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Photobleaching of the resonance Raman lines of cytochromes in living yeast cells
Journal of Photochemistry and Photobiology B: Biology 141 (2014) 269–274
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Photobleaching of the resonance Raman lines of cytochromes in living yeast cells Konstantin A. Okotrub, Nikolay V. Surovtsev ⇑ Institute of Automation and Electrometry, Russian Academy of Sciences, Novosibirsk 630090, Russia
a r t i c l e
i n f o
Article history: Received 4 July 2014 Received in revised form 17 September 2014 Accepted 11 October 2014 Available online 27 October 2014
a b s t r a c t The photobleaching of the resonance cytochrome Raman lines in living Saccharomyces cerevisiae cells was studied. The photobleaching rate versus the irradiation power was described by square function plus a constant in contrast to the linear dependence of the photoinjury rate. This difference distinguishes the cytochrome photooxidation from other processes of the cell photodamage. The square dependence is associated with the reaction involving two photogenerated intermediates while the constant with the dark redox balance rates. This work demonstrates a potential of Raman spectroscopy to characterize the native cytochrome reaction rates and to study the cell photodamage precursors. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Under intensive light irradiation of a living cell the photodamage effects can occur. Photodamage can result into the change of cell properties, metabolism, or even the cell death. In some cases the cell photodamage can be the goal of the cell illumination, e.g. in the case of the photodynamic therapy. In this case researchers are interested to enhance the photodamage effect by the addition of the photosensitizes [1,2]. On the other hand, the irradiation light is used in some experimental techniques of cell biology serving as probing or auxiliary irradiation (e.g. Raman spectroscopy or optical tweezers [3,4]). In this case the photodamage is an undesirable effect which put additional limitations on the irradiation power, the wavelength, and the exposure time. Independently whether the photodamage is desirable or undesirable effect the knowledge about microscopic photodamage mechanisms is of high importance. A lot of works are devoted to study the photodamage problem [3–10]. It is believed that in many cases the photodamaging factors are related to generation of the reactive oxygen species (ROS) [4,6,9]. Also the photodamage can occur via the direct light absorption by DNA and/or proteins [11,12]. Different techniques and methods are used to characterize the cell photodamage. The most commonly used approaches include the visual control of the cell state under illumination [3–8,13,14], photoluminescence methods [10,13–16], EPR techniques [9,17,18] and others. It was shown that for the low light intensity the photoinduced injury rate obeys the linear dependence on the ⇑ Corresponding author at: Pr. Ak. Koptyuga 1, 630090 Novosibirsk, Russia. Tel.: +7 383 3307978. E-mail address: [email protected] (N.V. Surovtsev). http://dx.doi.org/10.1016/j.jphotobiol.2014.10.008 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
irradiation power [4,6,8,13,16]. In this case the photoinduced injuries and the cell lifetime are defined by product of the irradiation power and the exposition time, or in other words by the exposition energy. The particular photodamage parameters depend on the irradiation wavelength [4,6,7]. The experimental techniques based on the visual characterization of cells or on integral characteristics do not provide the information about the microscopic mechanisms of the cell photodamage. Raman spectroscopy has capability to provide information about the chemical content [19]. The potential of Raman spectroscopy for characterization the cell activity was demonstrated recently by series of studies [20–23]. Another outstanding application of the Raman spectroscopy for cells is the studies of the Raman spectra of cytochromes [24–33]. The enhanced Raman intensity of reduced (Fe2+) state of b and c cytochromes under the green light excitation relates to the resonance scattering near the absorbance edge [34]. The Raman intensity of the oxidized cytochrome state (Fe3+) is lower by few orders for the green light excitation. Cytochromes take part in the mitochondrion electron transport chain (ETC) as electron carriers. In living cells there is the stationary balance between the reduced and oxidized states of cytochromes. Change of the redox balance means abnormalities in the ETC work. Thus, the photodamage effect on ETC work can be studied by the resonance Raman scattering of cytochromes. The present work is devoted to study the effect of photoinduced injuries in living yeast cells under the 532 nm irradiation by resonance Raman scattering technique. The photobleaching parameters of the cytochrome Raman spectra versus the laser intensity is studied. For the comparison the photoinduced injuries are characterized by the photoluminescence intensity. It will be shown that the laser power dependence of the photobleaching is different
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from the photoluminescence one. A simple model description of the photobleaching behavior is proposed. 2. Experimental 2.1. Sample preparation Commercially available Saccharomyces cerevisiae instant yeast cells pellets (20 mg) were added to 5 ml of isotonic saline solution (0.9 wt.% NaCl). For each Raman experiment, the cell suspension was placed on fused silica slide, covered with a piece of mica (5– 15 lm in thickness) and sealed with paraffin to prevent the sample desiccation. 2.2. Experimental setup A home-made confocal Raman setup, based on a modified microscope (Orthoplan, Leitz) and a monochromator (SP2500i, Princeton Instruments) equipped with a CCD detector (Spec10:256E/LN, Princeton Instruments) was used. The spectrometer wavelength calibration was done with a neon-discharge lamp. Raman scattering was excited by a solid-state laser (Millennia II, Spectra Physics) at 532.1 nm, the beam power was measured with a photodiode power meter (Lasercheck, Coherent). A 100 air objective with NA = 0.75 and working distance 4.6 mm (PL FLUOTAR L, Leica Microsystems) was used to focus the laser beam to 0.9 lm spot. For this spot the 1 mW laser irradiation corresponds to the intensity of about 0.13 MW/cm2 for the average intensity and about 0.29 MW/cm2 for the maximum. In Raman scattering experiment a single cell was exposed by a constant power of the laser beam, which simultaneously served as the excitation of Raman scattering. The sequence of Raman spectra (40 scans) was measured with the integration time optimized to the particular photobleaching rate, the integration time being 1 s for 20 mW of laser irradiation and 30 s for 1 mW. Evolution of the Raman spectrum of the cell under irradiation was studied. 3. Results Fig. 1 represents the Raman spectrum of the yeast cells and its evolution under the laser irradiation. In this spectrum the contributions of CH deformation (1440–1460 cm1), amide (1656, 1555 cm1), phenylalanine (1004 cm1) and nucleic acids (785 cm1) modes can be identified. The lines at 749, 1129, and 1585 cm1 seen in Fig. 1 are known as the most intensive lines of the resonance Raman spectrum of b and c cytochromes
Fig. 1. Time-resolved Raman spectra of living yeast cells for 10 mW laser power. The spectra are vertically shifted for convenience. The shadow stripes mark the Raman peaks of cytochrome. The asterisk denotes the contribution from the mica slice.
[35,36]. The peak at 703 cm1 in Fig. 1 is contributed by the mica slice covering the sample. As it is seen from Fig. 1 the contribution of the cytochrome lines decreases with the laser exposition, while the intensities of the rest Raman lines do not change. This decrease is associated with the decrease of the reduced cytochrome concentration. We followed the intensity of the line at 749 cm1 to estimate the amount of the reduced cytochrome in the yeast cell. This line corresponding to the heme perrole rings breathing mode [35] is intensive and placed apart the other cellular lines. For the quantitative analysis the intensity of the line at 749 cm1 was found taking into account the mica spectrum contribution and the background interpolated by a straight line. The relative contribution of cytochrome into the single cell Raman spectrum was characterized by the intensity ratio of the line at 749 cm1, I749, and of the deformational CH mode (dCH), ICH. This ratio can vary among the different yeast cells or even different cell parts, but it is independent of the absolute Raman intensity and, therefore, is convenient for the characterization of the single cell during the irradiation (for example, it eliminates the effect of change of the light absorption by the cell). The Raman spectra of yeast cells are placed on some photoluminescence background. Typically, the photoluminescence background is low at the beginning of the irradiation. However, after some laser exposition the photoluminescence background starts to increase. This increase is caused by some fluorophores resulted from photoinduced processes in the cell. Prolonged laser exposure leads in the cell degradation, resulting in the formation of visual defects such as holes or black points. The spectra from the photoinduced defects are characterized by the high level of the photoluminescence background. It is natural to relate the fluorophore increase under irradiation with increase of the photodegradation events. Thus we propose to follow the cell photoderagation by the study of the temporal dependence of the photoluminescence intensity. The photoluminescence intensity was characterized by the integral signal in the range 550–634 nm. The temporal evolution of the photoluminescence intensity of a yeast cell under the laser irradiation is represented in Fig. 2a. It is seen that the photoluminescence intensity is characterized by an abrupt increase at sPL associated with the photodegradation. Since sPL characterizes the time needed to reach the critical photoinjury level, inverse sPL reflects the rate of the photodamage accumulation.
Fig. 2. Representative time dependence of the photoluminescence intensity (a) and the I749/ICH ratio (b) under 4 mW laser irradiation. The onset of the drastic photoluminescence increase is denoted by the arrow in (a). The exponential decay fit is shown by the solid line in (b).
K.A. Okotrub, N.V. Surovtsev / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 269–274
The example of the 749 cm1 line intensity normalized to the dCH band versus the exposition time is shown in Fig. 2b. The photobleaching of Raman lines of the cytochromes is seen from this figure. The exponential decay fit was used to describe the photobleaching of the cytochrome line intensity:
I749 ¼ y0 þ A et=s ; IdCH
ð1Þ
where A is the photobleaching amplitude, s is the photobleaching time, and the constant y0 reflects the stationary amount of the reduced cytochromes under irradiation. The fits of the experimental data were done with the requirement of the non-negativity for y0. The representative fit is shown in Fig. 2b. It is seen that the exponential decay describes the experimental data quite well. In our study the time-resolved Raman spectra of a number of the irradiated yeast cells were measured for every laser power. The temporal evolution of I749/ICH ratio was extracted from the Raman spectra and fitted by Eq. (1). It was found that the fit parameters s, A, and y0 vary in the wide range from cell to cell. Fig. 3 shows the histograms of the fit parameters for the 2 mW irradiation power (statistics was done for the 129 yeast cells). The histograms of the parameter can be described by log-normal (s, A) and normal (y0) distributions (Fig. 3). These descriptions work well in irradiation power studied. The A and y0 variations are supposed to result from the cells heterogeneity, since the cytochromes placed mainly in mitochondria, while the dCH band intensity comes mainly from phospholipids. The s distribution is related to the photosensitivity variation. The average values of the parameters were found from the statistics over measurements with different yeast cells. Two procedures were used for this purpose either the arithmetical mean
Fig. 3. Distributions of the fit parameters s (a), A (b), y0 (c) for the 2 mW irradiation power. The lines in (a) and (b) are the log-normal distributions. The line in (c) is the normal distribution.
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over set of parameters for particular cell or averaging of Raman spectra corresponding to the same expose time, calculation I749/ ICH ratio and subsequent fitting by Eq. (1). The last procedure imitates the macroscopic experiment with the simultaneous integration of the Raman signal from all cells. Fig. 4a shows the power dependence of the cytochrome photobleaching rate in logarithmic scales. It is seen that both averaging procedures lead to significantly the same power dependence of s1. The experimental s1 data are well described by the quadratic function of the laser power P (Fig. 4a):
s1 ðPÞ ¼ a þ b P2 :
ð2Þ
The last term of Eq. (2) implies that the cytochromes photobleaching is likely to involve processes requiring two photons. To compare the power dependence of the cytochrome photobleaching rate with the inverse time of photoinduced injury, the data for s1 PL ðPÞ are also shown in Fig. 4a. It is seen that the photodamage rate has the different power dependence, which can be described by direct proportionality to the irradiation power. The question of the locality of the cytochrome photobleaching was considered. The time-resolved Raman spectra from two different parts of the same cell were studied. The delay between two measurements was less one minute. The Raman intensity of the cytochrome line at 749 cm1 is shown in Fig. 5 for two illuminated parts of the cell. As it is seen from Fig. 5 the previous irradiation of the first point has no influence on the temporal evolution of I749 of the second irradiated point of the cell. The experiment with five spatially different points of the cell showed similar results. Thus, the cytochrome photobleaching is the local process. Also, we studied whether the cytochrome photobleaching can be recovered in the dark. In this experiment two exposures of the same irradiation point by 2 mW laser power were done with the dark delay period between them. The one minute of the first exposure were followed by keeping the exposed cell in dark for 15 min, then the Raman experiment was continued. Such measure-
Fig. 4. (a) Power dependence of the photobleaching cytochrome rate and photodamage rate. The red circles are s1 from the average of the dataset of different yeast cells, the blue squares are s1 from the averaged Raman spectra. The solid line is the parabolic fit for the red circles. The green triangles are the photodamage rate, 1 s1 PL ; at 0.5 mW only the upper limitation of sPL was determined. The dashed line is the linear fit of s1 PL . (b) Power dependence of y0 parameter (red circles). The line is the fit by Eq. (8). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Exposition time dependence of I749 for two irradiated areas of the same cell. Inset shows the first (left) and second (right) irradiated areas in the photo of the yeast cell.
ments were done with 80 yeast cells. The representative time dependence of the I749/ICH ratio is shown in Fig. 6. It is seen that the first exposure reduces the cytochrome line intensity, which is recovered during the dark delay. The recovered I749/ICH ratio is slightly lower than the initial one. It is worth to note that the more intense or prolonged first exposure leading to the significant change of the I749/ICH ratio (the change of the ratio by about or more than 30%) results in the unrecoverable photodamage. The control experiment (Fig. 6) was done to eliminate the effect of the focusing mismatch during the dark delay. In this case the delay was less than 1 min, but in this time the laser beam was defocused in three dimensions and focused again at the same point of the cell. As it is seen from Fig. 6 the refocusing has no effect on the I749/ICH ratio. 4. Discussion Our study of the irradiation effect on the cytochromes redox state in living yeast cells was carried out with the intensive irradiation (0.07–3.15 MW/cm2). The photodamage rate, controlled by the photoluminescence intensity, demonstrates the linear power dependence (Fig. 4a) in the agreement with the previous studies [4,6,9,13,16] However the power dependence of the cytochrome photobleaching rate differs significantly (Fig. 4a). This means that the bioprocesses involved in the ETC photosensitivity and the cell photodamage are different. At the high irradiation intensities the
photobleaching rate shows the quadratic power dependence, while it becomes almost constant in the low intensity range. The quadratic dependence implies two-photon nature of the cytochrome photobleaching process. Two kinds of the cytochrome photobleaching processes requiring two photons can be considered. The first one relates to the direct two-photon absorption or to the consecutive absorption through the intermediate state, and the following transformation of the reduced cytochrome into another state. The second one is the cytochrome oxidation via interaction with two molecules of intermediates (for example, the reactive oxygen species, ROS) generated by the intensive green light irradiation. In particular, the cytochromes themselves can be the source of photogenerated ROS, since porphyrines are known to be photosensitizers [1,2]. The photobleaching localization effect (Fig. 5) can be interpreted as the space localization of the intermediates and of cytochromes binding to mitochondria internal membrane. In other words the intermediates have to be generated in the near proximity to ETC. It is worth to note that the square power dependence of the photobleaching rate turns into a constant at low irradiation intensities (Fig. 4a). To interpret this behavior we propose a simple model taking into account the cytochrome participation in ETC functioning. At dark the reduced and oxidized cytochrome concentrations is governed by the redox reactions kþ ;k
C oxi þ e ! C red ;
ð3Þ
where k+ and k are the direct and back reaction rate constants, e is the concentration of electrons involved in ETC working, Cred and Coxi are the reduced and oxidized cytochrome concentrations, respectively. For the simplicity the electron concentration is assumed to be independent from the irradiation power. The irradiation leads to an additional cytochrome oxidation by the photogenerated intermediates, those stationary concentration is proportional to the irradiation intensity. For the sake of definiteness, we will associate the intermediates with ROS. According to photobleaching power dependence (Fig. 4a), it is assumed that photoinduced cytochrome oxidation involves two photogenerated ROS kROS
2 C ROS þ C red ! C oxi ;
ð4Þ
where kROS is the rate constant of the oxidation reaction induced by ROS, CROS is the photogenerated ROS concentration. From the reactions (3) and (4), the kinetic equations for the reduced and oxidized cytochrome concentrations can be written
dC red dC oxi ROS ¼ ¼ kþ C oxi k C red k aI2 C red : dt dt
ð5Þ
In this case we assume that the photochange in CROS is much faster than the cytochrome oxidation, and the stationary limit, pffiffiffi C ROS ¼ aI, where I is the irradiation intensity, is used in Eq. (5). The total amount of cytochrome is invariable in Eq. (5) (the system is closed). The solution of Eq. (5) is
C red ¼
kþ S kþ þ k þ kROS aI2
2
þ AðIÞ eðkþ þk þkROS aI Þt ;
ð6Þ
where the constant S is the sum of Cred and Coxi concentrations, and A(I) is determined by the initial conditions. For the case of the initial condition of the dark equilibrium between the reduced and oxidized cytochromes, Fig. 6. Representative time dependence of the I749/ICH ratio under the 2 mW laser irradiation interrupted by the 15 min dark delay. The red circles correspond to the exposition before the delay and the blue circles to the exposition after the delay. The squares correspond to the control experiment (see text). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
AðIÞ ¼
kþ kROS aI2 S ðkþ þ k Þðkþ þ k þ kROS aI2 Þ
:
ð7Þ
The expression (6) provides the power dependences of s1 and y0 (first term):
K.A. Okotrub, N.V. Surovtsev / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 269–274
s1 ðIÞ ¼ kþ þ k þ kROS aI2 ¼
kþ S : y0
ð8Þ
The expected power dependence of s1 is in agreement with the experiment (Eq. (2) and Fig. 4). The model interprets the lowintensity limit of s1 as the combination of the dark reaction rate constants. The power dependence of y0 is related with s and shown in Fig. 4. It is seen that the expected y0(I) has the qualitative agreement with the experiment. The above model also supports the process of cytochromes dark recovery observed in the experiment. After the irradiation exposition the dark redox reactions (Eq. (5)) recover the stationary cytochrome balance with the rate k+ + k. The lack of the dark recovery for the high exposition is related to the accumulation of the photoinduced injuries and/or additional cytochrome decay channels, which are not taken into account by the model. Moreover the model (Eq. (7)) predicts the strong decrease of A(I) at low irradiation intensity, which was not observed. The deficiency of the model in the description of y0(I) and A(I) behavior is related to its simplicity; the real system seems to be open and should include more kinetic terms and equations. The present results and the model interpretation extend the capabilities of resonance Raman spectroscopy to provide the important information about the ETC work. The photobleaching rate at relatively low irradiation power (below 0.4 MW/cm2) provides the information about the cytochromes redox reaction rates. The parameter y0 characterizes the irradiation effect on the redox reaction balance and the irradiation invasiveness to ETC. The photobleaching rates mirror the cellular biochemical reactions, providing information about the cellular activity in different conditions. For example, the recent Raman studies [37,38] reveal the phase transitions during the cell freezing, and the cytochrome photobleaching rates could characterize the changes of cellular activity during these transitions and the anabiosis state. The interrelations of the cytochrome photobleaching and the large-scale photodegradation are not clear. But we can see that they obey the different power dependences and have the different time scales. The role of the cytochrome photobleaching in the cellular photoinjuries needs further investigations. 5. Conclusion The effect of intensive 532 nm irradiation on b and c cytochromes redox balance in living yeast cells was studied by resonance Raman spectroscopy. This irradiation leads to the photobleaching of the reduced cytochrome Raman lines. The photobleaching is local and the reduced cytochrome concentration can be recovered at dark in case of low exposition. The power dependence of the cytochromes oxidation rate was found to be nonlinear, while the estimation of the cell photoinjury rates based on the photoluminescence growth indicates the linear power dependence. This difference means that the cytochrome oxidation mechanism differs essentially from other processes related to the cell photodamage. The power dependence of the cytochromes oxidation rate was described by the square dependence plus a constant. A simple model was proposed, in the frame of which the square dependence is associated with two intermediates generated by the irradiation and the constant with the dark redox balance rates. Thus, the resonance Raman spectroscopy can characterize not only the cytochrome redox state, but also the native cytochrome reaction rates of ETC. Acknowledgment This work was supported by RFBR (Grants No. 14-04-31451).
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Photobleaching of the resonance Raman lines of cytochromes in living yeast cells Konstantin A. Okotrub, Nikolay V. Surovtsev ⇑ Institute of Automation and Electrometry, Russian Academy of Sciences, Novosibirsk 630090, Russia
a r t i c l e
i n f o
Article history: Received 4 July 2014 Received in revised form 17 September 2014 Accepted 11 October 2014 Available online 27 October 2014
a b s t r a c t The photobleaching of the resonance cytochrome Raman lines in living Saccharomyces cerevisiae cells was studied. The photobleaching rate versus the irradiation power was described by square function plus a constant in contrast to the linear dependence of the photoinjury rate. This difference distinguishes the cytochrome photooxidation from other processes of the cell photodamage. The square dependence is associated with the reaction involving two photogenerated intermediates while the constant with the dark redox balance rates. This work demonstrates a potential of Raman spectroscopy to characterize the native cytochrome reaction rates and to study the cell photodamage precursors. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Under intensive light irradiation of a living cell the photodamage effects can occur. Photodamage can result into the change of cell properties, metabolism, or even the cell death. In some cases the cell photodamage can be the goal of the cell illumination, e.g. in the case of the photodynamic therapy. In this case researchers are interested to enhance the photodamage effect by the addition of the photosensitizes [1,2]. On the other hand, the irradiation light is used in some experimental techniques of cell biology serving as probing or auxiliary irradiation (e.g. Raman spectroscopy or optical tweezers [3,4]). In this case the photodamage is an undesirable effect which put additional limitations on the irradiation power, the wavelength, and the exposure time. Independently whether the photodamage is desirable or undesirable effect the knowledge about microscopic photodamage mechanisms is of high importance. A lot of works are devoted to study the photodamage problem [3–10]. It is believed that in many cases the photodamaging factors are related to generation of the reactive oxygen species (ROS) [4,6,9]. Also the photodamage can occur via the direct light absorption by DNA and/or proteins [11,12]. Different techniques and methods are used to characterize the cell photodamage. The most commonly used approaches include the visual control of the cell state under illumination [3–8,13,14], photoluminescence methods [10,13–16], EPR techniques [9,17,18] and others. It was shown that for the low light intensity the photoinduced injury rate obeys the linear dependence on the ⇑ Corresponding author at: Pr. Ak. Koptyuga 1, 630090 Novosibirsk, Russia. Tel.: +7 383 3307978. E-mail address: [email protected] (N.V. Surovtsev). http://dx.doi.org/10.1016/j.jphotobiol.2014.10.008 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
irradiation power [4,6,8,13,16]. In this case the photoinduced injuries and the cell lifetime are defined by product of the irradiation power and the exposition time, or in other words by the exposition energy. The particular photodamage parameters depend on the irradiation wavelength [4,6,7]. The experimental techniques based on the visual characterization of cells or on integral characteristics do not provide the information about the microscopic mechanisms of the cell photodamage. Raman spectroscopy has capability to provide information about the chemical content [19]. The potential of Raman spectroscopy for characterization the cell activity was demonstrated recently by series of studies [20–23]. Another outstanding application of the Raman spectroscopy for cells is the studies of the Raman spectra of cytochromes [24–33]. The enhanced Raman intensity of reduced (Fe2+) state of b and c cytochromes under the green light excitation relates to the resonance scattering near the absorbance edge [34]. The Raman intensity of the oxidized cytochrome state (Fe3+) is lower by few orders for the green light excitation. Cytochromes take part in the mitochondrion electron transport chain (ETC) as electron carriers. In living cells there is the stationary balance between the reduced and oxidized states of cytochromes. Change of the redox balance means abnormalities in the ETC work. Thus, the photodamage effect on ETC work can be studied by the resonance Raman scattering of cytochromes. The present work is devoted to study the effect of photoinduced injuries in living yeast cells under the 532 nm irradiation by resonance Raman scattering technique. The photobleaching parameters of the cytochrome Raman spectra versus the laser intensity is studied. For the comparison the photoinduced injuries are characterized by the photoluminescence intensity. It will be shown that the laser power dependence of the photobleaching is different
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from the photoluminescence one. A simple model description of the photobleaching behavior is proposed. 2. Experimental 2.1. Sample preparation Commercially available Saccharomyces cerevisiae instant yeast cells pellets (20 mg) were added to 5 ml of isotonic saline solution (0.9 wt.% NaCl). For each Raman experiment, the cell suspension was placed on fused silica slide, covered with a piece of mica (5– 15 lm in thickness) and sealed with paraffin to prevent the sample desiccation. 2.2. Experimental setup A home-made confocal Raman setup, based on a modified microscope (Orthoplan, Leitz) and a monochromator (SP2500i, Princeton Instruments) equipped with a CCD detector (Spec10:256E/LN, Princeton Instruments) was used. The spectrometer wavelength calibration was done with a neon-discharge lamp. Raman scattering was excited by a solid-state laser (Millennia II, Spectra Physics) at 532.1 nm, the beam power was measured with a photodiode power meter (Lasercheck, Coherent). A 100 air objective with NA = 0.75 and working distance 4.6 mm (PL FLUOTAR L, Leica Microsystems) was used to focus the laser beam to 0.9 lm spot. For this spot the 1 mW laser irradiation corresponds to the intensity of about 0.13 MW/cm2 for the average intensity and about 0.29 MW/cm2 for the maximum. In Raman scattering experiment a single cell was exposed by a constant power of the laser beam, which simultaneously served as the excitation of Raman scattering. The sequence of Raman spectra (40 scans) was measured with the integration time optimized to the particular photobleaching rate, the integration time being 1 s for 20 mW of laser irradiation and 30 s for 1 mW. Evolution of the Raman spectrum of the cell under irradiation was studied. 3. Results Fig. 1 represents the Raman spectrum of the yeast cells and its evolution under the laser irradiation. In this spectrum the contributions of CH deformation (1440–1460 cm1), amide (1656, 1555 cm1), phenylalanine (1004 cm1) and nucleic acids (785 cm1) modes can be identified. The lines at 749, 1129, and 1585 cm1 seen in Fig. 1 are known as the most intensive lines of the resonance Raman spectrum of b and c cytochromes
Fig. 1. Time-resolved Raman spectra of living yeast cells for 10 mW laser power. The spectra are vertically shifted for convenience. The shadow stripes mark the Raman peaks of cytochrome. The asterisk denotes the contribution from the mica slice.
[35,36]. The peak at 703 cm1 in Fig. 1 is contributed by the mica slice covering the sample. As it is seen from Fig. 1 the contribution of the cytochrome lines decreases with the laser exposition, while the intensities of the rest Raman lines do not change. This decrease is associated with the decrease of the reduced cytochrome concentration. We followed the intensity of the line at 749 cm1 to estimate the amount of the reduced cytochrome in the yeast cell. This line corresponding to the heme perrole rings breathing mode [35] is intensive and placed apart the other cellular lines. For the quantitative analysis the intensity of the line at 749 cm1 was found taking into account the mica spectrum contribution and the background interpolated by a straight line. The relative contribution of cytochrome into the single cell Raman spectrum was characterized by the intensity ratio of the line at 749 cm1, I749, and of the deformational CH mode (dCH), ICH. This ratio can vary among the different yeast cells or even different cell parts, but it is independent of the absolute Raman intensity and, therefore, is convenient for the characterization of the single cell during the irradiation (for example, it eliminates the effect of change of the light absorption by the cell). The Raman spectra of yeast cells are placed on some photoluminescence background. Typically, the photoluminescence background is low at the beginning of the irradiation. However, after some laser exposition the photoluminescence background starts to increase. This increase is caused by some fluorophores resulted from photoinduced processes in the cell. Prolonged laser exposure leads in the cell degradation, resulting in the formation of visual defects such as holes or black points. The spectra from the photoinduced defects are characterized by the high level of the photoluminescence background. It is natural to relate the fluorophore increase under irradiation with increase of the photodegradation events. Thus we propose to follow the cell photoderagation by the study of the temporal dependence of the photoluminescence intensity. The photoluminescence intensity was characterized by the integral signal in the range 550–634 nm. The temporal evolution of the photoluminescence intensity of a yeast cell under the laser irradiation is represented in Fig. 2a. It is seen that the photoluminescence intensity is characterized by an abrupt increase at sPL associated with the photodegradation. Since sPL characterizes the time needed to reach the critical photoinjury level, inverse sPL reflects the rate of the photodamage accumulation.
Fig. 2. Representative time dependence of the photoluminescence intensity (a) and the I749/ICH ratio (b) under 4 mW laser irradiation. The onset of the drastic photoluminescence increase is denoted by the arrow in (a). The exponential decay fit is shown by the solid line in (b).
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The example of the 749 cm1 line intensity normalized to the dCH band versus the exposition time is shown in Fig. 2b. The photobleaching of Raman lines of the cytochromes is seen from this figure. The exponential decay fit was used to describe the photobleaching of the cytochrome line intensity:
I749 ¼ y0 þ A et=s ; IdCH
ð1Þ
where A is the photobleaching amplitude, s is the photobleaching time, and the constant y0 reflects the stationary amount of the reduced cytochromes under irradiation. The fits of the experimental data were done with the requirement of the non-negativity for y0. The representative fit is shown in Fig. 2b. It is seen that the exponential decay describes the experimental data quite well. In our study the time-resolved Raman spectra of a number of the irradiated yeast cells were measured for every laser power. The temporal evolution of I749/ICH ratio was extracted from the Raman spectra and fitted by Eq. (1). It was found that the fit parameters s, A, and y0 vary in the wide range from cell to cell. Fig. 3 shows the histograms of the fit parameters for the 2 mW irradiation power (statistics was done for the 129 yeast cells). The histograms of the parameter can be described by log-normal (s, A) and normal (y0) distributions (Fig. 3). These descriptions work well in irradiation power studied. The A and y0 variations are supposed to result from the cells heterogeneity, since the cytochromes placed mainly in mitochondria, while the dCH band intensity comes mainly from phospholipids. The s distribution is related to the photosensitivity variation. The average values of the parameters were found from the statistics over measurements with different yeast cells. Two procedures were used for this purpose either the arithmetical mean
Fig. 3. Distributions of the fit parameters s (a), A (b), y0 (c) for the 2 mW irradiation power. The lines in (a) and (b) are the log-normal distributions. The line in (c) is the normal distribution.
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over set of parameters for particular cell or averaging of Raman spectra corresponding to the same expose time, calculation I749/ ICH ratio and subsequent fitting by Eq. (1). The last procedure imitates the macroscopic experiment with the simultaneous integration of the Raman signal from all cells. Fig. 4a shows the power dependence of the cytochrome photobleaching rate in logarithmic scales. It is seen that both averaging procedures lead to significantly the same power dependence of s1. The experimental s1 data are well described by the quadratic function of the laser power P (Fig. 4a):
s1 ðPÞ ¼ a þ b P2 :
ð2Þ
The last term of Eq. (2) implies that the cytochromes photobleaching is likely to involve processes requiring two photons. To compare the power dependence of the cytochrome photobleaching rate with the inverse time of photoinduced injury, the data for s1 PL ðPÞ are also shown in Fig. 4a. It is seen that the photodamage rate has the different power dependence, which can be described by direct proportionality to the irradiation power. The question of the locality of the cytochrome photobleaching was considered. The time-resolved Raman spectra from two different parts of the same cell were studied. The delay between two measurements was less one minute. The Raman intensity of the cytochrome line at 749 cm1 is shown in Fig. 5 for two illuminated parts of the cell. As it is seen from Fig. 5 the previous irradiation of the first point has no influence on the temporal evolution of I749 of the second irradiated point of the cell. The experiment with five spatially different points of the cell showed similar results. Thus, the cytochrome photobleaching is the local process. Also, we studied whether the cytochrome photobleaching can be recovered in the dark. In this experiment two exposures of the same irradiation point by 2 mW laser power were done with the dark delay period between them. The one minute of the first exposure were followed by keeping the exposed cell in dark for 15 min, then the Raman experiment was continued. Such measure-
Fig. 4. (a) Power dependence of the photobleaching cytochrome rate and photodamage rate. The red circles are s1 from the average of the dataset of different yeast cells, the blue squares are s1 from the averaged Raman spectra. The solid line is the parabolic fit for the red circles. The green triangles are the photodamage rate, 1 s1 PL ; at 0.5 mW only the upper limitation of sPL was determined. The dashed line is the linear fit of s1 PL . (b) Power dependence of y0 parameter (red circles). The line is the fit by Eq. (8). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Exposition time dependence of I749 for two irradiated areas of the same cell. Inset shows the first (left) and second (right) irradiated areas in the photo of the yeast cell.
ments were done with 80 yeast cells. The representative time dependence of the I749/ICH ratio is shown in Fig. 6. It is seen that the first exposure reduces the cytochrome line intensity, which is recovered during the dark delay. The recovered I749/ICH ratio is slightly lower than the initial one. It is worth to note that the more intense or prolonged first exposure leading to the significant change of the I749/ICH ratio (the change of the ratio by about or more than 30%) results in the unrecoverable photodamage. The control experiment (Fig. 6) was done to eliminate the effect of the focusing mismatch during the dark delay. In this case the delay was less than 1 min, but in this time the laser beam was defocused in three dimensions and focused again at the same point of the cell. As it is seen from Fig. 6 the refocusing has no effect on the I749/ICH ratio. 4. Discussion Our study of the irradiation effect on the cytochromes redox state in living yeast cells was carried out with the intensive irradiation (0.07–3.15 MW/cm2). The photodamage rate, controlled by the photoluminescence intensity, demonstrates the linear power dependence (Fig. 4a) in the agreement with the previous studies [4,6,9,13,16] However the power dependence of the cytochrome photobleaching rate differs significantly (Fig. 4a). This means that the bioprocesses involved in the ETC photosensitivity and the cell photodamage are different. At the high irradiation intensities the
photobleaching rate shows the quadratic power dependence, while it becomes almost constant in the low intensity range. The quadratic dependence implies two-photon nature of the cytochrome photobleaching process. Two kinds of the cytochrome photobleaching processes requiring two photons can be considered. The first one relates to the direct two-photon absorption or to the consecutive absorption through the intermediate state, and the following transformation of the reduced cytochrome into another state. The second one is the cytochrome oxidation via interaction with two molecules of intermediates (for example, the reactive oxygen species, ROS) generated by the intensive green light irradiation. In particular, the cytochromes themselves can be the source of photogenerated ROS, since porphyrines are known to be photosensitizers [1,2]. The photobleaching localization effect (Fig. 5) can be interpreted as the space localization of the intermediates and of cytochromes binding to mitochondria internal membrane. In other words the intermediates have to be generated in the near proximity to ETC. It is worth to note that the square power dependence of the photobleaching rate turns into a constant at low irradiation intensities (Fig. 4a). To interpret this behavior we propose a simple model taking into account the cytochrome participation in ETC functioning. At dark the reduced and oxidized cytochrome concentrations is governed by the redox reactions kþ ;k
C oxi þ e ! C red ;
ð3Þ
where k+ and k are the direct and back reaction rate constants, e is the concentration of electrons involved in ETC working, Cred and Coxi are the reduced and oxidized cytochrome concentrations, respectively. For the simplicity the electron concentration is assumed to be independent from the irradiation power. The irradiation leads to an additional cytochrome oxidation by the photogenerated intermediates, those stationary concentration is proportional to the irradiation intensity. For the sake of definiteness, we will associate the intermediates with ROS. According to photobleaching power dependence (Fig. 4a), it is assumed that photoinduced cytochrome oxidation involves two photogenerated ROS kROS
2 C ROS þ C red ! C oxi ;
ð4Þ
where kROS is the rate constant of the oxidation reaction induced by ROS, CROS is the photogenerated ROS concentration. From the reactions (3) and (4), the kinetic equations for the reduced and oxidized cytochrome concentrations can be written
dC red dC oxi ROS ¼ ¼ kþ C oxi k C red k aI2 C red : dt dt
ð5Þ
In this case we assume that the photochange in CROS is much faster than the cytochrome oxidation, and the stationary limit, pffiffiffi C ROS ¼ aI, where I is the irradiation intensity, is used in Eq. (5). The total amount of cytochrome is invariable in Eq. (5) (the system is closed). The solution of Eq. (5) is
C red ¼
kþ S kþ þ k þ kROS aI2
2
þ AðIÞ eðkþ þk þkROS aI Þt ;
ð6Þ
where the constant S is the sum of Cred and Coxi concentrations, and A(I) is determined by the initial conditions. For the case of the initial condition of the dark equilibrium between the reduced and oxidized cytochromes, Fig. 6. Representative time dependence of the I749/ICH ratio under the 2 mW laser irradiation interrupted by the 15 min dark delay. The red circles correspond to the exposition before the delay and the blue circles to the exposition after the delay. The squares correspond to the control experiment (see text). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
AðIÞ ¼
kþ kROS aI2 S ðkþ þ k Þðkþ þ k þ kROS aI2 Þ
:
ð7Þ
The expression (6) provides the power dependences of s1 and y0 (first term):
K.A. Okotrub, N.V. Surovtsev / Journal of Photochemistry and Photobiology B: Biology 141 (2014) 269–274
s1 ðIÞ ¼ kþ þ k þ kROS aI2 ¼
kþ S : y0
ð8Þ
The expected power dependence of s1 is in agreement with the experiment (Eq. (2) and Fig. 4). The model interprets the lowintensity limit of s1 as the combination of the dark reaction rate constants. The power dependence of y0 is related with s and shown in Fig. 4. It is seen that the expected y0(I) has the qualitative agreement with the experiment. The above model also supports the process of cytochromes dark recovery observed in the experiment. After the irradiation exposition the dark redox reactions (Eq. (5)) recover the stationary cytochrome balance with the rate k+ + k. The lack of the dark recovery for the high exposition is related to the accumulation of the photoinduced injuries and/or additional cytochrome decay channels, which are not taken into account by the model. Moreover the model (Eq. (7)) predicts the strong decrease of A(I) at low irradiation intensity, which was not observed. The deficiency of the model in the description of y0(I) and A(I) behavior is related to its simplicity; the real system seems to be open and should include more kinetic terms and equations. The present results and the model interpretation extend the capabilities of resonance Raman spectroscopy to provide the important information about the ETC work. The photobleaching rate at relatively low irradiation power (below 0.4 MW/cm2) provides the information about the cytochromes redox reaction rates. The parameter y0 characterizes the irradiation effect on the redox reaction balance and the irradiation invasiveness to ETC. The photobleaching rates mirror the cellular biochemical reactions, providing information about the cellular activity in different conditions. For example, the recent Raman studies [37,38] reveal the phase transitions during the cell freezing, and the cytochrome photobleaching rates could characterize the changes of cellular activity during these transitions and the anabiosis state. The interrelations of the cytochrome photobleaching and the large-scale photodegradation are not clear. But we can see that they obey the different power dependences and have the different time scales. The role of the cytochrome photobleaching in the cellular photoinjuries needs further investigations. 5. Conclusion The effect of intensive 532 nm irradiation on b and c cytochromes redox balance in living yeast cells was studied by resonance Raman spectroscopy. This irradiation leads to the photobleaching of the reduced cytochrome Raman lines. The photobleaching is local and the reduced cytochrome concentration can be recovered at dark in case of low exposition. The power dependence of the cytochromes oxidation rate was found to be nonlinear, while the estimation of the cell photoinjury rates based on the photoluminescence growth indicates the linear power dependence. This difference means that the cytochrome oxidation mechanism differs essentially from other processes related to the cell photodamage. The power dependence of the cytochromes oxidation rate was described by the square dependence plus a constant. A simple model was proposed, in the frame of which the square dependence is associated with two intermediates generated by the irradiation and the constant with the dark redox balance rates. Thus, the resonance Raman spectroscopy can characterize not only the cytochrome redox state, but also the native cytochrome reaction rates of ETC. Acknowledgment This work was supported by RFBR (Grants No. 14-04-31451).
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