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:
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