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High spatial resolution microscope is desired for deep understanding of cellular functions, in order to develop medical technologies. We demonstrate high-resolution imaging of un-labelled organelles in living cells, in which live cells on a 50 nm thick silicon nitride membrane are imaged by autofluorescence excited with a focused electron beam through the membrane. Electron beam excitation enables ultrahigh spatial resolution imaging of organelles, such as mitochondria, nuclei, and various granules. Since the autofluorescence spectra represent molecular species, this microscopy allows fast and detailed investigations of cellular status in living cells.
© 2015 Optical Society of America
1. Introduction
High-resolution live-cell imaging has had considerable impact on the understanding of structures and functions. This knowledge is crucial for drug development and advances in medical technology. Fluorescence microscopy is widely used for the dynamic observation of specific, labelled cellular molecules, and super-resolution fluorescence techniques have been recently developed [1]. However, fluorescence staining requires pretreatments that may damage specimens and create artefacts that interfere with investigations of morphologic and cytologic structures. Label-free imaging techniques are highly desirable for the analysis of intrinsic features in biological specimens. Examples of label-free microscopy include Raman microscopy of cytochrome c dynamics from mitochondria during apoptosis [2], and second-harmonic generation microscopy of collagen fibre orientation in human tissues [3]. Third-harmonic generation microscopy has also been used to observe the Corpus Callosum, axon fibre bundles, white- and grey-matter structures, and blood vessels in living brain tissues [4], respectively. Another example is interferometric light microscopy [5] of membrane potential changes in single mammalian cells.
Autofluorescence from various endogenous regions is also label-free, and its spectrum represents distinct molecular constituents [6]. It can be used for quantitative analysis of metabolic mechanisms [7], and for multispectral imaging to distinguish normal tissues from metastatic ones [8]. Here, we discuss high-resolution autofluorescence microscopy of living cells using electron beam excitation. Specifically, we describe a direct-electron-beam-excitation-assisted optical (D-EXA) microscope for cathodoluminescence (CL) imaging of living cells with high spatial resolution [9]. CL can be applied for optical imaging methods with high spatial resolution [10–12]. The D-EXA microscope is a fluorescence microscope combined with an inverted scanning electron microscope. Living cells cultured on a silicon nitride (SiN) membrane are directly excited with the focused electron beam, and the resulting CL is imaged [9]. Electron-beam excitation enables resolution at the nanometre scale, and the electron-transparent SiN allows observation of specimens in fluids with an electron beam [13, 14]. This configuration in the D-EXA microscope has accessible space around the specimens to enable cellular response analysis to external stimulation, similar to a conventional optical microscope. Thus it enables correlative light-electron microscopy (CLEM) [15], and analysis of dynamic reactions of biochemical systems by fine electron beam processing [16]. Previously, we demonstrated the dynamic autofluorescence observation of intracellular granules in living cells without any pretreatments [17], and multicolour CL imaging in living cells using fluorescent nano diamonds [18].
Here, we demonstrate that various organelles can be observed with spatial resolution <100 nm and without staining. We detected the autofluorescence from unstained cells and we partially identified cell structures by comparison with the fluorescent microscope images. Firstly specimens stained with fluorescent dye were observed to investigate on the organelles distribution in cells. Then, the same regions were found without fluorescent signal from the dye, but with autofluorescence signal in the D-EXA. These organelles were identified by comparison to phase contrast and fluorescence images. In second experiments, cells without fluorescent dyes were studied to claim autofluorescence imaging in D-EXA microscope. Organelles were clearly imaged without any pre-treatments.
2. Experimental setup and sample preparations
The D-EXA microscope combines a fluorescence microscope and a compact scanning electron microscope (SEM, APCO Ltd.) [9]. The SEM is used for scanning electron beam excitation of the samples. The fluorescence microscope collects CL excited by the electron beam and detects it with a mounted photomultiplier tube (PMT, H10721-110, Hamamatsu Photonics K.K.). CL images are reconstructed with the raster scan of the electron beam. An electron-transparent silicon nitride (SiN) membrane window (Silson) is used as the sample substrate. The membrane thickness is 50 nm. This technique doesn’t need any sample treatments such as fixation, slicing, and staining. Biological specimens are directly cultured on the SiN membrane fixed into the glass culture dish [17]. Since a 50 nm thick SiN membrane is optically transmissive, specimens are also imaged with a phase contrast and fluorescence microscope in this culture dish. Specimens are observed with the D-EXA microscope in this culture dish, after the dish is set to the D-EXA stage, and vacuum is formed under the SiN membrane [18]. In this manuscript, all wavelengths of autofluorescence were detected for D-EXA microscope imaging.
MC3T3-E1 cells were cultured on the SiN membrane in a culture dish [17]. After sterilization of the culture dish with ethanol, cells were cultured in MEM-α (12571, Life technologies) with 10% fetal bovine serum (FBS, Life technologies) and 1% penicillin-streptomycin (15140, Life technologies) at 37°C in an environment of 5% of CO2. For autofluorescence imaging with the D-EXA microscope, cells can be imaged in culture medium without any pretreatments including replacements of culture solutions.
For confocal microscopy (SP8, Leica) of adiposomes, cells were stained with a fluorescent dye (H34476, Life technologies), following fixation with 4.0% paraformaldehyde in PBS solution for 30 min, in order to compare the D-EXA. The stained adiposomes were excited by 552-nm laser light and the 600-650-nm emission was collected with an objective lens (HCX PL APO CS 20X, N.A. 0.70, dry).
Mitochondria, actin filaments, and nuclei were respectively stained with Mito tracker orange CMTMRos (M7510, Life technologies), ATTO-488 (AD488-81, ATTO TEC), and DAPI (D1306, Life technologies), after cells adhered on the SiN membrane. For staining of mitochondria, the culture medium was replaced with new medium containing 1µM Mito tracker orange CMTMRos. (1 µl of 1 mM stock solution in Dimethyl sulfoxide (DMSO) was added to 1 ml of culture medium.) Cells were incubated for 30 min at 37°C in an environment of 5% CO2. For following staining of actin filaments, cells were fixed with 4.0% paraformaldehyde in PBS solutions for 20 min, and permeabilized with 0.25% Triton X (Triton X-100, T8787, Sigma) in PBS solution for 40 min. Actin filaments were stained with 1 µM of ATTO-488 in PBS solution for 4 hours (1 µl of 0.1 mM stock solution in DMSO was added to 100 µl of PBS solution). Finally, nuclei were stained with 300 nM of DAPI in PBS solution for 5 min. Before every operation, cells were rinsed with PBS solution. Stained cells were imaged with an epifluorescence microscope (IX-71, Olympus). Stained mitochondria, actin filaments, and nuclei were observed with U-MWIG3, U-MNIBA3, and U-MNU2 optical filters, respectively.
3. Observation results
Figure 1 compares D-EXA images of MC3T3-E1 cells with phase contrast and confocal fluorescence images. The MC3T3-E1 cells were cultured on a SiN membrane. After adhesion, they were fixed with 4.0% paraformaldehyde in phosphate-buffered saline (PBS) solution. The lipid droplet adiposomes were stained with fluorescent dye (H34476, Life technologies). The cells were imaged in the order phase contrast, confocal, and D-EXA.
Fig. 1 Stained adiposomes in MC3T3-E1 cells observed with the D-EXA, confocal, and phase contrast. (a) Raw D-EXA microscope image without processing. Granules were observed with high spatial resolution. The D-EXA image is primarily autofluorescence. (b) Confocal image of adiposomes. (c) Phase-contrast microscope image. (d, e, f) Magnified image of the area indicated with white rectangle shown in (a), (b), and (c), respectively. Adiposomes were observed as a bright spots in (d) and (e), and they were observed as dark spots in (f). In (d), particles other than adiposomes were also imaged by autofluorescence, as indicated by arrows. D-EXA and confocal images have pseudocolour.
Biological lipid droplets emit autofluorescence by electron beam excitation and the spectrum can be used to identify chemical constituents [19]. The D-EXA image was acquired with an acceleration voltage of 4.8 kV and a probe current of 2.4 nA. The dwell time and pixel size are 9.5 µs and 40 nm, respectively. The average electron dose was calculated to 0.89 e-/Å2. All images are raw data without any filter processing. The confocal and D-EXA images are pseudocoloured.
Figures 1(d)-1(f) show the magnified area outlined by a white rectangle in Fig. 1(a). Bright spots in the D-EXA image correspond to stained adiposomes in the confocal image. These adiposomes were also imaged in the phase contrast image as dark spots. The D-EXA image is primarily autofluorescence because the CL from the fluorescent dye in the adiposome is weak. It indicates that adiposomes emit autofluorescence via direct electron beam excitation. Other granules also emit CL, some of which (white arrows) are not observed in confocal fluorescence image.
Figure 2 shows comparison images of actin filaments, mitochondria, and nuclei. Cells were fixed with 4.0% paraformaldehyde in a PBS solution, and the mitochondria, actin filaments, and nuclei were respectively stained with Mito tracker orange CMTMRos (M7510, Life technologies), ATTO-488 (AD488-81, ATTO TEC), and DAPI (D1306, Life technologies). The cells were imaged in the order phase contrast, epi-fluorescence, and D-EXA microscope.
Fig. 2 Actin filaments, mitochondria, and nuclei observed with a D-EXA microscope, epifluorescence, and phase-contrast. D-EXA and epifluorescence images are in pseudocolour. In the epifluorescence images, mitochondrial, actin filaments, and nuclei are red, green, and blue, respectively. (a-d) Comparison of actin filament images. (a) D-EXA image. Filamentous structures were clearly observed. Granules were also observed without staining, but with autofluorescence excited by electron beam irradiation, as indicated with the arrowhead. (b, c) Epifluorescence image (d) Phase contrast image. (e-h) Comparison of mitochondria images. (e) D-EXA image. Mitochondria were imaged as indicated with the arrows. The arrowhead shows a granule observed without staining, but with autofluorescence excited by an electron beam. (f, g) Epi-fluorescence image (h) Phase contrast image. (i-l) Comparison of nuclei images. (i) D-EXA image. DNA dense area in nucleus was observed indicated with arrows in (i), (k), and (l). (j, k) Epi-fluorescence image (l) Phase contrast image.
Figures 2(a)-2(d) compare filamentous structures observed with D-EXA and the same structures observed with epifluorescence and phase contrast. Filamentous structures imaged along the edge of cells in Fig. 2(a) correspond to actin structures in Figs. 2(b) and 2(c). Figure 2(b) is a combined image with pseudocolor of actin (green), mitochondria (red), and nucleus (blue). Figure 2(c) is a fluorescence image of actin structures. In Fig. 2(d), black granules were observed with phase contrast. These unstained granules were also clearly observed in Fig. 2(a) as indicated with arrowheads, as well as in Fig. 2(e) and Fig. 2(i). Thus, the D-EXA images show primarily autofluorescence excited with electron beam irradiation. Figures 2(e)-(h) compare images of mitochondria. The mitochondria were observed with the D-EXA microscope, as indicated by the arrow in Figs. 2(e) and 2(g). In Figs. 2(i)-2(l), images of the nucleus are compared. The nucleus and fine structures inside the nucleus were observed with the D-EXA [Fig. 2(i)]. These structures correspond to the bright spots in Fig. 2(k) and the black spots in Fig. 2(l), as indicated by the arrows. Since the dye DAPI binds to the adenine-thymine cluster in the minor groove of double-stranded DNA [20], the D-EXA image reveals the DNA dense regions as observed in the epifluorescence and phase contrast images. Mitochondria also contain mtDNA.
D-EXA images show the actin filamentous structures, mitochondria, and fine structures in nuclei corresponding to epi-fluorescent images. Additionally CL is also detected from unstained regions. Thus, the D-EXA image primarily shows autofluorescence excited with electron beam. These results indicate that the membrane, mitochondria, and the DNA dense regions inside the nuclei emit cathodoluminescence. All the images are unprocessed, except for pseudocolouring in the D-EXA and epifluorescence images. The D-EXA images were acquired with an acceleration voltage of 4.8 kV and a probe current of 2.4−3.8 nA. The dwell time and pixel size were 19 µs and 66 nm, respectively. Calculated average electron doses were 0.66−1.04 e-/Å2.
Living MC3T3-E1 cells were also imaged with the D-EXA microscope. They were cultured on the SiN membrane, and imaged in the culture solution without pretreatments. Figures 3(a) and 3(b) are corresponding D-EXA and phase contrast images, respectively. Nuclei, nuclear membranes, intracellular granules, mitochondria, and DNA dense regions in the nuclei are identified. In the D-EXA microscope, only cells closely attached to the substrate can be resolved, even though cells are stacked due to longer period of incubation, because the electron beam scatters during propagation through the SiN and the cells [17]. Figure 3(c) is a high magnification D-EXA image of other cells with fine structures inside the nucleus and membrane.
Fig. 3 D-EXA imaging of live MC3T3-E1 cells. (a) D-EXA image in pseudocolour without processing. Cells were observed without staining. The nucleus, nucleus membrane, intracellular granules, mitochondria, and DNA dense regions in the nucleus are identified. Only cells closely attached to the substrate were observed. (b) Phase contrast image. (c) D-EXA image of other cells with high magnification. Fine structures inside nucleus and membrane are observed.
In summary, the D-EXA microscope enables us to observe cellular organelles without pretreatments. No surface modification of the SiN membrane was necessary for cell adhesion of MC3T3-E1 cells, HeLa cells and MARCO-expressing CHO cells [9, 17, 18]. Fine structures sized 100 nm can be seen in the D-EXA microscope, so the spatial resolution for the live cells is estimated to less than 100 nm. D-EXA images were acquired with an acceleration voltage of 4.8 kV and a probe current of 2.7 nA. The dwell time was 19 µs. Each pixel size and average electron dose was 59 nm, 0.93 e-/Å2 for Fig. 3(a), and 35 nm, 2.60 e-/Å2 for Fig. 3(c), respectively.
Bleaching of autofluorescence was also examined, as shown in Fig. 4. Figure 4(a) shows a second scan of the same region acquired in Fig. 3(a). Figures 4(b) and 4(c) show first and second frame image of magnified regions denoted by the white rectangle in Fig. 4(a). Figure 4(d) plots CL intensity distributions along marked lines (line width of 5 pixels) in the magnified images. The peak CL intensity of the granules and mitochondria, and the averaged CL intensity from the cell membrane, are reduced by 5.8%, 19.1%, and 37.1%, respectively, during image acquisitions. Since CL from the membranes was the most diminished, the contrast of these fine structures in the cells, nuclei, mitochondria, and intracellular granules, was enhanced.
Fig. 4 Bleaching in the D-EXA imaging. (a) Second frame image of Fig. 3(a). Components inside cells were observed clearly. (b) Magnified area indicated with white rectangle of the first frame image. (c) Magnified area of the second frame image. (d) CL intensity distributions indicated with lines in fig. (b) and (c). CL from cell membrane is more decreased than CL from organelles during image acquisition.
Spatial resolution of the D-EXA microscope was estimated with MC3T3-E1 cells fixed in 1% glutaraldehyde in PBS solution. They were imaged in PBS solution. Figures 5(a) and (b) show a raw D-EXA image with pseudocolor and a phase contrast image of the same area. The shape of the nucleus was observed in both images, and fine particles were resolved in Fig. 5(a). Figure 5(c) shows the CL intensity distribution of the particle between arrows shown in Fig. 5(a). The full width at half maximum (FWHM) of the Gaussian fitting is 105 nm. The signal intensity between top of the peak and the averaged background is 10.62 times higher than the standard deviation of the background measured in the region where cells were detached. A signal intensity 5 times higher than the standard deviation of the background signal is required for reliable identification of an image object, according to Rose criterion [21].
Fig. 5 Imaging of fixed cells for evaluation of spatial resolution. (a) D-EXA image in pseudocolour without processing. The outline of the nucleus and fine particles are observed. (b) Phase contrast image. (c) CL intensity distribution of the particle indicated between white arrows in (a). The FWHM of the Gaussian fit is 105 nm.
Thus autofluorescence of cells excited with an electron probe is imaged with higher resolution than that obtainable by diffraction-limited optical microscopy. The D-EXA image was acquired with an acceleration voltage of 4.8 kV, and a probe current of 3.5 nA. Dwell time and pixel size are 19 µs, and 40 nm, respectively. The average electron dose was calculated to 2.61 e-/Å2.
4. Conclusion and discussion
We have demonstrated high-resolution autofluorescence imaging of MC3T3-E1 cells using a D-EXA microscope. CL emitting organelles were identified by comparing corresponding structures in phase contrast and fluorescence images. Adiposomes, actin filaments, mitochondria, nuclei, and DNA dense area inside nuclei can be recognized in living cells in culture medium without pretreatments. Autofluorescence images with 100-nm resolution were also acquired. Bleaching of autofluorescence was also examined, measuring the reduction of autofluorescence intensity during acquisitions. Although the averaged CL intensity of the membrane reduced by 37.1%, the fine structures inside cells are still observed clearly in second acquired image.
In this technique, staining of specimens is not necessary. Thus the D-EXA microscope has a potential to observe intact cells with autofluorescence imaging. However, at present long term imaging is limited because the irradiance of the electron beam gives damage to the cells during the observation. For analysis of cell function in time-lapse imaging, additional optimization of irradiation condition is necessary, such as reduction of irradiation amount of electrons.
Since the CL spectrum depends on chemical constituents [22], the D-EXA microscope has the potential for multicolour dynamic imaging of endogenous compounds to analyze cell functions in liquid conditions. Spectroscopic analysis can be performed for CL ported via an optical fibre to a spectrometer. The investigation of CL from endogenous cell features is necessary for the developments of new, biocompatible fluorophores used in CL microscopy and correlative light electron microscopy.
In summary, D-EXA microscopy provides high-resolution live cell imaging for process analysis, and can be used to assess new fluorophores as well.
References and links
1. M. Heilemann, “Fluorescence microscopy beyond the diffraction limit,” J. Biotechnol. 149(4), 243–251 (2010). [CrossRef] [PubMed]
2. M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka, and K. Fujita, “Label-free Raman observation of cytochrome c dynamics during apoptosis,” Proc. Natl. Acad. Sci. U.S.A. 109(1), 28–32 (2012). [CrossRef] [PubMed]
3. T. Yasui, Y. Tohno, and T. Araki, “Determination of collagen fiber orientation in human tissue by use of polarization measurement of molecular second-harmonic-generation light,” Appl. Opt. 43(14), 2861–2867 (2004). [CrossRef] [PubMed]
4. S. Witte, A. Negrean, J. C. Lodder, C. P. J. de Kock, G. Testa Silva, H. D. Mansvelder, and M. Louise Groot, “Label-free live brain imaging and targeted patching with third-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 108(15), 5970–5975 (2011). [CrossRef] [PubMed]
5. S. Oh, C. Fang-Yen, W. Choi, Z. Yaqoob, D. Fu, Y. Park, R. R. Dassari, and M. S. Feld, “Label-free imaging of membrane potential using membrane electromotility,” Biophys. J. 103(1), 11–18 (2012). [CrossRef] [PubMed]
6. F. Jamme, S. Kascakova, S. Villette, F. Allouche, S. Pallu, V. Rouam, and M. Réfrégiers, “Deep UV autofluorescence microscopy for cell biology and tissue histology,” Biol. Cell 105(7), 277–288 (2013). [CrossRef] [PubMed]
7. J. V. Rocheleau, W. S. Head, and D. W. Piston, “Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response,” J. Biol. Chem. 279(30), 31780–31787 (2004). [CrossRef] [PubMed]
8. D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007). [CrossRef] [PubMed]
9. Y. Nawa, W. Inami, A. Chiba, A. Ono, A. Miyakawa, Y. Kawata, S. Lin, and S. Terakawa, “Dynamic and high-resolution live cell imaging by direct electron beam excitation,” Opt. Express 20(5), 5629–5635 (2012). [CrossRef] [PubMed]
10. W. Inami, K. Nakajima, A. Miyakawa, and Y. Kawata, “Electron beam excitation assisted optical microscope with ultra-high resolution,” Opt. Express 18(12), 12897–12902 (2010). [CrossRef] [PubMed]
11. D. M. Kaz, C. G. Bischak, C. L. Hetherington, H. H. Howard, X. Marti, J. D. Clarkson, C. Adamo, D. G. Schlom, R. Ramesh, S. Aloni, D. F. Ogletree, and N. S. Ginsberg, “Bright cathodoluminescent thin films for scanning nano-optical excitation and imaging,” ACS Nano 7(11), 10397–10404 (2013). [CrossRef] [PubMed]
12. H. T. Miyazaki, T. Kasaya, T. Takemura, N. Hanagata, T. Yasuda, and H. Miyazaki, “Diffraction-unlimited optical imaging of unstained living cells in liquid by electron beam scanning of luminescent environmental cells,” Opt. Express 21(23), 28198–28218 (2013). [CrossRef] [PubMed]
13. D. B. Peckys and N. de Jonge, “Liquid scanning transmission electron microscopy: imaging protein complexes in their native environment in whole eukaryotic cells,” Microsc. Microanal. 20(2), 346–365 (2014). [CrossRef] [PubMed]
14. U. M. Mirsaidov, H. Zheng, D. Bhattacharya, Y. Casana, and P. Matsudaira, “Direct observation of stick-slip movements of water nanodroplets induced by an electron beam,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7187–7190 (2012). [CrossRef] [PubMed]
15. K. Hirano, T. Kinoshita, T. Uemura, H. Motohashi, Y. Watanabe, T. Ebihara, H. Nishiyama, M. Sato, M. Suga, Y. Maruyama, N. M. Tsuji, M. Yamamoto, S. Nishihara, and C. Sato, “Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM,” Ultramicroscopy 143, 52–66 (2014). [PubMed]
16. T. Hoshino and K. Mabuchi, “Closed-looped in situ nano processing on a culturing cell using an inverted electron beam lithography system,” Biochem. Biophys. Res. Commun. 432(2), 345–349 (2013). [CrossRef] [PubMed]
17. Y. Nawa, W. Inami, A. Miyake, A. Ono, Y. Kawata, S. Lin, and S. Terakawa, “Dynamic autofluorescence imaging of intracellular components inside living cells using direct electron beam excitation,” Biomed. Opt. Express 5(2), 378–386 (2014). [CrossRef] [PubMed]
18. Y. Nawa, W. Inami, S. Lin, Y. Kawata, S. Terakawa, C.-Y. Fang, and H.-C. Chang, “Multi-color imaging of fluorescent nanodiamonds in living HeLa cells using direct electron-beam excitation,” ChemPhysChem 15(4), 721–726 (2014). [CrossRef] [PubMed]
19. G. Ning, T. Fujimoto, H. Koike, and K. Ogawa, “Cathodoluminescence-emitting lipid droplets in rat testis: a study by analytical color fluorescence electron microscopy,” Cell Tissue Res. 271(2), 217–225 (1993). [CrossRef] [PubMed]
20. M. Kubista, B. Aakerman, and B. Norden, “Characterization of Interaction between DNA and 4′,6-diamidino-2-phenylindole by optical spectroscopy,” Biochemistry 26(14), 4545–4553 (1987). [CrossRef] [PubMed]
21. A. Rose, Advances in Electronics vol.1 (Academic Press Inc. New York, 1948).
22. P. V. C. Hough, W. R. McKinney, M. C. Ledbeter, R. E. Pollack, and H. W. Moos, “Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam,” Proc. Natl. Acad. Sci. U.S.A. 73(2), 317–321 (1976). [CrossRef] [PubMed]
