- Email: [email protected]
Micro-spectroscopy of chloroplasts in protoplasts from Arabidopsis thaliana under single- and multi-photon excitations
Journal of Luminescence 98 (2002) 107–114
Micro-spectroscopy of chloroplasts in protoplasts from Arabidopsis thaliana under single- and multi-photon excitations Fu-Jen Kaoa,*, Yi-Min Wanga, Jian-Cheng Chena, Ping-Chin Chengb, Rung-Wu Chenc, Bai-Ling Linc a
Center for Neuroscience and Department of Physics, National Sun Yat-sen University, Kaohsiung 80424, Taiwan b Department of Electrical Engineering, State University of New York, Buffalo, NY 14260, USA c Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan
Abstract Ultrafast laser pulses are known to generate impacts on specimens in ways much different from cw illumination in the applications of confocal microscopy. In this study, protoplasts from Arabidopsis thaliana are employed as the samples to compare the effects induced by single- and multi-photon excitations under confocal imaging configuration. Through time-lapsed micro-spectroscopy we are able to characterize the differences in photo-bleaching and the evolution of fluorescence spectra of chloroplasts thus induced. These differences may be attributed to the disruption of chained reaction as a result of multi-photon excitation in photosynthesis. r 2002 Elsevier Science B.V. All rights reserved. PACS: 07.60.R; 87.64.V; 82.50.P Keywords: Micro-spectroscopy; Multi-photon excitation; Photo-bleaching; Chloroplasts
1. Introduction Laser scanning fluorescence confocal microscopy has established itself as one of the most important tools in various disciplines. In which, high-brightness laser beam is tightly focused on a sample through a high-numerical objective to obtain sectional images. However, the high intensity thus formed under an objective often causes fluorescent dyes to fade within minutes of continuous scanning. Therefore, one must limit the scanning time or light intensity if the specimen’s *Corresponding author. Tel.: +886-7-5253720; fax: +886-75253709. E-mail address: [email protected] (F.-J. Kao).
integrity has to be kept. The employment of two-photon excitation in fluorescent microscopy with ultrafast laser illumination was expected to solve this problem. In which, fluorescence induced by two-photon excitation depends on the square of the incident light intensity and thus decreases approximately as the square of the distance from the focus, forming a spatial distribution that can be described by the square of the objective lens’ point spread function assuming Stokes’ shift can be ignored. This highly nonlinear behavior causes only those dye molecules very near the focus of the beam to become excited effectively and limits the excitation volume accordingly. The sample above and below the plane of focus is merely subjected to infrared light that results in neither
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photo-bleaching nor photo-damage. It was believed that although the peak amplitude of the IR pulses is large, the mean power of the beam is only a few tens of milliwatts, insufficient to cause localized heating of the specimen. It is also generally accepted that two-photon excitation in confocal microscopy has the additional advantages of deeper penetration and less spherical aberration [1–3]. Nevertheless, the very high peak power of short laser pulses for two-photon excitation may generate unexpected excitation over the samples and induce nonlinear photobleaching or photo-chemical processes [4]. The effects of a pulsed laser on sample integrity or even cell viability have become an interesting and much debated topic in the applications of multi-photon fluorescence microscopy [5–8]. In this study, the protoplasts prepared from a model plant, Arabidopsis thaliana, are used as the specimen to demonstrate the difference due to multi-photon excitations [9–11]. They are chosen because of their importance as the organelle in plant cells that performs photosynthesis, and also
because they contain highly fluorescent pigments such as chlorophyll. We have found that the spectral responses of chloroplasts under cw and pulsed laser illumination exhibit very different characteristics, as reported previously [12]. In addition, the corresponding photo-bleaching cross sections are also different, as revealed by time-lapsed micro-spectroscopy. These differences may be accounted for by the interplay between chlorophyll and carotenoids [13], which is known to absorb excess energy to protect chloroplasts from photo-damages under intense illumination.
2. Experimental Fig. 1 shows the setup for two-photon timelapse micro-spectroscopy [14]. A mode-locked Ti:sapphire laser (Tsunami, Spectra-Physics) pumped by a frequency-doubled all-solid-state laser (Verdi, Coherent) provides ultrafast laser pulses centered at 780 nm with a pulse width of
Fig. 1. Schematics of the two-photon time-lapsed micro-spectroscopy setup. The combination of fiber bundle coupling and highly sensitive CCD-based spectrometer allows rapid acquisition of spectra with high signal-to-noise ratio.
F.-J. Kao et al. / Journal of Luminescence 98 (2002) 107–114
approximately 150 fs and a repetition rate of 82 MHz. The laser beam is directed into a modified fluorescence microscope (BX50, Olympus) via a dichroic mirror. The fluorescence from the samples is coupled to the spectrometer (77250, Oriel) via a special slit-outlet quartz fiber bundle. A 300groove grating is used so that a spectral range from 400 to 720 nm can be observed simultaneously. A highly sensitive back-illuminated liquid nitrogen cooled CCD camera (Orbis II, SpectraSource) is used to acquire the time-lapse spectra. The very high quantum efficiency and extremely low noise of the cooled CCD allow rapid acquisition of fluorescence spectra with high signal-tonoise ratio. A 10X objective (Plan, 0.25NA, Olympus) is used to focus the laser beam into chloroplasts in a cell. Peak intensity reached for Ti:sapphire laser illumination which is estimated to be 3.7 1010 W/cm2 under the objective, which approximates the intensity commonly used in twophoton confocal microscopy. In micro-spectroscopic measurements, two-photon excitation has the added advantages of limited excitation volume and thus has better rejection to background noise in spectral acquisition. For comparison, an aircooled argon ion laser is used to provide cw laser illumination with 1.2 mW (measured under the objective) at 488 nm. The average intensity from argon ion laser illumination is 2.65 104 W/cm2. Fluorescence microscopy is also performed on a modified Olympus Fluoview confocal microscope for both single- and multiple-photon excitations. The details regarding modification of the confocal microscope can be found in the work of Majewska et al. [15]. Plants of A. thaliana were grown from seeds sown in soil in 20 8 4 cm3 (L W H) flats in a growth chamber with day/night temperatures at 25/221C and 16/8 h day/night cycle, illuminated with fluorescent tubes (FL40D-Ex, Mitsubishi) emitting a near-sunlight spectrum to reach 160 mmol m2 s2 at the rosette level. Leaves from four to five weeks old plants were used. Mesophyll protoplasts were isolated from leaves through enzymatic digestion of the cell wall, and suspended in the culture medium [16], and were prepared freshly for each use. For microscopic observation, the protoplast suspension was placed in a cham-
109
bered coverglass (Lab-Tek, Illinois, USA). Aqueous solutions of riboflavin, FMN FAD, and b-carotene and acetone solution of chlorophyll a; chlorophyll b were used in this study.
3. Results and discussion Fig. 2 shows the time-lapse auto-fluorescence spectra of chloroplasts under cw laser excitation at 488 nm. The time-sequenced plot of the spectra is shown in Fig. 2(a). The red auto-fluorescence of chloroplasts centered around 690 nm exhibits exponential decay at cw laser illumination. Good fitting of the decaying curve in Fig. 2(b) to a
Fig. 2. Time-lapse fluorescence spectra from chloroplasts obtained with micro-spectroscopy excited with 488 nm laser beam from an argon ion laser: (a) the time-sequenced spectra; and (b) the 690 nm fluorescence decaying curve. A good fit to a double exponential decaying function indicates that there are at least two photo-bleaching processes.
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double exponential decaying function indicates that at least two different photo-bleaching processes can be identified. We found that the singlephoton bleaching cross sections for these two processes are 8.1 1024 and 5.4 1025 cm2, respectively. If the absorption cross section for chloroplast excitation is assumed to be 1016 cm2, in the order of a dye molecule, then the corresponding quantum efficiencies of the photobleaching processes are approximately 108. The relatively low bleaching rate indicates that chlorophyll is more stable than many dyes [17]. For comparison, a set of typical time-lapsed spectra from chloroplasts under multi-photon excitation is shown in Fig. 3. The more extendedly exposed spectra (T > 6 s) are in good agreement with previous two-photon spectra, which were acquired in static conditions [13]. In contrast to the monochromatic spectra from one-photon excitation, the multi-photon spectra exhibit colorful distribution of fluorescence from chloroplasts in the range 450–700 nm. The fluorescence at 495 nm was associated with the 11Bu state. The fluorescence peak centered at 525 nm was identified as the n1Ag state, while the feature at 620 nm was identified as the 21Ag state of the carotenoids. The state symbols are assigned by analogy to true C2h polyenes [13]. It is well known that the photosynthesis process of chloroplasts is a cascaded energy transfer chain reaction [18,19]. It can also be seen in the spectra that the rise of green fluorescence (500 nm) is accompanied by the decay of red fluorescence, which reflects the interplay between carotenoids and chlorophyll [13]. Carotenoids can absorb excess energy to protect chloroplasts from photo-damages under intense illumination [18,19]. Pure chlorophyll does not exhibit colorful spectrum as shown in Fig. 3. The emergence of green fluorescence may reflect this disrupted cascaded chain reaction. A model to account for photo-bleaching under multi-photon excitation is described in Appendix. Data shown in Fig. 3(c) are then fitted to the decaying function described by Eq. (A.3) in appendix using nonlinear least-square algorithm. The corresponding equivalent cross sections as defined by Eq. (A.5) are 2.8 1026, 2.6 1026, 2.3 1026, 1.0 1025 and 2.0 1025 cm2 for fluorescence
wavelengths at 665, 637, 600, 515, and 495, respectively [14]. The lower cross sections of twophoton excitation indicate that two-photon excitation is less destructive in this case. However, substantially higher average NIR intensity has to be used to obtain the same amount of autofluorescence from chloroplasts as in single-photon excitation. The resulted multi-photon photobleaching rate are thus higher than those induced by single-photon excitation. These effects shown in Fig. 3 are verified as multi-photon excitation in nature by changing the mode-locking status of the Ti:sapphire laser. The colorful characteristics in spectra can only be observed when the laser is mode-locked and generating ultrafast pulses. There are many chemicals emitting fluorescence in plants. The major categories of these chemicals include NAD(P)H, carotenoids, flavins, and chlorophyll. These chemicals are abundant in plants that perform photosynthesis. The two-photon spectra of NAD(P)H can be found in the work of Cheng et al. [10]. NAD(PH) plays an important role in mediating electron transport. The twophoton fluorescence spectra of a number of other chemicals are presented in Fig. 4. The spectra of chlorophyll a and chlorophyll b; shown in Fig. 4(a) and (b), exhibit peaks at 653 and 646 nm, respectively. The spectra of b-carotene, Riboflavin, FMN, and FAD are shown in Fig. 4(c)–(f), respectively. b-carotene is one of the carotenoids that assist in light absorption by chloroplasts. Riboflavin, FMN, and FAD are flavins that also play an important role in mediating electron transport. Other than chlorophyll, these chemicals emit two-photon fluorescence ranging from 450 to 630 nm. Fluorescence from NAD(P)H covers broader range than flavins and carotenoids. The spectral range of these chemicals matches quite well with those presented in Fig. 3. Under singlephoton excitation, only red fluorescence generated by chlorophyll can be observed, reflecting the normal light absorbing condition. In contrast, under multi-photon excitation, colorful spectral responses that may result from various chemicals can be observed, indicating a disrupted chain reaction. Fig. 5 shows a sequence of images of the irradiated protoplasts under pulsed laser
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Fig. 3. Time-lapse fluorescence spectra obtained with two-photon excitation: (a) microscopic image of protoplasts being excited with ultrafast laser pulses; (b) time-sequenced plot of spectra; and (c) the 495, 515, 600, 637, and 665 nm fluorescence decaying curves. (c) and (d) the 3D surface-shaded plot of spectra at two different viewing angles.
illumination. It was noted that a significant reduction in red fluorescence occurred after the first few scans. The emergence of green autofluorescence in chloroplasts is closely related to the decay of red-fluorescence. These changes of color in scanned images also reflect the spectral evolution as a result of multi-photon excitation shown
in Fig. 3. Exposure to pulsed laser would also cause rapid and severe damages to cells, as further evidenced by illuminating and observing the protoplasts loaded with Calcein AM dye. In which, failure to retain Calcein AM was also observed after approximately three imaging scans at a rate of 3.3 s per frame [12]. Multiple scan
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Fig. 4. Two-photon excited (Ex ¼ 780 nm) fluorescence spectra of (a) chlorophyll a (b) chlorophyll b (c) b-carotene (d) riboflavin (e) FAD (f) FMN.
results in a rapid decrease in overall fluorescence intensity, lowering the red-to-green fluorescence intensity ratio in chloroplasts, and finally causing visible structural damages such as shriveling or bursting of the cell [12]. In contrast, for a comparable red fluorescence signal level in chloroplasts, single-photon excitation using 488 nm allows significantly more scans before the cell fails to retain Calcein AM.
4. Conclusion In conclusion, we have used time-lapsed microspectroscopy to demonstrate that multi-photon excitation can induce nonlinear photo-bleaching processes in chloroplasts and accelerated photodamage on cells. It appears that multi-photon excitation does not necessarily induce higher equivalent bleaching cross sections than those by single-photon excitation. However, the much
Fig. 5. A sequence of two-photon fluorescence images of protoplasts, (a)–(d), showing changes of color as a result of multi-photon-induced photo-bleaching. (e) A lower magnification scan was followed to obtain this image. The center rectangular area appears heavily photo-bleached and exhibits green fluorescence after being repetitively scanned. Structural damages, such as shriveling and cell bursting, are also apparent after accumulated exposure.
higher peak power and average intensity applied for multi-photon excitation would nevertheless result in faster photo-bleaching within the focused volume, which is revealed in this work as well as the one by Patterson and Piston [4]. Nevertheless, the faster photo-bleaching rate observed does not necessarily imply higher overall rate to the sample since the volume affected is different under singleand multi-photon excitations. It is well known that single-photon excitation can affect a volume far
F.-J. Kao et al. / Journal of Luminescence 98 (2002) 107–114
greater than that by multi-photon excitation. Thus, integrated over volume, the accumulated photo-bleaching may still be higher for singlephoton excitation during scanning imaging. Multiphoton excitation is also found to generate unexpected and accelerated photo-damage by . Konig et al. In their work, a number of indicators were employed to assess cellular damage under illumination from various light sources. The indicators include cell cloning efficiency, cellular autofluorescence patterns and lifetime [5–8]. It was found that cell damage induced, being defined as cell cloning efficiency, was also accompanied by changes in fluorescence. It has long been recognized that the photosynthesis is a cascaded charge-transfer process. Interrupting the energy transfer in the electrontransport process prevents fluorescence emission from the molecule located at the end of the chain. Considering the energy-transfer nature within the cascaded chain, the fluorescence emitted from this disrupted chain is likely blue-shifted. The extent of blue-shift may reflect the degree of disruption experienced by the cascaded chain process. Overall reduction in the fluorescence intensity is the result of removing available fluorescencing molecules. The photo-bleaching and/or photo-damage to photosynthetic molecules are responsible for both intensity and spectral changes. In addition, the excitation pathway resulted by multi-photon excitation can be very different from single-photon excitation due to the different selection rules applied in optical transition. For example, the emergence of green fluorescence from chloroplasts due to multi-photon excitation has overlapped spectral distribution with that of GFP, a frequently used fluorescence probe. Care must be exercised in interpreting the results so obtained. The judicial employment of micro-spectroscopic techniques would nevertheless enable better understanding regarding the nature of such excitation.
Acknowledgements We are grateful for the generous research support from National Science Council (NSC89-
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2112-M-110-016 and NSC-89-2216-E-110-003) and Ministry of Education (89-B-FA08-1-4).
Appendix Photo-bleaching is modeled in the following way: If it is assumed that the change of fluorescence signal, DP; on the incident laser pulses is proportional to the leftover fluorescent material, which is proportional to the fluorescence signal, P; one then has [20,21] DP ¼ PgðF Þ:
ðA:1Þ
In which, gðF Þ is a general functional dependence of the decaying fluorescence on the accumulated incident fluence, F ; that has the dimension of photons/cm2. In the case of cw illumination and single-photon dependence, F ¼ I Dt and gðF Þ is simply sF =hn; where I is the incident laser beam intensity, s is the cross section of the photobleaching process, and hn is the incident photon energy. Eq. (A.1) can then be integrated against time to yield PðtÞ ¼ P0 eðsI=hnÞt :
ðA:2Þ
In the case of pulsed laser illumination with twophoton or multi-photon dependence, F represents number of photons per unit area in a single laser pulse and gðF Þ ¼ bðF =hnÞM ; where M is the exponent of the power law and b represents the multi-photon bleaching cross section with dimension (1/cm2)M. Eq. (A.1) can then be integrated against the number of incident laser pulses, N; to yield PðNÞ ¼ P0 ebðF =hnÞ
M
N
:
ðA:3Þ
For a mode-locked laser, the fluence of a single pulse, F ; can be expressed as It; where I is the average laser intensity and t is the period between pulses. The accumulated number of pulses is thus N ¼ ft; with f ¼ 1=t representing the repetition rate of the laser. Eq. (A.3) can then be rewritten as PðtÞ ¼ P0 ebðIt=hnÞ
M
ft
:
ðA:4Þ
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For comparison, the equivalent cross section of multi-photon excitation can be defined as seq ¼
bðF =hnÞM bðIt=hnÞM ¼ ¼ bðIt=hnÞM1 : ðF =hnÞ ðIt=hnÞ ðA:5Þ
It is apparent that the equivalent cross section for multi-photon excitation defined in this way will depend on the incident intensity or pulse fluence. Thus photo-bleaching under two-photon excitation will increase more rapidly as the incident intensity is increased than in singlephoton excitation.
References [1] W. Denk, J.H. Strickler, W.W. Webb, Science 248 (1990) 73. [2] M. Gu, Three Dimensional Confocal Microscopy, World Scientific, Singapore, 1996. [3] D. Day, M. Gu, Appl. Opt. 37 (1998) 6299. [4] G.H. Patterson, D.W. Piston, Biophys. J. 78 (2000) 2159. . [5] K. Konig, H. Liang, M.W. Berns, B.J. Tromberg, Opt. Lett. 21 (1996) 1090. . [6] K. Konig, H. Liang, M.W. Berns, B.J. Tromberg, Nature 377 (1995) 20.
. [7] K. Konig, P.T.C. So, W.W. Mantulin, E. Gratton, Opt. Lett. 22 (1997) 135. . [8] K. Konig, P.T.C. So, W.W. Mantulin, B.J. Tromberg, E. Gratton, J. Microscopy 183 (1996) 197. [9] B.L. Lin, F.J. Kao, P.C. Cheng, C.K. Sun, et al., SPIE Proc. 4082 (2000) 100. [10] P.C. Cheng, B.L. Lin, F.J. Kao, C.K. Sun, et al., SPIE Proc. 4082 (2000) 87. [11] P.C. Cheng, B.L. Lin, F.J. Kao, C.K. Sun, SPIE Proc. 4082 (2000) 134. [12] P.C. Cheng, B.L. Lin, F.J. Kao, M. Gu, et al., Micron 32 (2001) 661. [13] T.G. Owens, A.P. Shreve, A.C. Albrecht, Research in Photosynthesis, Vol. I, Kluwer Academic Publishers, Dordrecht, 1992. pp. 179–186. [14] F.J. Kao, Y.M. Wang, J.C. Chen, P.C. Cheng, R.W. Chen, B.L. Lin, Opt. Comm. 201 (2002) 85. [15] A. Majewska, G. Yiu, R. Yuste, Pfulgers Arch. Eur. J. Physiol. 441 (2000) 398. [16] H.-C. Huang, C.-C. Chen, J. Heredity 79 (1988) 28. [17] J. Pawley, Handbook of Biological Confocal Microscopy, Plenum Press, New York, 1995 (Chapter 16). [18] R. Buchanan, W. Gruissem, R.L. Jones, Biochemistry & Molecular Biology of Plants, American Society of Plant Physiologists, Rockville, 2000, pp. 573–580. [19] H.A. Frank, A. Cua, V. Chynwat, A. Young, D. Gosztola, M.R. Wasielewski, Photosynthesis Res. 41 (1994) 389. [20] F.J. Kao, D.G. Busch, D. Cohen, D. Gomes da Costa, W. Ho, Phys. Rev. Lett. 71 (1993) 2094. [21] F.J. Kao, D.G. Busch, D. Gomes da Costa, W. Ho, Phys. Rev. Lett. 70 (1993) 4098.
Micro-spectroscopy of chloroplasts in protoplasts from Arabidopsis thaliana under single- and multi-photon excitations Fu-Jen Kaoa,*, Yi-Min Wanga, Jian-Cheng Chena, Ping-Chin Chengb, Rung-Wu Chenc, Bai-Ling Linc a
Center for Neuroscience and Department of Physics, National Sun Yat-sen University, Kaohsiung 80424, Taiwan b Department of Electrical Engineering, State University of New York, Buffalo, NY 14260, USA c Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan
Abstract Ultrafast laser pulses are known to generate impacts on specimens in ways much different from cw illumination in the applications of confocal microscopy. In this study, protoplasts from Arabidopsis thaliana are employed as the samples to compare the effects induced by single- and multi-photon excitations under confocal imaging configuration. Through time-lapsed micro-spectroscopy we are able to characterize the differences in photo-bleaching and the evolution of fluorescence spectra of chloroplasts thus induced. These differences may be attributed to the disruption of chained reaction as a result of multi-photon excitation in photosynthesis. r 2002 Elsevier Science B.V. All rights reserved. PACS: 07.60.R; 87.64.V; 82.50.P Keywords: Micro-spectroscopy; Multi-photon excitation; Photo-bleaching; Chloroplasts
1. Introduction Laser scanning fluorescence confocal microscopy has established itself as one of the most important tools in various disciplines. In which, high-brightness laser beam is tightly focused on a sample through a high-numerical objective to obtain sectional images. However, the high intensity thus formed under an objective often causes fluorescent dyes to fade within minutes of continuous scanning. Therefore, one must limit the scanning time or light intensity if the specimen’s *Corresponding author. Tel.: +886-7-5253720; fax: +886-75253709. E-mail address: [email protected] (F.-J. Kao).
integrity has to be kept. The employment of two-photon excitation in fluorescent microscopy with ultrafast laser illumination was expected to solve this problem. In which, fluorescence induced by two-photon excitation depends on the square of the incident light intensity and thus decreases approximately as the square of the distance from the focus, forming a spatial distribution that can be described by the square of the objective lens’ point spread function assuming Stokes’ shift can be ignored. This highly nonlinear behavior causes only those dye molecules very near the focus of the beam to become excited effectively and limits the excitation volume accordingly. The sample above and below the plane of focus is merely subjected to infrared light that results in neither
0022-2313/02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 2 3 1 3 ( 0 2 ) 0 0 2 5 8 - 2
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photo-bleaching nor photo-damage. It was believed that although the peak amplitude of the IR pulses is large, the mean power of the beam is only a few tens of milliwatts, insufficient to cause localized heating of the specimen. It is also generally accepted that two-photon excitation in confocal microscopy has the additional advantages of deeper penetration and less spherical aberration [1–3]. Nevertheless, the very high peak power of short laser pulses for two-photon excitation may generate unexpected excitation over the samples and induce nonlinear photobleaching or photo-chemical processes [4]. The effects of a pulsed laser on sample integrity or even cell viability have become an interesting and much debated topic in the applications of multi-photon fluorescence microscopy [5–8]. In this study, the protoplasts prepared from a model plant, Arabidopsis thaliana, are used as the specimen to demonstrate the difference due to multi-photon excitations [9–11]. They are chosen because of their importance as the organelle in plant cells that performs photosynthesis, and also
because they contain highly fluorescent pigments such as chlorophyll. We have found that the spectral responses of chloroplasts under cw and pulsed laser illumination exhibit very different characteristics, as reported previously [12]. In addition, the corresponding photo-bleaching cross sections are also different, as revealed by time-lapsed micro-spectroscopy. These differences may be accounted for by the interplay between chlorophyll and carotenoids [13], which is known to absorb excess energy to protect chloroplasts from photo-damages under intense illumination.
2. Experimental Fig. 1 shows the setup for two-photon timelapse micro-spectroscopy [14]. A mode-locked Ti:sapphire laser (Tsunami, Spectra-Physics) pumped by a frequency-doubled all-solid-state laser (Verdi, Coherent) provides ultrafast laser pulses centered at 780 nm with a pulse width of
Fig. 1. Schematics of the two-photon time-lapsed micro-spectroscopy setup. The combination of fiber bundle coupling and highly sensitive CCD-based spectrometer allows rapid acquisition of spectra with high signal-to-noise ratio.
F.-J. Kao et al. / Journal of Luminescence 98 (2002) 107–114
approximately 150 fs and a repetition rate of 82 MHz. The laser beam is directed into a modified fluorescence microscope (BX50, Olympus) via a dichroic mirror. The fluorescence from the samples is coupled to the spectrometer (77250, Oriel) via a special slit-outlet quartz fiber bundle. A 300groove grating is used so that a spectral range from 400 to 720 nm can be observed simultaneously. A highly sensitive back-illuminated liquid nitrogen cooled CCD camera (Orbis II, SpectraSource) is used to acquire the time-lapse spectra. The very high quantum efficiency and extremely low noise of the cooled CCD allow rapid acquisition of fluorescence spectra with high signal-tonoise ratio. A 10X objective (Plan, 0.25NA, Olympus) is used to focus the laser beam into chloroplasts in a cell. Peak intensity reached for Ti:sapphire laser illumination which is estimated to be 3.7 1010 W/cm2 under the objective, which approximates the intensity commonly used in twophoton confocal microscopy. In micro-spectroscopic measurements, two-photon excitation has the added advantages of limited excitation volume and thus has better rejection to background noise in spectral acquisition. For comparison, an aircooled argon ion laser is used to provide cw laser illumination with 1.2 mW (measured under the objective) at 488 nm. The average intensity from argon ion laser illumination is 2.65 104 W/cm2. Fluorescence microscopy is also performed on a modified Olympus Fluoview confocal microscope for both single- and multiple-photon excitations. The details regarding modification of the confocal microscope can be found in the work of Majewska et al. [15]. Plants of A. thaliana were grown from seeds sown in soil in 20 8 4 cm3 (L W H) flats in a growth chamber with day/night temperatures at 25/221C and 16/8 h day/night cycle, illuminated with fluorescent tubes (FL40D-Ex, Mitsubishi) emitting a near-sunlight spectrum to reach 160 mmol m2 s2 at the rosette level. Leaves from four to five weeks old plants were used. Mesophyll protoplasts were isolated from leaves through enzymatic digestion of the cell wall, and suspended in the culture medium [16], and were prepared freshly for each use. For microscopic observation, the protoplast suspension was placed in a cham-
109
bered coverglass (Lab-Tek, Illinois, USA). Aqueous solutions of riboflavin, FMN FAD, and b-carotene and acetone solution of chlorophyll a; chlorophyll b were used in this study.
3. Results and discussion Fig. 2 shows the time-lapse auto-fluorescence spectra of chloroplasts under cw laser excitation at 488 nm. The time-sequenced plot of the spectra is shown in Fig. 2(a). The red auto-fluorescence of chloroplasts centered around 690 nm exhibits exponential decay at cw laser illumination. Good fitting of the decaying curve in Fig. 2(b) to a
Fig. 2. Time-lapse fluorescence spectra from chloroplasts obtained with micro-spectroscopy excited with 488 nm laser beam from an argon ion laser: (a) the time-sequenced spectra; and (b) the 690 nm fluorescence decaying curve. A good fit to a double exponential decaying function indicates that there are at least two photo-bleaching processes.
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double exponential decaying function indicates that at least two different photo-bleaching processes can be identified. We found that the singlephoton bleaching cross sections for these two processes are 8.1 1024 and 5.4 1025 cm2, respectively. If the absorption cross section for chloroplast excitation is assumed to be 1016 cm2, in the order of a dye molecule, then the corresponding quantum efficiencies of the photobleaching processes are approximately 108. The relatively low bleaching rate indicates that chlorophyll is more stable than many dyes [17]. For comparison, a set of typical time-lapsed spectra from chloroplasts under multi-photon excitation is shown in Fig. 3. The more extendedly exposed spectra (T > 6 s) are in good agreement with previous two-photon spectra, which were acquired in static conditions [13]. In contrast to the monochromatic spectra from one-photon excitation, the multi-photon spectra exhibit colorful distribution of fluorescence from chloroplasts in the range 450–700 nm. The fluorescence at 495 nm was associated with the 11Bu state. The fluorescence peak centered at 525 nm was identified as the n1Ag state, while the feature at 620 nm was identified as the 21Ag state of the carotenoids. The state symbols are assigned by analogy to true C2h polyenes [13]. It is well known that the photosynthesis process of chloroplasts is a cascaded energy transfer chain reaction [18,19]. It can also be seen in the spectra that the rise of green fluorescence (500 nm) is accompanied by the decay of red fluorescence, which reflects the interplay between carotenoids and chlorophyll [13]. Carotenoids can absorb excess energy to protect chloroplasts from photo-damages under intense illumination [18,19]. Pure chlorophyll does not exhibit colorful spectrum as shown in Fig. 3. The emergence of green fluorescence may reflect this disrupted cascaded chain reaction. A model to account for photo-bleaching under multi-photon excitation is described in Appendix. Data shown in Fig. 3(c) are then fitted to the decaying function described by Eq. (A.3) in appendix using nonlinear least-square algorithm. The corresponding equivalent cross sections as defined by Eq. (A.5) are 2.8 1026, 2.6 1026, 2.3 1026, 1.0 1025 and 2.0 1025 cm2 for fluorescence
wavelengths at 665, 637, 600, 515, and 495, respectively [14]. The lower cross sections of twophoton excitation indicate that two-photon excitation is less destructive in this case. However, substantially higher average NIR intensity has to be used to obtain the same amount of autofluorescence from chloroplasts as in single-photon excitation. The resulted multi-photon photobleaching rate are thus higher than those induced by single-photon excitation. These effects shown in Fig. 3 are verified as multi-photon excitation in nature by changing the mode-locking status of the Ti:sapphire laser. The colorful characteristics in spectra can only be observed when the laser is mode-locked and generating ultrafast pulses. There are many chemicals emitting fluorescence in plants. The major categories of these chemicals include NAD(P)H, carotenoids, flavins, and chlorophyll. These chemicals are abundant in plants that perform photosynthesis. The two-photon spectra of NAD(P)H can be found in the work of Cheng et al. [10]. NAD(PH) plays an important role in mediating electron transport. The twophoton fluorescence spectra of a number of other chemicals are presented in Fig. 4. The spectra of chlorophyll a and chlorophyll b; shown in Fig. 4(a) and (b), exhibit peaks at 653 and 646 nm, respectively. The spectra of b-carotene, Riboflavin, FMN, and FAD are shown in Fig. 4(c)–(f), respectively. b-carotene is one of the carotenoids that assist in light absorption by chloroplasts. Riboflavin, FMN, and FAD are flavins that also play an important role in mediating electron transport. Other than chlorophyll, these chemicals emit two-photon fluorescence ranging from 450 to 630 nm. Fluorescence from NAD(P)H covers broader range than flavins and carotenoids. The spectral range of these chemicals matches quite well with those presented in Fig. 3. Under singlephoton excitation, only red fluorescence generated by chlorophyll can be observed, reflecting the normal light absorbing condition. In contrast, under multi-photon excitation, colorful spectral responses that may result from various chemicals can be observed, indicating a disrupted chain reaction. Fig. 5 shows a sequence of images of the irradiated protoplasts under pulsed laser
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Fig. 3. Time-lapse fluorescence spectra obtained with two-photon excitation: (a) microscopic image of protoplasts being excited with ultrafast laser pulses; (b) time-sequenced plot of spectra; and (c) the 495, 515, 600, 637, and 665 nm fluorescence decaying curves. (c) and (d) the 3D surface-shaded plot of spectra at two different viewing angles.
illumination. It was noted that a significant reduction in red fluorescence occurred after the first few scans. The emergence of green autofluorescence in chloroplasts is closely related to the decay of red-fluorescence. These changes of color in scanned images also reflect the spectral evolution as a result of multi-photon excitation shown
in Fig. 3. Exposure to pulsed laser would also cause rapid and severe damages to cells, as further evidenced by illuminating and observing the protoplasts loaded with Calcein AM dye. In which, failure to retain Calcein AM was also observed after approximately three imaging scans at a rate of 3.3 s per frame [12]. Multiple scan
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Fig. 4. Two-photon excited (Ex ¼ 780 nm) fluorescence spectra of (a) chlorophyll a (b) chlorophyll b (c) b-carotene (d) riboflavin (e) FAD (f) FMN.
results in a rapid decrease in overall fluorescence intensity, lowering the red-to-green fluorescence intensity ratio in chloroplasts, and finally causing visible structural damages such as shriveling or bursting of the cell [12]. In contrast, for a comparable red fluorescence signal level in chloroplasts, single-photon excitation using 488 nm allows significantly more scans before the cell fails to retain Calcein AM.
4. Conclusion In conclusion, we have used time-lapsed microspectroscopy to demonstrate that multi-photon excitation can induce nonlinear photo-bleaching processes in chloroplasts and accelerated photodamage on cells. It appears that multi-photon excitation does not necessarily induce higher equivalent bleaching cross sections than those by single-photon excitation. However, the much
Fig. 5. A sequence of two-photon fluorescence images of protoplasts, (a)–(d), showing changes of color as a result of multi-photon-induced photo-bleaching. (e) A lower magnification scan was followed to obtain this image. The center rectangular area appears heavily photo-bleached and exhibits green fluorescence after being repetitively scanned. Structural damages, such as shriveling and cell bursting, are also apparent after accumulated exposure.
higher peak power and average intensity applied for multi-photon excitation would nevertheless result in faster photo-bleaching within the focused volume, which is revealed in this work as well as the one by Patterson and Piston [4]. Nevertheless, the faster photo-bleaching rate observed does not necessarily imply higher overall rate to the sample since the volume affected is different under singleand multi-photon excitations. It is well known that single-photon excitation can affect a volume far
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greater than that by multi-photon excitation. Thus, integrated over volume, the accumulated photo-bleaching may still be higher for singlephoton excitation during scanning imaging. Multiphoton excitation is also found to generate unexpected and accelerated photo-damage by . Konig et al. In their work, a number of indicators were employed to assess cellular damage under illumination from various light sources. The indicators include cell cloning efficiency, cellular autofluorescence patterns and lifetime [5–8]. It was found that cell damage induced, being defined as cell cloning efficiency, was also accompanied by changes in fluorescence. It has long been recognized that the photosynthesis is a cascaded charge-transfer process. Interrupting the energy transfer in the electrontransport process prevents fluorescence emission from the molecule located at the end of the chain. Considering the energy-transfer nature within the cascaded chain, the fluorescence emitted from this disrupted chain is likely blue-shifted. The extent of blue-shift may reflect the degree of disruption experienced by the cascaded chain process. Overall reduction in the fluorescence intensity is the result of removing available fluorescencing molecules. The photo-bleaching and/or photo-damage to photosynthetic molecules are responsible for both intensity and spectral changes. In addition, the excitation pathway resulted by multi-photon excitation can be very different from single-photon excitation due to the different selection rules applied in optical transition. For example, the emergence of green fluorescence from chloroplasts due to multi-photon excitation has overlapped spectral distribution with that of GFP, a frequently used fluorescence probe. Care must be exercised in interpreting the results so obtained. The judicial employment of micro-spectroscopic techniques would nevertheless enable better understanding regarding the nature of such excitation.
Acknowledgements We are grateful for the generous research support from National Science Council (NSC89-
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2112-M-110-016 and NSC-89-2216-E-110-003) and Ministry of Education (89-B-FA08-1-4).
Appendix Photo-bleaching is modeled in the following way: If it is assumed that the change of fluorescence signal, DP; on the incident laser pulses is proportional to the leftover fluorescent material, which is proportional to the fluorescence signal, P; one then has [20,21] DP ¼ PgðF Þ:
ðA:1Þ
In which, gðF Þ is a general functional dependence of the decaying fluorescence on the accumulated incident fluence, F ; that has the dimension of photons/cm2. In the case of cw illumination and single-photon dependence, F ¼ I Dt and gðF Þ is simply sF =hn; where I is the incident laser beam intensity, s is the cross section of the photobleaching process, and hn is the incident photon energy. Eq. (A.1) can then be integrated against time to yield PðtÞ ¼ P0 eðsI=hnÞt :
ðA:2Þ
In the case of pulsed laser illumination with twophoton or multi-photon dependence, F represents number of photons per unit area in a single laser pulse and gðF Þ ¼ bðF =hnÞM ; where M is the exponent of the power law and b represents the multi-photon bleaching cross section with dimension (1/cm2)M. Eq. (A.1) can then be integrated against the number of incident laser pulses, N; to yield PðNÞ ¼ P0 ebðF =hnÞ
M
N
:
ðA:3Þ
For a mode-locked laser, the fluence of a single pulse, F ; can be expressed as It; where I is the average laser intensity and t is the period between pulses. The accumulated number of pulses is thus N ¼ ft; with f ¼ 1=t representing the repetition rate of the laser. Eq. (A.3) can then be rewritten as PðtÞ ¼ P0 ebðIt=hnÞ
M
ft
:
ðA:4Þ
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For comparison, the equivalent cross section of multi-photon excitation can be defined as seq ¼
bðF =hnÞM bðIt=hnÞM ¼ ¼ bðIt=hnÞM1 : ðF =hnÞ ðIt=hnÞ ðA:5Þ
It is apparent that the equivalent cross section for multi-photon excitation defined in this way will depend on the incident intensity or pulse fluence. Thus photo-bleaching under two-photon excitation will increase more rapidly as the incident intensity is increased than in singlephoton excitation.
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