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APPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTSAPPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTS


Participating Experts:


Sriram Subramaniam, Ph.D.National Cancer Institute, NIHBethesda, MD

Hari Shroff, Ph.D.National Institute of Biomedical Imaging and Bioengineering, NIHBethesda, MD

Clare M. Waterman, Ph.D.National Heart, Lung and Blood Institute, NIHBethesda, MD

Sponsored by:

Visualizing Cells and Viruses at Molecular Resolution:Visualizing Cells and Viruses at Molecular Resolution:

Progress, Challenges and Future Prospects

Sriram Subramaniam

Imaging gaps in biology and medicine

Subramaniam, Curr. Opin. Microbiol. (2005)

Topics

Dynamic protein retroviruses bacteria mammalian

complexes cells

~ 15 nm ~150 nm ~1500 nm ~15000 nm

Structure determination using cryo-electron microscopy

GroEL molecular complexes

Structure determination using cryo-electron microscopy

Density map at ~ 7 Å resolution

Comparison between X-ray and cryo-electron microscopic

structures provides insight into protein dynamics

Bartesaghi, Schauder, Borgnia, de la Cruz, Milne and Subramaniam (2012)

Cryo-electron tomography

Cryo-electron tomography of HIV

Meyerson et al., JoVE (2011)

3D structure of native HIV-1 Env trimers

Liu et al., Nature (2008)White et al., PLoS Pathogens (2010)

Harris et al Proc Natl Acad Sci USA (2011)Harris et al., Proc. Natl. Acad. Sci. USA (2011) White et al., J. Virology (2011)

Catching HIV in the act

Structural biology of chemotaxis receptors in intact cells

Zhang et al., Proc. Natl. Acad. Sci. USA (2007)Khursigara et al., Proc. Natl. Acad. Sci. USA (2008)g , ( )

Khursigara et al., EMBO J. (2011)

Visualizing the 3D architecture of bacterial cells

October 2007 March 2011

Milne and S bramaniam Nat re Re ie s Microbiolog (2009)

Butan et al 2011Liu et al 2007

Milne and Subramaniam, Nature Reviews Microbiology (2009)

Ion-abrasion Scanning Electron Microscopy:

A lt ti h f 3D i i f ll d tiAn alternative approach for 3D imaging of cells and tissue

iteration of ion beam milling with SEM imaging

Ion-abrasion Scanning Electron Microscopy:

A lt ti h f 3D i i f ll d tiAn alternative approach for 3D imaging of cells and tissue

Ion-abrasion Scanning Electron Microscopy:A lt ti h f 3D i i f ll d tiAn alternative approach for 3D imaging of cells and tissue

it ti f i b illiiteration of ion beam milling with SEM imaging

Heymann et al J Struct Biol (2006)Heymann et al., J. Struct. Biol. (2006)Heymann et al., J. Struct. Biol. (2009)

Rate of imaging: A whole cell every few hours

~ 10,000 µm3 /day for 3D resolution of ~15 nm

The surface of antigen-presenting cells: A different perspective

Bennett et al., PLos Pathogens (2009) Felts et al., Proc. Natl. Acad. Sci. USA (2010), ( )Nikolic et al., Blood (2011)

The surface of antigen-presenting cells: A different perspective

Correlative live confocal and ion-abrasion SEM imaging

3D image of entire T-cell

Murphy, Narayan et al., J. Struct. Biol. (2011)

Correlative live confocal and ion-abrasion SEM imaging

3D image of entire T-cell

Murphy, Narayan et al., J. Struct. Biol. (2011)

3D chemical imaging of whole cells

3D SIMS imaging (Szakal et al., Anal. Chem. (2011))

Atom probe tomography (Narayan et al., J. Struct. Biol. (2012))

The changing landscape of 3D electron microscopy

The conventional methodThe conventional method…

The changing landscape of 3D electron microscopy

…and the new frontier

SummaryEmerging methods in 3D electron microscopy offer great

i f i i t i l i d llpromise for imaging protein complexes, viruses and cells

at resolutions in the nanometer range

l t i ihhttp://electron.nci.nih.gov










Sponsored by:

Super-resolution Optical Imaging in Biology

0.2 m0.5 m2 m

Hari Shroff, NIBIBFebruary 22, 2012

Biological imaging spans many length scales

Why fluorescence?

Excellent contrast and specificity, multiple colors,

High speed live imaging

Torsten WittmannWinner, 2003 Nikon Small World Competition

Hideo Otsuna2nd place, 2010 Nikon Small World Competition

The Problem: Diffraction limit 100x larger than molecular scaleDiffraction limit 100x larger than molecular scale

Green Fluorescent Protein

1 nm

The Problem: Diffraction limit 100x larger than molecular scaleDiffraction limit 100x larger than molecular scale

Green Fluorescent Protein Diffraction Limited Region: ‘PSF’

1 nm 100 nm

Diffraction limited imaging

McEvoy et al. BMC Biology 2010 8:106

Many choices for super-resolution…

PALMSTORMF-PALMiPALMiPALMspt PALMDSTORMGSDIM

LOCALIZATION BASED, SAME UNDERLYING PRINCIPLEGSDIM

BIPLANE PALM3B microscopyDAOSTORM

SAME UNDERLYING PRINCIPLE

DAOSTORMBALMPALMIRA…

STEDRESOLFT

SIMSSIMNONLINEAR SIM

Individual emitters can be localized with arbitrary precision

2D 3D

M.Speidel, A. Jonas, E.L. Florin,

Opt Lett 28 69 (2003)Opt. Lett. 28, 69 (2003)

Ability to find mean position limited only by SNR

Isolation followed by Localizationso

latio

nIs

izat

ion

Adapted from McEvoy et al. BMC Biology 2010 8:106 Loca

li

Photoactivate/ Photoswitch OFF -> ON

How to isolate?Photoactivate/ Photoswitch, OFF > ON

Patterson Lippincott-Schwartz

R. Ando, et al., PNAS 99, 12651 (2002)

Patterson, Lippincott Schwartz Science 297, 1873 (2002)

Temporarily shelve fluorophore in dark state, ON -> OFF

J. Folling et al,

M. Heilemann et al, Angew. Chem. Int. Ed. 47 6172-6176 (2008)

J. Folling et al, Nat Methods 5 943-945 (2008)

Structure/function in bacteria

Bacteria are small!

Clusters of differentreceptor sizes revealed with PALM

Greenfield et. al.PLoS Biology 7

1000137 (2009)

1m

e1000137 (2009)

50 nm

Size/spatial distribution of clusters explained by simple model

1m

Random insertion/diffusion/capture explains size and spatial cluster location:

50 nm

Random insertion/diffusion/capture explains size and spatial cluster location:‘Stochastic self-assembly’

Cytoskeletal elements/active transport not required

Actin dynamics in neuronal spines with particle tracking PALM

20 µm 250 nm4 µm

Combine particle tracking with PALM to circumvent diffraction limit

0 2 4 6 8 100 s 2 s 4 s 6 s 8 s 10 s

200 nm

Initial Particle Position

Successive Particle Positions

actin-mEos2 displacements

Velocity vector analysis in single dendritic spines

25 Molec

100 nm/s2

200 nm/s Actin mEos2PSD95 ceruleancules w

ithi

200 nm

250 nm

n 111 nm r

0

radiusInward motion away from spine edge

Molecular velocity elevated at subdomains throughout spine

Rapid assembly both near and away from PSD

y g p

Heterogenous actin organization may permit regulation of diverse spine functionsFrost et. al. Neuron 67, 86-99 (2010)

Instrumentation: present and future B i L li ti B d S l tiBasic Localization-Based Super-resolutionbuild (~ $200k)buy (Zeiss, Nikon, Leica, Applied Precision, Vutara) *

Deeper Imaging Higher Resolution

2 m2 m

Confining illumination

Dual objective STORM10 nm lateral, 20 nm axial resolutionXu et. al. Nat Methods 9, 185 (2012)

* NIH does not endorse or recommend any commercial products, processes, or services

Confining illuminationZanacchi et. al. Nat Methods 8, 1049 (2011)York et. al. Nat Methods 8, 327-333 (2011)

Careful sample preparation key for super-resolutionDoes sample prep indicate where protein is at diffraction-limited level?Does sample prep indicate where protein is at diffraction-limited level?

T h t t t d l t b lt t t ?

20 m

To what extent does sample prep perturb ultrastructure?

2 m

2% PF 4% PF 2% glutaraldehyde

Schnell et al. Nat. Methods 9, 152-158 (2012)

Interpret and perform live experiments cautiously

Focal adhesions in live CHO cell, ~50 s/frame, tdEos paxillin

2 m

PALM Conventional

Shroff et. al. Nat. Methods 5, 417-423 (2008)

Interpret and perform live experiments cautiously

Is physiology affected? Avoid cell torture!

17 m17 m

Extreme resolution requires extreme label densityPixels/line

sure

d el

s m

eas

ion

of p

ixFr

acti

Shroff et. al. Nat. Methods 5, 417 423 (2008)

Pick your dye carefully – single most important factor in maximizing resolution- attachment/specificity?

label densit ?

417-423 (2008)

- label density?- sample perturbation?- contrast?

Other commercial super-resolution techniques

Fast over small field of viewsAdjustable resolution

Fast over large field of viewsOnly ~2x increase in resolution

Ji et. al., Curr Opin Neurobiol. 18, 605-616 (2008)

High intensities for highest resolutionChoice of dye critical

Relatively low intensities requiredNo label restrictions

NIBIB/NIHHank Eden

Thanks!OthersMike Davidson (FSU)

Shroff LabAndrew YorkHank Eden

Richard LeapmanSheila BarrettLeah Baskin

Mike Davidson (FSU)Alipasha Vaziri (Univ. of Vienna)Tom Blanpied (UMD) Nick Frost (UMD)

Andrew YorkYicong WuPeter WinterAlireza Ghitani

Rosemary JacksonTruc LeChristopher WanjekMi h l G tt

Nick Frost (UMD)Jan Liphardt (UCB)Derek Greenfield (UCB)Ann McEvoy (UCB)

Peter WawruszinKelsey Temprine

Michael GottesmanJim and Cathy GalbraithJennifer Lippincott-Schwartz

Eric Betzig (JFRC)Harald Hess (JFRC)










Sponsored by:

Fluorescent Speckle MicroscopyFluorescent Speckle Microscopyyy

Clare M. WatermanCell Biology and Physiology Center

Clare M. WatermanCell Biology and Physiology CenterCell Biology and Physiology Center

National Heart Lung and Blood InstituteNIH Bethesda MD

Cell Biology and Physiology CenterNational Heart Lung and Blood Institute

NIH Bethesda MDNIH, Bethesda [email protected]

NIH, Bethesda [email protected]

Inflammatory response and epidermal repair i d d b fi h t ilin a wounded zebrafish tail.

Mike Redd, Paul Martin; University of Bristol, UK.

Light Microscopy: The Key to Time d S i Li i C ll

Light Microscopy: The Key to Time d S i Li i C lland Space in Living Cellsand Space in Living Cells

– Multiple molecules within cellular hi

– Multiple molecules within cellular himachines

– Multiple cellular machinesmachines

– Multiple cellular machinesp– Cell behavior

p– Cell behavior

Spatial Resolution: Diffraction Barrier

> 500 nm

~ 200-250 nm

Diffraction sets limit for smallest focal volume: Point Spread Function (PSF)

Davidson, Wikipediadx,y = 0.61/ NA

Rayleigh’s Criterion of Resolution

Fluorescent Speckle Microscopy: Marking i i ll h t tmicroscopically homogeneous structures

Waterman-Storer et al., 1998 Curr. Biol. 8:1227-1230

The POWER of a SPECKLE:A local probe of biochemistry and physics in a live cellA local probe of biochemistry and physics in a live cell.

• Intensity: rates of binding/ dissociation

• Motion: trajectory and velocity, materialvelocity, material properties

• Resolution of ~10 millisecond ~10millisecond, ~10 nanometer.

• ~50,000 datapoints per time point.

• Amenable to computer vision analysis.vision analysis.

Danuser Lab FSM Center computer visionsoftware for quantitative analysis of FSM images

Raw Image Data

q y g

Speckle Detection

Flow Tracking by Adaptive Multi-frameCorrelation

Gaudenz Danuser

Problem-SpecificSingle Particle

Speckle ParticlesLow Resolution Speed Maps

Coherent FlowComponents

Gaudenz Danuserhttp://lccb.hms.harvard.edu/index.html

pPost-ProcessingTexture Flow

FilteringTracking

Speckle Trajectories

High ResolutionSpeed Maps

Mechanistic

Analysis of Signal

Particle Flow FilteringClassification of

Trajectory Endpoints

jLow ResolutionAssembly/DisassMaps

Hi h R l ti

Information

Score Filtering

ConservationKinetic Scores

High ResolutionAssembly/DisassMaps

Valloton et al., 2003 PNAS101:9660-9665.

Computational FSM analysis reveals distinct zones of actin dynamicsdistinct zones of actin dynamics

Actin Myosin IIRLC

ContractileModule

FSM RLC Lamella

ConvergenceZone

CentralRegion

SpeedAssemblyDisassembly

Ponti et al., 2004. Science 305:1782-1786.

How to make a Speckle:Specimen RequirementsSpecimen Requirements

• Well labeled, functional protein., p– Multiple fluorophores/subunit?

• LOW expression level of fluorescent-labeled protein.– ~99% endogenous unlabeled, ~1% fluorescent labeled

• Difference in fluorescence intensity between dj t diff ti li it d i iadjacent diffraction-limited image regions

• Stability of such differences in the time frame of an image acquisitionimage acquisition. – Diffusing molecules move too fast (63 pixels/sec for our

microscope system)microscope system)– Fluorophores must be immobilized.

Wittmann et al., 2004 In: “Live Cell Imaging: A Laboratory Manual,”

FSM using a crippled promoter to drive low-level expression of fluorescent protein-level expression of fluorescent protein

tagged proteins

Total endogenous vinculin (immunofluorescence)

Expressed Vinculin-GFP(driven by CMV)

Adams et al 2004 J. Microscopy. 216:138-152

How to make a Speckle:Hardware RequirementsHardware Requirements

• Prevention of photobleaching. – Illumination shutters. – Oxygen scavengers.

Highly efficient photon collection• Highly efficient photon collection. – Low noise, high dynamic range, high QE camera. – Simple light path and lenses. p g p– Removal of DIC components.

• Focus stability. Hi h ifi ti d hi h l ti• High magnification and high resolution.– >1.4 NA Optics– Resolution of microscope and detector matchedResolution of microscope and detector matched

Wittmann et al., 2004 In: “Live Cell Imaging: A Laboratory Manual,”

FSM is amenable to any mode of fluorescence microscopy capablefluorescence microscopy capable of high magnification, diffraction-

S/limited, high S/N imaging

TIR-FSM imaging optimizes the speckle t t t th li fcontrast at the coverslip surface

TIR-FSMWide-field-FSM TIRFGFP-

vinculin

FSMGFP-vinculin

TIRFanti-vinculin

Adams et al 2004 J. Microscopy. 216:138-152

Multicolor FSMMulticolor FSM

Actin and Microtubule dynamicsSalmon et al., 2002 J. Cell Biol. 158: 31-37.

Correlational TIR-FSM measures molecular coupling betwwn actin and adhesions.

GFP-FAcoupling betwwn actin and adhesions.

• Dual-wavelength TIR-FSM of actin and adhesion

X-Rhodamine-actin

of actin and adhesion.• Track flow of actin and

adhesion speckles.• Segment adhesions.• Interpolate vectors on

idcommon grid.• Correlate direction and

velocity within vector pairs.e oc ty t ecto pa s

Directional Correlation score = Cos()

V• Actin vectors RED• FA molecule vectors yellow

Velocity Coupling Score = VFA actin

Vactin

Hu et al., 2006 Science 315:111-115.

Vinculin is partially coupled to actin

-1-1

1

0

1

0

direction correlation 0.72

Velocity coupling 0.45Hu et al., 2006 Science 315:111-115.

Future Directions for FSMFuture Directions for FSM

• Calibration of the qFSM software to giveCalibration of the qFSM software to give absolute kinetics.

• Application of qFSM to study other pp q ymacromolecular assemblies such as intermediate filaments, DNA binding proteins, cell surface receptors, signaling molecules.

• 3D speckles• Superresolution Speckles

Thanks toThanks to

Collaborators Waterman Lab MembersCollaborators• Ted Salmon University of

North Carolina

Waterman Lab Members• Margaret Gardel, University of

ChicagoK H Ph D I di U i it

• Gaudenz DanuserHarvard Medical School

Aaron Ponti

• Ke Hu, Ph.D. Indiana University• Stephanie Gupton, University of

North Carolina – Aaron Ponti– Andre Vallotton– Lin Ji

• Michael C. Adams, Sempra Energy.

• Torsten Wittmann, University of – Kathryn Applegate– Alex Matov– Matthias Macachek

California, San Francisco,• Wendy C. Salmon,

Massachusetts Institute of Technology.










Sponsored by:





k f bi i h i


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Science Webinar Series Slides_ NIH IRP... · Science Webinar Series APPLYING NEW IMAGING TECHNIQUES TO YOUR RESEARCH: ADVICE FROM THE EXPERTS 29 February, 2012 Change the size of