Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
Webinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScienceAPPLYING NEW IMAGING TECHNIQUESAPPLYING NEW IMAGING TECHNIQUESTO YOUR RESEARCH: ADVICE FROM THE EXPERTSTO YOUR RESEARCH: ADVICE FROM THE EXPERTS
29 February, 201229 February, 2012
Change the size of any window by dragging the lower left corner. Use controls in top right corner to close or maximize each window.
shows speaker bios
What each widget does:
shows the video screen shows speaker bios
download slides and more info
shows slide window
shows the video screen
search Wikipedia
view closed captioning
Facebook login
download slides and more info
LinkedIn login
to login to Twitter and send tweetsif you need helpTwitter login (#ScienceWebinar)
Webinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScienceAPPLYING NEW IMAGING TECHNIQUES TOAPPLYING NEW IMAGING TECHNIQUES TO
Brought to you by the Science/AAAS Custom Publishing Office
APPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTSAPPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTS
29 February, 201229 February, 2012
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
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
Webinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScienceAPPLYING NEW IMAGING TECHNIQUES TOAPPLYING NEW IMAGING TECHNIQUES TO
Brought to you by the Science/AAAS Custom Publishing Office
APPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTSAPPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTS
29 February, 201229 February, 2012
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
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:
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)
Webinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScienceAPPLYING NEW IMAGING TECHNIQUES TOAPPLYING NEW IMAGING TECHNIQUES TO
Brought to you by the Science/AAAS Custom Publishing Office
APPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTSAPPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTS
29 February, 201229 February, 2012
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
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:
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.
Webinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScienceAPPLYING NEW IMAGING TECHNIQUES TOAPPLYING NEW IMAGING TECHNIQUES TO
Brought to you by the Science/AAAS Custom Publishing Office
APPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTSAPPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTS
29 February, 201229 February, 2012
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
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:
Webinar SeriesWebinar SeriesWebinar SeriesWebinar SeriesScienceScienceScienceScienceAPPLYING NEW IMAGING TECHNIQUES TOAPPLYING NEW IMAGING TECHNIQUES TO
Brought to you by the Science/AAAS Custom Publishing Office
APPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTSAPPLYING NEW IMAGING TECHNIQUES TOYOUR RESEARCH: ADVICE FROM THE EXPERTS
29 February, 201229 February, 2012
k f bi i h i
Brought to you by the Science/AAAS Custom Publishing Office
Look out for more webinars in the series at:
www.sciencemag.org/webinar
To provide feedback on this webinar, please e‐mail
your comments to [email protected] @ g
For related information about the Intramural Research Program:irp.nih.gov
Sponsored by: