X-ray Imaging & Microscopy Applications and Future...

Shen – October 20, 2003

XX--ray Imaging & Microscopy Applicationsray Imaging & Microscopy Applicationsand Future Opportunitiesand Future Opportunities

⇒⇒ Motivation: Motivation: proposed energy recovery proposed energy recovery linaclinac (ERL) x(ERL) x--ray sourceray sourcescientific needs in imaging & in synchrotron sciencescientific needs in imaging & in synchrotron science

⇒⇒ Discussion: Discussion: why ERL ideal for advancing xwhy ERL ideal for advancing x--ray microscopyray microscopy

⇒⇒ New frontier of xNew frontier of x--ray science: structure of ray science: structure of nonperiodicnonperiodic specimenspecimenby imaging & microscopyby imaging & microscopy

Qun Shen Cornell High Energy Synchrotron Source (CHESS)

and Department of Materials Science & EngineeringCornell University, Ithaca, NY

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⇒⇒ Overview & examples:Overview & examples: xx--ray imaging & microscopy applicationsray imaging & microscopy applications


XX--ray Scienceray Science

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~85% structures by ~85% structures by xx--ray crystallographyray crystallography

2003 Nobel Prize 2003 Nobel Prize in Chemistry:in Chemistry:

Roderick Roderick MacKinnonMacKinnon(Rockefeller Univ.)(Rockefeller Univ.)

11stst KK++ channel structure channel structure by xby x--ray crystallography ray crystallography

based on CHESS data (1998)based on CHESS data (1998)

Ion channel proteinIon channel protein

CHESSCHESS


XX--ray Imaging & Microscopy ray Imaging & Microscopy

⇒ Century-old (since 1895), yet young scientific discipline

⇒ Highly dependent on x-ray source & optics

⇒ Rapid development after 3rd

generation SR sources

(search in INSPEC physics literature)

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Wilhelm Röntgen1st Physics Nobel (1901)


Energy Recovery Energy Recovery LinacLinac (ERL)(ERL)

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Georg HoffstaetterLEPP Journal Club311 Newman Lab.Friday, 3:45 pmOct. 24, 2003

One option:ERL @ CESR


ERL PropertiesERL Properties

• ERL would be the world's first high-intensitydiffraction-limited hard x-ray source

• ERL would produce a round point source of hard x-rays, ideal for imaging & microscopic applications

• ERL would bring high coherence to hard x-ray regime, enabling new opportunities for imaging science

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ERL emittance (0.15Å)ESRF emittance(4nm x 0.01nm)

Diffraction limited @ 8keV (0.123Å)

Diffraction limited source: 2πσ'σ = λ/2 or ε = λ/4π

εx = σx σx’

εy = σy σy’


Nature's DimensionsNature's Dimensions

100 µm

Size (m)

10 − 910 − 8 10 −1010 − 710 − 610 − 510 − 410 − 310 − 210 − 1

H2O

Micro-organisms

Cells

Organelles

Macromolecules

Small molecules

Plants & Animals

Atoms

Reovirus

Ribosome

Ge/Si dots

Si pillars

microchiphuman hair

ant

Helisoma

plants

onion cell

cheek cell

yeast

Cystine

Si (111)7x7

Naked eye

Optical microscopy

Electron microscopy

X-ray radiology

X-ray crystallography

TEM

Ultrasound, MRIAFM

STM

NMR

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Atoms

Three Imaging CategoriesThree Imaging Categories

100 µm

Size (m)

10 − 910 − 8 10 −1010 − 710 − 610 − 510 − 410 − 310 − 210 − 1

H2O

Organisms

Cells

Organelles

Macromolecules

Small molecules

Plants & Animals

Reovirus

Ribosome

Ge/Si dots

Si pillars

microchiphuman hair

ant

Helisoma

plants

onion cell

cheek cell

yeast

Cystine

Si (111)7x7

Medical Imagingà low contrast tissuesà real time imagingà high resolution

Cellular Imagingà subcellular organellesà protein locationsà natural state

Molecular Imagingàwithout need of crystalsà atomic resolutionà less damage

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XX--ray Microscopy Basicsray Microscopy Basics

Holography Holography

Direct imaging (radiography)Direct imaging (radiography)

FarFar--field diffraction field diffraction

ρ(x,y)

Scanning microscope (SXM) Scanning microscope (SXM)

Transmission microscope (TXM)Transmission microscope (TXM)

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XX--ray Contrastray Contrast

⇒⇒ Absorption contrast: Absorption contrast: µµzz = 4= 4πβπβzz/λ ∼ λ/λ ∼ λ33

⇒⇒ Phase contrast: Phase contrast: φφ(z) = 2(z) = 2πδπδzz/λ ∼ λ/λ ∼ λ

Refraction index: n = 1 Refraction index: n = 1 −− δδ −− iiββz

ρ(x,y)

E(z) ~ EE(z) ~ E00 ee−−ii22ππ((−−δδ−−iiββ)z/)z/λλ ~ E~ E00 eeii22πδπδz/z/λλ−−22πβπβz/z/λλ

I(z) ~ |E(z)|I(z) ~ |E(z)|22 ~ I~ I00 ee−−44πβπβz/z/λλ

ρ(x,y)

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Mori et al. (2002): broken rib with surrounding soft tissue


Phase vs. Absorption ContrastPhase vs. Absorption Contrast

• Phase contrast is x104 higher than absorption contrast for protein in water @ 8keV

Kagoshima et al. (2001): protein C94H139N24O31Sρ=1.35g/cm3, t=0.1µm in 10µm water

Phase contrast(with Ta phase plate)

Absorption contrast

• Required dose reduced due to phase contrast

C94H139N24O31S

1010

108

106

104

103102 104

Kirz (1995): 0.05µm protein in 10µm thick ice

X-ray Energy (eV)

Do

se (G

ray)

absorption contrast

phase contrast

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Examples of XExamples of X--ray Imaging ray Imaging -- II

100 µm

Size (m)

10 − 910 − 8 10 −1010 − 710 − 610 − 510 − 410 − 310 − 210 − 1

H2O

Organisms

Cells

Organelles

Macromolecules

Plants & Animals

Reovirus

Ribosome

Ge/Si dots

Si pillars

microchiphuman hair

ant

Helisoma

plants

onion cell

cheek cell

yeast

Cystine

Si (111)7x7




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Atoms

Small molecules


Phase Enhanced XPhase Enhanced X--ray Imagingray Imaging

• Phase radiography

• Diffraction enhanced imaging

• Interferometric imaging

Three Ways to See Phases ….Three Ways to See Phases ….

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a

Direct Phase ImagingDirect Phase Imaging

a

z

ρ(x,y)

λ Ι(u,v)

Propagation based method:(near-field)

Nugent et al., PRL 77, 2961 (1996)Paganin & Nugent, PRL 80, 2586 (1998)

Image interpretation: I(u, v) è ρ(x, y) = ?

Fresnel diffraction method:(intermediate-field)

Snigerev et al, RSI 66, 5486 (1996)Cloetens et al, J. Phys. D29, 133 (1996)Wilkins et al, Nature 384, 335 (1996)

Mayo et al. J. Microscopy (2002) 207, 79-96

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Phase Imaging with InterferometerPhase Imaging with Interferometer

A. Momose, J. Synch. Rad. (2002) 9, 136-142.

Phase image

Blood vessels in mouse liver

w/o contrast agent!

Phase-contrast x-ray CT of a rabbit liver

tissue (5mm in diameter)

Absorption imageCancerous lesion

Photon Factory 17.7 keV

rat cerebellum12.4 keVsame dose

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2a Sclerotic blood vessel

Tendon calcification

Air bubbles

PP

SDP

M

Bloodvessel

Tendon of flexor hallucis longus

Air bubbles

Fat pad

skin

Tendon of Extensor hallucis longus

Nail plate

*Tendon calcification

bDiffraction enhanced image

Conventional absorption imageof the great toe

M: metatarsalS: sesamoid bonesPP: proximal phalanxDP: distal phalanx

The major soft tissue structures that can be identified in DEI (not visible in absorption radiograph) include the two major tendons of the toe, the fat pad under the ball of the foot and the skin.

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Journal of Anatomy (2003)


RealReal--Time: Insect BreathingTime: Insect Breathing

Science (2003) 299, 598-599.

Field museum of Chicago & APS, Argonne National Lab.

wood beetle

•• Animal functionsAnimal functions•• BiomechanicsBiomechanics•• Internal movements Internal movements •• New findings not known beforeNew findings not known before

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• ERL would extend these studies to much higher lateral resolution and faster time scales


50m 0.5m4mm

8m

S=10µm

s=0.1µm

30 mrad

Resolution d = 0.1µmRequired pixel size: <0.2mm

M = 8m/4mm= 2000x

Image size: 24cmField view: 0.12mm

Ultimate resolution d = 6µm/2000 = 3nmif focal spot size of 3nm can be achieved

Focusing optic: 1m-long ML KB mirrors @ 1o

double triangle Si (111) @ 10o

0.3 mradERL: 3cm-long '2 + 2' ID

PointPoint--Source Projection MicroscopySource Projection Microscopywith ERLwith ERL

ERL has the potential to improve lateral resolution in direct phase imaging by two orders of magnitude

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Summary on Summary on Medical & Small Animal ImagingMedical & Small Animal Imaging

•• With fast detectors such as pixelWith fast detectors such as pixel--arrays, would offer ultraarrays, would offer ultra--fast fast highhigh--resolution xresolution x--ray radiography at video frequency of MHz.ray radiography at video frequency of MHz.

•• PhasePhase--enhanced xenhanced x--ray imaging allows observation of ray imaging allows observation of weakweak--absorbing features that are otherwise not observable absorbing features that are otherwise not observable with conventional radiography, with less radiation dosage.with conventional radiography, with less radiation dosage.

•• Many experiments can be done at existing synchrotron Many experiments can be done at existing synchrotron sources, but realsources, but real--time (or faster) imaging at high spatial time (or faster) imaging at high spatial resolution of ~1resolution of ~1µµm or less appears to be difficult.m or less appears to be difficult.

Projection Microscopy with ERL:Projection Microscopy with ERL:

•• Would improve lateral resolution by 2Would improve lateral resolution by 2--3 orders of magnitude, 3 orders of magnitude, down to subdown to sub--µµm scales.m scales.

•• With short xWith short x--ray pulses, ERL offers the potential for flash ray pulses, ERL offers the potential for flash imaging at time scales of < 1 imaging at time scales of < 1 psps..

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Examples of XExamples of X--ray Imaging ray Imaging -- IIII

100 µm

Size (m)

10 − 910 − 8 10 −1010 − 710 − 610 − 510 − 410 − 310 − 210 − 1

H2O

Organisms

Cells

Organelles

Macromolecules

Plants & Animals

Reovirus

Ribosome

Ge/Si dots

Si pillars

microchiphuman hair

ant

Helisoma

plants

onion cell

cheek cell

yeast

Cystine

Si (111)7x7




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Atoms

Small molecules


Challenges in Cell BiologyChallenges in Cell Biology

•• Cellular imaging is of critical importance in the post-genomic era as we face the daunting task of determining the function of the vast number of genes and gene products identified as a result of modern molecular biology techniques.

• The basic information about the organization of cells and subcellular structures is critical for our understanding of cellular functions – central theme in cell biology.

•• The challenge in cell biology has been to obtain the best resolution 3D morphological information about cells that are examined in a state most closely resembling their natural environment.

•• X-ray microscopy is proving to be a powerful method in that (a) it provides far better resolution than confocal laser microscopy, and (b) one can examine whole, fully-hydrated cells, avoiding potential artifacts introduced by the dehydration, embedding and sectioning that is required for electron microscopy.

Transmission xTransmission x--ray microscope image of mouse ray microscope image of mouse 3T3 fibroblasts, a type of connective3T3 fibroblasts, a type of connective--tissue cell, tissue cell, with spatial resolution of 36 nm, clearly shows with spatial resolution of 36 nm, clearly shows features features ---- such as nucleoli and the sharp such as nucleoli and the sharp nuclear membrane nuclear membrane ---- not resolvable with opticalnot resolvable with opticalconfocalconfocal microscopy.microscopy.

C. Larabell (LBNL) ALS XM-1

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SubcellularSubcellular Imaging with LabelsImaging with Labels

C.C. LarabellLarabell (LBNL): (LBNL): cell cell biologist, usingbiologist, using confocalconfocal & & electron microscopy electron microscopy

•• Immunocytochemistry: a method for identifying structure-function relation-ships of cells and proteins in cells by looking at the sub-cellular location of these proteins

•• Critical proteins inside cells are labeled so that X-rays could be used to identify them

• X-ray microscopy gives cell biologists a whole new way of looking at their samples

•• Nuclear pore complex (NPC): a large (50-100 MD) collection of proteins which organize the ~9 nm openings in nuclear membranes of eukaryotic cells.

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CryoCryo--TomographyTomography of Whole Hydrated Cellsof Whole Hydrated Cells

Drosophila embryonic cell (G. Schneider, LBNL)

Green = nucleolusGold = sex-determining protein

(labeled with 1nm Au & Ag-enhanced)22

• Soft x-ray microscope, depth of field ~10µm

=> Unique 3D information about cells and interactions of intracellular organelles

• Flash-frozen, whole, fully hydrated cells


Summary on Cellular MicroscopySummary on Cellular Microscopy

•• bring high brilliance xbring high brilliance x--ray microscopy to hard xray microscopy to hard x--ray regime, allowing ray regime, allowing (a) possibility with phasing contrast and less dose requirem(a) possibility with phasing contrast and less dose requirement,ent,(b) substantial increase in depth of view for 3D (b) substantial increase in depth of view for 3D tomographytomography, and, and(c) extended range of elemental labels in the higher energy (c) extended range of elemental labels in the higher energy region. region.

•• deliver an ultradeliver an ultra--small, round, isotropic resolution functionsmall, round, isotropic resolution function

•• XX--ray microscopy is beginning to show its potential ray microscopy is beginning to show its potential as a as a powerful toolpowerful tool in cell biologyin cell biology

•• XX--ray ray microscopymicroscopy could have high impact on could have high impact on cell cell biologybiology in a way that is similar to what synchrotron in a way that is similar to what synchrotron xx--ray ray crystallographycrystallography is doing on is doing on molecular biologymolecular biology

•• Substantial increase in demand for highSubstantial increase in demand for high--brightness brightness xx--ray microscopesray microscopes is expected for the next decadeis expected for the next decade

Cellular Microscopy with ERL:Cellular Microscopy with ERL:

ERL would be an ideal xERL would be an ideal x--ray source to satisfy the need for ray source to satisfy the need for highhigh--quality cellular and quality cellular and subcellularsubcellular imaging. It would:imaging. It would:

•• improve soft ximprove soft x--ray microscope brightness by 2ray microscope brightness by 2--3 orders of magnitude3 orders of magnitude

~85% structures by ~85% structures by xx--ray crystallographyray crystallography

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Examples of XExamples of X--ray Imaging ray Imaging -- IIIIII

100 µm

Size (m)

10 − 910 − 8 10 −1010 − 710 − 610 − 510 − 410 − 310 − 210 − 1

H2O

Organisms

Cells

Organelles

Macromolecules

Plants & Animals

Reovirus

Ribosome

Ge/Si dots

Si pillars

microchiphuman hair

ant

Helisoma

plants

onion cell

cheek cell

yeast

Cystine

Si (111)7x7




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Atoms

Small molecules


Molecular Imaging & MicroscopyMolecular Imaging & Microscopy

• Spatial resolution: essentially no limit.(only limited by ∆λ/λ and weak signals at large angles)

• Molecular imaging requires lateral resolution << 10nm => current limit on optics

• Present limitations: Lack of intense, coherent microbeams. ERL would change this dramatically.

• Coherent diffraction from noncrystalline specimen => continuous diffraction pattern

Coherent X-rays

Miao et al. Nature (1999) >>>soft x-ray diffraction reconstruction to 75 nm

• Diffraction imaging is analogous to crystallography, but for noncrystallinematerials

• To move beyond the limit, lensless imaging or diffraction imaging using coherent beam is an attractive alternative

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Imaging Whole Escherichia Coli Bacteria Using Single Particle X-ray Diffraction

Jianwei Miao*†, Keith O. Hodgson*‡, Tetsuya Ishikawa§,

Carolyn A. Larabell¶?, Mark A. LeGros**, and Yoshinori Nishino§

Diffraction image to ~30nm resolution

Total dose to specimen ~ 8x106 Gray

E. Coli bacteria ~ 0.5 µm by 2 µm

SPring-8, λ = 2 Å, pinhole 20 µm

Miao et al., Proc. Nat. Acad. Sci. (2003)

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Labeled with maganese oxide


Simulations Using ERL BeamSimulations Using ERL Beam

V. Elser, Acta Cryst. A (2003)

• Simulation of diffraction pattern using coherent beam from ERL, with statistical noise & missing beam-stop region.

• Image retrieval in collaboration with Elser (Physics, Cornell)

• Assembly of 2900 gold atoms in 10nm box

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counts/120s

Hi-coh ERL with 30:1 focus: I0= 5x1014 ph/s/µm2


Summary on Summary on Molecular ImagingMolecular Imaging

•• ERL would offer 2 orders of magnitude increase in ERL would offer 2 orders of magnitude increase in coherent flux compared to presentcoherent flux compared to present--day synchrotron day synchrotron sources.sources.

•• Coherent xCoherent x--ray diffraction imaging offers the potential to go beyond ray diffraction imaging offers the potential to go beyond the spatial resolution limit due to fabrication difficulties on the spatial resolution limit due to fabrication difficulties on xx--ray optics. ray optics. It could become the fundamental technique in molecular imaging.It could become the fundamental technique in molecular imaging.

•• It would, in principle, allow atomic resolution imaging It would, in principle, allow atomic resolution imaging on on noncrystalline noncrystalline materials and do "crystallography" materials and do "crystallography" without the need for crystals. Resolution is only limited without the need for crystals. Resolution is only limited by radiation damage.by radiation damage.

Molecular Imaging with ERL:Molecular Imaging with ERL:

•• It would be an ideal and essential hard xIt would be an ideal and essential hard x--ray source ray source for molecular imaging using coherent diffraction.for molecular imaging using coherent diffraction.

•• This type of experiments is completely limited by This type of experiments is completely limited by coherent fluxcoherent flux available at existing sources, and will not available at existing sources, and will not become "bread & butter" measurements until the next become "bread & butter" measurements until the next generation of sources such as ERL comes along.generation of sources such as ERL comes along.

coherentcoherent

3rd SR

ERL

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Scanning XScanning X--ray Microscoperay Microscope

• Lateral resolution of most existing scanning x-ray microscopes (micro-probes) is limited by horizontal source size of synchrotron radiation.

ESRF ID21: SXMSXM 2-10 keV

⇒ transmission⇒ fluorescence⇒ photoemission electron⇒ x-ray diffraction

• ERL would completely remove this limitation, allowing diffraction-limited focal area 100-1000x smaller than currently available.

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FluorescenceFluorescence Microtomography Microtomography

C.G. Schroer (Aachen) A. Snigirev (ESRF)

ID22 ESRF:

parabolic Al refractive lensfocal spot: 3 µm x 0.9 µm scan step: 1 µm 132 projections in 360o

Specimen:

mycorrhyzal root of tomato plant, grown on heavy metal polluted soil

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DifferentialDifferential--Aperture Aperture XX--ray Microscopy (DAXM)ray Microscopy (DAXM)

Cargill (intro to Larson), Nature, 2002

Larson, et al., Nature, 2002

"The availability of ... DAXM... provides a direct, - and previously missing - link between the actual microstructure and evolution in materials and the results of numerical simulations ...on mesoscopic length scales"

These experiments are microbeam flux limited. ERL

would extend realm.

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ConclusionsConclusions

Size (m)

10 − 910 − 8 10 −1010 − 710 − 610 − 510 − 410 − 310 − 210 − 1

Organisms

Cells

Organelles

Macromolecules

Small molecules

Plants & Animals Atoms




Synchrotron x-ray imaging & microscopy has made substantial progresses in all three areas of imaging science. With future sources such as ERL:

• it would open up molecular structural science from crystal-based today to noncrystalline & nanocrystalline materials

• It would make XRM a powerful tool in cellular biology, just like what x-ray crystallography is today to molecular biology

• It would allow real-time, phase-contrast x-ray imaging at <0.1µm resolution in medical & small animal imaging as well as in other biological specimens

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AcknowledgmentsAcknowledgments

ERL Project (Cornell):ERL Project (Cornell): Sol Sol GrunerGruner, Don , Don BilderbackBilderback, , Maury TignerMaury Tigner, , Ivan Ivan BazarovBazarov, Charlie Sinclair, Richard , Charlie Sinclair, Richard TalmanTalman, , Hasan PadamseeHasan Padamsee, , Georg HoffstaetterGeorg Hoffstaetter, , Ken Finkelstein, Alex Ken Finkelstein, Alex KazimirovKazimirov

Phasing diffraction pattern:Phasing diffraction pattern: Pierre Pierre ThibaultThibault, , Veit Elser Veit Elser (Cornell)(Cornell)

XX--ray microscopy:ray microscopy: Chris Jacobsen (Stony Brook)Chris Jacobsen (Stony Brook)Janos Kirz Janos Kirz (Stony Brook)(Stony Brook)Ian McNulty (APS)Ian McNulty (APS)

Imaging examples:Imaging examples: Dean Chapman (Illinois Inst. Tech.)Dean Chapman (Illinois Inst. Tech.)Carolyn Carolyn Larabell Larabell (LBNL)(LBNL)Gerd Gerd Schneider (LBNL)Schneider (LBNL)Malcolm Howells (LBNL)Malcolm Howells (LBNL)John John Miao Miao (SSRL, Stanford)(SSRL, Stanford)Peter Peter Cloetens Cloetens (ESRF)(ESRF)WahWah--Keat Keat Lee (APS)Lee (APS)

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Thank You !Thank You !

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