In Vivo Hyperspectral Confocal Fluorescence Imaging to Determine Pigment Localization and Distribution in Cyanobacterial Cells
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In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells Wim F. J. Vermaas*†, Jerilyn A. Timlin‡, Howland D. T. Jones‡, Michael B. Sinclair‡, Linda T. Nieman‡§, Sawsan W. Hamad*, David K. Melgaard‡, and David M. Haaland‡ *School of Life Sciences and Center for Bioenergy and Photosynthesis, Arizona State University, Box 874501, Tempe, AZ 85287-4501; and ‡Sandia National Laboratories, MS0895, Albuquerque, NM 87185 Edited by Elisabeth Gantt, University of Maryland, College Park, MD, and approved January 25, 2008 (received for review August 27, 2007) Hyperspectral confocal fluorescence imaging provides the oppor- cytoplasmic membrane of cyanobacteria, whereas Chl is not (6, tunity to obtain individual fluorescence emission spectra in small 7). Additional light-harvesting capability, primarily for PS II, is (Ϸ0.03-m3) volumes. Using multivariate curve resolution, individ- provided by phycobilisomes, which are pigment-binding com- ual fluorescence components can be resolved, and their intensities plexes in the cytoplasm that associate with thylakoids to enable can be calculated. Here we localize, in vivo, photosynthesis-related energy transfer to Chl (8, 9). Phycocyanin (PC), allophycocyanin pigments (chlorophylls, phycobilins, and carotenoids) in wild-type (APC), and allophycocyanin-B (APC-B) are the main phyco- and mutant cells of the cyanobacterium Synechocystis sp. PCC bilisome pigments in Synechocystis sp. PCC 6803 (10). 6803. Cells were excited at 488 nm, exciting primarily phycobilins Chl and phycobilisome pigments fluoresce at room tempera- and carotenoids. Fluorescence from phycocyanin, allophycocyanin, ture with spectral maxima in the 640- to 700-nm range. PC emits allophycocyanin-B/terminal emitter, and chlorophyll a was re- fluorescence with an Ϸ650-nm maximum, APC at 665 nm, and solved. Moreover, resonance-enhanced Raman signals and very APC-B at 675 nm, and the main emission wavelength of Chl is weak fluorescence from carotenoids were observed. Phycobilin at 685 nm (11). Phycobilisomes are highly fluorescent in isolated emission was most intense along the periphery of the cell whereas form, but the fluorescence yield is decreased in intact systems chlorophyll fluorescence was distributed more evenly through- because in vivo the excitation energy is transferred efficiently out the cell, suggesting that fluorescing phycobilisomes are from PC to APC to APC-B or to long-wavelength APC associ- more prevalent along the outer thylakoids. Carotenoids were ated with the ApcE protein (terminal emitter) (12) and even- prevalent in the cell wall and also were present in thylakoids. Two tually to Chl in the thylakoid membranes. chlorophyll fluorescence components were resolved: the short- Much is known regarding cyanobacterial cell architecture and wavelength component originates primarily from photosystem II thylakoid organization (2, 13–15), the structure of individual and is most intense near the periphery of the cell; and the pigment-binding complexes (16, 17), the distribution of photo- long-wavelength component that is attributed to photosystem I synthetic complexes in fixed thylakoid membranes (18), and the because it disappears in mutants lacking this photosystem is of ability of phycobilisomes to dynamically associate with photo- higher relative intensity toward the inner rings of the thylakoids. synthetic complexes in the membrane (19–21). However, pho- Together, the results suggest compositional heterogeneity be- tosynthetic pigments and their interactions have not yet been tween thylakoid rings, with the inner thylakoids enriched in visualized distinctly in vivo because of their spectral overlap. photosystem I. In cells depleted in chlorophyll, the amount of both Spectral congestion (fluorescence emission maxima that are chlorophyll emission components was decreased, confirming the different by Ͻ20–30 nm) is common in photosynthetic systems accuracy of the spectral assignments. These results show that that depend on spectral overlap for efficient energy transfer and hyperspectral fluorescence imaging can provide unique informa- presents a major problem in data analysis/interpretation. Con- tion regarding pigment organization and localization in the cell. focal laser scanning microscopy coupled with spectral imaging techniques has the potential to visualize photosynthetic pigments cyanobacteria ͉ photosynthetic pigments ͉ multivariate curve resolution even amidst spectral overlap. However, because of low spectral resolution of current commercial instrumentation and the ab- yanobacteria convert light energy to chemical energy by sence of methods available for robust analysis of spectrally and Cmeans of photosynthesis, using water as a source of electrons spatially overlapped spectral images, application of this tech- for CO2 reduction and O2 production. A key part of the nique has been limited to systems with relatively few fluorescent photosynthesis process is light absorption (harvesting) by pig- pigments that are spectrally distinct and spatially isolated (22). ments, followed by excitation transfer to reaction center chlo- The recent development of a high-resolution hyperspectral rophyll (Chl) a of photosystems (PS) II and I (1). These processes confocal fluorescence imaging microscope (23) and correspond- take place in thylakoid membranes that in cyanobacteria gen- erally form an extensive internal membrane complex of several layers along the periphery of the cytoplasm, with thylakoids Author contributions: W.F.J.V., J.A.T., H.D.T.J., M.B.S., and D.M.H. designed research; W.F.J.V., J.A.T., H.D.T.J., M.B.S., L.T.N., S.W.H., and D.M.H. performed research; W.F.J.V., found less frequently toward the center of the cell (2). J.A.T., H.D.T.J., M.B.S., S.W.H., D.K.M., and D.M.H. contributed new reagents/analytic tools; The pigments associated with the photosynthetic apparatus W.F.J.V., J.A.T., H.D.T.J., M.B.S., L.T.N., S.W.H., D.K.M., and D.M.H. analyzed data; and are bound to thylakoid proteins, modifying their spectral prop- W.F.J.V., J.A.T., H.D.T.J., and D.M.H. wrote the paper. erties and providing a spatial distribution that aids in the The authors declare no conflict of interest. efficiency of light harvesting and energy transfer to reaction This article is a PNAS Direct Submission. center Chls. Pigments bound to integral membrane proteins in †To whom correspondence should be addressed. E-mail: [email protected]. reaction center complexes in thylakoids of cyanobacteria include §Present address: Department of Biomedical Engineering, University of Texas M. D. Ander- Chl a [Ϸ40 per PS II (3) and Ϸ100 per PS I (4)] and carotenoids; son Cancer Center, Houston, TX 77030. 3 the latter act in photoprotection and Chl quenching but do not This article contains supporting information online at www.pnas.org/cgi/content/full/ effectively transfer energy to Chl in cyanobacterial PS II (5). 0708090105/DC1. Carotenoids are also present in the outer cell membrane and © 2008 by The National Academy of Sciences of the USA 4050–4055 ͉ PNAS ͉ March 11, 2008 ͉ vol. 105 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708090105 Downloaded by guest on September 29, 2021 ing robust multivariate curve resolution (MCR) algorithms (24–28) provide a unique opportunity to reliably resolve pig- ments with rather similar fluorescence emission spectra in a living cell with diffraction-limited spatial resolution (Ϸ250 ϫ 250 ϫ 600 nm). By using MCR, spectra representing pure fluorescent components can be extracted without reference spectra, provided that such components are not present in the same ratio relative to each other in all voxels. This approach allows a detailed determination of pure individual fluorescent components and their relative intensities in three dimensions within a single cell. Synechocystis sp. PCC 6803 is a model cyanobacterium with a known genome sequence and a facile and reliable transforma- tion system (29, 30). Mutants lacking genes for specific photo- systems or pigment biosynthesis pathways have been created. One mutant strain used in this study lacks the genes for PsaA and PsaB, the major subunits of PS I, with which Ϸ75% of the Chl in the cell is associated (31). Consequently, the PS I-less strain contains only Ϸ25% of the usual amount of Chl a, without affecting the phycobilisome content. In addition, we use a strain that lacks chlL, which codes for a component of the light- independent NADPH/protochlorophyllide oxidoreductase (32). As a consequence, this strain cannot synthesize Chl in darkness, but Chl synthesis can occur in the light because of the presence of a light-dependent protochlorophyllide oxidoreductase (33). Moreover, we have used the double mutant (PS I-less/ChlL-less) (34) and the wild-type strain in this study. Together, these strains represent a collection with different pigment compositions and PLANT BIOLOGY properties and allow validation of spectral assignments. Fig. 1. MCR analysis of a single optical section from a 3D hyperspectral image of live wild-type Synechocystis cells. (A) Mathematically extracted pure- Results component spectra of fluorescent components in Synechocystis using the pure Hyperspectral Imaging of Intact Synechocystis Cells. Using 488-nm emission spectra obtained from the composite image in SI Fig. 5. The spectra laser excitation, fluorescence was elicited from intact, live were normalized to unit length as required for MCR algorithm stability. (B Synechocystis cells of three strains grown under normal and Upper) Fit between