Dynamic autofluorescence imaging of intracellular ... - OSA Publishing

Dynamic autofluorescence imaging of intracellular components inside living cells using direct electron beam excitation Yasunori Nawa,1,2 Wataru Inami,3,4 Aki Miyake,3,4 Atsushi Ono,4,5 Yoshimasa Kawata,1,3,4,5,* Sheng Lin,3 and Susumu Terakawa4,6 1

Graduate School of Science and Technology, Shizuoka University, Johoku, Naka, Hamamatsu 4328561, Japan 2 Research Fellow of the Japan Society for the Promotion of Science, Chiyoda, Tokyo 102-0083, Japan 3 Faculty of Engineering, Shizuoka University, Johoku, Naka, Hamamatsu 4328561, Japan 4 CREST, Japan Science and Technology Agency, Japan 5 Research Institute of Electronics, Shizuoka University, Johoku, Naka, Hamamatsu 4328011, Japan 6 Photon Medical Research Center, Hamamatsu University School of Medicine, Handayama, Higashi, Hamamatsu 4313192, Japan * [email protected]

Abstract: We developed a high-resolution fluorescence microscope in which fluorescent materials are directly excited using a focused electron beam. Electron beam excitation enables detailed observations on the nanometer scale. Real-time live-cell observation is also possible using a thin film to separate the environment under study from the vacuum region required for electron beam propagation. In this study, we demonstrated observation of cellular components by autofluorescence excited with a focused electron beam and performed dynamic observations of intracellular granules. Since autofluorescence is associated with endogenous substances in cells, this microscope can also be used to investigate the intrinsic properties of organelles. ©2014 Optical Society of America OCIS codes: (110.0180) Microscopy; (170.0180) Microscopy; (170.2520) Fluorescence microscopy; (180.2520) Fluorescence microscopy; (180.5810) Scanning microscopy.

References and links 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

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Received 16 Dec 2013; revised 19 Dec 2013; accepted 21 Dec 2013; published 7 Jan 2014

1 February 2014 | Vol. 5, No. 2 | DOI:10.1364/BOE.5.000378 | BIOMEDICAL OPTICS EXPRESS 378

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1. Introduction In biological cells, cellular functions emerge as a result of localization and dynamic interaction of molecules. To investigate the distribution and movement of such molecules in real time, several high-resolution microscopy techniques using light or electrons have been developed. Light microscopy has the advantage of allowing noninvasive examination of living biological specimens. The spatial distribution of specific cellular components can be determined using fluorescence microscopy. Use of immunostaining with various fluorescent dyes, including potential sensitive dyes and pH- and ion-dependent fluorophores, enables investigation of cellular states and functions [1–5]. Autofluorescence analysis, in which cellular molecules are optically excited without the need for a fluorescent dye, is also useful for investigating the chemical composition of organelles. When the excitation and emission spectra of cellular molecules have been established [6], it can be used to identify specific components in cells. Cellular states can be investigated because physiological or pathological processes produce changes in the distribution and intensity of autofluorescence. The absence of any need for a fluorescent dye eliminates cell damage that may occur during labeling, which sometimes limits the types of observations that can be made on living biological specimens. Scanning electron microscopy (SEM) is also a powerful tool for cell analysis because of its extremely high spatial resolution. In SEM analysis, several signals can be obtained, including secondary electrons, backscattered electrons, transmitted electrons, Auger electrons, X-rays, and cathodoluminescence (CL). Since CL is produced by loosely bound π-electrons in organic compounds under electron beam excitation [7], it can be used to investigate chemical bonding in specimens. CL analysis using a focused electron beam is more advantageous than conventional fluorescence microscopy [8–10] in that a higher spatial resolution can be achieved and it can be applied to a wider variety of substances because of the higher energy associated with accelerated electrons. However, CL analysis of living cells is limited because of the requirement of a vacuum environment and the need for sample preparation steps such as thin slicing, metal staining, and freezing.

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Recent development of an electron-transparent membrane has made it possible to apply EM techniques for the analysis of hydrated specimens. This leads to various applications in biological analysis [11–15]. We have previously reported the development of a very-highresolution direct electron-beam excitation assisted (D-EXA) optical microscope for dynamic observation of living cells [16]. Fluorescent materials attached as labels to specific cellular molecules or endogenous fluorophores are directly irradiated with an electron beam, and the resulting CL is detected. Such electron beam excitation enables achievement of nanometerscale resolution, beyond the diffraction limit of light. In the present study, autofluorescence from HeLa cells in aqueous solutions was observed, and we demonstrated that intracellular granules and cytoskeletal structures can be seen without need for any staining. Dynamic movement of intracellular granules was observed using time-lapse imaging. Thus, the D-EXA microscope is extremely useful for autofluorescence studies of cells and for its ability to accomplish label-free imaging of intracellular structures. 2. Principle of autofluorescence imaging with the D-EXA microscope Figure 1 shows the principle of live-cell imaging under electron beam excitation. Cells are directly cultured on an electron-transparent thin film and irradiated through the film using a focused electron beam without need for pretreatment such as fixation or staining. The autofluorescence produced by cellular components is emitted as CL, which is detected under atmospheric pressure. Use of a focused electron beam allows nanometer-scale resolution because the electron beam can be focused to a diameter of a few tens of nanometers after penetrating the film. The amount of electron scattering occurring in the film can be calculated by Monte Carlo simulation [16–18]. Images can be formed by raster scanning the electron beam, and a frame rate corresponding to real-time video can be achieved. Since some cellular substances emit CL, the D-EXA microscope enables label-free imaging in addition to qualitative chemical analysis using the CL spectrum.

Fig. 1. Principle of live-cell imaging with direct electron beam excitation. Biological cells are cultured on a thin film, and the focused electron beam directly excites cathodoluminescence through the film. Because the film separates the vacuum region from an air or a liquid environment, live cells can be observed. The direct electron beam excitation permits nanoscale resolution beyond the diffraction limit of light.

3. Materials and methods The D-EXA microscope is a combination of a scanning electron microscope and a fluorescence microscope [16,18], and open environment around specimens is available in this configuration [12,13,16,18]. A scanning electron microscope is used for excitation of specimens and scanning the electron beam to reconstruct images, and a fluorescence

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microscope is used for detection of CL excited with the electron beam. CL is detected using a photomultiplier tube (Hamamatsu Photonics K.K., H10721-110). In the present study, a 50-nm thick silicon nitride (SiN) membrane window (Silson) was used as an electron-transparent film to support specimens. A 50 µm × 50 µm aperture was produced in the silicon substrate supporting the silicon nitride film. Biological cells were directly cultured on a SiN membrane fixed to a culture dish [16]. The culture dish for the DEXA microscope consisted of a glass dish, a SiN membrane, and a metal plate, as shown in Fig. 2. First, a SiN membrane was affixed to the metal plate, which was used to avoid a charging effect resulting from the electron beam irradiation. The metal plate had a hole in its center through which the electron beam was passed to excite specimens. A glass dish with a hole larger than the SiN membrane was then affixed to the metal plate. This dish was for containing the culture medium during incubation and observation. Epoxy resin was used for all fixation procedures. After the dish was sterilized for 21 min at 121°C in an autoclave (Super Clave FX-220, Hillson), the cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) until they adhered to the film. After incubation, DMEM was removed and phosphate-buffered saline was placed in the dish before observations were made. Silicon nitride membrane window

Glass dish Epoxy resin

Metal plate

Epoxy resin

Fig. 2. Preparation of the culture dish used for the D-EXA microscope with a SiN membrane. First, a SiN membrane was fixed to the metal plate, reducing the charging effect of electron beam irradiation. The metal plate had a hole in its center through which the electron beam was passed to excite specimens. A glass dish with a hole larger than the SiN membrane was affixed to the metal plate for hold the culture medium during incubation and observation. Epoxy resin was used for all fixation procedures.

4. Observation results The spatial resolution of our microscope depends on the excitation spot size formed by scattering of the electron beam. First, to evaluate the actual electron scattering in the substrate and the spot size used in the D-EXA microscope, 20-nm gold spheres (EMGC20, BBI solution) were observed using the D-EXA microscope and a conventional field emission scanning electron microscope (FE-SEM) (JSM 7001, JEOL). Figures 3(a) and 3(b) show the experimental setup for the D-EXA microscope and FE-SEM observations. In the D-EXA imaging, gold spheres under atmospheric pressure were observed through the SiN membrane. Specimens consisting of heavy elements were observed detecting the backscattered electrons (BE) through the film [12,13]. Figure 3(c) shows the BE image acquired using an acceleration voltage of 5 kV. The same area was observed under vacuum using FE-SEM. Figure 3(d) shows the secondary electron (SE) image of the gold spheres acquired using an acceleration voltage of 15 kV. In both images, gold spheres were visible as bright spots. Figure 3(e) and 3(f) show the line profile of each gold sphere, indicated with arrows. The intensity distribution was averaged with the width of the line, 5 pixels. Full widths at half maximum (FWHMs) of the fitting curves were about 63 and 18 nm. Figure 3(f) shows the actual size of the gold spheres, whereas FWHMs in Fig. 3(e) were broadened owing to electron scattering in the substrate.

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Fig. 3. Observation results of 20-nm gold spheres. (a, b) Experimental set-up for the D-EXA and FE-SEM imaging. (c) Backscattered electron image of 20-nm gold spheres acquired with the D-EXA microscope through the SiN membrane. (d) Secondary electron image of 20-nm gold spheres acquired with FE-SEM. (e, f) Line profiles of individual particles indicated with arrows in (c) and (d). Each FWHM of fitting lines was approximately 63 nm and 18 nm.

Microscope images are formed as a convolution of the object function with the pointspread function (PSF) of the microscope. Given that the image function and the object function are the fitting Gaussian functions in Fig. 3(e) and 3(f) respectively, the FWHM of the PSF can be calculated as 60 nm. Thus, a spot size of several tens of nanometers is realized in the D-EXA microscope under an acceleration voltage of 5 kV after the electron beam penetrates the 50-nm thick SiN membrane. In this estimation, a BE image was employed for the image function. BE are scattered in the SiN membrane before they reach the detector. For more accurate evaluation, it is necessary to observe CL from phosphors smaller than a few tens of nanometers. A spot size of several tens of nanometers was also demonstrated by observation of zinc oxide (ZnO) nanoparticles smaller than 50 nm (Sigma-Aldrich; 677450). ZnO is well known as a brilliant emitter of photoluminescence and CL [19,20] and can be applied for bioimaging [21]. Figure 4(a) shows a secondary electron image of ZnO nanoparticles acquired with an FE-SEM. ZnO nanoparticles were dispersed on the substrate and dried. Figure 4(b) shows a D-EXA image of an isolated ZnO nanoparticle in aqueous solution. The acceleration voltage is 5 kV. A ZnO nanoparticle is visible as a bright spot. A line profile between the arrows is shown in Fig. 4(c). The intensity distribution was averaged with the width of the line, 10 pixels. The FWHM of the Gaussian fitting curve is about 57 nm. The average signalto-noise ratio (SNR) was 8.52. The SNR was determined from the ratio of the peak signal height and the standard deviation of the background signals. The peak signal height is the difference between the maximum signal and the average of the background signal. The average and standard deviation of the background signal were determined at horizontal positions between 0 and 500 nm in 5 areas. According to the Rose criterion [11,22], an SNR larger than 5 is required for reliable identification of an image object. Given that the SNR of this image is 8.52, larger than 5, the observation of ZnO nanoparticles indicated that a spot size of the electron beam was smaller than 60 nm after penetrating the SiN membrane in this experiment.

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Fig. 4. Observation results of ZnO nanoparticles (