Induction kinetics of heat emission before and after photoinhibition in cotyledons of Raphanus sativus

Heat emitted during non-radiative de-excitation was determined in vivo by the photoacoustic method. The dependence of the photoacoustic signal on the length of the pulses (modulation frequency) of the excitation light and the effect of continuous light, which saturates photosynthesis but does not directly contribute to the signal, are described. The induction kinetic of heat emission measured with intact leaves differed only slightly from the induction kinetic of fluorescence (Kautsky effect) detected in parallel. The photoacoustic signal at high modulation frequencies (279 Hz), which represents the signal of heat emission, and the photoacoustic signal at low modulation frequencies (17 Hz), interpreted as a signal of pulsed oxygen evolution superimposed on the heat emission, were measured with leaves before and after photoinhibition. It was demonstrated that after photoinhibition the decrease in fluorescence yield and in photosynthetic activity (here detected as photoacoustic signal at 17 Hz) are paralleled by an increase in the yield of non-radiative deexcitation (photoacoustic signal at 279 Hz). The increase of heat emission, which has been hypothized for photoinhibited leaves, could now be proved by measuring the induction kinetics of the photoacoustic signal.     

In vivo studies of gross photosynthesis in attached leaves by means of photothermal radiometry

In photothermal radiometry, heat radiation from an illuminated object, in synchronism with incident chopped light, is observed using an infrared detector with suitable electronics. By thus measuring the heat released during pulse-wise irradiation of leaves, conclusions can be drawn as to the gross efficiency of photosynthesis: More heat means less photochemically stored energy. Saturation of photosynthesis, by employing additional strong continuous-wave background light, affords an internal photothermal radiometry signal reference corresponding to the photochemical zero efficiency level, against which the signal in the absence of saturation can be compared. Through such means, gross energy storage efficiencies approaching 30% have been observed in Caragana arborescens Lam. at low light intensities. Several other examples are given, including measurements on dark-adapted leaves and leaves infiltrated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, to support our conclusion that photothermal radiometry provides a powerful new method for in vivo studies of photosynthesis in whole, attached leaves.     

In vivo measurement of spectroscopic and photochemical properties of intact leaves using the ‘mirage effect’

This paper describes a new, highly sensitive, method for in vivo studies of photosynthesis based on the ‘mirage effect’ in which thermal energy dissipation from intact leaves, illuminated with intensity-modulated light, is sensed through the periodic deflection of a laser beam propagating along the leaf surface. The photothermal deflection technique allows one to rapidly estimate the gross efficiency of photochemical energy storage by comparing the heat emission signal with and without an additional strong, photosynthetically saturating, non-modulated light. In pea leaves, the maximal storage efficiency at low light intensities was shown to approach 55%. The general utility of this simple photothermal method is illustrated by examining the variation of the deflection signal under different conditions. The spectral resolution of this new method is shown to be much higher than that of the photoacoustic method.     

Light-induced heat production correlated with fluorescence and its quenching mechanisms

Light-induced heat produced by the non-radiative decay represents one way of de-excitation after excitation by light absorption. It was detected in vivo with cotyledons of radish seedlings (Raphanus sativus L.) by measuring the photoacoustic signal at a modulation frequency of 279 Hz. During the induction kinetic of photosynthesis the photoacoustic signal, the chlorophyll fluorescence as well as the photochemical and the non-photochemical quenching of fluorescence were simultaneously determined in order to get information about the correlation of heat production, fluorescence and its quenching mechanisms. Our results demonstrate that the changes of the photoacoustic signal can in most cases be related directly or indirectly to changes in the photochemical activity. However the kinetic of the photoacoustic signal differs from that of the fluorescence and from that of the non-photochemical quenching. This indicates that the sum of energy dissipation processes resulting in the production of light-induced heat and measured by the high-frequency photoacoustic signal must be taken into account when judging photosynthetic activity.Abbreviations LED light-emitting diode - PA photoacoustic - PAM pulse-amplitude-modulated     

The use of photothermal radiometry in assessing leaf photosynthesis: I. General properties and correlation of energy storage to P700 redox state

Energy storage measurements by modulated photothermal radiometry (PTR) were carried out on intact leaves to assess the value of the PTR method for photosynthesis research. In particular, correlations to the redox state of P₇₀₀ under various conditions were examined. PTR monitors modulated light conversion to heat by sensing the resulting modulated infra-red radiation emitted from the leaf. It is, therefore, a complementary method to photoacoustics for estimating energy storage and its time variation, particularly under controlled leaf atmosphere.With modulated light-1 (>690 nm) the energy storage approached zero and P₇₀₀ was maximally oxidized. When background light of shorter wavelength (<690 nm-light-2) was added, energy storage momentarily increased (a manifestation of Emerson enhancement) while P₇₀₀ was reduced. The values of both parameters varied as a function of the background light intensity, keeping a mutual linear relationship. Following the initial change, there was a slow reversal transient of P₇₀₀ oxidation with a parallel decrease in energy storage. Temporal correlation to P₇₀₀ redox state after dark adaptation was observed also for the energy storage measured in modulated light 2 when combined with background actinic light of medium intensity (about 50 W m²). Under these circumstances P₇₀₀ was almost totally oxidized initially and then gradually reduced while energy storage was initially low and then increased parallel to P₇₀₀ reduction.A comparison between the maximum energy storage in modulated light 1, enhanced by background light 2, to the energy storage with short wavelength light (where light tends to be more evenly distributed) indicates a comparable contribution to energy storage from each active photosystem. The above experiments indicate that energy storage contribution from PS I is directly related to the extent of openness of its reaction-centers.While some aspects of the data call for more experimentation, these experiments already establish PTR as a valuable method to monitor photosynthetic energy storage activity in vivo, particularly when used simultaneously with other non-invasive methods.Abbreviations ES energy storage - light 1 or light 2 light of spectral distribution which favors absorption in PS I or PS II, resp. - PTR photothermal radiometry - P₇₀₀ the primary donor in PS I reaction center     

Photoacoustic detection of photosynthetic oxygen evolution from leaves. Quantitative analysis by phase and amplitude measurements

The photoacoustic signal from an intact leaf was analyzed as a vectorial summation of photothermal and photosynthetic oxygen-evolution contributions. A method is outlined to estimate each contribution separately. The amplitude of the oxygen-evolution component relative to that of the photothermal singnal decreases as the modulation frequency increases due to two processes which specifically damp the oxygen-evolution modulation: (1) diffusion of oxygen from the chloroplasts to the cell boundary, and (2) electron-transfer reactions occurring between the photochemical act and oxygen evolution. The effects of the two processes are well separated and are observed over different ranges of modulation frequency. Analysis of the data leads to a consistent estimation of the oxygen diffusion coefficient and also to a preliminary idea on the limiting time constant on the donor side of Photosystem II. The dependence of the photoacoustic oxygen-evolution signal on the intensity of added nonmodulated background light is used to construct the light saturation curve of (gross) Photsynthesis, with an estimation of the ratio maximal rate / maximal quantum yield. The photoacoustic method is distinguished by its sensitivity and rapidity (a single measurement takes approx. 1 s), far better than any other method to measure gross photosynthesis. The only disadvantage is in the fact that the quantum yield of oxygen evolution is determined in a relative basis only. Attempts to calibrate the photoacoustic measurements in an absolute sense are underway.     

Correlation between chlorophyll fluorescence and photoacoustic signal transients in spinach leaves

Chlorophyll fluorescence and photoacoustic transients from dark adapted spinach leaves were measured and analyzed using the saturating pulse technique. Except for the first 30 s of photosynthetic induction, a good correlation was found between photoacoustically detected oxygen evolution at 35 Hz modulation frequency and electron flow calculated from the fluorescence quenching coefficients q_P and q_N. The induction kinetics of the photothermal signal, i.e., the photoacoustic signal at 370 Hz, reveal a fast (t _r <10 ms) and a slow (t _r 1 s) rise component. The fast component is suggested to be composed of the minimal thermal losses in photosynthesis and thermal losses from non-photosynthetic processes. The slow phase is attributed to variable thermal losses in photosynthesis. The variable thermal losses were normalized by measuring the minimal photothermal signal (H₀) in the dark-adapted state and the maximal photothermal signal (H_m) during a saturating light pulse. The kinetics of the normalized photochemical loss (H-H₀)/(H_m-H₀) obtained from high-frequency PA measurements were found to correlate with the kinetics of oxygen evolution measured at low frequency.Abbreviations F_m maximum fluorescence - F₀ initial fluorescence - F_v variable fluorescence - H photothermal signal - I in-phase - LED light emitting diode - PA photoacoustic - PL photochemical loss - Q quadrature - q_N non-photochemical quenching - q_P photochemical quenching - VCLS voltage controlled light source     

A photoacoustic study of water infiltrated leaves

Photoacoustic measurements of photosynthetic energy storage were conducted on water infiltrated pea and sugar maple leaves. The samples were vacuum infiltrated with pure water or with a suitable buffer. The use of such methodology permitted an accurate determination of the energy storage parameter at low modulation frequencies, where in non-infiltrated leaves oxygen evolution dominates the photoacoustic signal and does not allow energy storage measurements. Differences between infiltration media were not essential, however the use of pure water as infiltration medium sometimes caused instability of the measured energy storage, particularly at longer experimental time. Values of energy storage in individual samples ranged mostly between 0.2 to 0.35. Measured as a function of the modulation frequency, energy storage was found to be constant from about 10 to 200 Hz for pea leaves. In sugar maple leaves, the energy storage slightly increased between 100 and 500 Hz. Obtaining an accurate value for energy storage also allowed an accurate estimation of the O₂ evolution contribution to the photoacoustic signal of an unfiltrated leaf. In a maple leaf its frequency dependence showed only the effect of diffusion in the entire frequency range (10–500 Hz). Energy storage transients were observed after long periods (ca. 1/4-2 hrs) of dark adaptation upon the transition to light. In this case the initial energy storage was roughly about 1/2 that of the steady state value indicating strong PS I activity, while PS II was transiently incompetent. Energy-storage increased during illumination in a way to correspond to photosynthetic induction events as previously measured by fluorescence and O₂ evolution. Transients in energy storage were also found following high light to low light transitions (i.e., switch off of the saturating background light), that paralleled similar transients in oxygen evolution, showing initial transient inactivation followed by progressive reactivation of PS II.Abbreviations ES energy storage - PA photoacoustic(s) - PTR photothermal radiometry     

Increased thermal deactivation of excited pigments in pea leaves subjected to photoinhibitory treatments

The photoacoustic technique was used to monitor thermal deexcitation of the photosynthetic pigments in intact pea leaves (Pisum sativum L.) submitted to photoinhibitory treatments. When the leaves were exposed to photon flux densities above 1000 micromoles per square meter per second, the amplitude of the photothermal component of the in vivo photoacoustic signal strongly increased. This high-light-induced stimulation of nonradiative energy dissipation (heat emission) was accompanied by an inverse change in the O₂ evolution activity and in the steady state emission of 685 nanometer chlorophyll fluorescence. The time course of these effects was shown to be very rapid, with a t_1/2 of around 15 minutes. When high-light-treated leaves were readapted to the dark, the heat emission changes were reversed, following somewhat slower kinetics. A reversible increase in the rate of light energy dissipation via radiationless transitions could be a photoprotective mechanism eliminating excess excitation energy from the photosynthetic reaction centers. Interestingly, this process does not operate at temperatures below about 12°C.     

Photoacoustic study of the green alga Trebouxia in the lichen Ramalina duriaei in vivo

Photosynthetic parameters of the lichen Ramalina duriael were investigated in vivo, by the photoacoustic method using intensity—modulated exciting light (frequency range 5–300 Hz). The photoacoustic signal in intact lichens was similar in its general characteristics to that obtained from intact leaves of higher plants (Poulet et al., Biochim. Biophys. Acta 724, 433–446, 1983). It included two components interpreted to be due to modulated heat and modulated oxygen evolution. The quantum yield of the oxygen evolution component was maximal in the red spectral region and exhibited the red drop decrease at wavelengths larger than 680 nm, similar to observations in higher plants. In contrast to those however, there was a pronounced decrease in this yield in the region below about 600 nm, indicating that pigments absorbing at shorter wavelengths are inefficient energy transfer agents. Similar results were observed for the quantum yield spectrum of photochemical energy storage. Analysis of oxygen diffusion in the symbiont alga, from the modulation frequency dependence of the ratio of oxygen evolution to photothermal signal component is consistent with an average diffusion path of about 4 m, compared to a smaller, 1 m, average diffusion path obtained in green leaves.     

New applications of photoacoustics to the study of photosynthesis

Photoacoustic methods offer unique capabilities for photosynthesis research. Phenomena that are readily observed by photoacoustics include the storage of energy by electron transport, oxygen evolution by leaf tissue at microsecond time resolution, and the conformational changes of photosystems caused by charge separation. Despite these capabilities, photoacoustic methods have not been widely exploited in photosynthesis research. One factor that has contributed to their slow adoption is uncertainty in the interpretation of photoacoustic signals. Careful experimentation is resolving this uncertainty, however, and technical refinements of photoacoustic methods continue to be made. This review provides an overview of the application of photoacoustics to the study of photosynthesis with an emphasis on the resolution of uncertainties in the interpretation of photoacoustic signals. Recent developments in photoacoustic technology are also presented, including a microphotoacoustic spectrometer, gas permeable photoacoustic cells, the use of photoacoustics to monitor phytoplankton populations, and the use of photoacoustics to study protein dynamics. This revised version was published online in June 2006 with corrections to the Cover Date.     

Computer-controlled pulse modulation system for analysis of photoacoustic signals in the time domain

A newly developed photoacoustic system for measurement of photosynthetic reactions in intact leaves is described. The system is based on pulsed light-emitting diodes, the pulse program and pulse response analysis being computer controlled. Separation of various components in the overall photoacoustic signal is achieved by curve fitting analysis of the responses following individual measuring light pulses in the millisecond time domain. This procedure is in distinction to the conventionally used analysis in the frequency domain, with the advantage that various signal components are obtained by on-line deconvolution, yielding simultaneous recordings of photothermal (complement of energy storage) and photobaric (evolution and uptake) signals. The basic components of the new system are described by block diagrams and the principal steps for deconvolution of the overall photoacoustic response are outlined. An example of application with simultaneous recording of chlorophyll fluorescence is given. It is apparent that the photobaric uptake component represents a significant part of the overall signal, particularly during induction of photosynthesis after dark-adaptation. This component probably contains not only O₂-uptake but uptake of CO₂ as well.Abbreviations PA photoacoustic - LED light-emitting-diode - RAM random access memory     

Photoacoustic spectroscopy of Anacystis nidulans. III. Detection of photosynthetic activities

Photosynthetic activities of Anacystis nidulans can be detected by photoacoustic spectroscopy. Algae treated by a photosynthetic inhibitor are used to provide the signal from the photochemically inactive sample. The results of these measurements correspond well with the activities which can be monitored by conventional biochemical assays. Acoustic data from A. nidulans are used to obtain the action spectrum for photochemical energy storage. It is concluded that phycocyanin harvests light for both photoreactions but that chlorophyll alpha molecules convey most of their excitation energy to photoreaction I. As judged from the relationship between the modulation frequency and the acoustic signal intensity, at least 60% of the photons absorbed at 630 nm perform photochemical work and about half of the useful energy is stored at stable products. Although it cannot be separated from the purely thermal effect, the contribution of modulated oxygen evolution to the acoustic signal of algae is estimated to be relatively small. Due to structural peculiarities, the opposite situation predominates in low frequency measurements performed with leaves from Impatiens petersiana.     

Photosynthetic responses of leaves to water stress, expressed by photoacoustics and related methods : I. Probing the photoacoustic method as an indicator for water stress in vivo

The effect of leaf desiccation on the photosynthetic activities in vivo was probed by the photoacoustic method. The aim of this research was: (a) To study the photoacoustic signal per se in varied conditions in order to develop this tool as a probe for stress conditions in vivo. (b) To obtain results pertaining to electron transport activities in vivo, and confirm conclusions based on work with isolated chloroplasts, which could otherwise be the result of nonspecific damage occurring during their isolation. Leaf discs from tobacco (Nicotiana tabacum L.) were routinely used, with other species tested also for comparison. Rapid leaf desiccation caused changes in the low frequency photoacoustic signal, attributed both to the mechanism of signal transduction, influenced by changes in the structural parameters of the leaf, and to the direct (nonstomatal) inhibition of gross photosynthesis. The dependence of the photothermal part of the signal on the frequency indicated the presence of two photothermal components, one of which persisted only at low modulation frequencies (below about 100 Hz) and which largely increased with the desiccation treatment. This component was ascribed to a thermal wave which reaches the leaf surface. The other nonvariable photothermal component was ascribed to a thermal wave propagating from the chloroplasts to the surface of the mesophyll cell. Only this component is considered in the ratio of the O₂ signal to the photothermal signal, which is used to estimate the quantum yield of photosynthesis. The specific dependence of the latter ratio on the frequency yielded a comparative quantum yield parameter from its extrapolation to zero frequency, and also indicated stress induced changes in the diffusion of O₂ through the mesophyll cell, reflected by changes in its characteristic slope. The (zero frequency extrapolated) quantum yield was markedly reduced with the progression of the water stress, indicating the inhibition of (gross) phototosynthetic electron transport in vivo. This result was expressed even more emphatically by the stronger inhibition of the photochemical energy storage, obtained by photoacoustic measurements at a high modulation frequency.     

Inhibition of photosynthetic activities under slow water stress measured in vivo by the photoacoustic method

A slow water stress over several days was imposed on tobacco plants (Nicotiana tabacum L. var. Xanthi) by withholding water from the soil. Photosynthesis was measured in leaves from those water-stressed plants by the photoacoustic method. Slow drought induced marked changes in the photoacoustic signals, which were largely similar to those observed previously in leaves subjected to rapid desiccation in air (over 3–4 h), reflecting two simultaneous changes: 1) Modification of the heat and oxygen diffusion characteristics of the leaves due to changes in their anatomical structure [shown by the change in the slope of the plot of the oxygen (A_OX) to photothermal signal (A_PT) ratio vs the square root of the modulation frequency]; 2) Inhibition of gross photosynthesis measured by the extrapolation of the A_OX/A_PT ratio to zero frequency. However, in contrast to rapid water stress in detached leaves, where it was shown that mainly the oxidizing side of photosystem II (PS II) was damaged, we found a slower and more complex phenomenology having largely biphasic kinetics. During the first 6 days, there was a strong reduction in the photochemical energy storage, but the inhibition of oxygen evolution was relatively mild. The Emerson enhancement in state 1 dropped considerably, indicating lowering of the apparent absorption cross-section of PS II. Fluorescence measurements suggest that PS II reaction center itseIf may be the primary site of the damage. PS I activity, judged by cytochrome f photooxidation, remained largely intact. The subsequent days were associated with a further spectacular decrease in the oxygen evolution quantum yield with both photosystems damaged. The photochemical energy storage continued to decrease further. The Emerson enhancement ratio of the remaining activities in both State 1 and 2 showed a marked increase, indicating the reestablishment of a strong imbalance in the distribution of excitation energy within the photochemical apparatus in favor of PS II. All the photoacoustic changes observed in response to drought were completely reversible within 2–3 days upon rewatering of the soil.     

Bill Arnold and calorimetric measurements of the quantum requirement of photosynthesis—once again ahead of his time

The approach of photocalorimetry to decide on the true quantum requirement of photosynthesis — one of the main issues of the research in the first half of the century and a source of a bitter debate — is described. Bill Arnold's original approach to get into the true answer is reflected from the point of view of present day calorimetric techniques.Abbreviations PA photoacoustic(s) - PTR photothermal radiometry - PBD probe beam deflection (thermal measurement) - ES energy storage - QR quantum requirement This is CIW/DPB Contribution No. 1286.     

Probing Red Blood Cell Morphology Using High-Frequency Photoacoustics

A method that can rapidly quantify variations in the morphology of single red blood cells (RBCs) using light and sound is presented. When irradiated with a laser pulse, an RBC absorbs the optical energy and emits an ultrasonic pressure wave called a photoacoustic wave. The power spectrum of the resulting photoacoustic wave contains distinctive features that can be used to identify the RBC size and morphology. When particles 5–10 μm in diameter (such as RBCs) are probed with high-frequency photoacoustics, unique periodically varying minima and maxima occur throughout the photoacoustic signal power spectrum at frequencies >100 MHz. The location and distance between spectral minima scale with the size and morphology of the RBC; these shifts can be used to quantify small changes in the morphology of RBCs. Morphological deviations from the normal biconcave RBC shape are commonly associated with disease or infection. Using a single wide-bandwidth transducer sensitive to frequencies between 100 and 500 MHz, we were able to differentiate healthy RBCs from irregularly shaped RBCs (such as echinocytes, spherocytes, and swollen RBCs) with high confidence using a sample size of just 21 RBCs. As each measurement takes only seconds, these methods could eventually be translated to an automated device for rapid characterization of RBC morphology and deployed in a clinical setting to help diagnose RBC pathology.     

Photothermal beam deflection: a new method for in vivo measurements of thermal energy dissipation and photochemical energy conversion in intact leaves

A novel photosynthetic technique, photothermal deflection spectroscopy, is presented which is based on the mirage effect and allows the rapid measurement of thermal deactivation of excited pigments in leaf samples placed in an open cell. Modulated heat emission from leaves illuminated with intensity-modulated light was measured via the detection of the periodic deflection of a laser beam parallel to the sample surface. Photothermal deflection signals can be monitored in vivo in leaves placed in various, liquid or gaseous, environments with a satisfactory signal-to-noise ratio close to 60–80 (in distilled water) at low modulation frequencies (below 50 Hz). Using this new and simple photothermal method, it was possible to easily obtain useful information on the leaf photochemical activity and its light-saturation characteristics under normal or stress conditions, suggesting that in vivo deflection signals could be used for assaying the photosynthetic state of health of crop plants. The beam deflection method presented in this paper appears to be a potentially useful photosynthetic tool complementary to the related photoacoustic technique.Abbreviations DCMU dichlorophenyldimethylurea - PD photothermal deflection - PL photochemical energy storage - S/N ratio signal-to-noise ratio     

动脉粥样硬化斑块中脂肪沉积的扫频光热波成像

动脉粥样硬化和易损斑块破裂在全球范围内具有最高的死亡率,超过传染病和癌症导致的死亡率的总和。动脉粥样硬化斑块是由一层很薄的”纤维帽”和导致血栓形成的脂质核心构成。光热波成像是基于对被目标发色团（本文中指脂肪沉积）吸收的光信号强度进行周期调制,从而实现对目标发色团释放的热（红外）信号的调制。这里,我们利用光热波成像来检测来自兔子动脉硬化模型的粥样硬化斑块中脂肪沉积的三维分布。波长为1210纳米的激光被用来靶向检测脂肪。动脉粥样硬化斑块组织在0．1到5赫兹连续扫频的激光的激发下发出光热波,光热波传播到样品表面形成红外辐射温度并被红外相机以25．6帧／秒的速度接收并录制20秒。红外相机上的每一个像素（总共256~256像素）在进行时域傅里叶变换以后得到强度和相位的频域光热波图像。某一特定频率的强度和相位光热波图像对应着脂肪沉积在动脉粥样硬化斑块样品中的横向和纵向分布。对强度和相位光热波图像的分析指出：光热波成像能够用来检测脂肪在动脉粥样硬化斑块中的三维分布,并且脂肪的分布和动脉粥样硬化斑块的形状特征有着紧密联系。     

金纳米棒的表面修饰及其应用于肿瘤诊疗的研究进展

金纳米棒具有独特的光学性质、表面易修饰性、较低的生物毒性和良好的生物相容性,因而在成像、光热治疗和药物载带等方面具有极高的潜在应用价值.本文综述了典型的金纳米棒表面修饰方法及其在生物成像、光热治疗和药物治疗中的应用,重点阐述了通过金纳米棒同时实现肿瘤诊断和治疗相结合的研究进展.