Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
Sandia National Laboratories
22
In Vivo Observation of the Native Pigments in Synechocystis sp. PCC 6803 Using a New Hyperspectral Confocal Microscope
Michael B. Sinclair1* (mbsincl@sandia.gov), Jerilyn A. Timlin1, David M. Haaland1, Sawsan Hamad2, and Wim F.J. Vermaas2
1Sandia National Laboratories, Albuquerque, NM and 2Arizona State University, Tempe, AZ
We have developed a new hyperspectral confocal microscope that combines the attributes of high spatial resolution (<0.5 µm), high speed acquisition (>8 MB/s) and single photon sensitivity. The new instrument records the emission spectrum from 500 nm to 800 nm for each voxel within the 3-dimensional sample. The acquisition of full spectral information, when coupled with modern multivariate data analysis techniques allows for quantification of the contribution of each of the emitting components present within any voxel. To demonstrate the advantages of this approach, we have obtained and analyzed in vivo hyperspectral images of wild type Synechocystis sp. PCC 6803, as well as two mutant strains: chlL–, which is incapable of light-independent synthesis of chlorophyll, and PS1-less/chlL–, which in addition to being incapable of light-independent synthesis of chlorophyll does not assemble photosystem I. The raw emission spectra obtained from these specimens are quite complex, containing overlapping signatures from many pigments. Multivariate curve resolution analysis of the spectral images shows that each spectrum can be decomposed into independently varying contributions from phycocyanin, allophycocyanin, chlorophyll, a specific pool of “low-energy” chlorophyll associated with photosystem I, and protochlorophyllide. The relative contribution of each of these components varies from species to species in a manner consistent with expectations based on the genetic composition of the mutant strains. For example, we observe significant protochlorophyllide emission from the chlL– mutant which was grown in virtual darkness, while this emission is absent in the wild type. To our knowledge, this is the first ever demonstration of the coupling of rigorous deconvolution methods with hyperspectral confocal microscopy to reveal multiple overlapping native pigment emissions from in vivo specimens. We have also observed evidence for an inhomogeneous distribution of the emitting compounds within the cyanobacterium and are currently quantitatively exploring this inhomogeneity in the concentration distributions both within and between cells. This presentation will describe the design, construction and performance of the hyperspectral confocal microscope. Our results for Synechocystis will be described in detail.
Sandia is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DE-ACO4-94AL85000. This work was funded in part by the U.S.Department of Energy’s Genomics: GTL program (genomicsgtl.energy.gov) under project, “Carbon Sequestration in Synechococcus sp: From Molecular Machines to Hierarchical Modeling,” (www.genomes-to-life.org).
* Presenting author
