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S-Nitrosoglutathione-induced mouse thymocyte apoptosis studied by fluorescence near-field scanning optical microscopy
Immunology Letters 85 (2003) 225 /229 www.elsevier.com/locate/
S-Nitrosoglutathione-induced mouse thymocyte apoptosis studied by fluorescence near-field scanning optical microscopy A.F. Xie a,b, S.J. Duan a,, Z.B. Zhang b, Y.X. Chen a, L.H. Xue a, G.Z. Yang b a
b
Guang An Men Hospital, China Academy of Traditional Chinese Medicine, Beijing 100053, China Laboratory of Optical Physics, Institute of Physics, Chinese Academy of Sciences, P.O. Box 603, Beijing 100080, China Received 4 March 2002; received in revised form 11 August 2002; accepted 14 August 2002
Abstract This study is an attempt to deeply understand the mechanisms ensuring self-tolerance of T cells via clonal deletion of thymocytes and exploring T lymophocyte homeostasis by observing the apoptosis of single mouse thymocyte induced by S -nitrosoglutathione (GSNO, a nitric oxide donor) using fluorescence near-field scanning optical microscopy (NSOM) in illumination mode. The GSNOinduced thymocytes were stained with propidium iodide containing 0.01% Triton X-100 and excited with light of 488 nm and the emitting fluorescence at 525 nm. According to the NSOM fluorescence image and the simultaneously obtained topography image, the feature of mouse thymocyte apoptosis was characterized by scattering pattern of the fluorescence spots with the size 0.2 /2.1 mm at the full width at half-maximum of fluorescence intensity 78 /80 kHz in the GSNO-treated thymocyte nucleus. Whereas there is no fluorescence from the untreated thymocyte. The intensity of the fluorescence from the dexamethasone-treated thymocyte was much stronger than that from GSNO-induced thymocytes. Furthermore, the fluorescence distribution in the latter were concentrated in the nucleus. Those results also demonstrate the advantages of NSOM such as high spatial resolution and the topography of biology samples. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Near-field scanning optical microscopy; Apoptosis; S -Nitrosoglutathione; Dexamethasone; Propidium iodide; DNA fragments
1. Introduction Apoptosis is a structurally distinct ‘naturally’ programmed cell death pathway, which is involved in a variety of pathologic processes and is of vital importance for tissue to normally develop and homeostasis in living systems, especially, in immunological system [1]. Studying thymocyte apoptosis is beneficial for elaborating the T cell homeostatic mechanisms, and the study of apoptosis ensuring self-tolerance of T cells via clonal deletion of thymocytes will be useful to understand how to control autoimmune diseases. In the past decade, nitric oxide (NO) and NO donors have been recognized
Abbreviations: NSOM, near-field scanning optical microscopy; GSNO, S -nitrosoglutathione; DXM, dexamethasone; PI, propidium iodide; NO, nitric oxide; FWHM, the full width at half maximum. Corresponding author. Tel.: /86-10-88001173; fax: /86-1063014195 E-mail address: [email protected] (S.J. Duan).
as triggers and factors which can induce apoptosis [2]. S -Nitrosoglutathione (GSNO), as one of NO donors, can induce apoptosis via nitrosylation, where nitrosylation functions intermediate, regulation, transduction and cytoxicity in physiological and pathological processes [3,4]. It has been found that GSNO is able to induce mouse thymocyte apoptosis via up-regulation of p53 expression [5], and during apoptosis, GSNO plays the dual role of both inducing and prolonging the apoptosis and NO serves as a messenger molecule in signaling [6]. In the past, the techniques and methods used for detecting apoptosis have their distinct limitations such as (1) transmission electron microscopy, which can only detect the morphological charges of cell membrane, nuclei and organelle undergoing apoptosis and whose sample is limited to the section format at given time, (2) DNA agarose gel electrophoresis-DNA ladder, which can only denote the broken DNA fragments of the apoptotic thymocyte [7], (3) laser scanning confocal
0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 2 ) 0 0 1 9 7 - 9
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optical microscopy whose spatial resolution still can not overcome the refractive limit [3], and (4) flow cytometry with various fluorescence probes and the approach of TdT-mediated dUTP nick end labeling, which can only determine the percentage and the fluorescence image of lots of apoptotic thymocytes, respectively [8]. Now, we successfully investigate the features of a single thymocyte undergoing apoptosis using a novel kind of technique */fluorescence near-field scanning optical microscopy (NSOM). NSOM has the ability to achieve a high spatial resolution and to simultaneously measure the topography of a sample [9,10]. In this technique, a very small aluminum-coated single-mode fiber tip with an aperture smaller than the wavelength of incident light is scanned near the sample surface of interest. The light from the tip illuminates the sample. The light transmitting the sample and the fluorescence emitting from the sample are collected by an objective and then a photo detector records its intensity. NSOM with fluorescence detection is a suitable technique for studying macromolecules and signal cells [11 /14], and has advantages over atomic force microscopy, electron microscopy, and other techniques. In illumination mode as used in our present experiments, NSOM eliminates the necessity of removing the cell membrane because the incident light can be more powerful in this mode than the other modes when the sample was stained with absorption fluorescence probes [9]. This is the first study using near-field optical microscopy to reveal the feature of apoptosis of GSNOtreated mouse thymocyte, a well-characterized model system for apoptosis [15].
2. Materials and methods 2.1. Materials Glutathione, propidium iodide (PI), Triton X-100, dexamethasone (DXM) and SWIM’S S-77 medium were purchased from Sigma (St. Louis, MO). And all of the other materials used in our expriments are of the highest grade of purity (AR and GR) commercially available and were obtained from the chemical plants in China. 2.2. GSNO synthesis GSNO is the S -nitroso derivative of glutathione and must be freshly synthesized prior to experiments as described previously [16]. Here, the brief procedures for GSNO synthesis were described as follows. Glutathione was dissolved in 0.1 N HCl to a final concentration 60 mM, and then an equimolar amount of sodium nitrite were fully mixed by shaking the mixture on an vortex. During the whole procedures, the mixture should be
protected from light. The final GSNO was characterized using ultraviolet-spectroscopy. 2.3. Preparation of thymocytes Thymocytes were obtained from the thymus of 3 /4week-old BALB/C mouse (12 /13.5 gm B.W.). The fresh thymus was gently and repeatedly pressed against a piece of nylon net which submerged in the SWIM’S-77 medium without any fetal calf serum and sulfhydryl. By spinning the suspension, the bits of broken tissue were removed out and there were only the thymocytes in the final suspension with 107 cells/ml. The final thymocyte suspension was equally divided into three aliquots (with the respective volume 0.5 ml) and put into three sterilized flasks, respectively. The thymocytes in two of three flasks were treated with 0.3 mM GSNO and 0.1 mM DXM (final concentration), respectively, and then cultured at 37 8C in a humidified incubator in an atmosphere of 5% CO2 for 3 h. The thymocytes in the third flask were not treated with any factors or triggers and kept at 4 8C. 2.4. Labeling procedures and preparation of the samples for image analysis It is well-known that PI (one kind of fluorescence probe) is capable of binding to the nick ends of the DNA fragments and, once excited with light at 488 nm, emits fluorescence at 525 nm [17,18]. Just before being scanned using NSOM, the thymocytes in each of three flasks were stained with the same mount of PI and 0.01% Triton X-100 for 30 min. Twenty microliter thymocyte suspension from one flask was dripped onto a special 3% polylysine-coated slide to adhere and fix the thymocytes onto it. Shortly after, the slide was washed twice and scanned using fluorescence NSOM immediately. 2.5. Fluorescence NSOM A commercial near-field scanning optical microscope (RHK Technology, USA) was used in this work and its block diagram is shown in Fig. 1. The incident light was 488 nm from an Argon-ion laser (MELLES GRIOT, USA). The diameter of the cantilevered fiber tip in this NSOM is 100 nm. Fluorescence detection was obtained by a single photon counting avalanche photodiode (SPCM-AQR-15, EG&G, USA). The typical pixel dwell time in our experiments was 10 ms. Optical filter 5239/5 nm between the objective and detector separated the fluorescence from the excitation light and the background noise. The optical images and the simultaneously obtained topography images were acquired and processed by using the software of SPM32 5.06 version (RHK Technology, USA). For all the 2-dimen-
A.F. Xie et al. / Immunology Letters 85 (2003) 225 /229
Fig. 1. Block diagram of NSOM. The light of 488 nm coming from the Ar laser and transmitting from the tip illuminates the sample. The light from the sample is collected by the objective, filtered by a passband filter at 5259/5 nm, and then detected by APD. FC, fiber coupler; APD, avalanche photodiode; PD, position detector; LD, laser diode.
sional NSOM images in our experiments, the scan area and the scan pixels is 12/12 mm2 and 256/256, respectively.
3. Results 3.1. NSOM images of a single untreated mouse thymocyte
227
respectively, and both lines are at the same scanned place in the sample. In Fig. 2A, the light intensity in the cell nucleus is poor and the position of the cell nucleus can be decided by the topography image of Fig. 2B. This result can be shown quantitatively in Fig. 2C and D, where the fluorescence intensity is 30 kHz in the nucleus and 54 kHz in the place outside the cell. Here, it should be pointed out that the light intensity recorded by the single photon counting avalanche photodiode was 56 kHz when the tip was placed near to the glass slide surface which coated with polylysine and the thymocyte suspension. This intense background noise mainly resulted from the filter used in the NSOM whose cutoff ratio is low at the excitation laser light (the background noise was the same for the fluorescence NSOM images in all of our experiments because the incident source, the filter and the optical routine remained unchanged). Moreover, the nucleus has a higher refractive index than cytoplasm of thymocyte and the part around the cell, so the transmitted background light intensity should be weaker in the nucleus just as shown in Fig. 2A. So it is certain that there was no fluorescence emitting from the cell nucleus of the untreated thymocyte, further speaking, there was no PI binding to the intact DNA just as expected. Consequently, the untreated thymocyte was not undergoing apoptosis in our experimental conditions according to the NSOM images. 3.2. NSOM images of a single DXM-treated mouse thymocyte
The untreated thymocytes stained with Triton X-100 and PI were used as a control in the present experiments. The NSOM 2-dimensional optical image and simultaneously obtained topography image of a single thymocyte is, respectively, shown in Fig. 2A and B. The plot in Fig. 2C and D corresponds to the line in Fig. 2A and B,
DXM, a kind of glucocorticoid, can induce thymocytes apoptosis, which has been generally recognized in area of cell biology [2,3]. Here, we employed DXMtreated thymocyte apoptosis as the positive control to characterize the GSNO-treated mouse thymocytes in
Fig. 2. NSOM images of DXM-treated thymocyte. The incident light is 488 nm. A, the fluorescence (525 nm) image; B, the topography image simultaneously obtained with A; C, the plot corresponding to the line in A; D, the plot corresponding to the line in B.
Fig. 3. NSOM images of DXM-treated thymocyte. The incident light is 488 nm. A, the fluorescence (525 nm) image; B, the topography image simultaneously obtained with A; C, the plot corresponding to the line in A; D, the plot corresponding to the line in B.
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our experiments. Fig. 3A and B are the NSOM 2dimensional optical image and the corresponding topography image of a single GSNO-treated mouse thymocyte, respectively. The plot in Fig. 3C and D corresponds to the line in Fig. 3A and B, respectively. Both lines are at the same scanned place in the same sample. As clearly shown in Fig. 3A, the light intensity in the nucleus is much stronger than those in the other parts. The nucleus can be located by the topography image of Fig. 3B. The plot in Fig. 3C indicates that the light intensity in the nucleus is up to 180 kHz and the background noise outside the cell is about 50 kHz (near up to 56 kHz). Therefore, it is clear that there was strong fluorescence from the nucleus of the DXM-treated thymocyte, which not only indicates that there was PI binding to the nick end in broken DNA, but also proves that NSOM can detect the apoptosis of DXM-treated thymocyte by imaging the intensity distribution of fluorescence at 525 nm from the DNA fragments in the treated thymocyte nucleus. 3.3. NSOM images of a single GSNO-treated mouse thymocyte Fig. 4A and B are, respectively, the 2-dimensional optical image and the simultaneously obtained topography image of a single GSNO-treated mouse thymocyte scanned by fluorescence NSOM under the same experimental conditions as those for the control and the positive control. The plot in Fig. 4C and D corresponds to the line in Fig. 4A and B, respectively. Both lines are at the same scanned place in the sample. In the optical image Fig. 4A, there are some spots with high light intensity scattering within the nucleus, whose location can be determined by the topography image of Fig. 4B. The plot in Fig. 4C shows that the full width at halfmaximum (FWHM) of the light intensity in these spots
Fig. 4. NSOM images of one GSNO-treated thymocyte. The incident light is 488 nm. A, the fluorescence (525 nm) image; B, the topography image simultaneously obtained with A; C, the plot corresponding to the line in A; D, the plot corresponding to the line in B.
ranges from 0.2 to 2.1 mm and their intensity is from 73 to 80 kHz. The light intensity in the fluorescence image where the cell was not located is 45 kHz. As analysed for the results from untreated thymocyte and DXM-treated thymocyte and based on the PI function, the light spots in Fig. 4A must be fluorescence spots as the result of the formation of nicked DNA fragments excited at 488 nm. So there is the fluorescence scattering pattern in the NSOM image under the GSNO-treated thymocyte undergoing apoptosis.
4. Discussion Here, the results from the untreated thymocyte as a control and the DXM-treated one as a positive control using fluorescence NSOM are exactly consistent with those using other methods as mentioned in the introduction, therefore the potential fluorescence spots with scattering pattern in the fluorescence NSOM image of GSNO-treated thymocyte can mark the feature of GSNO-induced apoptosis. The size of some of scattering spots (from 0.2 to 2.1 mm) at FWHM of the light intensity as shown in Fig. 4A is less than half of the wavelength of incident light, which indicates that the spatial resolution of the NSOM image overcomes the refraction limit. This is the understanding advanteage of NSOM over the previous regular methods in the field of cell apoptosis. Another advangage of NSOM is that the topography images can not only show the wrinkled morphology of the membrane but also locate the features in the fluorescence images. Of course, using various probes in the samples, NSOM can reveal the different features of thymocyte undergoing apoptosis in the spatial distribution. Therefore, we know that NSOM has immense potential for observing biological samples. Although both GSNO and DXM were able to induce mouse thymocyte apoptosis as shown in our results, the patterns of the fluorescence intensity distribution in their NSOM fluorescence images are obviously different. For the former, the distribution of fluorescence spots is scattered in the nucleus, but for the latter, the distribution is concentrated in the nucleus. This feature can be reasonably attributed to the different target molecules which NO and DXM bind to, the different signal transudation pathways and the different expressed genes to control programmed cell death [19]. The mechanism of DXM-induced apoptosis via stimulation of restriction endonuclease activity was generally recognized [2,3,20 /22], although Lei et al. reported that the mechanism of DXM induced thymocyte apoptosis is still unclear [23]. Generally, during GSNO-induced apoptosis, GSNO is decomposed and releases NO, and then induces mouse thymocyte apoptosis either via nitrosylation and p53 gene expression. In the other pathway, NO reacts with superoxide to form peroxyni-
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trite, which produces cellular stress and damages thymocyte chromasomal DNA to generate DNA fragments [24 /26]. It is unclear whether the scattering fluorescence spots were derived from the oxidative damage to mitochondrial DNA by peroxynitrite to form DNA fragments with processing GSNO-induced thymocyte apoptosis. The research in this area is currently under investigation in our laboratory. It is believed that apoptotic mechanism by NO will be elaborated with advancing development of NSOM integrated with other techniques.
Acknowledgements This project is supported by a grant from the national Natural Science Foundation of China (39770202) and from the key laboratory of optical physics of Ministry of Education of PR China.
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[6] K. Sandau, B. Bru¨ne, Cell Signal 8 (1996) 173 /177. [7] J.T. Wolfe, D. Ross, G.M. Cohen, FEBS Lett. 352 (1994) 58 /62. [8] G. Filippini, S. Griffin, M. Uhr, H. Eppenberger, J. Bonilla, F. Cavalli, G. Soldati, Cytometry 31 (1998) 180 /186. [9] M. Ohtsu, H. Hori, Near-field Nano-optics, from Basic Principles to Nano-Fabrication and Nano-Photonics, Kluwer Academic/ Plenum Publishers, New York, 1999. [10] R.C. Dunn, Chem. Rev. 99 (1999) 2891 /2927. [11] W.P. Ambrose, P.M. Goodwin, J.C. Martin, R.A. Keller, Science 265 (1994) 364 /367. [12] M.H.P. Moers, A.G.T. Ruiter, A. Jalocha, N.F. Van Hulst, Ultramic. 61 (1995) 279 /283. [13] C.W. Hollars, R.C. Dunn, J. Chem. Phys. 112 (2000) 7822 /7830. [14] N.F. van Hulst, J.A. Veerman, M.F. Garcia-Parajo, L.K. Kuipers, J. Chem. Phys. 112 (2000) 7799 /7810. [15] H. Kizaki, T. Tadakuma, Microbiol. Immunol. 37 (1993) 917 / 925. [16] T.W. Hart, Tetrahedron Lett. 26 (1985) 2013 /2016. [17] I. Nicoletti, G. Migliorati, M.C. Pagliacci, F. Grignani, C. Riccardi, J. Immunol. Meth. 139 (1991) 271 /279. [18] I. Schmid, C.H. Uitenbogaart, J.V. Giorgi, Cytometry 15 (1994) 12 /20. [19] J.S. Stamler, Cell 78 (1994) 931 /936. [20] M.J. Arends, A.H. Wyllie, Int. Rev. Exp. Path. 32 (1991) 223 / 254. [21] J.J. Cohen, R.C. Duke, K.S. Sellins, Annu. Rev. Immunol. 10 (1992) 267 /293. [22] A.H. Wyllie, J.F.R. Kerr, A.R. Currie, Int. Rev. Cytol. 68 (1980) 251 /306. [23] H.Y. Lei, M.J. Tang, N. Tsao, Apoptosis 2 (1997) 304 /312. [24] A.W. David, J.B. Mrrchell, Free Rad. Biol. Med. 25 (1998) 434 / 456. [25] W.H. Koppenol, Free Rad. Biol. Med. 25 (1998) 335 /351. [26] I. Lizasoan, M.A. Moro, R.G. Knowles, V. Darley-Usmar, S. Moncada, J. Biochem. 314 (1996) 877 /886.
S-Nitrosoglutathione-induced mouse thymocyte apoptosis studied by fluorescence near-field scanning optical microscopy A.F. Xie a,b, S.J. Duan a,, Z.B. Zhang b, Y.X. Chen a, L.H. Xue a, G.Z. Yang b a
b
Guang An Men Hospital, China Academy of Traditional Chinese Medicine, Beijing 100053, China Laboratory of Optical Physics, Institute of Physics, Chinese Academy of Sciences, P.O. Box 603, Beijing 100080, China Received 4 March 2002; received in revised form 11 August 2002; accepted 14 August 2002
Abstract This study is an attempt to deeply understand the mechanisms ensuring self-tolerance of T cells via clonal deletion of thymocytes and exploring T lymophocyte homeostasis by observing the apoptosis of single mouse thymocyte induced by S -nitrosoglutathione (GSNO, a nitric oxide donor) using fluorescence near-field scanning optical microscopy (NSOM) in illumination mode. The GSNOinduced thymocytes were stained with propidium iodide containing 0.01% Triton X-100 and excited with light of 488 nm and the emitting fluorescence at 525 nm. According to the NSOM fluorescence image and the simultaneously obtained topography image, the feature of mouse thymocyte apoptosis was characterized by scattering pattern of the fluorescence spots with the size 0.2 /2.1 mm at the full width at half-maximum of fluorescence intensity 78 /80 kHz in the GSNO-treated thymocyte nucleus. Whereas there is no fluorescence from the untreated thymocyte. The intensity of the fluorescence from the dexamethasone-treated thymocyte was much stronger than that from GSNO-induced thymocytes. Furthermore, the fluorescence distribution in the latter were concentrated in the nucleus. Those results also demonstrate the advantages of NSOM such as high spatial resolution and the topography of biology samples. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Near-field scanning optical microscopy; Apoptosis; S -Nitrosoglutathione; Dexamethasone; Propidium iodide; DNA fragments
1. Introduction Apoptosis is a structurally distinct ‘naturally’ programmed cell death pathway, which is involved in a variety of pathologic processes and is of vital importance for tissue to normally develop and homeostasis in living systems, especially, in immunological system [1]. Studying thymocyte apoptosis is beneficial for elaborating the T cell homeostatic mechanisms, and the study of apoptosis ensuring self-tolerance of T cells via clonal deletion of thymocytes will be useful to understand how to control autoimmune diseases. In the past decade, nitric oxide (NO) and NO donors have been recognized
Abbreviations: NSOM, near-field scanning optical microscopy; GSNO, S -nitrosoglutathione; DXM, dexamethasone; PI, propidium iodide; NO, nitric oxide; FWHM, the full width at half maximum. Corresponding author. Tel.: /86-10-88001173; fax: /86-1063014195 E-mail address: [email protected] (S.J. Duan).
as triggers and factors which can induce apoptosis [2]. S -Nitrosoglutathione (GSNO), as one of NO donors, can induce apoptosis via nitrosylation, where nitrosylation functions intermediate, regulation, transduction and cytoxicity in physiological and pathological processes [3,4]. It has been found that GSNO is able to induce mouse thymocyte apoptosis via up-regulation of p53 expression [5], and during apoptosis, GSNO plays the dual role of both inducing and prolonging the apoptosis and NO serves as a messenger molecule in signaling [6]. In the past, the techniques and methods used for detecting apoptosis have their distinct limitations such as (1) transmission electron microscopy, which can only detect the morphological charges of cell membrane, nuclei and organelle undergoing apoptosis and whose sample is limited to the section format at given time, (2) DNA agarose gel electrophoresis-DNA ladder, which can only denote the broken DNA fragments of the apoptotic thymocyte [7], (3) laser scanning confocal
0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 2 ) 0 0 1 9 7 - 9
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optical microscopy whose spatial resolution still can not overcome the refractive limit [3], and (4) flow cytometry with various fluorescence probes and the approach of TdT-mediated dUTP nick end labeling, which can only determine the percentage and the fluorescence image of lots of apoptotic thymocytes, respectively [8]. Now, we successfully investigate the features of a single thymocyte undergoing apoptosis using a novel kind of technique */fluorescence near-field scanning optical microscopy (NSOM). NSOM has the ability to achieve a high spatial resolution and to simultaneously measure the topography of a sample [9,10]. In this technique, a very small aluminum-coated single-mode fiber tip with an aperture smaller than the wavelength of incident light is scanned near the sample surface of interest. The light from the tip illuminates the sample. The light transmitting the sample and the fluorescence emitting from the sample are collected by an objective and then a photo detector records its intensity. NSOM with fluorescence detection is a suitable technique for studying macromolecules and signal cells [11 /14], and has advantages over atomic force microscopy, electron microscopy, and other techniques. In illumination mode as used in our present experiments, NSOM eliminates the necessity of removing the cell membrane because the incident light can be more powerful in this mode than the other modes when the sample was stained with absorption fluorescence probes [9]. This is the first study using near-field optical microscopy to reveal the feature of apoptosis of GSNOtreated mouse thymocyte, a well-characterized model system for apoptosis [15].
2. Materials and methods 2.1. Materials Glutathione, propidium iodide (PI), Triton X-100, dexamethasone (DXM) and SWIM’S S-77 medium were purchased from Sigma (St. Louis, MO). And all of the other materials used in our expriments are of the highest grade of purity (AR and GR) commercially available and were obtained from the chemical plants in China. 2.2. GSNO synthesis GSNO is the S -nitroso derivative of glutathione and must be freshly synthesized prior to experiments as described previously [16]. Here, the brief procedures for GSNO synthesis were described as follows. Glutathione was dissolved in 0.1 N HCl to a final concentration 60 mM, and then an equimolar amount of sodium nitrite were fully mixed by shaking the mixture on an vortex. During the whole procedures, the mixture should be
protected from light. The final GSNO was characterized using ultraviolet-spectroscopy. 2.3. Preparation of thymocytes Thymocytes were obtained from the thymus of 3 /4week-old BALB/C mouse (12 /13.5 gm B.W.). The fresh thymus was gently and repeatedly pressed against a piece of nylon net which submerged in the SWIM’S-77 medium without any fetal calf serum and sulfhydryl. By spinning the suspension, the bits of broken tissue were removed out and there were only the thymocytes in the final suspension with 107 cells/ml. The final thymocyte suspension was equally divided into three aliquots (with the respective volume 0.5 ml) and put into three sterilized flasks, respectively. The thymocytes in two of three flasks were treated with 0.3 mM GSNO and 0.1 mM DXM (final concentration), respectively, and then cultured at 37 8C in a humidified incubator in an atmosphere of 5% CO2 for 3 h. The thymocytes in the third flask were not treated with any factors or triggers and kept at 4 8C. 2.4. Labeling procedures and preparation of the samples for image analysis It is well-known that PI (one kind of fluorescence probe) is capable of binding to the nick ends of the DNA fragments and, once excited with light at 488 nm, emits fluorescence at 525 nm [17,18]. Just before being scanned using NSOM, the thymocytes in each of three flasks were stained with the same mount of PI and 0.01% Triton X-100 for 30 min. Twenty microliter thymocyte suspension from one flask was dripped onto a special 3% polylysine-coated slide to adhere and fix the thymocytes onto it. Shortly after, the slide was washed twice and scanned using fluorescence NSOM immediately. 2.5. Fluorescence NSOM A commercial near-field scanning optical microscope (RHK Technology, USA) was used in this work and its block diagram is shown in Fig. 1. The incident light was 488 nm from an Argon-ion laser (MELLES GRIOT, USA). The diameter of the cantilevered fiber tip in this NSOM is 100 nm. Fluorescence detection was obtained by a single photon counting avalanche photodiode (SPCM-AQR-15, EG&G, USA). The typical pixel dwell time in our experiments was 10 ms. Optical filter 5239/5 nm between the objective and detector separated the fluorescence from the excitation light and the background noise. The optical images and the simultaneously obtained topography images were acquired and processed by using the software of SPM32 5.06 version (RHK Technology, USA). For all the 2-dimen-
A.F. Xie et al. / Immunology Letters 85 (2003) 225 /229
Fig. 1. Block diagram of NSOM. The light of 488 nm coming from the Ar laser and transmitting from the tip illuminates the sample. The light from the sample is collected by the objective, filtered by a passband filter at 5259/5 nm, and then detected by APD. FC, fiber coupler; APD, avalanche photodiode; PD, position detector; LD, laser diode.
sional NSOM images in our experiments, the scan area and the scan pixels is 12/12 mm2 and 256/256, respectively.
3. Results 3.1. NSOM images of a single untreated mouse thymocyte
227
respectively, and both lines are at the same scanned place in the sample. In Fig. 2A, the light intensity in the cell nucleus is poor and the position of the cell nucleus can be decided by the topography image of Fig. 2B. This result can be shown quantitatively in Fig. 2C and D, where the fluorescence intensity is 30 kHz in the nucleus and 54 kHz in the place outside the cell. Here, it should be pointed out that the light intensity recorded by the single photon counting avalanche photodiode was 56 kHz when the tip was placed near to the glass slide surface which coated with polylysine and the thymocyte suspension. This intense background noise mainly resulted from the filter used in the NSOM whose cutoff ratio is low at the excitation laser light (the background noise was the same for the fluorescence NSOM images in all of our experiments because the incident source, the filter and the optical routine remained unchanged). Moreover, the nucleus has a higher refractive index than cytoplasm of thymocyte and the part around the cell, so the transmitted background light intensity should be weaker in the nucleus just as shown in Fig. 2A. So it is certain that there was no fluorescence emitting from the cell nucleus of the untreated thymocyte, further speaking, there was no PI binding to the intact DNA just as expected. Consequently, the untreated thymocyte was not undergoing apoptosis in our experimental conditions according to the NSOM images. 3.2. NSOM images of a single DXM-treated mouse thymocyte
The untreated thymocytes stained with Triton X-100 and PI were used as a control in the present experiments. The NSOM 2-dimensional optical image and simultaneously obtained topography image of a single thymocyte is, respectively, shown in Fig. 2A and B. The plot in Fig. 2C and D corresponds to the line in Fig. 2A and B,
DXM, a kind of glucocorticoid, can induce thymocytes apoptosis, which has been generally recognized in area of cell biology [2,3]. Here, we employed DXMtreated thymocyte apoptosis as the positive control to characterize the GSNO-treated mouse thymocytes in
Fig. 2. NSOM images of DXM-treated thymocyte. The incident light is 488 nm. A, the fluorescence (525 nm) image; B, the topography image simultaneously obtained with A; C, the plot corresponding to the line in A; D, the plot corresponding to the line in B.
Fig. 3. NSOM images of DXM-treated thymocyte. The incident light is 488 nm. A, the fluorescence (525 nm) image; B, the topography image simultaneously obtained with A; C, the plot corresponding to the line in A; D, the plot corresponding to the line in B.
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our experiments. Fig. 3A and B are the NSOM 2dimensional optical image and the corresponding topography image of a single GSNO-treated mouse thymocyte, respectively. The plot in Fig. 3C and D corresponds to the line in Fig. 3A and B, respectively. Both lines are at the same scanned place in the same sample. As clearly shown in Fig. 3A, the light intensity in the nucleus is much stronger than those in the other parts. The nucleus can be located by the topography image of Fig. 3B. The plot in Fig. 3C indicates that the light intensity in the nucleus is up to 180 kHz and the background noise outside the cell is about 50 kHz (near up to 56 kHz). Therefore, it is clear that there was strong fluorescence from the nucleus of the DXM-treated thymocyte, which not only indicates that there was PI binding to the nick end in broken DNA, but also proves that NSOM can detect the apoptosis of DXM-treated thymocyte by imaging the intensity distribution of fluorescence at 525 nm from the DNA fragments in the treated thymocyte nucleus. 3.3. NSOM images of a single GSNO-treated mouse thymocyte Fig. 4A and B are, respectively, the 2-dimensional optical image and the simultaneously obtained topography image of a single GSNO-treated mouse thymocyte scanned by fluorescence NSOM under the same experimental conditions as those for the control and the positive control. The plot in Fig. 4C and D corresponds to the line in Fig. 4A and B, respectively. Both lines are at the same scanned place in the sample. In the optical image Fig. 4A, there are some spots with high light intensity scattering within the nucleus, whose location can be determined by the topography image of Fig. 4B. The plot in Fig. 4C shows that the full width at halfmaximum (FWHM) of the light intensity in these spots
Fig. 4. NSOM images of one GSNO-treated thymocyte. The incident light is 488 nm. A, the fluorescence (525 nm) image; B, the topography image simultaneously obtained with A; C, the plot corresponding to the line in A; D, the plot corresponding to the line in B.
ranges from 0.2 to 2.1 mm and their intensity is from 73 to 80 kHz. The light intensity in the fluorescence image where the cell was not located is 45 kHz. As analysed for the results from untreated thymocyte and DXM-treated thymocyte and based on the PI function, the light spots in Fig. 4A must be fluorescence spots as the result of the formation of nicked DNA fragments excited at 488 nm. So there is the fluorescence scattering pattern in the NSOM image under the GSNO-treated thymocyte undergoing apoptosis.
4. Discussion Here, the results from the untreated thymocyte as a control and the DXM-treated one as a positive control using fluorescence NSOM are exactly consistent with those using other methods as mentioned in the introduction, therefore the potential fluorescence spots with scattering pattern in the fluorescence NSOM image of GSNO-treated thymocyte can mark the feature of GSNO-induced apoptosis. The size of some of scattering spots (from 0.2 to 2.1 mm) at FWHM of the light intensity as shown in Fig. 4A is less than half of the wavelength of incident light, which indicates that the spatial resolution of the NSOM image overcomes the refraction limit. This is the understanding advanteage of NSOM over the previous regular methods in the field of cell apoptosis. Another advangage of NSOM is that the topography images can not only show the wrinkled morphology of the membrane but also locate the features in the fluorescence images. Of course, using various probes in the samples, NSOM can reveal the different features of thymocyte undergoing apoptosis in the spatial distribution. Therefore, we know that NSOM has immense potential for observing biological samples. Although both GSNO and DXM were able to induce mouse thymocyte apoptosis as shown in our results, the patterns of the fluorescence intensity distribution in their NSOM fluorescence images are obviously different. For the former, the distribution of fluorescence spots is scattered in the nucleus, but for the latter, the distribution is concentrated in the nucleus. This feature can be reasonably attributed to the different target molecules which NO and DXM bind to, the different signal transudation pathways and the different expressed genes to control programmed cell death [19]. The mechanism of DXM-induced apoptosis via stimulation of restriction endonuclease activity was generally recognized [2,3,20 /22], although Lei et al. reported that the mechanism of DXM induced thymocyte apoptosis is still unclear [23]. Generally, during GSNO-induced apoptosis, GSNO is decomposed and releases NO, and then induces mouse thymocyte apoptosis either via nitrosylation and p53 gene expression. In the other pathway, NO reacts with superoxide to form peroxyni-
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trite, which produces cellular stress and damages thymocyte chromasomal DNA to generate DNA fragments [24 /26]. It is unclear whether the scattering fluorescence spots were derived from the oxidative damage to mitochondrial DNA by peroxynitrite to form DNA fragments with processing GSNO-induced thymocyte apoptosis. The research in this area is currently under investigation in our laboratory. It is believed that apoptotic mechanism by NO will be elaborated with advancing development of NSOM integrated with other techniques.
Acknowledgements This project is supported by a grant from the national Natural Science Foundation of China (39770202) and from the key laboratory of optical physics of Ministry of Education of PR China.
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