CENTRAL LASER FACILITY Annual Report 2010 - 201136
LASERS FOR SCIENCE FACILITY PROGRAMME Biology
A photoacoustic (PA) spectroscopy systemhas been built to study the differencesbetween the PA spectra of oxygenated anddeoxygenated blood, and various PAcontrast agents, with a view to optimisingthe identification of these media in clinicalPA images. A tunable laser delivers pulses(ns) of light into a sample causing it tomomentarily expand and emit a pressurewave, the energy of which is measured bythe computer using a digital oscilloscopethat samples the signal from a focused 7.5 MHz ultrasound transducer. Oncescanned at multiple wavelengths (400-700nm) the resulting optical spectra arecorrected for some system variables,
including the wavelength-dependent laserenergy. The measured PA spectrum ofoxygenated blood strongly resemblespublished optical absorption spectrum.Also the PA absorption spectrum of goldnano-particles compares well withstandard spectrophotometermeasurements. This system may haveapplications as a laboratoryspectrophotometer for absorption spectra.
D. Birtill1, A. Shah1,2, M. Jaeger1,2, A. Gertsch1,J. Bamber1
(1Joint Department of Physics)
(2CRUK-EPSRC Cancer Imaging Centre,Institute of Cancer Research, UK)
Two-photon excited fluorescence lifetime imaging of theintracellular uptake of (E)-combretastatin derivatives
Intracellular uptake by prostate cancercells of the anticancer drug combretastatinA4 and analogues has been measured usingmultiphoton excited fluorescence lifetimeimaging. Fluorescence from the less activetrans isomers has been used to produceimages (A) of the intracellular distributionof these compounds. The fluorescencelifetime image (B) shows a distribution oflifetimes (C, D) centred around 1.2nanoseconds. Complementary studies in arange of solvents indicate that thisfluorescence lifetime reports a viscousenvironment. Co-localisation studies with
the lipid probe, nile red, suggest the drug isaccumulated within cellular membranesand lipid droplets. Uptake of thecombretastatin is rapid (E), occurring inminutes at room temperature. Combiningfluorescence intensities and lifetimesshows that the intracellular concentrations(these are mapped within a field of cells (F))exceed those in the surrounding mediumby between 2 and 3 orders of magnitude,indicating accumulation is likely based onlipophilicity of the combretastatinmolecule.
R. H. Bisby, J. A. Hadfield, A. T. McGown, K. M. Scherer (University of Salford, UK)
S. W. Botchway, A. W. Parker (STFC, Rutherford Appleton Laboratory, UK)
R. H. [email protected]
Diagram of thephotoacoustic setup
Two-photon fluorescence lifetimeimaging of a fluorinatedcombretastatin in PC-3 cells. The intensity (A) and lifetime (B)images are shown after incubationwith drug (10 M) for 10 minutes.The lifetime distribution (C) is showntogether with a single pixelfluorescence decay (D). The time-dependent increase in intracellularfluorescence intensity after additionof E-CA4F to the cells is shown in E.A map of intracellular concentration(in M) is shown in F. (The imagesshow a field of 70 x 70 m)
ler74Typewritten [email protected]
LASERS FOR SCIENCE FACILITY PROGRAMME Biology
CENTRAL LASER FACILITY Annual Report 2010 - 2011 37
The plant secretoryome: protein-protein interactionsin the higher plant secretory pathway
We have been continuing to look atprotein-protein interactions in the plantGolgi apparatus, concentrating oninteractions between glycosyl transferasesin the cisternal membranes of the Golgistack. Initial results from FLIM dataindicate that FRET can occur betweenfluorescent proteins tagged to a range ofcis-Golgi enzymes. Our data suggests forthe first time homo- and hetero-oligomerisation between plant Golgiprocessing enzymes.
In a new project with a collaborator fromINRA, Nantes, we have been looking at theformation of wheat storage protein bodiesin tobacco cells. FRET-FLIM (Figure) was
used to show the interaction between N- and C- terminal domains of the storageproteins -gliadin, thus confirming theconfocal and biochemical data fromexpression of the protein in tobacco.
During the year we obtained the first datafrom a newly constructed TIRF-based lasertrapping microscope and successfullycaptured Golgi bodies targeted withfluorescent proteins expressed in tobaccoleaves. The instrument will be used for astudy of the interactions of the plant Golgiapparatus with the endoplasmic reticulumand cytoskeleton.
C. Hawes, I. Sparkes, A. Osterrieder, J. Schoberer (Oxford Brookes University)
S. Botchway, and A. Ward (STFC, Rutherford Appleton Laboratory, UK)
C. Hawes [email protected]
A .Expression of the C terminal domain of-gliadin fused to-GFP and N terminaldomainfused to RFP in tobacco leaves showing co-localisation of the proteinbodies. B-F. FRET-FLIM analysis in tobacco leaf epidermal cells. Lifetime imagesof Cter--gliadin-GFP (B), and Cter--gliadin-GFP in a cell coexpressing Nter--gliadin-mRFP (D) are shown. Insets are confocal images showingexpression of Nter--gliadin-mRFP (red) and or Cter--gliadin-GFP (green) in thecells shown. C,E Curves of B & E showing reduction in GFP lifetime in co-expressing cells.
Flavoproteins are a large family of proteinsthat contain a covalently or non-covalentlybound flavin cofactor. Although theisoalloxazine ring of the flavin functionspredominantly as an electron transferintermediate in biochemical oxidation-reduction reactions, there are at least threesubfamilies of flavoproteins that functionas photoreceptors: the light-oxygen-voltage (LOV) domain proteins, thecryptochromes, and the Blue Light UsingFAD (BLUF)-domain proteins.Thechromophores in other well knownphotoreceptors such as the rhodopsins,xanthopsins and phytochromes undergo anisomerization when light is absorbed.However, this is not the case forflavoprotein receptors. Consequently thereis substantial interest in understanding howabsorption of light by the flavin is coupledto the conformational change(s) that leadto the signaling state. In the LOV domain
proteins a cysteinyl-flavin adduct istransiently formed in the signaling state.However, in the cryptochromes and BLUFproteins, the initial structural changesresulting from photoexcitation are less wellestablished.
A. Lukacs, R-K. Zhao, S. R. Meech(University of East Anglia)
R. Brust, A. Haigney, P. J. Tonge(Stony Brook University, USA)
G. M. Greetham, M. Towrie(STFC, Rutherford Appleton Laboratory, UK)
S. R. [email protected]
TRIR spectra of dAppABLUF (black), lAppABLUF(red) and Q63E AppABLUF (green) bound toFAD. Protein concentration was 2 mM in pD 8phosphate buffer and the TRIR spectra wererecorded with a time delay of 3 ps. Thespectra have been normalized to the intenseFAD ring bleach mode at 1547 cm-1.
Probing the mechanism of blue light sensingBLUF domain proteins: A Study through transient infra-redspectroscopy, isotope editing and mutagenesis
CENTRAL LASER FACILITY Annual Report 2010 - 201138
LASERS FOR SCIENCE FACILITY PROGRAMME Biology
Kinetically stable metal complexes for multimodality PET/SPECTand optical fluorescence microscopy probed in vitro by FLIM
J. R. Dilworth, S. Faulkner M. W. Jones and P. A. Waghorn(University of Oxford, UK)
S. I. Pascu and R. Arrowsmith(University of Bath, UK)
S. W. Botchway and A. Parker
(STFC, Rutherford Appleton Laboratory, UK)
S. I. [email protected]
Molecular imaging is a rapidly expandingfield of global importance for both thediagnosis and personalised therapy of arange of disease states. There is anincreasing demand for molecular probesthat can be used for imaging and earlydetection of specific cancers whichrepresent major life risks worldwide, suchas breast, colon, and prostate cancer.Shortage of oxygen (hypoxia) is a commoncause of cancer treatment failure: reliableimaging tools for tumour hypoxia would be
invaluable in planning treatment regimensand predicting clinical outcomes. Confocalfluorescence microscopy has been usedextensively to track compounds and followprocesses in cells. We have designed andtested in vitro a series of kinetically stablefluorescent metal complexes, that can beused for whole body imaging using gammaor positron emission and may be monitoredin cells by virtue of their 1 or 2-photonexcited fluorescence and fluorescencelifetime imaging (FLIM).
Two-Photon Fluorescence lifetimeimaging map (ex 910 nm, 20 minincubation) in prostate cancer cellsfor a new gallium aromatic complexwith relevance to positron emissiontomography of cancer cells
Porous carbon microspheres: Solution phenomenaand cellular uptake
Carbon based micro and nanomaterials areattractive for biological applicationsbecause of their excellent biocompatibilityprofile. Porous microparticles, prepared viaa routine synthesis, are easy to handle andpurify, and combine a high specific surfacearea with carbons high functionality. Wewish to leverage these properties for thepurposes of cell imaging and delivery. Inthis work, porous carbon microsphereswith a high specific surface area wereprepared and their fundamental propertiesstudied using Raman optical tweezers.These experiments showed that 532 nmexcitation of microspheres trapped insolvents that display poor heat conduction
resulted in graphitization andincandescence. These phenomena werealso observed for microspheres in thewater presence of a cationic lipid (DOTAP).The uptake of the particles by cells wasdemonstrated by fluorescence confocalmicroscopy ima