In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells

Willem Vermaas, Jerilyn A. Timlin, Howland D T Jones, Michael B. Sinclair, Linda T. Nieman, Sawsan W. Hamad, David K. Melgaard, David M. Haaland

Research output: Contribution to journalArticle

109 Citations (Scopus)

Abstract

Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (≈0.03-μm 3) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exciting primarily phycobilins and carotenoids. Fluorescence from phycocyanin, allophycocyanin, allophycocyanin-B/terminal emitter, and chlorophyll a was resolved. Moreover, resonance-enhanced Raman signals and very weak fluorescence from carotenoids were observed. Phycobilin emission was most intense along the periphery of the cell whereas chlorophyll fluorescence was distributed more evenly throughout the cell, suggesting that fluorescing phycobilisomes are more prevalent along the outer thylakoids. Carotenoids were prevalent in the cell wall and also were present in thylakoids. Two chlorophyll fluorescence components were resolved: the shortwavelength component originates primarily from photosystem II and is most intense near the periphery of the cell; and the long-wavelength component that is attributed to photosystem I because it disappears in mutants lacking this photosystem is of higher relative intensity toward the inner rings of the thylakoids. Together, the results suggest compositional heterogeneity between thylakoid rings, with the inner thylakoids enriched in photosystem I. In cells depleted in chlorophyll, the amount of both chlorophyll emission components was decreased, confirming the accuracy of the spectral assignments. These results show that hyperspectral fluorescence imaging can provide unique information regarding pigment organization and localization in the cell.

Original languageEnglish (US)
Pages (from-to)4050-4055
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number10
DOIs
StatePublished - Mar 25 2008

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Optical Imaging
Thylakoids
Chlorophyll
Phycobilins
Fluorescence
Carotenoids
Photosystem I Protein Complex
Phycobilisomes
Phycocyanin
Synechocystis
Photosystem II Protein Complex
Photosynthesis
Cyanobacteria
Cell Wall

Keywords

  • Cyanobacteria
  • Multivariate curve resolution
  • Photosynthetic pigments

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells. / Vermaas, Willem; Timlin, Jerilyn A.; Jones, Howland D T; Sinclair, Michael B.; Nieman, Linda T.; Hamad, Sawsan W.; Melgaard, David K.; Haaland, David M.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 10, 25.03.2008, p. 4050-4055.

Research output: Contribution to journalArticle

Vermaas, Willem ; Timlin, Jerilyn A. ; Jones, Howland D T ; Sinclair, Michael B. ; Nieman, Linda T. ; Hamad, Sawsan W. ; Melgaard, David K. ; Haaland, David M. / In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells. In: Proceedings of the National Academy of Sciences of the United States of America. 2008 ; Vol. 105, No. 10. pp. 4050-4055.
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AU - Sinclair, Michael B.

AU - Nieman, Linda T.

AU - Hamad, Sawsan W.

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AU - Haaland, David M.

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AB - Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (≈0.03-μm 3) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exciting primarily phycobilins and carotenoids. Fluorescence from phycocyanin, allophycocyanin, allophycocyanin-B/terminal emitter, and chlorophyll a was resolved. Moreover, resonance-enhanced Raman signals and very weak fluorescence from carotenoids were observed. Phycobilin emission was most intense along the periphery of the cell whereas chlorophyll fluorescence was distributed more evenly throughout the cell, suggesting that fluorescing phycobilisomes are more prevalent along the outer thylakoids. Carotenoids were prevalent in the cell wall and also were present in thylakoids. Two chlorophyll fluorescence components were resolved: the shortwavelength component originates primarily from photosystem II and is most intense near the periphery of the cell; and the long-wavelength component that is attributed to photosystem I because it disappears in mutants lacking this photosystem is of higher relative intensity toward the inner rings of the thylakoids. Together, the results suggest compositional heterogeneity between thylakoid rings, with the inner thylakoids enriched in photosystem I. In cells depleted in chlorophyll, the amount of both chlorophyll emission components was decreased, confirming the accuracy of the spectral assignments. These results show that hyperspectral fluorescence imaging can provide unique information regarding pigment organization and localization in the cell.

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