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