Plasmon-Enhanced Autofluorescence Imaging of Organelles in Label

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Plasmon-Enhanced Autofluorescence Imaging of Organelles in Label-Free Cells by Deep-Ultraviolet Excitation Masakazu Kikawada,† Atushi Ono,‡,§ Wataru Inami,†,‡ and Yoshimasa Kawata*,†,‡ †

Graduate School of Science and Technology, Shizuoka University 3-5-1, Johoku, Naka, Hamamatsu 432-8561, Japan Research Institute of Electronics, Shizuoka University 3-5-1, Johoku, Naka, Hamamatsu 432-8561, Japan § Department of Electronics and Materials Science, Shizuoka University 3-5-1, Johoku, Naka, Hamamatsu 432-8561, Japan ‡

ABSTRACT: We demonstrate the observation of organelles in label-free cells on an aluminum thin film using deep-ultraviolet surface plasmon resonance (DUV-SPR). In particular, the Kretschmann configuration is used for the excitation of DUV-SPR. MC3T3-E1 cells are directly cultured on the aluminum thin film, and DUV-SPR leads to autofluorescence of in the label-free MC3T3-E1. We found that nucleic acid and mitochondria in these label-free MC3T3-E1 cells quite strongly emit the autofluorescence as a result of DUV-SPR. Yeast cells are also deposited on the aluminum thin film. Tryptophan and mitochondrial nicotinamide adenine dinucleotide (NADH) in the yeast cells are subsequently excited, and their autofluorescence is spectrally analyzed in the UV region. On the basis of these results, we conclude that DUV-SPR constitutes a promising technique for the acquisition of highly sensitive autofluorescence images of various organelles in the cells.

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organelles in cells using SPR in the DUV region (DUVSPR).37,38 In this paper, we demonstrate the enhanced autofluorescence and high-sensitivity bioimaging of intracellular organelles using DUV-SPR. More specifically, MC3T3-E1 and yeast cells are observed. MC3T3-E1 cells are one of the osteoblast cells of a mouse. They have been investigated for the analysis of expression for mouse bones by proteins. MC3T3-E1 cells have many organelles such as mitochondria, actin, and so on. We used MC3T3-E1 cells to evaluate and identify the organelles that generate autofluorescence with deep-UV excitation. While, we also used yeast cells to evaluate the damage by deep-UV light irradiation. Yeast cells have the simple textures and are resistant to damage. They are suitable for observation of living cells using DUV excitation.

ioimaging using optical microscopes represents a powerful approach for observing of intracellular organelles such as mitochondria,1,2 ocular structures,3,4 cancers,5,6 and so on. Recently, label-free imaging techniques, including autofluorescence microscopy,7,8 Raman microscopy,9,10 coherent antiStokes Raman scattering (CARS) microscopy,11 near-infrared reduced-illuminance autofluorescence imaging (NIR-RAFI),12 surface-enhanced Raman scattering (SERS),13−15 and SPR based high resolution imaging,16−19 have attracted considerable attention because they can be used to observe living cells without the need for staining. In this context, autofluorescence refers to the inherent emission of intracellular organelles.20 Recently, the autofluorescence-excited deep-ultraviolet (DUV) region has been studied because the high photon energy of DUV light excites many kinds of biological structures in cells.21−24 In particular, DNA25 and proteins26 absorb in the DUV region. In fact, Zeskind et al. demonstrated the observation of label-free human and mouse cells using a DUV microscope. They were able to quantitatively estimate the mass of proteins and nucleic acids in label-free living cells as well as their fluorescence yields. Additionally, the autofluorescence signal excited by DUV light can provide important information regarding the distribution of tissue histology and cell biology. Surface plasmon resonance (SPR) excited by DUV light has also been investigated in the context of fluorescence enhancement,27−29 enhancement of photoelectron emission,30−32 localized surface plasmon resonance (LSPR),33−36 and so on, because it can enhance the electric field of incident light by a factor of 10 or more. Against this background, we have demonstrated the highly sensitive observation of dye-labeled © XXXX American Chemical Society

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HIGH-SENSITIVITY OBSERVATION USING DUV-SPR Figure 1 shows a schematic diagram of label-free observation using DUV-SPR. Label-free biological samples are directly cultured or deposited on an aluminum thin film. DUV-SPR then excites the label-free biological samples using the Kretschmann configuration. At this stage, the autofluorescence signal is detected from the top of the biological samples. In order to excite SPR in the DUV region, a metal with a negative permittivity and a minimal loss coefficient in the DUV region is required. Aluminum satisfies these requirements, as its permittivity is −9.79 + 1.32i at a wavelength of 266 nm.39 We calculated the optimum aluminum thickness using the Fresnel Received: October 27, 2015 Accepted: December 15, 2015

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DOI: 10.1021/acs.analchem.5b04060 Anal. Chem. XXXX, XXX, XXX−XXX

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DUV laser. We used an aluminum thickness of 24 nm because the aluminum surface becomes oxidized into alumina. The alumina surface was estimated to be 6 nm by comparison between the experimental and calculated results. The aluminum thickness of 18 nm is different from the optimum aluminum thickness because we cannot control the thickness of oxidation, and it is enough efficiently to excite DUV-SPR. The measured reflectance was then compared with the calculated reflectance. The solid and long-dashed lines indicate reflectance before and after the biological samples are cultured, respectively. The short-dashed line indicates the measured reflectance using MC3T3-E1 cells on the glass. DUV-SPR is excited at an incident angle of 52° after the biological samples are cultured, and the excitation angle of DUV-SPR is shifted to a higher angle. Figure 2b shows the autofluorescence spectra of the label-free MC3T3-E1 cells. The solid and dashed lines indicate the autofluorescence intensity of the label-free MC3T3-E1 cells with and without DUV-SPR, respectively. The autofluorescence intensity for the aluminum is higher than that for the glass. It can be seen that MC3T3-E1 cells exhibit two fluorescence peaks, which are located around 330 and 500 nm. These 330 and 500 nm emissions indicate nucleic acid 23,26 and mitochondria,40 respectively. The difference of fluorescence intensity between 330 nm emission and 500 nm emission is due to the concentration of nucleic acid and mitochondrial NADH. Both of the enhancement factors are 8 times. The fluorescence peak of nucleic acid is clearly observed because of the fluorescence enhancement by DUV-SPR. From these results, we may conclude that DUV-SPR enhances the autofluorescence intensity of the label-free MC3T3-E1 cells. Figure 2c−f shows spectral autofluorescence images of MC3T3-E1 cells using a charge-coupled device (CCD) (iXon Ultra 888: ANDOR) with laser power of 1 mW, an CCD exposure time of 1 s, and a numerical aperture (NA) of 0.4 for the objective lens. Filters for the 330 nm emission (FF01-325/ 40-25, Semrock) and the 500 nm emission (LM01-427-25, Semrock) are also used. Figure 2c,d indicates autofluorescence images of nucleic acid in label-free MC3T3-E1 cells cultured on the aluminum and glass surfaces, respectively. Figures 2e,f shows images of mitochondrial NADH in the cells cultured on the aluminum and glass surfaces, respectively. In the autofluorescence images with DUV-SPR, organelles can be clearly observed in the MC3T3-E1 cells. On the other hand, the autofluorescence intensity is very weak in the images without DUV-SPR. In the 330 nm fluorescence image, the autofluorescence intensity of the structures in the nucleus is particularly strong compared with the 500 nm fluorescence image. More specifically, the distribution of nucleic acid in the nucleus is efficiently excited with 266 nm excitation light. In addition to the nucleic acid, DUV-SPR excites the membrane, actin, and tubulin (i.e., fibrous structures). Fine structures around the nucleus are clearly observed in the 330 nm fluorescence image. However, for the autofluorescence images without DUV-SPR, it is difficult to distinguish the structures of the nucleic acid, mitochondria, and so on. Hence, autofluorescence in the 330 and 500 nm emissions is enhanced by DUV-SPR, and spectral autofluorescence images are clearly obtained with high sensitivity. DUV-SPR enhances more structures from UV to visible emissions.

Figure 1. Schematic diagram of label-free observation using deepultraviolet surface plasmon resonance (DUV-SPR).

equations for high-efficiency SPR excitation and high electric field intensity, this value is 21 nm.37

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LABEL-FREE IMAGING OF MC3T3-E1 CELLS MC3T3-E1 cells are chosen as a biological sample, and they are cultured on an aluminum and glass surfaces. The aluminum and glass substrates were sterilized dipping in ethanol. MC3T3-E1 cells were cultured on the both substrates in an incubator for 2 days. After culturing, MC3T3-E1 cells were fixed on the aluminum and glass surfaces. Figure 2a shows the measured relationship between the reflectance and incident angle under the excitation of a 266 nm

Figure 2. (a) Measured reflectance as a function of the incident angle. (b) Measured autofluorescence spectra of MC3T3-E1 cells. (c−f) Autofluorescence images of MC3T3-E1 cells: (c) nucleic acid at the 330 nm emission with deep-ultraviolet surface plasmon resonance (DUV-SPR) and (d) without DUV-SPR; (e) mitochondria at the 500 nm emission with DUV-SPR and (f) without DUV-SPR. B

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INVESTIGATION OF ORGANELLES IN THE CELLS The autofluorescence image was then compared against the image of a stained cell image in order to investigate what kinds of organelles generate autofluorescence (Figure 2e). In order to make a such a comparison, we prepared the biological samples according to the following procedure. The label-free MC3T3E1 cells were observed using DUV-SPR as a first step. We then stained mitochondria in the same MC3T3-E1 cells with MitoTracker. Stained mitochondria were then observed using a conventional epifluorescence microscope. At this stage, the autofluorescence image was compared against the image of the stained mitochondria. Figure 3a shows the autofluorescence image of label-free MC3T3-E1 cells excited by DUV-SPR. The autofluorescence of

incident angle of 52°, and the DUV-SPR is excited after depositing the yeast cells. Figure 4b shows the autofluorescence spectra of label-free yeast cells. The autofluorescence intensity obtained by using DUV-SPR is 4 times higher than that without DUV-SPR. The yeast cells exhibit two emission peaks, which are located around 330 and 450 nm. The two emissions indicate the autofluorescence of tryptophan, which is a type of protein41 and mitochondrial NADH42 in yeast cells, respectively. The fluorescence intensities of tryptophan and mitochondrial NADH were four times enhanced by the excitation of DUV-SPR. We then evaluated the damage caused by DUV light irradiation. Figure 4c shows the autofluorescence spectra of the yeast cells after 10 min of DUV-SPR exposure. It can be seen that the autofluorescence intensity of tryptophan decreases. On the other hand, the autofluorescence intensity of mitochondrial NADH almost remains constant. Fluorescence intensity of the tryptophan on the aluminum and glass decreases 91 and 73%, respectively. Absorption of tryptophan is higher than that of mitochondrial NADH at 266 nm.43 The maximum absorptions of tryptophan and mitochondrial NADH are 270 and 340 nm, respectively. DUV light causes severe damage to proteins because proteins absorb DUV light well.44 Even though the observation of living cells with DUV light is limited by the damage, DUV-SPR enhances fluorescence of living cells without staining and shorten the observation time. DUV-SPR reduced exposure energy during observation compared with the conventional microscope.38 Figure 4d,e shows the spectral images of tryptophan in labelfree yeast cells just after DUV-SPR exposure and 10 min exposure thereafter. Figure 4f,g shows mitochondrial NADH under the same respective conditions. The inset in each image indicates the spectral autofluorescence images of yeast cells without DUV-SPR. The particles represent yeast cells, which clump together in these images. On the other hand, the autofluorescence intensity without DUV-SPR is very weak. In fact, it is difficult to recognize the particles in these images. By comparing parts d and f of Figure 4, one can see that the fluorescence positions are almost identical; accordingly, we can conclude that tryptophan and mitochondrial NADH in the yeast cells are almost uniformly distributed. The autofluorescence intensity of tryptophan decreases in Figure 4e, and the particle structures cannot be clearly recognized. The signal-tonoise ratio of mitochondrial NADH is almost constant after 10 min of exposure, as shown in Figure 4g. The signal-to-noise ratios in Figure 4f,g are 3.2 and 2.9, respectively. It is worth noting that tryptophan and mitochondrial NADH in yeast cells generated the autofluorescence at emissions of 335 and 450 nm, respectively, and the NADH was less damaged compared with tryptophan. In conclusion, label-free MC3T3-E1 and yeast cells were observed using DUV-SPR. DUV-SPR was excited after culturing the MC3T3-E1 and yeast cells on the aluminum. The autofluorescence spectra of MC3T3-E1 cells exhibited two emission peaks: one at 330 nm, which derives from nucleic acid, and another at 500 nm, which derives from mitochondria. DUV-SPR enhanced the autofluorescence of the structures, and the nucleic acid and mitochondria in MC3T3-E1 cells were separately observed with high sensitivity. Futhermore, mitochondria in the free-labeled MC3T3-E1 cells were identified by comparison with stained mitochondria. DUV-SPR enhanced the autofluorescence of living yeast cells. The autofluorescence intensity of tryptophan, which is

Figure 3. Fluorescence images of mitochondria in the MC3T3-E1 cells. (a) Autofluorescence image of label-free mitochondria excited by deep-ultraviolet surface plasmon resonance (DUV-SPR). (b) Fluorescence image of stained mitochondria using a conventional fluorescence microscope. (c) Magnified image of the dashed box in Figure 2a. (d) Magnified image of the dashed box in Figure 2b.

mitochondria can be clearly observed. Figure 3b shows the image of stained mitochondria in MC3T3-E1 cells using the conventional epifluorescence microscope. The number of the particles between two images is different. We believe that the difference is caused by nonuniformity of staining process and the damage because mitochondria were stained after DUV-SPR irradiation. Figure 3c,d shows magnified images of the areas marked with dashed boxes in Figure 3a,b. The features of the autofluorescence agree well with those of the image of the stained mitochondria. From these results, we conclude that the autofluorescence of fine structures is generated from mitochondria in the label-free MC3T3-E1 cells as a result of the excitation of DUV-SPR.

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LABEL-FREE IMAGING OF YEAST CELLS Living yeast cells were also observed using DUV-SPR. It is easy to analyze yeast cells because the components of yeast cells have been thoroughly investigated and are resistant to damage. Label-free yeast cells were dissolved in pure water with sugar and were then deposited on the aluminum thin film and glass after fermentation. Figure 4a shows the measured reflectance after depositing of the label-free yeast cells. The solid and broken lines indicate the reflectance of yeast cells on the aluminum and glass, respectively. The reflectance drastically decreases at the C

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Figure 4. (a) Measured reflectance as a function of the incident angle after depositing yeast cells on the aluminum. (b) Measured autofluorescence spectra of yeast cells. (c) Measured autofluorescence spectra of yeast cells after deep-ultraviolet surface plasmon resonance (DUV-SPR) exposure for 10 min. (d−g) Autofluorescence images of yeast cells excited by DUV-SPR: (d) autofluorescence image of tryptophan at the 335 nm emission and (e) after 10 min exposure; (f) autofluorescence image of mitochondrial NADH at the 440 nm emission and (g) after 10 min exposure. Inserts indicate autofluorescence images of yeast cells without DUV-SPR. (2) Lopez-Otin, C.; Blasco, M. A.; Partridge, L.; Serrano, M.; Kroemer, G. Cell 2013, 153, 1194−1217. (3) Keilhauer, C. N.; Delori, F. C. Invest. Ophthalmol. Visual Sci. 2006, 47, 3556−3564. (4) Schweitzer, D.; Gaillard, E. R.; Dillon, J.; Mullins, R. F.; Rus-sell, S.; Hoffmann, B.; Peters, S.; Hammer, M.; Biskup, C. Invest. Ophthalmol. Visual Sci. 2012, 53, 3376−3386. (5) Audo, I.; Tsang, S. H.; Fu, A. D.; Barnes, J. A.; Holder, G. E.; Moore, A. T. Arch. Ophthalmol. 2007, 125, 714−715. (6) Zellweger, M.; Grosjean, P.; Goujon, D.; Monnier, P.; van den Bergh, H.; Wagnières, G. J. Biomed. Opt. 2001, 6, 41−51. (7) Shibuki, K.; Hishida, R.; Murakami, H.; Kudoh, M.; Kawagu-chi, T.; Watanabe, M.; Watanabe, S.; Kouuchi, T.; Tanaka, R. J. J. Physiol. 2003, 549, 919−927. (8) De Veld, D. C. G. D.; Witjes, M. F. H.; Sterenborg, H. J. C. M.; Roodenburg, J. L. N. Oral Oncol. 2005, 41, 117−131. (9) Ni, Z.; Wang, Y.; Yu, T.; Shen, Z. Nano Res. 2008, 1, 273−291. (10) Chernenko, T.; Matthaüs, C.; Milane, C. L.; Quintero, L.; Amiji, M.; Diem, M. ACS Nano 2009, 3, 3552−3559. (11) Zumbusch, A.; Holtom, G. R.; Xie, X. S. Phys. Rev. Lett. 1999, 82, 4142−4145. (12) Cideciyan, A. V.; Swider, M.; Aleman, T. S.; Roman, M. I.; Sumaroka, A.; Schwartz, S. B.; Stone, E. M.; Jacobson, S. G. J. Opt. Soc. Am. A 2007, 24, 1457−1467. (13) Nie, S.; Emory, S. R. Science 1997, 275, 1102−1106. (14) Taguchi, A.; Hayazawa, N.; Furusawa, K.; Ishitobi, H.; Kawata, S. J. Raman Spectrosc. 2009, 40, 1324−1330.

essential proteins as a precursor, and mitochondrial NADH were dominant at 335 and 440 nm, respectively. The autofluorescence intensity of tryptophan in the yeast cells decreased after DUV-SPR exposure. The tryptophan and mitochondrial NADH were observed with different contrasts after exposure, even though their distributions were almost identical in the label-free yeast cells. Accordingly, DUV-SPR can facilitate the observation of proteins, DNA in nucleic acids, and other structures that cannot be excited by visible light. On the basis of these results, DUV-SPR is shown to be a powerful technique for acquiring high-sensitivity label-free observation of biological samples as well as distribution analyses of proteins and RNA.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81-53-478-1069. Fax: +81-53-478-1128. Notes

The authors declare no competing financial interest.

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