1.

Introduction

Light microscopy (LM) and electron microscopy (EM) are widely used cellular imaging methods. LM is a useful method for imaging biomolecules stained with fluorescent probes in the fields of biology and medicine. Staining with multiple fluorescent probes such as dyes,¹ quantum dots (QDs),^2,3 and fluorescent proteins^4,5 allows simultaneous visualization of the distributions of individual biomolecular species with different emission spectra, and imaging with multiple labeling provides information about biological molecular interactions. On the other hand, EM gives us fine structural information about cellular components, such as the membrane, mitochondria, and endoplasmic reticulum.

The spatial resolution of EM is higher than that of LM because the spot size of a focused electron beam is on the order of nanometers. Furthermore, EM allows us to observe a molecular species at nanoscale resolution through immunostaining with gold nanoparticles (GNPs).^6,7 More than one kind of molecular species can be identified by using GNPs of different sizes.⁸9.^–10 There is also a method capable of identifying different types of biomolecules by using the sizes and shapes of QDs.¹¹

The spatial resolution achievable with LM is limited to submicrometer order by the diffraction limit of light,¹² and discrimination of different kinds of biomolecules using EM is still difficult because EM images are monochromatic, not color. These limitations of LM and EM hamper the detailed understanding of cellular functions in life processes, especially in the case of complex distributions of molecules.

Cathodoluminescence (CL) is one candidate that fills this functional gap between LM and EM. CL is the light emission from a material induced by accelerated electrons. The spatial resolution of CL microscopy reaches the nanometer scale because the spot size of an electron beam is on the order of nanometers, and the CL color depends on the material.¹³ Therefore, by using suitable imaging probes, CL microscopy enables multicolor biological imaging with nanoscale spatial resolution. To exploit these benefits, we have previously reported biological CL imaging with rare-earth-doped nanophosphors (RE-NPs) based on a combined scanning electron microscopy–cathodoluminescence (SEM-CL) technique.^14,15 Several research groups have also studied SEM-CL imaging.¹⁶17.18.^–19 However, SEM-CL imaging has difficulty in imaging cellular structural information due to a low contrast image, even samples stained with heavy metals such as osmium and uranium.

The scanning transmission electron microscopy–cathodoluminescence (STEM-CL) method^20,21 is also a promising technique for realizing multicolor biological imaging with nanoscale spatial resolution. It is known that images of cellular structure (e.g., organelles, membranes, and macromolecules) obtained with STEM using thin cell sections stained with heavy metals have high contrast. To reveal the detailed cellular functions at nanoscale resolution, simultaneous observation of both structural information and protein distributions is quite important. Furthermore, higher spatial resolution and higher contrast are obtained compared with SEM-CL because the acceleration voltage of STEM (80 to 200 kV) reduces the electron scattering volume compared with SEM (typical acceleration voltage: $\sim 30\mathrm{kV}$).

Here, we describe multicolor biological imaging using CL microscopy based on STEM, with ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ RE-NPs, which have high resistance against electron beam irradiation and narrow spectral bandwidth. Using these nanophosphors allows simultaneous cellular structural imaging by STEM and multicolor imaging by CL. In addition, multimodal imaging using LM is also demonstrated using the same nanophosphors. To the best of our knowledge, this is the first demonstration of an imaging technique in which RE-NPs are applied to CL biological imaging using STEM.

2.

Imaging System

2.1.

STEM-CL System

Our CL system based on an STEM instrument (JEM-2100F) is illustrated in Fig. 1. CL is collected by a parabolic mirror, forming parallel rays. The parallel rays exit the STEM instrument through a quartz window. The CL is focused onto a slit of a spectrometer (Andor, SR163, $f=163\mathrm{mm}$, $F/3.6$) by a lens and is detected by an electron multiplying (EM)-CCD detector (Andor, DU920p-BU) for spectral imaging.

Fig. 1

Cathodoluminescence (CL) detection system combined with a scanning transmission electron microscopy (STEM) instrument.

The electron beam is controlled via the beam scanning system of the transmission electron microscopy/STEM instrument using an external PC and is scanned on the specimen. CL from the specimen is obtained by the EM-CCD for a certain duration at each point. Using the EM-CCD detector, the emission spectrum is detected for each pixel, realizing spectral imaging at each wavelength. Since the STEM and CL signals are simultaneously obtained by the external PC and the electron beam is scanned step-by-step to observe these images, the position of each pixel in the STEM image is identical to that in the CL image.

3.

Preparation of Specimens

4.

STEM-CL Imaging and Characterization of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ Nanophosphors

First, we measured the CL spectra of the synthesized ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ nanophosphors on the elastic carbon-coated copper grids using the STEM-CL system in Fig. 1. Figure 2 shows normalized CL intensity spectra of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ (red) and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ (green) nanophosphors. The emission at around 600 nm from ${\mathrm{Eu}}^{3+}$ ions is due to the magnetic dipole transition ${}^5{\mathrm{D}}_0\Rightarrow {}^7{\mathrm{F}}_1$, which is insensitive to the symmetry of the lattice field, and the most intense emission around 615 nm is derived from the electric dipole transition ${}^5{\mathrm{D}}_0\Rightarrow {}^7{\mathrm{F}}_2$.²² In the ${\mathrm{Tb}}^{3+}$ ions, electric and magnetic dipole transitions are induced regardless of the symmetry of the lattice field, and the peak in the luminescence intensity is maximum around 545 nm (${}^5{\mathrm{D}}_4\Rightarrow {}^7{\mathrm{F}}_5$).²² Since the most intense emission bands between ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ had almost no overlap and were very narrow, these two kinds of spectra are easy to distinguish.

Fig. 2

Spectra of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ nanophosphors synthesized by homogeneous precipitation (HP) method (slit size: 0.1 nm, blaze wavelength: 400 nm, groove density: $150\mathrm{L}/\mathrm{mm}$).

In biological CL imaging, bleaching of probes due to high-energy electron exposure has been a crucial problem especially for organic fluorophors.²⁴ In inorganic materials, although QDs have high quantum efficiency and high resistance against light exposure,²⁵ they are easily bleached by electron beam exposure (data not shown). To confirm the stability under electron beam exposure, we examined the CL intensity of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ as a function of time (Fig. 3). The electron beam was focused at one point on each phosphor, and the CL intensity was monitored. The acceleration voltage of the electrons was set to 80 kV. Intensity decreases of about 1% and 3% were observed for 60 s exposure in the ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ samples, respectively. The intensity decrease of the phosphors includes the effect of hydrocarbon contamination from the elastic carbon-supported copper grid and the epoxy resin, thus the actual intensity decay of these phosphors will be smaller.

Fig. 3

Stability of CL intensity against electron beam exposure (${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ nanophosphors synthesized by homogeneous method).

The CL stability of these phosphors was high enough to achieve imaging even after long-term exposure with high acceleration voltage electrons. This suggests that ${\mathrm{Y}}_2{\mathrm{O}}_3$-based RE-NPs are suitable phosphors for biological CL imaging using the STEM-CL method. It is known that these types of phosphors are stable for low acceleration voltages (about 3 kV).²⁶ So far, use of these phosphors for biological measurement of CL intensity using a STEM-CL system has not been reported yet. Our experiments showed that these phosphors can also be used at high acceleration voltage (80 kV). Moreover, our results show the possibility of repeatable observation using STEM with multiple colors.

Figure 4 shows STEM and CL images of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ nanophosphors. The two contacting particles were spatially resolved, indicating that the spatial resolution of the CL images was a few tens of nanometers, which is obviously beyond the diffraction limit of light. A possible reason for the high spatial resolution is the high acceleration voltage of this STEM system. Since a higher acceleration voltage prevents scattering of electrons in both the membrane and the phosphors, the excitation volume of CL is close to the size of the focused electron beam spot. The obtained CL images had high contrast compared with typical SEM-CL images because of the high CL intensity induced by the high acceleration voltage and the high beam current of the electron beam. The low scattering volume also gives high-contrast images since it prevents undesired CL from nonphosphor areas.¹⁵ The aforementioned results demonstrate the suitability of these phosphors and our STEM-CL system for biological CL imaging.

Fig. 4

STEM and CL images of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ nanophosphors synthesized by HP method. (a) and (b) Interlaced STEM image and CL image of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$. (c) and (d) Interlaced STEM image and CL image of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$.

5.

Cellular STEM-CL and Fluorescence Imaging of HeLa Cells Using ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ Nanophosphors

We demonstrated multicolor biological STEM-CL imaging with nanoscale spatial resolution by using RE-NPs. Figure 5 shows STEM and color CL images of a HeLa cell containing ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ introduced by endocytosis. The acceleration voltage and current were 80 kV and 2 nA, and the exposure time for CL images was set to $100\mathrm{ms}/\mathrm{pixel}$. From the STEM image, nanophosphors were observed in the endocytotic vesicle, and mitochondria and nanophosphors were both clearly observed in the cell structure. The sizes of the introduced nanophosphors were 300 nm (${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$) and 500 nm (${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$). Although it was not possible to discriminate the two kinds of nanophosphors from the STEM image [Fig. 5(a)], the nanophosphor species could be identified from the CL spectra [Figs. 5(c) and 5(d)]. The spatial resolution of the CL images was beyond the diffraction limit of light.

Fig. 5

STEM and CL images of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ nanophosphors in 100 nm thin section of HeLa cell. (a) STEM image of 100 nm thin section of HeLa cell with introduced nanophosphors, (b) partial view of STEM image of HeLa cell with introduced nanophosphors, (c) and (d) CL images of nanophosphors (${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$) in a cell.

Since the contrast of these CL images was similar to that of the CL images of phosphors dispersed on elastic carbon-coated copper grid [Figs. 4(b) and 4(d)], it was found that light obstruction due to the biological composition was negligible. In this multicolor biological imaging, crosstalk of the emission from ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ did not hinder discrimination of the phosphor emission. Although cellular regions stained with heavy metals (osmium and uranium) are considered to cause scattering of the electron beam, almost no undesired CL due to scattering from the cellular region was observed in the CL images. The reason for this is the sufficiently high acceleration voltage and sufficiently thin cellular section. It is known that some types of proteins and lipids emit autofluorescence,²⁷28.^–29 but such fluorescence did not affect the image contrast in this experiment. Since the CL signal from the cellular region was negligibly small, the contrast was maintained compared with CL images of phosphors dispersed on the elastic carbon-supported copper grid. These results imply that our biological STEM-CL imaging might be suitable for color EM. In recent years, synthesis of RE-NPs of 20 to 60 nm in diameter using the hydrothermal method has been reported.³⁰ It may be possible to apply methods of this kind to the synthesis of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ nanophosphors.

It is possible to excite ${\mathrm{Y}}_2{\mathrm{O}}_3$-based RE-NPs with not only an electron beam but also UV light.³¹ Thus, multimodal multiscale imaging using CL microscopy and fluorescence microscopy is possible using the same probes. Figure 6(a) shows photoluminescence spectra of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ nanophosphors under 254-nm UV excitation. The inset shows a photograph of the photoluminescence. The shapes of the spectra are similar to those of the CL spectra, and the most intense peaks did not spectrally overlap. Figures 6(b) to 6(d) show transmission, photoluminescence, and merged (transmission, red fluorescence, and green fluorescence) images of a HeLa cell containing ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$. These nanophosphors were identified with their emission wavelengths. Although some aggregated phosphors were not resolved spatially, imaging of a large area in wet conditions was possible.

Fig. 6

(a) Emission spectra of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$, ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$, (b) transmission, (c) green fluorescence, (d) red fluorescence, and (e) merged images of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ nanophosphors contained in a HeLa cells. ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$, and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ nanophosphors were excited by 254-nm UV light.

These nanophosphors can be used in correlative light and electron microscopy (CLEM). CLEM is one kind of bimodal imaging method for biological observation, which combines the advantages of both LM and EM.³²33.^–34 The combination of LM and EM offers more information about biological specimens than either method alone. However, in typical CLEM, multicycle observation of a thin-sectioned sample using LM and EM is not possible due to bleaching of fluorescence probes after irradiation with an electron beam. Our probes make it possible to store stained biological samples, like immunogold-stained samples, which can then be repeatedly used. In addition, fluorescence imaging in wet conditions makes it easy to check the sample condition before carrying out the time-consuming sample preparation process required for CL imaging (e.g., molding, sectioning).

For imaging with deeper penetration and less autofluorescence using LM, upconversion fluorescence imaging is an effective approach.^35,36 We have already proposed multimodal/correlative microscopy using ${\mathrm{Y}}_2{\mathrm{O}}_3$:Tm, Yb (thulium and ytterbium doped yttria) nanophosphors, which are excited by both an electron beam and near-infrared light.³⁷ This multimodal microscopy, allowing observation at different scales using LM and CL, may contribute to the discovery of new cellular and molecular functions and will improve the effectiveness of our imaging technique.

6.

Summary

Multicolor high-resolution microscopy of biological specimens was performed using a STEM-CL system. CL images of ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ nanophosphors introduced into cells and heavy-metal-stained cellular structure were observed using the STEM-CL system. The CL spectra of the synthesized nanophosphors were sufficiently narrow to distinguish them from each other, and the spatial resolution of the CL images was sufficiently high to image biological molecules at a resolution beyond the diffraction limit of light. The ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ RE-NPs showed remarkable resistance against electron beam exposure even at a high acceleration voltage (80 kV) and kept a high CL intensity for 1 min. CL images of the RE-NPs nanophosphors in cells and the cellular structure, such as mitochondria and endocytotic vesicle, were clearly observed from the STEM image with high contrast. The spatial resolution of cellular CL imaging using RE-NPs was not reduced much, even though heavy-metal-stained cells are usually regarded as highly scattering media.

${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Eu}$ and ${\mathrm{Y}}_2{\mathrm{O}}_3:\mathrm{Tb}$ RE-NPs will be useful phosphors for biological STEM-CL imaging at high spatial resolution beyond the diffraction limit of light, and the application of this immunostaining method will allow us to identify protein species at high spatial resolution with cellular structural information.