Letter pubs.acs.org/ac
Photothermal Microscopy: Imaging of Energy Dissipation From Photosynthetic Complexes Wieslaw I. Gruszecki,* Rafal Luchowski, Monika Zubik, and Wojciech Grudzinski Department of Biophysics, Institute of Physics, Maria Curie-Skłodowska University, 20-031 Lublin, Poland S Supporting Information *
ABSTRACT: An idea of a photothermal imaging microscopy (PTIM) is proposed, along with its realization based on a dependence of fluorescence anisotropy of dye molecules on heat emission in their nearest vicinity. Erythrosine B was selected as a fluorophore convenient to report thermal deactivation of the excited pigment−protein complex isolated from the photosynthetic apparatus of plants (LHCII), owing to the relatively large spectral gap between the fluorescence emission bands of chlorophyll a and a probe. Comparison of the simultaneously recorded images based on fluorescence lifetime of LHCII and fluorescence anisotropy of erythrosine shows a high rate of thermal energy dissipation from the aggregated forms of the complex and, possibly, thermal energy transmission along the protein supramolecular structures. Relatively high resolution of this novel microscopic technique, comparable to the fluorescence lifetime microscopy, enables its application in a nanoscale imaging and in nanothermography.
A
pathways.1 Fluorescence emission and thermal energy dissipation are the major de-excitation channels therefore, very often, a decrease in a fluorescence quantum yield is interpreted in terms of an increase in efficiency of thermal deactivation of excited molecules. Such an approach is a gold standard in examination of photosynthetic systems. In this case, chlorophyll fluorescence measurements are used as a routine.2 Novel fluorescence techniques, such as fluorescence lifetime imaging microscopy (FLIM), are also, recently, more frequently applied in photosynthesis research.3,4 On the other hand, other than fluorescence and heat emission energy deactivation channels can be active in several molecular systems. A prominent example of such deactivation of singlet excited states is intersystem crossing, leading to formation of triplet states.1 This particular deactivation channel of the singlet excited molecule, in organic solvents, may exceed 60% in the case of chlorophyll a and even exceed 80% in the case of chlorophyll b.5 Energy of excited triplet states can be dissipated as a heat, as in the case of singlet excitations, but also can be used to drive other processes, such as sensitized oxidation. In order to assess direct information on thermal energy dissipation from excited molecular systems, several variants of photothermal spectroscopic techniques have been developed. Among these techniques are photoacoustic spectroscopy,6,7 photothermal beam deflection,8,9 laser-induced optoacoustic studies,10 or even direct calorimetric measurement of heat emission.11 These
bsorption of light by molecules brings them to one of the excited states which can be depopulated by several
Received: August 19, 2015 Accepted: September 22, 2015 Published: September 22, 2015
Figure 1. Scheme presenting an idea of the photothermal imaging microscope. © 2015 American Chemical Society
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Figure 2. Calibration of a response of fluorescence anisotropy of probe molecules to heat emission in their environment. Left hand panel presents exemplary microscopic images recorded from the polylysine covered glass slides at which deposited has been a glycerol solution of erythrosine B (ErB) or the same solution supplemented with carbon black nanoparticles (ErB + CB). Images were recorded with a 470 nm laser tuned to different power, indicated. Images are colored with false colors representing the fluorescence anisotropy level (the scale presented below the images). Carbon black nanoparticles located in the focal plane absorb maximum light energy which is converted to heat and dissipated into the environment of the nearest ErB solution, covering nanoparticles. These regions of the samples, characterized by a decreased fluorescence anisotropy level (displayed with red) were subjected to a quantitative analysis. The ROI (regions of interest) analyzed are marked (white circles). The ROI of the sample imaged with different laser power was the same. The results of analyses are presented in the form of histograms in the central panel. The experimental points shown in the right-hand panel represent the arithmetic mean from the histograms, obtained for different laser powers. The error bars represent standard errors of the arithmetic mean.
medium. The fluorescence signal is separated, by dichromatic mirrors, to the channels collecting fluorescence of a sample and fluorescence of a probe. Fluorescence signal of a sample can be analyzed as in the conventional fluorescence or fluorescence lifetime imaging microscopes, while fluorescence signal of a probe is split into the beams with the electric vector of electromagnetic radiation polarized parallel and perpendicular with respect to the polarization of the excitation light beam. Analysis of number of photons in these two channels enables calculation of the fluorescence anisotropy parameter (see the Supporting Information) which is strictly related to the motional freedom of fluorescence emitting molecules in their environment.1 Heating of the fluorescence probe solution can, in principle, affect both the local environment temperature and viscosity which, according to the Perrin’s theory, are related to rotational correlation time.1 The rotational correlation time is proportional to viscosity and in inverse proportion to temperature.1 Owing to this fact, the dependency of
techniques are very powerful and they brought great progress in molecular research, including research on photosynthesis.12,13 Despite several advantages of the photothermal techniques, it has to be pointed out that, unfortunately, they are not suitable for research at a nanoscale. Owing to this fact, conclusions regarding thermal energy dissipation in microscopic research have to be drawn based on fluorescence measurements. Recently, activity of several research groups has been concentrated on a design of a technique suitable for imaging of heat emission on a microscopic level.14−17 In the present work, we propose an original idea of a photothermal imaging microscopy (PTIM) which can fill the gap in the arsenal of advanced techniques addressed to study molecular objects and processes associated with energy conversion and transmission. The approach is based on the confocal laser scanning fluorescence microscopy (Figure 1). A scanning laser wavelength is selected to excite simultaneously molecules of a sample and molecules of a heat emission probe dissolved in the 9573
DOI: 10.1021/acs.analchem.5b03197 Anal. Chem. 2015, 87, 9572−9575
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Analytical Chemistry
Figure 3. Comparison of the fluorescence lifetime image of the LHCII sample and the fluorescence anisotropy image of ErB. Both the fluorescence lifetime image of LHCII sample (panel a) and the fluorescence anisotropy image of ErB (panel b) were recorded simultaneously from the same area of the sample composed of supramolecular structures of LHCII deposited on a polylysine covered glass slide at which the glycerol solution of ErB was added. The images were recorded based on a 500 × 500 pixel projection. The images are displayed in the false color scales presented. The images displayed in panels a and b represent two detection channels schematically shown in Figure 1. Fluorescence excitation was with a 470 nm pulse laser and emission was detected after the band-pass filters: 680/13 nm (panel a) and 520/35 nm (panel b), selective for the emission of LHCII and ErB, respectively (see Supporting Information, Figure S1). A single molecular organization form of LHCII, characterized by relatively high fluorescence lifetimes, is framed by a white line (the same area in both panels). This structure is discussed in the text in the aspect of a hypothetical thermal energy transmission along a protein.
fluorescence anisotropy on local heat emission could be complex but, on the other hand, can be relatively easily calibrated. An example of such a calibration experiment is presented in Figure 2. In the experiment, carbon black (CB) particles, placed in the erythrosine B (ErB) solution in glycerol were scanned with a laser at different light power density. As can be seen, heat emission by CB influences a fluorescence anisotropy level of ErB in the surrounding solution. The same heat emission probe system (ErB in glycerol) can be used to monitor thermal energy dissipation from the photosynthetic systems, e.g., the major antenna complex of plants, LHCII. This is owing to the fact that both the photosynthetic pigments in LHCII (chlorophyll b and xanthophylls) and ErB absorb light at 470 nm. Moreover, the fluorescence emission bands from LHCII (maximum at 680 nm) and from ErB (maximum at 560 nm) are clearly separated, which makes it easy to analyze independently both the fluorescence signals (see the Supporting Information, Figure S1). The fact that ErB has a relatively short fluorescence lifetime (below 500 ps) makes this fluorophore particularly suitable to monitor changes in a fluorescence anisotropy. An idea of the photothermal imaging microscopy (PTIM) was tested on supramolecular structures of LHCII. Differences in molecular organization of this complex result in different rate of thermal energy dissipation.3,4,18 Comparison of the simultaneously recorded images of LHCII particles placed in the environment of a glycerol solution of ErB, based on fluorescence lifetimes of the sample and fluorescence anisotropy of a heat emission probe, is presented in Figure 3. As can be seen, spontaneous organization of LHCII in the sample results in a spatial heterogeneity in the fluorescence emission decay channel. This lets one anticipate that also rates of thermal energy dissipation are not homogeneously distributed over the structure examined. Indeed, the image based on a heat emission probe shows that efficiency of thermal energy dissipation varies in different places of the sample. Such a result demonstrates high sensitivity of the method. A close correspondence of the several regions characterized by short fluorescence lifetimes (displayed in blue color) and the regions characterized by a high rate of thermal energy dissipation (lower fluorescence anisotropy,
displayed in red color) can be observed. An inverse correlation can be also clearly seen. The regions of the sample characterized by relatively long chlorophyll fluorescence lifetimes (less efficient energy dissipation, displayed in red color) and high anisotropy regions (not elevated temperatures, displayed in blue color) can be noticed, in both images in Figure 3. One of such structures is framed by a white line. As can be seen, the central part of the structure, characterized by relatively high fluorescence lifetimes, appears uniformly colored in red. On the other hand, the same area imaged with the PTIM technique demonstrates certain heterogeneity, the central part appears colored in blue while the edges in green. Such a difference is not observed in the peripheral region of the protein sample (e.g., beyond the right-hand side edge of the marked structure). This means that the difference in the same protein structure imaged in the fluorescence lifetime and heat emission channels is not due to heat propagation in ErB solution but rather in the protein moiety. The comparison of the fluorescence emission and the heat emission channels suggests, therefore, the possibility of thermal energy transmission along the protein structures. The results confirm applicability of the PTIM technique in monitoring thermal energy dissipation and transmission at the microscopic and submicroscopic scale. Moreover, the resolution of the image based on a photothermal effect is at least as good as in the case of the image based on the fluorescence lifetime (Figure 3). This opens avenues for possible application of the PTIM technique in nanoimaging and nanothermography.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03197. Fluorescence spectra of the samples and the description of experimental details (PDF) 9574
DOI: 10.1021/acs.analchem.5b03197 Anal. Chem. 2015, 87, 9572−9575
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Analytical Chemistry
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AUTHOR INFORMATION
Corresponding Author
*Phone: + (48 81) 537 62 52. Fax: + (48 81) 537 61 91. Email:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research has been performed within the framework of the project “Molecular Spectroscopy for BioMedical Studies” financed by the Foundation for Polish Science within the TEAM program (TEAM/2011-7/2). The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Development of the Eastern Poland Operational Programme.
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ABBREVIATIONS LHCII, Light-harvesting pigment−protein complex of Photosystem II; ErB, erythrosine B; FLIM, fluorescence lifetime imaging microscopy
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REFERENCES
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