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1.  Geranylgeranyl Reductase and Ferredoxin from Methanosarcina acetivorans Are Required for the Synthesis of Fully Reduced Archaeal Membrane Lipid in Escherichia coli Cells 
Journal of Bacteriology  2014;196(2):417-423.
Archaea produce membrane lipids that typically possess fully saturated isoprenoid hydrocarbon chains attached to the glycerol moiety via ether bonds. They are functionally similar to, but structurally and biosynthetically distinct from, the fatty acid-based membrane lipids of bacteria and eukaryotes. It is believed that the characteristic lipid structure helps archaea survive under severe conditions such as extremely low or high pH, high salt concentrations, and/or high temperatures. We detail here the first successful production of an intact archaeal membrane lipid, which has fully saturated isoprenoid chains, in bacterial cells. The introduction of six phospholipid biosynthetic genes from a methanogenic archaeon, Methanosarcina acetivorans, in Escherichia coli enabled the host bacterium to synthesize the archaeal lipid, i.e., diphytanylglyceryl phosphoglycerol, while a glycerol modification of the phosphate group was probably catalyzed by endogenous E. coli enzymes. Reduction of the isoprenoid chains occurred only when archaeal ferredoxin was expressed with geranylgeranyl reductase, suggesting the role of ferredoxin as a specific electron donor for the reductase. This report is the first identification of a physiological reducer for archaeal geranylgeranyl reductase. On the other hand, geranylgeranyl reductase from the thermoacidophilic archaeon Sulfolobus acidocaldarius could, by itself, replace both its orthologue and ferredoxin from M. acetivorans, which indicated that an endogenous redox system of E. coli reduced the enzyme.
doi:10.1128/JB.00927-13
PMCID: PMC3911245  PMID: 24214941
2.  Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination 
Biomedical Optics Express  2013;4(11):2396-2410.
We demonstrate super-resolution imaging with background fluorescence rejection by interferometric temporal focusing microscopy, in which temporal focusing is combined with structured illumination. The lateral resolution and the optical sectioning capability are simultaneously improved by factors of 1.6 and 1.4, respectively, compared to conventional temporal focusing microscopy. Fluorescent beads (200 nm diameter) that are difficult to distinguish from the background fluorescence in conventional temporal focusing microscopy, are clearly visualized by interferometric temporal focusing microscopy.
doi:10.1364/BOE.4.002396
PMCID: PMC3829536  PMID: 24298403
(180.4315) Nonlinear microscopy; (190.4180) Multiphoton processes
3.  Implementation of spatial overlap modulation nonlinear optical microscopy using an electro-optic deflector 
Biomedical Optics Express  2013;4(10):1937-1945.
A spatial overlap modulation (SPOM) technique is a nonlinear optical microscopy technique which enhances the three-dimensional spatial resolution and rejects the out-of-focus background limiting the imaging depth inside a highly scattering sample. Here, we report on the implementation of SPOM in which beam pointing modulation is achieved by an electro-optic deflector. The modulation and demodulation frequencies are enhanced to 200 kHz and 400 kHz, respectively, resulting in a 200-fold enhancement compared with the previously reported system. The resolution enhancement and suppression of the out-of-focus background are demonstrated by sum-frequency-generation imaging of pounded granulated sugar and deep imaging of fluorescent beads in a tissue-like phantom, respectively.
doi:10.1364/BOE.4.001937
PMCID: PMC3799657  PMID: 24156055
(180.4315) Nonlinear microscopy; (180.2520) Fluorescence microscopy; (190.4180) Multiphoton processes
4.  Simultaneous imaging of two-photon absorption and stimulated Raman scattering by spatial overlap modulation nonlinear optical microscopy 
Biomedical Optics Express  2013;4(9):1548-1558.
Imaging of simultaneous two-photon absorption and stimulated Raman scattering is accomplished by detecting the intensity changes of the two-color pulses simultaneously and the mathematical operations of addition and subtraction. The stimulated Raman scattering is quantitatively separated from the two-photon absorption, generated in a mixed solution in which a glycerin solution is miscible in various proportions with a quantum dot solution. Our technique is applied to simultaneous two-photon absorption and stimulated Raman scattering imaging.
doi:10.1364/BOE.4.001548
PMCID: PMC3771826  PMID: 24049676
(180.4315) Nonlinear microscopy; (180.5655) Raman microscopy; (190.4180) Multiphoton processes
5.  Background-free deep imaging by spatial overlap modulation nonlinear optical microscopy 
Biomedical Optics Express  2012;3(7):1594-1608.
We demonstrate how the resolution and imaging depth limitations of nonlinear optical microscopy can be overcome by modulating the spatial overlap between two-color pulses. We suppress out-of-focus signals, which limit the imaging depth, by a factor of 100, and enhance the lateral and axial resolution by factors of 1.6 and 1.4–1.8 respectively. Using spatial overlap modulation, we demonstrate background-free three-dimensional imaging of fixed mouse brain tissue at depths for which the signals of the conventional technique are swamped by background noise from out-of-focus regions.
doi:10.1364/BOE.3.001594
PMCID: PMC3395484  PMID: 22808431
(180.4315) Nonlinear microscopy; (180.2520) Fluorescence microscopy; (170.5660) Raman spectroscopy; (190.4180) Multiphoton processes
6.  Archaeal Phospholipid Biosynthetic Pathway Reconstructed in Escherichia coli 
Archaea  2012;2012:438931.
A part of the biosynthetic pathway of archaeal membrane lipids, comprised of 4 archaeal enzymes, was reconstructed in the cells of Escherichia coli. The genes of the enzymes were cloned from a mesophilic methanogen, Methanosarcina acetivorans, and the activity of each enzyme was confirmed using recombinant proteins. In vitro radioassay showed that the 4 enzymes are sufficient to synthesize an intermediate of archaeal membrane lipid biosynthesis, that is, 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate, from precursors that can be produced endogenously in E. coli. Introduction of the 4 genes into E. coli resulted in the production of archaeal-type lipids. Detailed liquid chromatography/electron spray ionization-mass spectrometry analyses showed that they are metabolites from the expected intermediate, that is, 2,3-di-O-geranylgeranyl-sn-glycerol and 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphoglycerol. The metabolic processes, that is, dephosphorylation and glycerol modification, are likely catalyzed by endogenous enzymes of E. coli.
doi:10.1155/2012/438931
PMCID: PMC3357500  PMID: 22645416
7.  High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process 
Biomedical Optics Express  2010;1(3):791-797.
We demonstrate high-resolution fluorescence microscopy based on a cyclic sequential multiphoton (CSM) process, which gives rise to fluorescence emission following a sequence of cyclic transitions between the bright and dark states of a fluorophore induced by pump and reverse light. By temporally modulating the reverse intensity, we can extract the fluorescence signal generated through the CSM process. We show that the demodulated fluorescence signal is nonlinearly proportional to the excitation intensities and it gives a higher spatial resolution than that of a confocal microscope.
doi:10.1364/BOE.1.000791
PMCID: PMC3018047  PMID: 21258510
(180.2520) Fluorescence microscopy; (180.6900) Three-dimensional microscopy; (120.1880) Detection
8.  Measurement of two-photon excitation spectrum used to photoconvert a fluorescent protein (Kaede) by nonlinear Fourier-transform spectroscopy 
Biomedical Optics Express  2010;1(2):687-693.
We demonstrate the measurement of two-photon excitation (TPE) spectra, used not only for fluorescence but also for photoconversion in green-to-red photoconvertible Kaede, using nonlinear Fourier-transform spectroscopy. It was found that in unphotoconverted Kaede, the TPE spectrum for photoconversion is much different to that for green-fluorescence. This is similar to the difference between the one-photon excitation of photoconversion in the neutral form and that of green-fluorescence in the ionized form.
doi:10.1364/BOE.1.000687
PMCID: PMC3017996  PMID: 21258500
(300.6420) Spectroscopy, nonlinear; (170.2520) Fluorescence microscopy

Results 1-8 (8)