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Ultra-Bright and Stable Luminescent Labels for Correlative Cathodoluminescence Electron Microscopy (CCLEM) Bioimaging Kerda Keevend, Laurits Puust, Karoliine Kurvits, Lukas R.H. Gerken, Fabian H.L. Starsich, Jian-Hao Li, Martin T Matter, Anastasia Spyrogianni, Georgios A. Sotiriou, Michael Stiefel, and Inge K Herrmann Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b01819 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019
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Ultra-Bright and Stable Luminescent Labels for
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Correlative Cathodoluminescence Electron
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Microscopy (CCLEM) Bioimaging
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Kerda Keevend1,2,3, Laurits Puust4, Karoliine Kurvits4, Lukas R.H. Gerken1,3, Fabian H.L.
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Starsich5, Jian-Hao Li1,3, Martin T. Matter1,3,5, Anastasia Spyrogianni6, Georgios A. Sotiriou7,
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Michael Stiefel8,º, and Inge K. Herrmann1,3,º,*
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1
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Laboratories for Materials Science and Technology (Empa), Lerchenfeldstrasse 5, CH-9014 St.
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Gallen, Switzerland. 2
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Laboratory for Particles Biology Interactions, Department Materials Meet Life, Swiss Federal
Optical Nanomaterial Group, Institute for Quantum Electronics, Department of Physics, ETH Zurich, Auguste-Piccard- Hof 1, CH-8093 Zurich, Switzerland.
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Laboratory for Nanoparticle Systems Engineering, Institute of Process Engineering, Department
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of Mechanical and Process Engineering, ETH Zurich, Sonneggstrasse 3, CH-8092 Zurich,
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Switzerland.
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Laboratory of Laser Spectroscopy, Institute of Physics, University of Tartu, W. Ostwaldi St 1, 50411 Tartu, Estonia.
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5
Particle Technology Laboratory, Institute of Process Engineering, Department of Mechanical
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and Process Engineering, ETH Zurich, Sonneggstrasse 3, CH-8092 Zurich, Switzerland. 6
Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH
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Zurich, Vladimir-Prelog-Weg 1-5, CH-8093 Zurich, Switzerland. 7
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Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-17177 Stockholm, Sweden.
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Laboratory for Transport at Nanoscale Interfaces, Department Materials Meet Life, Swiss
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Federal Laboratories for Materials Science and Technology (Empa), Überlandstrasse 129, CH-
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8600 Dübendorf, Switzerland.
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ºshared senior author
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ABSTRACT
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The mechanistic understanding of structure-function relationships in biological systems heavily
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relies on imaging. While fluorescence microscopy allows the study of specific proteins following
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their labelling with fluorophores, electron microscopy enables holistic ultrastructural analysis
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based on differences in electron density. To identify specific proteins in electron microscopy,
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immunogold labelling has become the method of choice. However, the distinction of
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immunogold-based protein-labels from naturally occurring electron dense granules, and the
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identification of several different proteins in the same sample remains challenging. Correlative
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cathodoluminescence electron microscopy (CCLEM) bioimaging has recently been suggested to
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provide an attractive alternative based on labels emitting characteristic light. While luminescence
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excitation by an electron beam enables sub-diffraction imaging, structural damage to the sample
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by high energy electrons has been identified as potential obstacle.
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Here, we investigate the feasibility of various commonly used luminescent labels for CCLEM
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bioimaging. We demonstrate that organic fluorophores and semiconductor quantum dots suffer
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from a considerable loss of emission intensity, even when using moderate beam voltages (2 kV)
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and currents (0.4 nA). Rare-earth (RE) element doped nanocrystals, in particular Y2O3:Tb3+ and
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YVO4:Bi3+,Eu3+ nanoparticles with green and orange-red emission, respectively, feature
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remarkably high brightness and stability in the CCLEM bioimaging setting. We further illustrate
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how these nanocrystals can be readily differentiated from morphologically similar naturally
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occurring dense granules based on optical emission, making them attractive nanoparticle core
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materials for molecular labelling and (multi)color CCLEM.
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Keywords: Super-resolution microscopy; Immunogold; Molecular labelling; Nanocrystals,
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Nanoscopy
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Biological systems are enormously complex and our understanding of them relies on the ability
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to examine relationships between structure and function at various levels of resolution, and
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within a functionally correlated context.1 In recent years, correlative light and electron
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microscopy (CLEM)2 has gained increasing importance as it combines imaging based on
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fluorescent labels and ultrastructural analysis by electron microscopy. However, the resolution
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mismatch3 between the optical and the electron signal constitutes a major obstacle.4 While recent
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developments in super-resolution microscopy techniques have enabled fascinating sub-
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diffraction fluorescence imaging,5-7 direct combination with electron microscopy remains
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challenging due to differences in sample preparation requirements.8 Recently, the use of
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(multi)color correlative cathodoluminescence electron microscopy (CCLEM) for the analysis of
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biological samples has been proposed by us9 and others.10-13 Cathodoluminescence (CL) is
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generated by an electron beam, which can be focused down to a few nanometers, thereby
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enabling luminescence data acquisition with deep-subwavelength spatial resolution.14,15 CL
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spectra can be acquired across a wide spectral range and thus open the possibility of (multi)color
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imaging, which in turn allows identification of structures directly based on spectral signatures
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rather than electron density (as in the case of immunogold labelling). Because CL emission from
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organic materials, including most biological tissues, is usually low, the use of exogenous
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luminescent labels has been suggested for CCLEM.16 However, the electron beam used for the
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excitation of the luminescent probes is potentially damaging to the biological samples17 and the
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labels themselves.18
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Here, we investigate the feasibility of different luminescent labels for cathodoluminescence
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bioimaging. We report on the optical properties of a selection of luminescent labels with wide-
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spread adoption in biological imaging in a photoluminescence (PL, excitation by photons) and a
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cathodoluminescence (excitation by accelerated electrons) bioimaging setting. Additionally, we
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show how inherent properties of the label (stability, brightness and emission lifetimes) and the
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electron beam characteristics (acceleration voltage and current) affect CL data quality, and we
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identify optimal settings for high-resolution CCLEM bioimaging. We then demonstrate that
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ultra-bright rare-earth (RE) element doped nanocrystals can be used for nanometric CCLEM
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bioimaging at low electron acceleration voltages (≤ 2 kV). High-resolution CCLEM allows
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straightforward differentiation between naturally occurring dense granules and nanocrystals,
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which is pivotal for prospective molecular labelling applications.
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The labels investigated in the present study include the widely used organic fluorophore 4'-6-
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diamidino-2-phenylindole
(DAPI),
conjugated
polymer-based
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semiconductor quantum dots (CdTe and CdSe/CdS), and two types of RE3+ doped nanocrystals
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(Y2O3:Tb3+ and YVO4:Bi3+,Eu3+), which have previously been employed for photoluminescence
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bioimaging (Figure 1a,b and see ESI, Figure S1 for structures, Figure S2 for corresponding
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photo-excitation and emission spectra, Table S1 for PL spectra acquisition parameters, and
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Figure S3 for RE3+ doped nanocrystals phase identification).19-21 While the organic fluorophores
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exhibit broad emission, the rare-earth element doped nanocrystals present narrow emission lines
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with main emission peaks in the green (545 nm, Y2O3:Tb3+) and the orange-red spectral region
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(620 nm, YVO4:Bi3+,Eu3+) (see ESI, Figure S2). Both organic labels (DAPI and CPN) show
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rapid bleaching under laser excitation, and the luminescence emission intensity decreased by
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more than 40% within the first three minutes of illumination (corresponding to a dose of 360
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J/cm2) (Figure 1c). Such photo bleaching of organic molecules is well-known from fluorescence
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microscopy and has been attributed to structural damage to the organic molecules.22,23 CdTe and
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CdSe/CdS semiconductor quantum dots on the other hand show a moderate reduction in
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quantum
dots
(CPN),
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luminescence intensity of ~25% and 10%, respectively. Both RE3+ doped nanocrystals present
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fluorescence emission stability over time superior to the other labels investigated, in line with
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previous fluorescence imaging studies.20,21 While YVO4:Bi3+,Eu3+ nanoparticles showed stable
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luminescence emission over the full duration of the experiment (15 mins), Y2O3:Tb3+
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nanocrystals presented a minor increase in the luminescence emission intensity, which can be
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attributed to desorption of surface-bound oxygen species (see ESI, Figure S4).24
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In addition to brightness and emission stability, short emission lifetimes are crucial for high-
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resolution imaging in scanning mode. In case the emission lifetime is longer than the dwell time,
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image quality is strongly compromised by smearing.25 While the organic fluorophores and
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semiconductor quantum dots have short fluorescence lifetimes (e.g., 2.6 nanoseconds for DAPI,
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see ESI, Figure S5),26,27 the RE3+ doped nanocrystals have emission lifetimes in the range of
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milliseconds21,28 (see ESI, Figure S5). Based on the measured PL lifetimes, the dwell times for
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imaging YVO4:Bi3+,Eu3+and Y2O3:Tb3+ nanocrystals should be no shorter than 1 and 5 ms,
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respectively.21,28 Although the excitation mechanism for cathodoluminescence is comparable to
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photoluminescence,14,29 electron beam excitation generally leads to emission by all luminescence
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mechanisms present in the material, since the electrons act as a supercontinuum source15 and
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hence lifetimes measured in PL may be underestimating CL lifetimes. Using long dwell times is
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beneficial in terms of signal-to-noise (S/N) ratio, however, may cause structural damage to the
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fluorophore as well as specimen heating.30
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While all of the above labels can be employed for PL bioimaging despite the observed
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bleaching, exposure to accelerated electrons is typically even more damaging.17 To quantify the
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label stability in a CL setting, we acquired sequential CL spectra (Figure 1d) using optimized
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beam parameters and monitored the cathodoluminescence intensity over time (see ESI, Table S2
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for data acquisition parameters).
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Figure 1: a) Transmission electron micrographs of the two synthesized RE3+ doped nanocrystals
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with optical emission in green and orange-red upon exposure to UV light (λex = 254 nm)
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(inserts). b) Fluorescence microscopy image of YVO4:Bi3+,Eu3+ nanocrystals (red) in human
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vascular endothelial cells. The cytoskeleton is stained with Alexa 488 Phalloidin (green) and the
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nucleus with DRAQ5 (magenta). Scale bar: 25 µm. c) Photoluminescence emission stability over
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time under laser excitation for the different labels. d) Cathodoluminescence emission spectra, e)
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brightness, and f) emission stability over time for all the corresponding labels.
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The beam parameters were optimized in order to retain nanometric resolution but still produce
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sufficient luminescence emission. The resolution is driven by the highly localized excitation, and
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is influenced by three factors: (i) the spot size of the electron beam, (ii) the spreading of the
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electrons of the primary beam in the sample (generation volume), and (iii) the diffusion of the
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generated secondary carriers in the sample.29,31 The electron beam spot size is mostly determined
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by the beam current (lower electron beam current leads to smaller spot size).31 At a given
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current, higher acceleration voltage leads to smaller spot size, however, also increases the
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electron beam interaction volume (see Monte Carlo simulations for ESI, Figure S6), which in
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turn increases CL generation volume and therefore reduces spatial resolution. Based on the
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simulations and experiments at different beam voltages as shown later on Figure 2, 2 kV was
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found to be optimal in terms of signal/noise ratio. At 2 kV, the brightness was more than 10
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times higher for the RE3+ doped nanocrystals compared to the other labels (Figure 1e). The
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YVO4:Bi3+,Eu3+ nanocrystals showed the brightest emissions, followed by the Y2O3:Tb3+
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nanocrystals. While emission could be detected from all the labels investigated, both organic
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fluorophores bleached almost immediately under the electron beam excitation, even when using
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a 2 kV electron beam and the lowest possible current where an emission could still be detected
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(0.8 nA) (Figure 1f). DAPI and CPN lost around 80% of their initial emission intensity within
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the first 10 s of beam exposure (equivalent to a dose of 0.04 nC/μm2). A decrease in CL emission
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intensities over time has been widely reported for organic molecules due to structural damage
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(degradation of the label) by the electron beam.32 While semiconductor quantum dots were more
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stable under electron excitation compared to organic materials, CdTe quantum dots showed
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reduced emission (- 40%) within the first 10 seconds and - 80% after 60 seconds (equivalent to a
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dose of 0.24 nC/μm2). Similarly, CdSe/CdS core/shell quantum dots also exhibited a significant
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drop (30% reduction) within the first 10 s (equivalent to a dose of 0.08 nC/μm2), however, after
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60 s (equivalent to a dose of 0.48 nC/μm2) 50% of the initial intensity was retained indicating
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that the composition and architecture of the semiconductor quantum dots plays an important role
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in their emission stability. A loss in CL emission intensities of quantum dots has been attributed
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to electron ionization and trapping in the quantum dot structure.33 Despite the wide-spread
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adoption of semiconductor quantum dots in fluorescence imaging, the limited brightness and
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electron beam stability restricts their utility in CL bioimaging settings.14 RE3+ doped
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nanocrystals on the other hand presented comparatively high electron beam stability. The drop in
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emission intensity within the first 10 seconds (equivalent to a dose of 0.04 nC/μm2) was 10 -
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20%, showing total reduction of 40% after a dose of 0.48 nC/μm2 over 2 minutes. The minor loss
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in emission intensity observed for RE3+ doped nanocrystals may be attributed to the
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accumulation of active quenchers as indicated by the observed film (e.g., hydrocarbons, see ESI,
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Figure S7, S8).10,34 No evidence in support of label degradation,23,32 thermal effects,35 CL
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emission saturation (ESI, Figure S9)34 or charge accumulation for RE3+ doped nanocrystals was
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found in our setting. Charging effects were ruled out by discharging experiments indicating only
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incomplete recovery of the CL emission signal after sample grounding (see ESI, Figure S7).
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In order to further illustrate the feasibility of the nanocrystals for CCLEM bioimaging and
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especially high-resolution mapping, the different labels were incubated with human vascular
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endothelial cells, chemically fixed with formaldehyde and osmium tetroxide, embedded in epoxy
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resin, sectioned with a focused ion beam (FIB), and subsequently imaged with scanning electron
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microscope (SEM). For semiconductor quantum dot containing cells, no characteristic emission
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by the quantum dots could be detected (data not shown). Both RE3+ doped nanocrystals could be
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detected inside the osmium-stained cells based on backscattered electrons (BSE) (Figure 2a, see
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ESI, Figure S10a,d) and the CL signal (using a photomultiplier tube) (Figure 2b-d, see ESI,
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Figure 10b,e).
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Figure 2: a) Backscattering image of YVO4:Bi3+,Eu3+ nanocrystal-containing human vascular
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endothelial cells in a focused ion beam cross-section showing electron dense particles and the
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cellular ultrastructure. The regions of interest for BSE, SE, and CL co-localization studies have
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been indicated (low magnification, α-α' and high magnification, β-β'). b-d) Corresponding CL
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images acquired with 2 kV, 5 kV, and 10 kV acceleration voltages indicating strong voltage
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dependence of the CL resolution. Scale bars: 1 µm. e) Line profiles of SE and CL signals with
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different acceleration voltages for low magnification (α-α', A) and high magnification (β-β', B)
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settings. The profiles obtained with low acceleration voltage show the highest correlation
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between SE, (BSE) and CL signals.
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The cells containing YVO4:Bi3+,Eu3+ nanocrystals were imaged using different acceleration
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voltages (1, 2, 5, and 10 kV, Figure 2b-d and ESI, Figure S11, S12). The corresponding
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secondary electron (SE) micrographs (see ESI, Figure S11) present distinct differences in the
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interaction volume, resulting in less resolved features in the images acquired with higher
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acceleration voltages from the region of interest (ROI). The difference in the feature resolution is
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even more pronounced in CL maps (Figure 2b-d). The images acquired with 2 kV show well-
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resolved features with good signal-to-noise ratio and high correlation between CL and SE images
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(Figure 2b,e see ESI, Figure S13), while the images collected with both 5 kV and 10 kV show
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blurry and non-resolvable features (Figure 2c-e). The acquired CL map from regions containing
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small nanocrystal clusters (see ESI, Figure S14) indicates that emission can also be observed for
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the smallest nanoparticle features with a resolution similar to SE images and thus illustrates the
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potential of YVO4:Bi3+,Eu3+ nanocrystals for protein labelling studies. For the YVO4:Bi3+,Eu3+
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nanocrystals, it is even possible to obtain CL maps with acceleration voltages as low as 1 kV
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approaching a critical S/N ratio (see ESI, Figure S12). These experimental results indicate a sub-
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diffraction resolution of < 100 nm for acceleration voltages of ≤ 2 kV and are in excellent
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agreement with Monte Carlo (MC) simulations in osmium-containing epoxy resin (see ESI,
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Figure S6).36 The MC simulations illustrate that spatial resolution is strongly influenced by the
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interaction of the landing electrons and the sample. The CL generation volume in 7% osmium
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containing cells was approximated by simulating the electron penetration range as a function of
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acceleration voltage.37 Increased energy of the incident electrons leads to increased electron
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penetration depth (20, 60, 225, 700 nm for 1, 2, 5, 10 keV respectively) and maximum lateral
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spread (15, 40, 300, 800 nm for 1, 2, 5, 10 keV respectively), which results in different electron
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energy loss profiles in the material (ESI, Figure S6).
37
While low energy electrons (< 2 keV)
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mostly excite nanocrystals proximal to the surface, high energy electrons (5 and 10 keV)
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primarily interact with nanocrystals in deeper regions.
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features appear whereas others more proximal to the surface disappear when imaging samples
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with 10 keV compared to 2 keV electron beam (Figure 2b,e). At the same time, 5 and 10 keV
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electrons show broadening in the lateral in-plane electron energy depth loss profile with
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increased depth (see ESI, Figure S6c), explaining the progressive blurring of the CL signal with
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increased acceleration voltage (Figure 2c,d). These results illustrate necessity to reduce electron
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acceleration voltages to a minimum in a FIB/SEM setting using full-thickness epoxy embedded
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biological samples. While low beam energies are generally desirable, they come at the expense
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of lower signals (less energy transfer), introduction of surface effects (non-radiative decay may
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dominate) and high injection density, which may lead to saturation. As a consequence, both the
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acceleration voltage and the current should be reduced to a minimum, which in turn requires
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highly efficient (bright) CL labels, such as YVO4:Bi3+,Eu3+ nanocrystals which can be imaged at
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voltages as low as 1 kV and currents of ~0.4 nA.
38,39
As a direct consequence, additional
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Following optimization of the imaging conditions for nanocrystal-containing epoxy-embedded
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cells, the cells were imaged with low electron acceleration voltage also in electron backscattering
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mode, which visualizes the cellular ultrastructure, including mitochondria, vesicular and nuclear
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membranes and various populations of dense particles (e.g., Figure 3a and see ESI, Figure
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S10a,d, S15). Simultaneous acquisition of corresponding secondary electron images (Figure 3c
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and see ESI, Figure S10c,f) and cathodoluminescence maps (Figure 3b and ESI, Figure S10b,e)
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indicates that not all electron-dense particle-like structures in BSE show emission in CL. In order
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to identify the underlying reason, corresponding secondary electron image (Figure 3d) and
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energy-dispersive X-ray (EDX) maps from the same region were recorded (Figure 3e, maps
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shown for the YVO4:Bi3+,Eu3+ nanocrystal-containing cells), which reveal that these dense
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nanocrystal-like structures are devoid of any rare-earth elements and are endogenous granules.
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Importantly, while EDX has a slightly smaller generation volume than CL for the same
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acceleration voltage, the minimum energy required for detection of elements with high atomic
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number, such as europium, in EDX is significantly higher (typically 5-15 kV) than for CL. 40 The
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comparison of the SE images recorded at the two respective voltages used for EDX (10 kV,
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Figure 3d) and CL (2 kV, Figure 3c) illustrates the difference in the probing depth of the EDX
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and CL mapping, translating into different spatial resolution. Therefore, due to the higher
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acceleration voltage needed for EDX, additional deep-lying features appear on the EDX map
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originating from a higher signal generation volume. The lower electron beam acceleration
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voltages needed for CL mapping and the corresponding smaller signal generation volume further
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illustrate the potential of CL-based high-resolution imaging compared to other competing
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techniques, especially in a FIB/SEM setting.
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Similar non-emitting granules could also be observed in samples containing Y2O3:Tb3+
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nanocrystals (see ESI, Figure S10). In order to exclude quenching of the emitted photons by
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osmium-containing epoxy resin covering the nanocrystals, cross section containing Y2O3:Tb3+
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nanocrystals was polished with the focused ion beam and a thin (
