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We present label-free and high spatial-resolution imaging for specific cellular structures using an electron-beam excitation-assisted optical microscope (EXA microscope). Images of the actin filament and mitochondria of stained HeLa cells, obtained by fluorescence and EXA microscopy, were compared to identify cellular structures. Based on these results, we demonstrated the feasibility of identifying label-free cellular structures at a spatial resolution of 82 nm. Using numerical analysis, we calculated the imaging depth region and determined the spot size of a cathodoluminescent (CL) light source to be 83 nm at the membrane surface.
© 2016 Optical Society of America
1. Introduction
High-resolution imaging of cellular structures, such as actin filaments, mitochondria, and nuclei, is a key tools for analyzing cellular activities, including movement, organelle transportation, and energy generation [1,2]. Although imaging of cell structures by phase-contrast microscopes and fluorescent microscopes revealed the fundamental mechanisms of cellular movement and organelle transportation [3,4], many questions about the details of cellular functions or activities remain [5]. Since the spatial resolution of conventional optical microscopes is bounded by the diffraction limit, it is difficult to observe a cell’s detailed structures. To reveal new mechanisms of cellular function, improved optical microscopes are needed for achieving greater spatial resolution.
In recent years, super-resolution fluorescence microscopes, used in stimulated emission depletion microscopy [6], structured illumination microscopy [7], photoactivated localization microscopy [8], and stochastic optical reconstruction microscopy [9], have demonstrated nano-structure observations of less than 100 nm in lateral spatial resolution. These super-resolution microscopes have proven capable of imaging the detail strcuctures and dynamics of cellular structures, such as receptors, microtubules, and lysosomes [10–12].
Imaging with super-resolution microscopes is based on fluorescent labeling of cellular structures [13]. Fluorescent labelling permits high-contrast imaging of specific structures. In addition, biomolechlar interactions with other structures can be observed by multicolor imaging [14,15]. However, organic fluorescence dyes are toxic to cells, and the uniformity of the stain process always introduces the possibility of artifacts [16]. In addition, the fluorescent intensity of labeling bleaches the images under excitation light irradiation [17,18]. It is difficult, as well, to observe long-duration activities or the alteration of biological cells [18].
There many advantages of label-free technique, for example, non-preparation before observation, reduction of damage or deformation of biological cells due to fluorescence labeling and no photobleaching of fluorescent dye [18,19]. From these advantages, the label-free imaging techniques contribute long-term dynamic observation of biological cells [20–23]. In addition, label-free imaging technique is suited to observation for stem cells since the labeling of stem cells is undersirable [21,24,25]. The label-free imaging techniques are required in biomedical field and drug discovery [22,23].
We have developed an electron-beam excitation-assisted optical (EXA) microscope, which can observe label-free cells with high spatial resolution [26–28]. The cathodoluminescent (CL) light source is excited in the luminescent thin film by the irradiation of an electron beam. The EXA microscope observes the spacimen beyond the diffraction limit by raster scanning the CL light source, where the CL spot is a few tens nm of nanometers in diameter.
Using the EXA microscope, we demonstrated label-free biological cell imaging with 82 nm spatial resolution. We identified cellular structures specifically actin filaments, mitochondria, and nuclei by comparing the fluorescent images and EXA images for the labeled cellular structures. We also present calculations and estimates of the theoretical spatial resolution, where the depth of the observed region was estimated from the intensity distribution analysis of the CL light source.
2. Configuration of the EXA microscope
Figure 1(a) shows the schematic of the EXA microscope. The EXA microscope is constructed from an electron microscope, a culture dish, and an optical microscope. The inverted scanning electron microscope (APCO Ltd., MINI-EOC) is used to generate the nanometric light source in the EXA microscope. The electron beam from a field-emission-type electron gun irradiates the luminescent film at the culture dish. The CL intensity excited in the luminescent thin film is detected by the photomultiplier tube (Hamamatsu Photonics K. K., H10721-20). The observation image is obtained by raster scanning of the electron beam. In the EXA microscope, vacuum and atmosphere are separated by the substrate and fluorescent thin film at the culture dish [27,28]. Atmospheric pressure is maintained in the observation location of the specimens so that biological cells can be observed in a living state in the culture solution.
Fig. 1 (a) Schematic of the EXA microscope. The EXA microscope is constructed from optical microscope, culture dish and electron microscope. (b) Enlarged image of the highlighted square region in Fig. 1(a). The spot size of CL excited in the ZnO layer is less than 100 nm at the Si3N4 surface. The biological cell is directly cultured on the Si3N4 substrate. ZnO presents single emission peak at 380 nm. The single peak is due to the band gap emission of the ZnO. (c) CL spectrum of the ZnO luminescent thin film. (d) Surface morphology of the ZnO observed by an atmic force microscope (AFM). The ZnO surface is composed by 50 nm particles. The RMS surface roughness is 3.68 nm.
Figure 1(b) shows the detail of the square region in Fig. 1(a). In this study, the membrane substrate and fluorescent film materials are silicon nitride (Si3N4) and zinc oxide (ZnO), respectively. Si3N4 with a thickness of 30 nm to 50 nm can serve as a boundary between the vacuum and atmosphere because the Si3N4 has a high mechanical strength of 1GPa at room temperature [29]. A ZnO luminescent film is deposited to the back side of the Si3N4 because ZnO film causes toxicity to biological cell if the ZnO contacts with biological cell. The biological cells are cultured directly on the Si3N4. The cells are observed in CL light from the ZnO transmitted through of the Si3N4 membrane. We formed the as-deposited ZnO film by radio-frequency magnetron sputtering that utilized a ZnO target with an oxygen-and-argon mixed reactive plasma. After the sputtering process, the ZnO thin film was annealed at 800 °C in the presence of N2. The EXA microscope can observe the specimen with high spatial resolution because the CL spot size at the Si3N4 surface remains a few tens of nanometers in diameter. The image contrast is produced by the scattering or absorption by the specimens illuminated by the evanescent light at the Si3N4 surface.
Figure 1(c) shows the CL spectrum of ZnO in this study. The CL single-emission peak of the ZnO appears at 380 nm. This CL emission arises from the band gap of the ZnO [30,31]. As Fig. 1(c) illustrates, the ZnO luminescent thin film presents a monochromatic peak emission.
In this study, ZnO luminescent film was fabricated by annealing at 800 °C for 15 minutes in N2. In previous report in Ref [27,32], we utilized Zn2SiO4 as the luminescent film by annealing ZnO at 1000 °C for 60 minutes in N2. Although the Zn2SiO4 emits bright CL emission, CL from Zn2SiO4 includes intensity variability of 24.0% as shown in Fig. 2(a). This intensity variability is caused by inhomogeneous crystalline growth in the annealing process. To suppress the intensity variability of CL emission, we change the annealing temperature and annealing time from 1000 °C to 800 °C, and 60 minutes to 15 minutes, respectively. From these adjustments, we suppressed the intensity variability of CL. Figure 2(b) shows the CL image of ZnO by annealing at 800 °C for 15 minutes in N2. The CL from ZnO film includes 11.3% root mean square variation. In comparison with Zn2SiO4, the intensity variation of this ZnO is reduced from 24.0% to 11.3%. The intensity variation is two times higher than that of Zn2SiO4.
Fig. 2 Comparison of CL image. (a) CL image of the Zn2SiO4. RMS variability of CL intensity includes 24.0%. (b) CL image of the ZnO. RMS variability of CL intensity includes 11.3%.
To consider the reason of intensity variation reduction, we obtained the particle size of ZnO surface. Figure 3 shows the AFM image of ZnO surface. Figure 3(a) shows the as-deposited ZnO, and Fig. 3(b) shows the after annealing ZnO. The particle size of ZnO is 30 to 50 nm in Figs. 3(a) and 3(b). According to Ref [32], the particle size of Zn2SiO4 increase 2.5 times as compared with as-deposited ZnO. In this study, the crystalline growth was suppressed by controlling the annealing temperature and time. We consider that the suppression of crystalline growth was contributed to the homogeneous CL emission as shown in Fig. 2(b).
Fig. 3 AFM image of (a) as-deposited ZnO and (b) after annealing ZnO surface. Particle size of ZnO in both images is 30 to 50 nm. RMS of as deposited ZnO is 3.00 nm and that of after annealing ZnO is 3.68 nm.
3. Label-free cell imaging using the EXA microscope with high spatial resolution
Figure 4(a) shows the result of high spatial-resolution imaging of the HeLa cell. The acquisition time to obtaine Fig. 4(a) was 200 seconds. The acceleration voltage of electorn beam was 5 kV and current density was 1 nA. The inset shows the enlarged image of square region in Fig. 4(a).
Fig. 4 (a) High resolution EXA microscopic imaging of the cellular granules (b) Intensity profile of the cellular granules highlighted by arrows in Fig. 2(a). The full width at half maximum is 82 nm and signal-to-noise ratio is 10.5.
Figure 4(b) shows the intensity profile in the vicinity of the intracellular granules, highlighted by arrows in Fig. 4(a). The full width at half maximum (FWHM) of the intracellular granules is 82 nm and the signal-to-noise ratio (SNR) is 10.5. The SNR must exceed 5 to sustain a calim that the imaged object is the structure in question [33,34]. In this study, the achieved SNR of 10.5 is more than two times higher than the minimum neeeded. The EXA microscope observed HeLa cells with spatial resolution of 82 nm and high SNR due to the reduction of the CL intensity variation.
4. Estimation of CL spot size and observation depth region
We also estimated the spatial resolution and the depth of observation region of the EXA microscope by using numerical analysis. We analyzed the CL intensity inside and outside of the film. The CL spot size and observation region were calculated from the result of this analysis. The CL intensity distribution was analyzed by combining Monte Carlo simulation and finite-differential time-domain method [35,36].
Figure 5(a) shows the intensity distribution analysis result of the CL excited in the ZnO thin film. As Fig. 5(a) illustrates, the CL excited in the ZnO propagates in the Si3N4. Figure 3(b) shows the intensity profile of the CL along the Si3N4 surface (x-direction). In Fig. 5(b), the FWHM of the CL spot is 83 nm in Fig. 5(b). The FWHM of the CL spot corresponds to the observation result shown in Fig. 4.
Fig. 5 (a) Electric field intensity distribution of CL excited in ZnO. (b) Electric field intensity profile at the Si3N4 surface. The full width at half maximum (FWHM) of electric field intensity at the Si3N4 surface is 83 nm. (c) Electric field intensity profiles for various distances from the Si3N4 surface. In case of d = 30 nm, the peak intensity falls to 50% compared to the peak intensity of d = 0 nm. (d) Dependence of the FWHM of the electric field intensity on the distance from Si3N4 surface. The FWHM of electric field intensity spreads with increasing distance from Si3N4 surface. If the d = 20 nm, the FWHM of electric field intensity is 117 nm. In the EXA microscope, the high resolution imaging region along the depth direction is around 20 nm from Fig. 3(d).
Figure 5(c) shows the CL intensity for various distances from the Si3N4 membrane surface. The peak CL intensity decreases with increasing distance d from the Si3N4 surface. In the case of d = 20 nm, the peak intensity falls to 50% compared to the peak intensity when d = 0 nm.
Figure 5(d) indicates the dependence of the FWHM of the CL spot on the distance from the Si3N4 surface. The CL spot spreads as the distance from the Si3N4 surface increases. Because the FWHM of the CL spot is 117 nm if the distance from the Si3N4 surface is 20 nm, it is difficult to observe the specimen with high spatial resolution. According to Fig. 5(d), the limitation depth of high spatial-resolution imaging in the EXA microscope is 20 nm. To achieve high spatial-resolution imaging in the EXA microscope, it is recommended that the specimens be positioned 20 nm to the Si3N4 surface.
To observe more higher spatial resolution with higher SNR, the strong adhesion of biological cells to the Si3N4 surface is one of the effective technique. We already reported surface modification of the Si3N4 [37,38]. The biological cells are strongly adhered by modifying the carboxyl group to the Si3N4 surface.
5. Cell structure imaging using fluorescence and the EXA microscope
In order to identify the label-free cellular structures in the EXA microscope image, we observed the stained cells both with fluorescence and the EXA microscope to determine the points of correspondence between the imaging results.
Figure 6(a) shows the observation results, obtained with a fluorescence microscope (IX-71, Olympus America Inc.), of fixed HeLa cells with stained actin filaments and mitochondria. HeLa cells were fixed by 1% glutaraldehyde. The HeLa cells were placed in a phosphate-buffered saline solution. The actin filament and mitochondria were stained by AD48-81 (ATTO-488, ATTO-TEC GmbH) and Mitotracker Orange CMTMRos (M7510, Thermo Fisher Scientific Inc.), respectively. Stained actin filament and mitochondria were observed with fluorescence filter cubes of U-MNIBA3 and U-MWIG3 (Olympus America Inc.), respectively.
Fig. 6 (a) Fluorescent microscopic image of the HeLa cells with stained actin filament and mitochondria. (b) EXA microscope image for the HeLa cells. The observation region is the same as Fig. 6(a). The corresponding actin filaments and mitochondria are indicated by arrows and triangles.
Figure 6(b) shows the observation result, obtained with the EXA microscope, of the same area as shown in Fig. 6(a). The acceleration voltage was 4.8 kV and the irradiation current was 1 nA. The acquire time to obtain Fig. 4(b) was 200 secconds. In Fig. 6(b), the EXA image was obtained only with the CL from the ZnO transmited through the 390 nm band-path filter (FF01-390/40-25, IDEX Corpration). The cellular stuructrures in Fig. 6(b) can be identified by comparing them with the corresponding structures in Fig. 6(a). In Figs. 6(a) and 6(b), the corresponding actin filaments and mitochondria are indicated by arrows and triangles. The actin filaments and mitochondria are brighter than the background CL because the evanescent light is converted to propagation light by scattering with the actin filaments and mitochondria near the Si3N4 surface. As Fig. 6(b) displays, the EXA microscope successfully imaged the actin filaments and mitochondria by means of the CL from the ZnO; these cellular structures were obserebed as bright regions when compared to the background CL intensity.
Secondly, we observed the label-free HeLa cells with the EXA microscope and discerned the cellular structures based on the information from imaging results of stained HeLa cells, as shown in Figs. 6(a) and 6(b). Figures 7(a) and 7(b) show the imaging results obtained with the phase-contrast and EXA microscope, respectively. The label-free HeLa cells were fixed by 1% glutaraldehyde. The acceleration voltage was 4.8 kV and irradiation current was 1 nA. The acquisition time to obtain Fig. 7(b) was 200 seconds.
Fig. 7 (a) Phase-contrast microscopic image for label-free HeLa cells. (b) EXA microscope image for label-free HeLa cells. The actin filament, mitochondria, nucleus, filopodia, and intracellular granule are observed with label-free condition.
In Fig. 7(b), the actin filaments, nucleus, mitochondria, filopodia, and intracellular granules are clearly observed. The actin filaments are fiber structure inside the biological cell as shown in Fig. 6. We identified the fiber structure as actin filaments from based on comparison results in Fig. 6. The nucleus is indicated as circle region in Fig. 7. The nucleus can be identified from comparison result with phase-contrast microscope. The mitochondoria usually exists as rod structure. In Fig. 6, the mitochondoria can be found the rod structure. The longitudinally length of mitochondoria is 1 μm to 2 μm [39]. In Fig. 7(b), the lengths ofrod structures indicated by triangles are 1 μm to 2 μm. According to these references, we consider that the structures indicated by triangles are mitochondria. The filopodia is needle-like protrusions constructed by actin filaments [40]. The filopodia is expressed toward from cell membrane surface to outside the cell [41]. In Fig. 7, we found the needle-like structure from cell membrane surface. According to reference, we identified this needle-like structure is filopodia. The cellular granules are formed of grain shape [42,43]. We identified the cellular granules from phase-contrast microscope image as shown in Fig. 7(a). The cellular granules are indicated by arrows in Fig. 7.
Because the EXA microscope permits observations near the surface region only, the actin filament and filopodia present in the cell membrane, as depicted in Fig. 7(b), adhered to substrate surface. From Fig. 7(b), it is found that the EXA microscope can observe cellular structures under label-free conditions.
6. Conclusion
In this study, we observed the label-free cellular structures of HeLa cells at 82 nm spatial resolution by using the EXA microscope. We succeessed in reducing the CL intensity variation from 24.0% to 11.3%. This high spatial resolution imaging was achieved by suppressing CL intensity variation. As a result, cellular granules were observed at the same 82 nm spatial resolution. We also calculated and estimated the intensity distribution of the CL both inside and outside of the ZnO and Si3N4 and estimated the CL spot size and observation depth of the EXA microscope in this study. From the results of this analysis, we determined the CL spot size to be 83 nm at the Si3N4 surface. The CL spot size corresponded with the spatial resolution of the EXA microscope. The observation depth was 50 nm. According to the simulation results, the observation results of the cellular structures were constructed in the region 50 nm from the Si3N4 surface.
In addition, we identified cellular structures by comparing the EXA microscope images with the labeled observation results. The actin filaments and mitochondria of the HeLa cells were identified in the observation image of the EXA microscope. In the EXA image, the cellular structures were observed as brighter regions as compared with the background CL intensity. By comparing the EXA microscopic observed results with images of label-free HeLa cells, we confirmed that it was possible to indentify the cellular structures, such as actin filaments, mitochondria, filopodia, nuclei, and cellular granules.
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