and an ultraviolet-visible (UV-VIS) fluorescence spectrometer (Jasco, FP-6500) in the nanosecond and millisecond range, respectively.
EXA microscope observation for 100-nm Au NPs (Sigma-Aldrich, PN. 742031) was performed under the atmospheric condition. The inverted scanning electron microscope (SEM) (APCO, MINI-EOC) and optical microscope (Olympus, BXFM) were separated by a luminescent YAP membrane during imaging. The 60× objective lens (Olympus, LUMPLFLN60XW) was used to collect CL emission. Finally, CL emission was detected by a highly sensitive, subminiature PMT (R7400U-06, Hamamatsu Photonics) in analog mode.
3. Results and discussion
To determine the optimal film thickness for providing a narrow, near-field optical probe, we first performed Monte Carlo simulations [20,21]. Figure 1(c) illustrates the size of the localized CL spot excited by a focused electron beam and its energy distribution in the volume of the thin scintillating film (Fig. 6 in Appendix). When the electron beam passes through the Si membrane, the YAP film is excited, broadening the diameter of the electron beam and leading to the divergence of the CL spot. Such broadening of the primary electron beam was estimated to be up to 30-50 nm after passing through a Si membrane and YAP thin film. This process is dependent on the film thickness due to scattering within the film volume. Thus, the localized CL spot causes subwavelength optical excitation, which directly influences the spatial resolution. The spatial resolution is equal to the CL spot broadening. In this work, the 30-50 nm broadening being lesser than the diffraction limit of light (∼250 nm) leads to a far improved spatial resolution. This 30-50 nm spatial resolution is smaller or on the order of the general feature size of the biological samples. Therefore, it is sufficient enough for many bioimaging applications. Assuming a Si substrate with constant thickness and infinitely thick YAP, we estimated the optimal thickness for the luminescent material to be in the range of 30-50 nm. To confirm our theoretical calculations, we deposited thin films of 30-, 35-, 40-, and 50-nm thickness on 35-nm Si substrates with subsequent annealing as described in the experimental section. Measured CL spectra revealed the highest CL intensity for the 35-nm YAP thin film. Based on rigorous simulations, CL intensity with respect to the thickness of the thin films revealed a correlation between the experimental and theoretical results in Fig. 1(d). The dependence of CL intensity on accelerating voltage, as shown in Fig. 2(a), shows a maximum efficiency near 6 kV. Beyond 7 kV, the CL intensity starts to decay, indicating that the electron beam penetrates the YAP thin film, reducing the amount of interactions required for CL excitation.
Fig. 2. (a) Dependence of CL intensity on accelerating voltage. (b) Micromachined electron-transparent membrane with 9 single-crystal silicone (Si) windows. (c) Opposite side of the Si membrane with deposited YAP thin film. (d) Modified specimen holder for EXA microscope. (e) XRD pattern for a YAP thin film (red), reference pattern of ICDD (green) and simulated pattern (blue). (f) AFM image of the YAP thin film surface.
The surface morphology and luminescent properties of the deposited YAP scintillators are strongly dependent on the properties of the substrate. To find a suitable host with respect to optimum CL intensity and surface roughness, we studied YAP thin films deposited on different substrates. We found that silicon nitride/yttrium oxide (Si3N4/Y2O3) substrates are unsuitable for the deposition of YAP thin films because of a significant lattice mismatch and high residual stress. Single-crystal silicon (Si) membranes with a thickness of 35 nm were used as substrates for the thin film deposition. Figure 2(b) presents the SEM image of an imaging chip with electron-transparent windows defined by the Si membranes. Deposition of the luminescent film on the opposite side activates these windows for imaging with up to 9 different specimens at one time (Fig. 2(c)). It is of crucial technological importance that the electron-transparent membrane be capable of withstanding the vacuum inside the SEM, while still providing a uniform background to enable imaging with high resolution and contrast. We corroborate the single-crystal Si membranes are robust, with a maximum tolerated differential pressure of 2.34 × 105 Pa for a sealed vacuum chamber isolated from the specimen. For imaging at atmospheric pressure, we used a modified specimen holder composed of a glass dish, a metal plate, and the attached Si membrane with deposited YAP thin film (Fig. 2(d)). Both, the glass dish and the metal plate have holes in the middle for passing of the electron beam. The imaging chip and the metal plate are glued to the glass dish by using epoxy resin. A metal plate is aided in preventing a charging effect from the electron beam irradiation.
As near-field interactions between the nanoscale CL spot and the specimen imply distances smaller than 100 nm [22], an atomically smooth surface is preferable. Since the specimen is located directly on the thin film, the surface must be smooth, homogeneous and biocompatible and must emit persistent CL with high photostability. The thickness of the deposited films, measured using an Alpha-Step IQ (KLA-Tencor) profiler, was determined to be in the range of 30-50 ± 0.8 nm. The crystal structure for the annealed films was confirmed by X-ray spectroscopy. The X-ray diffraction pattern (XRD) shown in Fig. 2(e) corresponds to a perovskite stoichiometry with a single-phase orthorhombic structure. The observed XRD spectra agreed well with the ICDD 33-0041 diffraction pattern. The theoretical XRD pattern was calculated in CaRIne Crystallography and was found to coincide with the ICDD and experimental data. Lattice parameters were calculated from the experimental XRD peak angles and compared to XRD simulations of the YAP:Gd3+ crystal. In this manner, lattice constants for orthorhombic YAP with a 62Pnma space group were estimated as a = 5.4 Å, b = 7.375 Å, and c = 5.18 Å. The average grain size of ∼0.75 nm was estimated from the XRD pattern by the Scherrer equation:
where k = 0.89 is a numerical constant, λ is the wavelength of the X-rays radiation, β is the diffraction peak of full-width at half maximum, and θ is the Bragg angle. Surface analysis revealed atomically smooth YAP thin films with a surface root-mean-square (RMS) < 1 nm (Fig. 2(f)). In addition to atomic force microscopy (AFM) measurements, observation by SEM was carried out to verify the high surface quality without visible grains or other surface imperfections.Electron beam damage is one of the limiting factors in bioimaging. Accurate electron dose interpretation is needed to evaluate the threshold for cell damage. This parameter can affect the spatial resolution and image contrast with a detrimental effect on biological specimens due to electrons penetrating through the membrane and being absorbed by cells. Hence, a low electron dose is needed to prevent cell damage. The minimum probe current of 1.6 pA required for imaging with sufficient contrast based on the Rose criterion estimated by the threshold equation [23]:
where Ib is the probe current, ηs is the backscatter coefficient, T is the imaging time in seconds, and T is the contrast. In our experimental configuration for the EXA microscope, the acquisition time required to obtain an image of 1,024 × 1,024 pixels was 10 seconds, with a pixel dwell time of 5-10 microseconds. For the imaging mode, the accelerating voltage was fixed at 5 kV, with a probe current of 150-200 pA and a pixel size of 15-25 nm, which produced an average electron dose of approximately 9 e−/nm2. This dose is considerably lower than that used in several other imaging techniques [24–26]. In this case, 95% of the total deposited energy was transmitted through the Si membrane, after which 70% of the remaining electron flux passed through the YAP thin film. Further deposition of an additional conductive layer beneath the scintillating film can create an optical transducer, which would ultimately prevent electrons from reaching the specimen.To investigate the luminescence uniformity of the YAP thin films, we performed spectroscopy measurements by acquiring CL images and spectra. A monochromator was used for measuring the YAP emission spectra and for imaging at a selected wavelength (Fig. 3(a)). Focused at each point, the electron beam excites CL light from the YAP scintillator in a narrow area down to 30-50 nm. Light reflected from the CL mirror passes by a light guide through a monochromator before detection by a highly sensitive PMT. During imaging, the intensity of each pixel corresponds to the electron beam position during scanning. The monochromatic photon emission map shown in Fig. 3(b) was obtained by scanning the electron beam in the middle of the Si window over the YAP thin film. The average intensity per pixel was 202 mV, with a standard deviation estimated at 3.1 mV. The histogram in Fig. 3(b) (inset) shows the intensity variation as a function of electron beam position, with an evident normal distribution. Based on the calculated variability of the acquired images, we confirmed uniform, persistent luminescence for the YAP thin films within 1.5% variation of the CL emission. We assume that the suppression of the variation of CL intensity is caused by homogeneous crystalline growth and sub-nanometer crystallite size after the annealing process.
Fig. 3. (a) Schematic of spectroscopic CL measurements. (b) Monochromatic photon emission map. The inset shows the CL intensity distribution. (c) Monochromatic CL image of 100-nm Au NPs.
Electron scattering affects the spatial resolution, and a sufficient SNR (> 5) is required to obtain an image that can be used to discriminate an object from the background. The latter depends on the luminescent properties of the scintillating material. A low luminescence intensity, even with sufficient resolution, will result in the inability to distinguish observed objects because of the low emission with high background noise. To estimate the necessary detection limit and SNR for CL imaging, we consider the dark current and shot noise of the PMT in the photon-counting mode. The detection limit can be defined as the light level at which the SNR equals 5 according to the Rose criterion [27]. Thus, an estimated detection limit of 107 photons/s is required to obtain an image of 512 × 512 pixels within 0.05 fps. SNR was calculated by considering the dark current and shot noise of the PMT with the following formula:
where Ik is the cathode current and B is the bandwidth. The acquired monochromatic CL image for 100-nm Au NPs at 630 nm in Fig. 3(c) with SNR = 6 confirms the sufficient luminosity of the YAP thin films for spectrally selective CL imaging. The value of 630 nm for estimation of the SNR was obtained from CL spectra measurements, which will be discussed next.The luminescence efficiency of thin scintillating films was determined using CL spectroscopy. Figure 4(a) illustrates the CL spectra for YAP thin films measured at 5 kV in the UV-Vis region. The CL intensities, ascribed as PMT counts per second (counts/s), present two strong peaks at 317 nm and 630 nm. The detection efficiency ηdet in photon-counting mode corresponds to the ratio of the number of counted pulses Nd to the number of incident photons Np at a given wavelength:
where η is the PMT quantum efficiency, ηAl is the reflectivity of aluminum, ηSi is the fraction of light reflected from the Si substrate, ηΩ = 1.33π/4π is the fraction of mirror-reflected photons into a solid angle, and α is the collection efficiency of the PMT. Considering the quantum efficiency of the PMT at the corresponding wavelengths (Table 1), the YAP thin films emit a total of 2×107 photons/s, facilitating spectrally resolved imaging with an SNR ≥ 7.Fig. 4. (a) CL spectra of YAP thin film. (b) PL spectra for the YAP thin film. The inset depicts the lifetime decay. (c) Lifetime measurements of YAP thin film in the nanoscale range. The PL instrument response function (IRF) is illustrated in black.
Table 1. Detection efficiency of PMT in photon-counting mode.
The spectral data are characterized by narrow emission peaks of almost monochromatic light with a line-width of 5 nm due to weak vibronic coupling of the Gd3+ ions [19]. UV-Vis emission with sharp peaks centered at 317 nm and 630 nm corresponds to subsequent 6P7/2$\; \to \; $8S7/2, 6G7/2 $\to \; $6P3/2 transitions caused by QC. Many studies have reported VUV (8Sj$\to $6Gj), UV (6Pj$\to $8S7/2), Vis (6Gj$\to $6Pj) or IR emission from Gd3+ RE ions [28–30]. Also, strong UV and slight Vis emission were previously observed simultaneously in [19,31], we discovered emission lines in the UV-Vis region with the high-intensity peaks of the same order of magnitude. For comparison, we measured the CL spectra for previously used ZnO [32] and Zn2SiO4 [33] thin films under the same conditions described in the experimental section. Spectrally selected CL intensities for annealed YAP thin films give a total photon flux of 2×107 photons/s contributed from 6.5×106 photons/s in the UV type B (280-320 nm) region at 317 nm and 1.4×107 photons/s in the visible region at 630 nm. ZnO and Zn2SiO4 emissions at 382 nm give a total of 4.5×104 photons/s and 2.4×105 photons/s, respectively. Thus, according to spectral measurements, we report a very high CL intensity for YAP thin films with enhanced scintillating properties. Figure 4(b) shows the PL emission spectra excited at 210 nm, in which the CL peak at 317 nm is present. The absence of an emission peak at 630 nm is attributed to the 6G7/2 $\to \; $6P3/2 transition caused by multiphoton 6Gj $\to \; $6Dj, 6Dj $\to \; $6Ij, 6Ij $\to \; $6Pj relaxations.
Additional information regarding the origin of the UV-Vis emission obtained from PL lifetime measurements is presented in Figs. 4(c) and 4(b) (inset). Emission decay curves were recorded under excitation at 280 nm with emission monitored at 320 nm. The presence of a few decay components in the nanosecond range is evident. We assume these fast decays are due to nonradiative transitions. Single-exponent fitting for the emission decay curve in Fig. 4(b) (inset) determines the lifetime yield for the Gd3+ excited 6P7/2 state to be 0.6 ms, which is consistent with previously reported results [28,29].
To elucidate the spatial resolution and contrast of the EXA microscope, we obtained CL images of Au NPs dispersed directly on a YAP thin film. An EXA microscope is an integrated microscope, which consists of an inverted SEM and fluorescence microscope. This approach incorporates simultaneous CL and secondary electron (SE) imaging in which the PMT signal is coupled with the electron beam position. As previously mentioned, the CL light from the YAP thin film is scattered in the specimen and is then collected and detected with a highly sensitive photodetector. Near-field interactions involve illumination and require the distance between the optical source and the sample to be smaller than the excitation wavelength, resulting in high spatial resolution and contrast. Figure 5 shows dual SE and CL EXA images acquired in the same region of interest using a 5-kV electron beam. During SEM imaging, electron beam rastering over the luminescent membrane generates SEs. SEs scatter more on the NPs, producing an image with bright spots on a dark background. In contrast to SEM, EXA imaging involves electron-beam-excited CL in the luminescent film behind the specimen on the opposite side, forming a bright background with dark spots because CL emission is obscured by the Au NPs located on the top. These imaging modes result in an inversion of contrast between the SE and EXA images. As the full width at half maximum (FWHM) obtained from the intensity profile in Fig. 5(a) is 100 ± 2 nm, we confirm subdiffraction resolution for the acquired images. An intensity profile of the EXA image in Fig. 5(a) across a single Au NP revealed a high contrast between the signal from the NP and the background noise. SNR is commonly defined as the ratio of the peak signal value to the standard deviation of the background. The resulting SNR value of ∼20 is four times higher than the minimum required. Thus, we have demonstrated a spatial resolution of approximately 100 nm with a high SNR.
Fig. 5. (a) EXA image of 100-nm Au NPs. The Au NPs are depicted as black spots. (b) Corresponding SE image. The Au NPs are depicted as white spots.
4. Conclusion
In summary, we have introduced a new nanoimaging platform for super-resolution imaging with luminescence enhanced nanoprobe. Our approach combines the concept of electron beam excitation-assisted (EXA) optical microscopy with a luminescent thin film that converts electron beam energy to near-field, visible radiation at the nanoscale. YAlO3:Gd3+ thin films act as a near-field optical source, achieving resolution below the classical diffraction limit. The development of bright scintillator films was an inevitable step to CL-activated nanoimaging under the atmospheric condition in the air or liquid. We determined the most favorable parameters for manufacturing these thin films for super-resolution bioimaging, including film thickness, CL uniformity, and emission yield. The potential for application in single-molecule imaging attracts significant interest in biological and pharmacological research due to the ability to elucidate molecular structure and intracellular interactions. We have demonstrated dye-free imaging by using YAP thin films as an optical nanoprobe with subdiffraction spatial resolution and SNR of ∼20. These films are a promising illumination source for advanced imaging techniques such as environmental electron microscopy for live-cell imaging in liquid [7,24–26], atmospheric SEM (ASEM) [34], CL-activated nanoimaging [35], high-resolution CL spectroscopy [36], in situ dynamic nanoimaging [37] and EXA microscopy.
Appendix
Funding
Adaptable and Seamless Technology Transfer Program through Target-Driven R and D (A-STEP).
Acknowledgements
We acknowledge Prof. A. Ono, Prof. V. Mizeikis and G. Prabhudesai for helpful discussions.
References
1. R. Heintzmann and T. Huser, “Super-Resolution Structured Illumination Microscopy,” Chem. Rev. 117(23), 13890–13908 (2017). [CrossRef]
2. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006). [CrossRef]
3. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006). [CrossRef]
4. Z. Liu, L. D. Lavis, and E. Betzig, “Imaging Live-Cell Dynamics and Structure at the Single-Molecule Level,” Mol. Cell 58(4), 644–659 (2015). [CrossRef]
5. 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). [CrossRef]
6. M. Fernández-Suárez and A. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol. 9(12), 929–943 (2008). [CrossRef]
7. N. d. Jonge, D. B. Peckys, G. J. Kremers, and D. W. Piston, “Electron microscopy of whole cells in liquid with nanometer resolution,” Proc. Natl. Acad. Sci. 106(7), 2159–2164 (2009). [CrossRef]
8. S. Kawata, Y. Inouye, and P. Verma, “Plasmonics for near-field nano-imaging and superlensing,” Nat. Photonics 3(7), 388–394 (2009). [CrossRef]
9. W. Inami, K. Nakajima, A. Miyakawa, and Y. Kawata, “Electron beam excitation assisted optical microscope with ultra-high resolution,” Opt. Express 18(12), 12897–12902 (2010). [CrossRef]
10. B. C. Heng, G. K. Das, X. Zhao, L.-L. Ma, T. T.-Y. Tan, K. W. Ng, and J. S.-C. Loo, “Comparative cytotoxicity evaluation of lanthanide nanomaterials on mouse and human cell lines with metabolic and DNA-quantification assays,” Biointerphases 5(3), FA88–FA97 (2010). [CrossRef]
11. J. Zhou, Q. Liu, W. Feng, Y. Sun, and F. Li, “Upconversion luminescent materials: Advances and applications,” Chem. Rev. 115(1), 395–465 (2015). [CrossRef]
12. W. R. Algar, K. Susumu, J. B. Delehanty, and I. L. Medintz, “Semiconductor quantum dots in bioanalysis: Crossing the valley of death,” Anal. Chem. 83(23), 8826–8837 (2011). [CrossRef]
13. T. K. Nguyen, H. P. Phan, H. Kamble, R. Vadivelu, T. Dinh, A. Iacopi, G. Walker, L. Hold, N. T. Nguyen, and D. V. Dao, “Superior Robust Ultrathin Single-Crystalline Silicon Carbide Membrane as a Versatile Platform for Biological Applications,” ACS Appl. Mater. Interfaces 9(48), 41641–41647 (2017). [CrossRef]
14. C. W. E. Van Eijk, “Inorganic scintillators in medical imaging detectors,” Nucl. Instrum. Methods Phys. Res., Sect. A 509(1-3), 17–25 (2003). [CrossRef]
15. A. A. Talin, K. A. Dean, and J. E. Jaskie, “Field emission displays: A critical review,” Solid-State Electron. 45(6), 963–976 (2001). [CrossRef]
16. H. Lin, G. Zhang, P. A. Tanner, and H. Liang, “VUV-Vis luminescent properties of BaCaBO3F doped with Ce3+ and Tb3+,” J. Phys. Chem. C 117(24), 12769–12777 (2013). [CrossRef]
17. F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013). [CrossRef]
18. M. Kikawada, A. Ono, W. Inami, and Y. Kawata, “Plasmon-Enhanced Autofluorescence Imaging of Organelles in Label-Free Cells by Deep-Ultraviolet Excitation,” Anal. Chem. 88(2), 1407–1411 (2016). [CrossRef]
19. R. Wegh, H. Donker, A. Meijerink, R. Lamminmäki, and J. Hölsä, “Vacuum-ultraviolet spectroscopy and quantum cutting for Gd3+ in LiYF4,” Phys. Rev. B - Condens. Matter Mater. Phys. 56(21), 13841–13848 (1997). [CrossRef]
20. Y. Nawa, W. Inami, A. Chiba, A. Ono, A. Miyakawa, Y. Kawata, S. Lin, and S. Terakawa, “Dynamic and high-resolution live cell imaging by direct electron beam excitation,” Opt. Express 20(5), 5629–5635 (2012). [CrossRef]
21. D. Drouin, A. R. Couture, D. Joly, X. Tastet, V. Aimez, and R. Gauvin, “CASINO V2.42 - A fast and easy-to-use modeling tool for scanning electron microscopy and microanalysis users,” Scanning 29(3), 92–101 (2007). [CrossRef]
22. W. Inami, J. Fujiwara, F. Masahiro, A. Ono, and Y. Kawata, “Analysis of electron and light scattering in a fluorescent thin film by combination of Monte Carlo simulation and finite-difference time-domain method,” Appl. Phys. Lett. 101(15), 151104 (2012). [CrossRef]
23. S. Thiberge, O. Zik, and E. Moses, “An apparatus for imaging liquids, cells, and other wet samples in the scanning electron microscopy,” Rev. Sci. Instrum. 75(7), 2280–2289 (2004). [CrossRef]
24. E. Kennedy, E. M. Nelson, T. Tanaka, J. Damiano, and G. Timp, “Live Bacterial Physiology Visualized with 5 nm Resolution Using Scanning Transmission Electron Microscopy,” ACS Nano 10(2), 2669–2677 (2016). [CrossRef]
25. D. B. Peckys and N. De Jonge, “Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy,” Nano Lett. 11(4), 1733–1738 (2011). [CrossRef]
26. L. R. Parent, E. Bakalis, A. Ramírez-Hernández, J. K. Kammeyer, C. Park, J. De Pablo, F. Zerbetto, J. P. Patterson, and N. C. Gianneschi, “Directly Observing Micelle Fusion and Growth in Solution by Liquid-Cell Transmission Electron Microscopy,” J. Am. Chem. Soc. 139(47), 17140–17151 (2017). [CrossRef]
27. A. Rose, “Television Pickup Tubes and the Problem of Vision,” Adv. Electron. Electron Phys. 1, 131–166 (1948). [CrossRef]
28. Z. Yang, J. H. Lin, M. Z. Su, Y. Tao, and W. Wang, “Photon cascade luminescence of Gd3+ in GdBaB9O16,” J. Alloys Compd. 308(1-2), 94–97 (2000). [CrossRef]
29. J. Zhong, H. Liang, Q. Su, J. Zhou, Y. Huang, Z. Gao, Y. Tao, and J. Wang, “Luminescence properties of NaGd(PO3)4:Eu3+ and energy transfer from Gd3+ to Eu3+,” Appl. Phys. B: Lasers Opt. 98(1), 139–147 (2010). [CrossRef]
30. K. Zheng, W. Qin, C. Cao, D. Zhao, and L. Wang, “NIR to VUV: Seven-photon upconversion emissions from Gd3+ ions in fluoride nanocrystals,” J. Phys. Chem. Lett. 6(3), 556–560 (2015). [CrossRef]
31. Y. Shimizu, K. Ueda, and Y. Inaguma, “Photoluminescence excitation spectra of lanthanide doped YAlO3 in vacuum ultraviolet region,” Opt. Mater. 66, 327–331 (2017). [CrossRef]
32. A. Miyake, S. Kanamori, Y. Nawa, W. Inami, H. Kominami, Y. Kawata, and Y. Nakanishi, “Formation of ZnO luminescent films on SiN films for light source of high-resolution optical microscope,” Jpn. J. Appl. Phys. 53(3), 034101 (2014). [CrossRef]
33. T. Furukawa, S. Kanamori, M. Fukuta, Y. Nawa, H. Kominami, Y. Nakanishi, A. Sugita, W. Inami, and Y. Kawata, “Fabrication of bright and thin Zn2SiO4 luminescent film for electron beam excitationassisted optical microscope,” Opt. Express 23(14), 18630–18637 (2015). [CrossRef]
34. H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, S. Kitamura, and C. Sato, “Reprint of: Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film,” J. Struct. Biol. 172(2), 191–202 (2010). [CrossRef]
35. C. G. Bischak, C. L. Hetherington, Z. Wang, J. T. Precht, D. M. Kaz, D. G. Schlom, and N. S. Ginsberg, “Cathodoluminescence-activated nanoimaging: Noninvasive near-field optical microscopy in an electron microscope,” Nano Lett. 15(5), 3383–3390 (2015). [CrossRef]
36. P. Chaturvedi, K. H. Hsu, A. Kumar, K. H. Fung, J. C. Mabon, and N. X. Fang, “Imaging of plasmonic modes of silver nanoparticles using high-resolution cathodoluminescence spectroscopy,” ACS Nano 3(10), 2965–2974 (2009). [CrossRef]
37. N. Liv, D. S. B. Van Oosten Slingeland, J. P. Baudoin, P. Kruit, D. W. Piston, and J. P. Hoogenboom, “Electron microscopy of living cells during in situ fluorescence microscopy,” ACS Nano 10(1), 265–273 (2016). [CrossRef]
