lk: , Markus Osterhoff – Marten Bernhardt · We thank Jan-David Nicolas and Andrew Wittmeier for...
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References [1] T. Salditt, M. Osterhoff, M. Krenkel, R.N. Wilke, M. Priebe, M. Bartels, S. Kalbfleisch, M. Sprung: Compound focusing mirror and X-ray waveguide optics for coherent imaging and nano-diffraction; J. Synchr. Rad., 2015. [2] S. Kalbfleisch: A Dedicated Endstation for Waveguide-based X-Ray Imaging PhD Thesis, Uni Göttingen, 2012. [3] M. Bernhardt et al., in review. [4] M. Bernhardt, J.D. Nicolas, M. Eckermann, B. Eltzner, F. Rehfeldt, T. Salditt: Anisotropic x-ray scattering and orientation fields in cardiac tissue cells, New Journal of Physics 19, 2017. [5] M. Krenkel, M. Töpperwien, F. Alves, T. Salditt: Three-dimensional single-cell imaging with X-ray waveguides in the holographic regime, Acta Cryst. A, 73, 2017. [6] S. Hell, J. Wichmann: Breaking the diffraction resolution limit by stimulated emission: STED fluorescence microscopy, Optics Letters 19, 1994. [7] V. Westphal, S. Hell: Nanoscale Resolution in the Focal Plane of an Optical Microscope, Physical Review Letters 94, 2005. [8] M. Reuss, J. Engelhardt, S. Hell: Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation, Optics Express 18, 2010. Acknowledgements & Funding We gratefully acknowledge funding by BMBF Verbundforschung, grant No. 05K16MG2 and by Deutsche Forschungsgemein- schaft DFG, SFB 755 and SFB 937. We thank Stefan Hell, Haugen Mittelstädt, Matthias Reuss, and Benjamin Harke from Abberior Instruments for the design and fabrication of the STED microscope. We thank Jan-David Nicolas and Andrew Wittmeier for help during beamtime. We are grateful to Michael Sprung and his team for excellent working conditions. We thank Peter Luley, Bastian Hartmann, and Peter Nieschalk for engineering support, constructions, and (often last-minute) mechanical work. Scanning SAXS with micro and nano-focused X-rays nano-SAXS holography Instrumentation Methods Beating Abbe: Stimulated Emission Depletion easySTED: design and main parts [8] Algorithmically tracked filaments inside a neonatal cardiac tissue cell red and blue lines show filaments found in STED-micrograph and RAAR-reconstruction; common matches are shown in orange; inset: elongation extracted from STXM dataset [3] Reciprocal space local ordering in sample scatters X-rays onto far- field detector; access to “typical length scales” of 1 nm to 100 nm Real space focused beam measures local ordering at spatial resolution of sub-100 nm to few µm (a) excitation laser (b) STED laser (c) acustooptic modulators (d) apertures (e) adjustable mirrors (f,g) glass fibre cable (h) easySTED unit (i,j) mirrors (k) STED objective (l) xyz-translation (m) tubus lens (n) dichroic mirror (o) towards APD (p) APD (q) motorised mirror (r) epi-fluorescence LED (s) dichroic mirror (t) CCD camera sample in focus (~ 300 nm) detector: single-photon counting @ 5 m, e.g. Dectris Pilatus 300k, EigerX 4M pixel size 172 µm … 75 µm continuous scanning (“fly-scan”) using PI P–615 piezo scanner, 10 … 100 Hz; step scans on STED compatible stage sample in waveguide filtered defocus detector: imaging camera @ 5 m, e.g. Photonic Science sCMOS, Gadox scintillator, 6.5 µm pixel size full-field images in multiple distances, accumulation times ~ few seconds + tomographic rotation; Optical fluorescence specific labelling of bio-molecules (functions) with fluo-active markers; diffraction limit ~ 300 nm STED [6–8] annulus-shaped beam stimulates emission, depletes markers; resolution ~ 25 nm non-linear effect Experiment 1. X-ray holography resolution sub-100 nm 2. STED microscopy resolution ~ 100 nm 3. X-ray nano-SAXS real-space ~ 300 nm, reciprocal ~ 10 nm Shuttle between X-ray and STED ~ 60 s, after calibration of relative beam positions Integration into GINIX setup STED microscope is attached to granite block, looking anti-parallel to X-ray beam, horizontally shifted by ~ 300 mm Sample is mounted on SmarAct Hexapod, “shuttle stage” for transfer between X-ray and STED Hexapod rotations to align tip/tilt, translations to align sample’s position; on top: tomographic stage + centre translations X-ray waveguide is mounted on second Hexapod, directly attached to Kirkpatrick-Baez mirror vessel Cardiac tissue cells, scalebar: 10 µm; (b) and (c) show orientation angles of fibres, obtained from optical fluorescence (offline) and nano-SAXS [4]. Synchrotron beam focused to few µm or sub-100 nm; sample is raster- scanned (continuously, “fly-scan”) in 2D, far-field detector taking reciprocal diffraction per real- space position. I [ph./s] 0.00 0.40 lin. scale 1.00E5 1.80E7 lin. scale ω pa [1] 0 180 lin. scale 0 180 lin. scale θ pa [°] θ fs [°] a I [ph./s] 10^0.0 10^2.0 log. scale d 3 1 2 4 3 1 2 4 b c match STED & RAAR STED filaments RAAR filaments principal axes I [ph./s] 0.00E0 2.00E7 lin. scale optics box of STED-microscope safety cap x-ray beam axis STED pos. x-ray pos. sample beam direction automated shuttle transfer sample position k in k out waveguide sample detector focusing mirrors sample detector beamstop aperture focusing mirrors easy STED sample depletion Laser excitation Laser objective STED microscopy source, undulator & monochromator x-ray holography depletion effective spotsize excitation b a scanning SAXS c 1 2 3 z 1 z 2 detector x y z Three imaging modalities built into one synchrotron endstation. Complementary imaging schemes inform each each other; sub-100 nm spatial resolution. We visualise labelled bio- molecules and unlabelled structures of the same specimen in the same environment at the PETRA III / P10 / GINIX [1,2]. Holography using Waveguide-filtered X-rays X-ray waveguides coherence filter, quasi-point-source X-ray holography interference of scattered wave with spherical wave; numerical phase-retrieval Holo-tomography three-dimensional quantitative imaging Mouse alveolar macrophages, stained with BaSO 4 and OsO 4 , measured in four- distances holo-TIE; phase-retrieval + tomography to overcome inconsistencies [5]. Full-field mode Scanning mode brightfield epifluorescence confocal STED APD STED-Laser Excitation Laser absorber emitter absorber emitter mechanical safetyshutter attached to interlock system a b c d e f q glass fiber / towards position g k o g h i j l m n p r s t { comming from glass fiber & position a b o excitation depletion remaining signal scan Instrumentation for Correlative Imaging: Combining Scanning SAXS and X-ray Holography with Optical Fluorescence Institut für Röntgenphysik – Friedrich-Hund-Platz 1 – D–37077 Göttingen Markus Osterhoff – Marten Bernhardt – Matthias Meister – Sarah Köster – Tim Salditt Talk: M. Bernhardt, I2.3 Bioimaging Fri, 10:35, 101CD
Transcript of lk: , Markus Osterhoff – Marten Bernhardt · We thank Jan-David Nicolas and Andrew Wittmeier for...
References[1]T.Salditt,M.Osterhoff,M.Krenkel,R.N.Wilke,M.Priebe,M.Bartels,S.Kalbfleisch,M.Sprung:
CompoundfocusingmirrorandX-raywaveguideopticsforcoherentimagingandnano-diffraction;J.Synchr.Rad.,2015.[2]S.Kalbfleisch:ADedicatedEndstationforWaveguide-basedX-RayImaging
PhDThesis,UniGöttingen,2012.[3] M. Bernhardt et al., in review.[4]M.Bernhardt,J.D.Nicolas,M.Eckermann,B.Eltzner,F.Rehfeldt,T.Salditt:
Anisotropicx-rayscatteringandorientationfieldsincardiactissuecells,NewJournalofPhysics19,2017.[5]M.Krenkel,M.Töpperwien,F.Alves,T.Salditt:
Three-dimensionalsingle-cellimagingwithX-raywaveguidesintheholographicregime,ActaCryst.A,73,2017.[6]S.Hell,J.Wichmann:
Breakingthediffractionresolutionlimitbystimulatedemission:STEDfluorescencemicroscopy,OpticsLetters19,1994.[7]V.Westphal,S.Hell:
NanoscaleResolutionintheFocalPlaneofanOpticalMicroscope,PhysicalReviewLetters94,2005.[8]M.Reuss,J.Engelhardt,S.Hell:
BirefringentdeviceconvertsastandardscanningmicroscopeintoaSTEDmicroscope thatalsomapsmolecularorientation,OpticsExpress18,2010.
Acknowledgements & FundingWe gratefully acknowledge funding by BMBF Verbundforschung, grant No.
05K16MG2
and by Deutsche Forschungsgemein-schaft DFG, SFB 755 and SFB 937.
We thank Stefan Hell, Haugen Mittelstädt, Matthias Reuss, and Benjamin Harke from Abberior Instruments for the design and fabrication of the STED microscope.
We thank Jan-David Nicolas and Andrew Wittmeier for help during beamtime.We are grateful to Michael Sprung and his team for excellent working conditions.We thank Peter Luley, Bastian Hartmann, and Peter Nieschalk for engineering
support, constructions, and (often last-minute) mechanical work.
Scanning SAXS with micro and nano-focused X-rays nano-SAXS holography
InstrumentationMethods
Beating Abbe: Stimulated Emission Depletion
easySTED: design and main parts [8]
Algorithmically tracked filaments inside a neonatal cardiac tissue cell
redandbluelinesshowfilamentsfoundinSTED-micrographandRAAR-reconstruction;commonmatchesareshowninorange; inset:elongationextracted fromSTXMdataset[3]
Reciprocal space localorderinginsamplescattersX-raysontofar-fielddetector; accessto“typicallengthscales”of1 nm to 100 nm
Real space focusedbeammeasureslocalorderingatspatialresolutionof sub-100 nm to few µm (a)excitationlaser
(b)STEDlaser (c)acustoopticmodulators (d)apertures (e)adjustablemirrors(f,g)glassfibrecable (h)easySTEDunit (i,j) mirrors (k)STEDobjective (l)xyz-translation (m)tubuslens (n)dichroicmirror (o)towardsAPD (p)APD (q) motorised mirror (r)epi-fluorescenceLED (s)dichroicmirror (t)CCDcamera
sampleinfocus(~300nm)
detector:single-photon counting@5m, e.g.DectrisPilatus300k,EigerX4M pixelsize172µm…75µm
continuous scanning(“fly-scan”)using PIP–615piezoscanner,10…100Hz; stepscansonSTEDcompatiblestage
sampleinwaveguide filtered defocus
detector:imaging camera@5m, e.g.PhotonicSciencesCMOS, Gadoxscintillator,6.5µmpixelsize
full-field imagesinmultipledistances, accumulationtimes~fewseconds +tomographicrotation;
Optical fluorescence specificlabellingof bio-molecules(functions) withfluo-activemarkers; diffractionlimit~300nm
STED [6–8] annulus-shapedbeamstimulatesemission, depletesmarkers; resolution~25nm non-linear effect
Experiment 1. X-rayholography resolutionsub-100nm
2. STEDmicroscopy resolution ~100nm
3. X-raynano-SAXS real-space ~300nm, reciprocal ~10nm
Shuttle betweenX-rayandSTED ~60s,aftercalibration ofrelativebeampositions
Integration into GINIX setup
STEDmicroscopeisattachedtograniteblock, lookinganti-parallel to X-ray beam, horizontallyshiftedby~300mm
SampleismountedonSmarAct Hexapod, “shuttlestage”fortransferbetweenX-rayandSTED
Hexapodrotationstoalign tip/tilt, translationstoalignsample’sposition; ontop:tomographic stage+centretranslations
X-ray waveguideismountedonsecondHexapod, directlyattachedtoKirkpatrick-Baezmirrorvessel
Cardiactissuecells,scalebar:10µm;(b)and(c)showorientationanglesoffibres,obtainedfromopticalfluorescence(offline)andnano-SAXS[4].
Synchrotronbeamfocusedtofewµmorsub-100nm;sampleisraster-scanned(continuously,“fly-scan”)in2D,far-fielddetectortakingreciprocaldiffractionperreal-spaceposition.
I [ph./s]
0.00 0.40lin. scale
1.00E5 1.80E7lin. scale
ωpa [1]
0 180lin. scale
0 180lin. scale
θpa [°]θfs [°]
a
I [ph./s]
10^0.0 10^2.0log. scale
d
3
1
24
3
1 2
4
b c
match STED & RAARSTED �lamentsRAAR �laments
principal axes
I [ph./s]0.00E0 2.00E7lin. scale
optics box of STED-microscope
safety cap
x-ray beam axis
STED pos.
x-ray pos.
sample
beamdirection
autom
ated
shuttle
transf
er
sample position
kinkout
waveguide sample detectorfocusingmirrors
sample detectorbeamstopaperturefocusingmirrors
easySTED
sampledepletionLaser
excitationLaser objective
STED microscopy
source,undulator &monochromator
x-ray holography
depletion
effectivespotsize
excitation
b
a
scanning SAXS
c
1
2
3
z1 z2
detector
xy
z
Three imaging modalities built into one synchrotron endstation.
Complementary imaging schemes inform each each other; sub-100 nm spatial resolution.
We visualise labelled bio-molecules and unlabelled structures of the same specimen in the same environment at the PETRA III / P10 / GINIX [1,2].
Holography using Waveguide-filtered X-raysX-ray waveguides coherence filter, quasi-point-source
X-ray holography interferenceofscatteredwavewithsphericalwave; numericalphase-retrieval
Holo-tomography three-dimensional quantitativeimaging
Mousealveolarmacrophages,stainedwithBaSO4 and OsO4,measuredinfour-distancesholo-TIE;phase-retrieval+tomographytoovercomeinconsistencies[5].
Full-
field
mod
eSc
anni
ng m
ode
brightfield epifluorescence
confocal STED
APD
STED-Laser
Excitation Laserabsorber
emitter
absorber
emitter
mechanicalsafetyshutter
attached to interlock system
a
b
cd
e
f
q
glass fiber / towards position g
k
og
h
ij
lm
n
p
r
s
t
{
comming from glass fiber & positiona bo
excitation
depletion
remainingsignal
scan
Instrumentation for Correlative Imaging:Combining Scanning SAXS and X-ray Holography with Optical Fluorescence InstitutfürRöntgenphysik –Friedrich-Hund-Platz1–D–37077Göttingen
MarkusOsterhoff–MartenBernhardt– MatthiasMeister–SarahKöster–TimSaldittTalk: M. Bernhardt,
I2.3 Bioimaging
Fri, 10:35, 101CD
