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We propose a direct electron-beam excitation assisted optical microscope with a resolution of a few tens of nanometers and it can be applied for observation of dynamic movements of nanoparticles in liquid. The technique is also useful for live cell imaging under physiological conditions as well as observation of colloidal solution, microcrystal growth in solutions, etc. In the microscope, fluorescent materials are directly excited with a focused electron beam. The direct excitation with an electron beam yields high spatial resolution since the electron beam can be focused to a few tens of nanometers in the specimens. In order to demonstrate the potential of our proposed microscope, we observed the movements of fluorescent nanoparticles, which can be used for labelling specimens, in a water-based solution. We also demonstrated an observation result of living CHO cells.
©2012 Optical Society of America
1. Introduction
Nanoimaging of specimens in liquid conditions are highly required in various applications such as analysis of colloidal solutions, observation of microcrystal growth and self-assembly process, etc. Especially, imaging of biological specimens with high resolution is crucial for a deeper understanding of cell functions. Fluorescence microscopy has been widely used to analyze the dynamic behavior of cellular components in living cells, because stained molecules of interest in the specimens can be imaged with high contrast [1, 2]. In addition, the use of light for cell imaging enables relatively non-invasive observations under physiological conditions. However, many structures in cells are small to be resolved with a standard optical microscope as a consequence of the finite resolution due to the diffraction limit of light. Recently, several concepts for super-resolution techniques have been developed and proposed for observations of nanostructures in cells [3–6]. Fluorescence microscopy is required to become an even more useful tool for biological analysis with nanometer-scale resolution.
Electron microscopy has been traditionally used to investigate the structures of cells [7, 8], as another analytical tool with nanometer-scale resolution. In combination with specific labelling using immunogold spheres, electron microscopy will be useful to define the distribution of cellular molecules of interest [9]. Normally, these techniques require various sample preparation procedures such as thin slicing, staining with metal species and freezing the specimens. Such sample preparation procedures have always limited direct research on living cells in their active state. Recently environmental electron microscopy has been developed and applied to the observations of biological specimens [10, 11].
Here, we propose a direct electron-beam excitation assisted optical microscope (D-EXA microscope) combining a scanning electron microscope (SEM) with a fluorescence microscope [12]. The D-EXA microscope is one of the super-resolution fluorescence microscopes, which has the advantages of spatial resolution of a few tens of nanometers, and the ability to image dynamic behavior of nanopartciles in liquid conditions. In the microscope, flurorescent particles, which is can be used for labelling of biological specimens, are placed on the film used as the electron transparent window, and irradiated with a focused electron beam to directly excite luminescence. A high spatial resolution is achieved by direct electron beam excitation through the film, since the electron beam can be focused in a smaller region than that of light even if some of the electrons are scattered in the film. Specimens can be observed in their native state in a liquid environment, because the film is used to separate the environment of the specimen from the vacuum in the microscope. The excited luminescence with the electron beam is the so-called cathodoluminescence [13–16]. In this work, we observed the dynamic behavior of fluorescent nanoparticles in solution, and demonstrated the application to live cell imaging in culture solution using a prototype D-EXA microscope in order to demonstrate the potential for live cell imaging.
2. Principle of high resolution imaging with direct electron beam excitation
Figure 1 shows the principle of direct excitation with a focused electron beam in the D-EXA microscope. Specimens are directly placed on a thin film, and irradiated with an electron beam through the thin film. The focused electron beam directly excites luminescence. The electron beam can be focused to an area of approximately 2 nm in diameter in vacuum [16], and the focus spot was broaden in a few tens nanometers region by electron scattering in the film and specimens. The scattering was estimated with Monte-Carlo simulation [17]. High spatial resolution can thus be realized by exciting with this focused electron beam.
Fig. 1 Principle of the D-EXA microscope. Specimens are placed on the thin film and a focused electron beam excites luminescence in the specimens directly through the thin film. The electron beam can be focused to a few tens of nanometers in diameter in the specimen. The thin film is also used to separate vacuum from the environment of the specimen such as air or liquid. The dynamic behavior of living cells is thus imaged with nanometer-scale resolution.
Since the thin film separates the vacuum from the environment such as air or liquid where the specimens are placed, the specimens do not need to be in vacuum and it is possible to observe the dynamic behavior under various conditions. Luminescence images with high spatial resolution are reconstructed with raster scanning of the electron beam. Real-time imaging is thus possible, because the electron beam can be scanned with modulation of the magnetic or electric field without any mechanical moving parts. Qualitative analysis is also possible by the combination with spectroscopic techniques. The D-EXA microscope allows for the analysis of cell functions in the nanometer-scale under physiological conditions.
3. Materials and methods
Figure 2(a) shows a photograph of a prototype D-EXA microscope that we developed, while Fig. 2(b) shows a schematic of the D-EXA microscope. The D-EXA microscope is the combination of a scanning electron microscope (SEM) and a fluorescence microscope [12].
Fig. 2 Structure of the D-EXA microscope. (a) Prototype of the D-EXA microscope we have developed. (b) Schematic of the D-EXA microscope. A scanning electron microscope is used for excitation and scanning the electron beam. A fluorescence microscope is used to collect the luminescence from the specimens.
A thin film is used to combine the two type microscopes, separating the vacuum in the SEM from the other environment within which specimens are placed. The SEM is used for excitation of fluorescent materials and for scanning the electron beam.
Luminescence from the specimens is detected with the fluorescence microscope system. A photomultiplier tube (PMT) (Hamamatsu Photonics K.K., H5784-04) is used as a detector and mounted on the fluorescence microscope. Images are reconstructed from the signal intensity by raster scanning of the electron beam.
We selected 100 nm zinc oxide (ZnO) nanoparticles (SIGMA-ALDRICH, PN. 544906) as fluorescent materials that can be excited with an electron beam. The microscope also required an electron-transparent film to separate the vacuum section from atmospheric pressure to observe specimens in a liquid. In this work, a silicon nitride (Si3N4) film was employed as the thin film [12]. The thickness of the film was 50 nm with a size of 50 μm x 50 μm.
4. Observation results
We observed dried zinc oxide (ZnO) nanoparticles under atmospheric pressure [18, 19], in order to evaluate the resolution of the D-EXA microscope. ZnO nanoparticles (100 nm) in ultrapure water were placed on the thin film and dried at room temperature. The ZnO nanoparticles were observed with a conventional epi-fluorescence microscope and the D-EXA microscope.
Figures 3(a) and 3(b) show luminescence images acquired with the conventional epi-fluorescence microscope and the D-EXA microscope. Both images represent the same area of 9.7 μm x 9.7 μm. These results show that the D-EXA microscope has a higher spatial resolution than that of the epi-fluorescence microscope. The ZnO nanoparticles in Fig. 3(b) are resolved more clearly compared to Fig. 3(a). Figure 3(a) was obtained with an excitation filter (U-MNU2, Olympus) and a 60 x 0.70 NA objective lens. Figure 3(b) was acquired with an acceleration voltage of 5 kV and probe current of 1 nA.
Fig. 3 Luminescence images of 100 nm ZnO nanoparticles. (a) Fluorescence image excited with UV light in a conventional epi-fluorescence microscope. (b) Luminescence image acquired with the D-EXA microscope. The acceleration voltage was 5 kV and probe current was 1 nA. (d, e) Line profiles of luminescence intensity distributions between the arrows in Figs. 3(a) and 3(b) respectively. In Fig. 3(b), the ZnO nanoparticles can be clearly distinguished from each other. (c) Luminescence image of isolated ZnO nanoparticles acquired with the D-EXA microscope. (f) Line profile of one ZnO nanoparticle between the arrows in Fig. 3(c). The full width at half maximum is 100 nm.
Figures 3(d) and 3(e) show line-profiles of the regions between the arrows in Figs. 3(a) and 3(b), respectively. The data indicates that the D-EXA microscope is able to distinguish each ZnO nanoparticle. While, the ZnO nanoparticles in Fig. 3(a) cannot be resolved, since the spatial resolution of the epi-fluorescence microscope is limited to approximately 200 nm due to the diffraction limit of light.
Figure 3(c) shows a luminescence image of isolated ZnO nanoparticles acquired with the D-EXA microscope. Figure 3(f) is the line profile of an individual ZnO nanoparticle between the arrows in Fig. 3(c). The full width at half maximum of one ZnO nanoparticle is 100 nm. We may conclude that the spatial resolution of the D-EXA microscope is greater than 100 nm.
We also observed the movements of ZnO nanoparticles in ultrapure water to demonstrate the potential to observe the dynamic behavior of nanoparticles in a liquid. ZnO nanoparticles move by convective flow in the droplet. The observation was started just after the droplet containing ZnO nanoparticles was dropped on the thin film. The dynamic behavior of the moving nanoparticles was observed and tracked by time lapse imaging.
The observation result is shown in the video file, Media 1, and Figs. 4(a) -4(e) show five frames from the video. The video was acquired at 3 fps with the size of 256 x 256 pixels, and played at 60 fps, 20 times speed. Figures 4(f)-4(j) show magnified images of the respective areas indicated by the squares in Figs. 4(a)-4(e). In Figs. 4(b) and 4(c), the ZnO nanoparticles indicated by the dashed circle disappeared in the subsequent image. Since the electron beam was focused on the surface of the thin film, nanoparticles move away from the surface of the film and the nanoparticles disappeared from the observed image. On the contrary, when the ZnO nanoparticles moved close to the surface of the film, the nanoparticles were observed in the luminescence image as shown within the solid circles in Fig. 4(c). Figures 4(f)-4(j) show ZnO nanoparticles moving along the surface of the film. As shown in Figs. 4(f)-4(j), the ZnO nanoparticle, indicated by an arrow in the figures, moved in a planar direction with changing luminescence intensity. Figure 4(k) shows a track of the moving ZnO nanoparticle. The ZnO nanoparticle moved a total of 1 μm over 96 s. We also observed other nanoparticles randomly moving with the change in the luminescence intensity. The change in the luminescence intensity showed that the nanoparticles move up and down within the focal plane of the electron beam.
Fig. 4 Observation results of the movements of 100 nm ZnO nanoparticles in a water solution. (Media 1) (a)-(e) Time lapse images of the movements of ZnO nanoparticles. Since the electron beam was focused on the surface of the film, ZnO nanoparticles moving away from the surface of the film disappeared in the next image, as shown by the dashed circle, whereas ZnO nanoparticles moving closer to the surface of the film appeared, as shown within the solid circles. (f)-(j) Magnified images of the areas indicated by the squares in Figs. 4(a)-4(e). The ZnO nanoparticle indicated by the arrow moved along the surface of the film. (k) An analysis of the movement of the ZnO nanoparticle. The nanoparticle moved randomly with the change in luminescence intensity. The change in the intensity showed that the nanoparticle moved up and down in the direction away from the film plane within the focal plane of the electron beam.
These results demonstrated that fluorescent materials and their movements in a liquid can be observed with direct electron beam excitation in the D-EXA microscope.
We observed living MARCO-expressing CHO cells, using the D-EXA microscope to confirm the potential for live cell imaging. In order to observe cells in our method, cells were directly cultured on the Si3N4 film. Figure 5(a) shows a culture dish filled with culture solution. After incubation, the culture dish was set to the specimen holder of the D-EXA microscope and observed.
Fig. 5 Observation results of living cells using the D-EXA microscope. (a) A culture dish for the D-EXA microscope. Cells were directly cultured on the Si3N4 membrane. (b) A luminescence image of living MARCO-expressing CHO cells in culture solution without any treatments. The intracellular granules indicated with arrows are observed as white sports and the cell membranes are observed as light-gray contrast against the dark-gray background. The acceleration voltage was 5 kV and probe current was 1 nA. (c) A phase contrast microscope image of the living cells. It represents the same area as Fig. 5(b).
Figure 5(b) shows a luminescence image of the cells acquired with the D-EXA microscope, and Fig. 5(c) shows a phase contrast microscope image. Cells were observed in culture solution without any treatments, such as fixation and drying. The shape of each cell was clearly recognized and some bright spots were observed in cells. We believe that the bright spots indicated with arrows were auto-fluorescence of intracellular granules and light-grey regions were auto-fluorescence of cell membranes. It is clearly demonstrated that the D-EXA microscope is useful tool for observation of living biological cells in physiological conditions.
5. Conclusion and discussion
We proposed the D-EXA microscope as a technique with high spatial resolution beyond the diffraction limit of light. A spatial resolution greater than 100 nm was achieved for the D-EXA microscope and the dynamic behavior of moving nanoparticles in water was observed by time lapse imaging. We also demonstrated luminescence image of living cells in culture solution without any treatments. The D-EXA microscope can be applied for the analysis of cell functions in live cells stained with fluorescent materials such as ZnO.
The concept of the D-EXA microscope combines the advantage of a SEM with a few nanometers resolution and the advantage of an optical microscope suitable for observations of dynamic activities of live biological cells. In our proposed microscope, fluorescent materials, used for labelling of biological specimens, are directly excited with the focused electron beam. A higher spatial resolution is expected since the electron beam can be focused in a few tens of nanometers region in specimens. The film is also used to separate vacuum from other environments where biological specimens are placed, such that they can be observed in various surroundings without any treatments. The D-EXA microscope allows for live cell imaging under physiological conditions.
In this study, we employed and observed ZnO nanoparticles as the fluorescent material that can be excited with an electron beam. ZnO nanoparticles could be excited with both UV-light and an electron beam with high quantum efficiency. It is possible to observe specimens using both conventional fluorescent microscopes and D-EXA microscopy after only a single staining process using ZnO. In addition, it has been reported that quantum dots and other materials yield cathodoluminescence, and they are widely used to observe protein distributions in biological cells [20–22]. Using such materials, multistaining procedures can be applied for the D-EXA microscope observations. This is very promising to reveal interactions among several elements in cells with high resolution.
In this system, the spatial resolution depends on the spot size of the electron beam to excite luminescence. Decreasing electron scattering in the thin film and specimens can lead to higher spatial resolution. We simulated electron scattering in the film in order to evaluate the spatial resolution of the D-EXA microscope using Monte Carlo simulation [17].
A 2 nm spot size of electron beam is expected to extend about a 30 nm spot size by scattering caused during propagation in a 50 nm thick Si3N4 film with an acceleration voltage of 5 kV.
We also simulated the electrons scattering in water after penetrating the Si3N4 film of 50 nm thickness, since specimens in water are observed with our method. In Monte Carlo simulation, electrons scattering was determined by the atomic number of material components. From the simulation results, we found that in the case of the acceleration voltage of 25 kV, the spatial resolution was greater than 50 nm at the depth of 250 nm in water. We need more detailed discussions in the simulation for evaluating the spatial resolution of our method.
In future work, we need to evaluate the damage to the specimen by electron beam irradiation as well as optimization of the acceleration voltage of the electron beam, the film thickness and the material in order to minimize any damage to the sample and improve the spatial resolution.
The D-EXA microscope can be used for high-resolution imaging of living cells. We believe that the D-EXA microscope is a promising tool and opens up new applications for life sciences as well as other fields.
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