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Super‐Resolution Microscopy and its Applications in Fast and Complex Biological SystemsManasa V. Gudheti, Ph.D., Applications Scientist
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Outline
• Introduction to Super‐Resolution Microscopy• Single Molecule Localization (SML)• Vutara 350 Design & Features• Biological Applications
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Evolution of Super Resolution Microscopy
Z
X
100 nm
Resolutionxy‐20 nm; z‐50 nm
Single molecule Localization (SML)
Confocal SIM STED
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250 nm
Principle of Single Molecule Localization (SML)
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Principle of Single Molecule Localization (SML)
Images are captured from two focal planes simultaneously on the Vutara microscope
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Super resolutionConventional
Alexa 647 labeled microtubules in a BSC1 cell
Comparison
2 um
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Vutara 350: Video Rate Super‐Resolution Microscope
• Fastest 3D super‐resolution microscope on the market• Only 3D video‐rate super‐resolution microscope• Precise 3D super‐resolution (SML) : 20nm (x,y) & 50nm (z)• Designed by scientists for biologists • Easy to use, yet powerful software• Loaded with innovative and cutting edge features
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• ResEnhanced Technology• 3D w/ Biplane• Quadfield• Parallel Processing
Innovative Design
Vutara 350
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sCMOS
ResEnhanced
Pixel Specific Noise
ResEnhanced Technology for sCMOS
Adapted from Huang et al. Nat. Meth. 10, 653‐658 (2013)
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Adapted from Juette et al. Nat. Methods. 5, 527‐529 (2008)
1
405
488
561
640
750*2
Vutara Laser Launch
EMCCD sCMOS
Lens
Beam splittermirror
Dichroic
*750 on Vutara 350 Video Rate Super‐Resolution Microscope
Cutting Edge 3D
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x
z
camera camera
plane 2plane 1
0 nm
+125 nm
+250nm
‐125nm
‐250nm
1 µm
Joerg Bewersdorf
Biplane Approach
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Biplane Offers Superior Localization Over Astigmatism
Average localization precision (sigma) vs background (beta) comparison for x,y,z (a,b,c) for N=6,000 (top panel) and N= 1,000 photons (bottom panel) for all signal to background levels the biplane shows superior results. (Adapted from Badierireostami et al. Applied Physics Letters 97, 161103 (2010))
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Quadfield
Detector
Color1
Color2
Customized:• 2 Colors / 2 Planes• 4 colors
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Vutara SRX Software
• Software uses parallel processing• GPU + Multi‐threaded CPU• Fast real‐time localization with acquisition• Advanced 3D visualization• Measured point spread function• Integrated drift correction• Open source data export
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750
750 nm laser
Even illumination
3D particle tracking
Ease of use Multi‐color Custom design
Speed Live‐cell High power lasers
Vutara 350 Cutting Edge Features
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Speed
800fps 800fps 2500fps
Alexafluor‐647 labeled alpha‐tubulin (Red) and Cy3B labeled TOM20 (Green)in BSC1 cells
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Speed Comparison
Betzig2006
2 hr‐12 hr
20 min
SRsystems
Vutara200
Vutara350
1‐15 sec10 min
Faster Imaging:1. More accurate data
• Lower drift• Better colocalization
2. Higher throughput• More experiments in less time
Betzig et al. Science. 313, 1642‐1645 (2006)
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Live‐cell Imaging
• Biological System: Live HeLa Cell• Label: mEos3.2‐clathrin light chain• Imaged at 600 fps for 58 s• 2 seconds per SR image
Adapted from Huang et al. Nat. Meth. 10, 653‐658 (2013)
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Live‐cell Fast Imaging using Organic Dyes
• Biological System: LiveEA.Hy926 Cell• Label: AlexaFluor 647 labeled transferrin• Imaged at 1600 fps • Super‐resolution images were reconstructed
from sequential sets of 50 frames (31‐ms acquisition time or 32 super‐resolution images per second )
Adapted from Huang et al. Nat. Meth. 2013
Adapted from Huang et al. Nat. Meth. 10, 653‐658 (2013)
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Live Cell Imaging of BSC1 cells Labeled with AF647 Transferrin
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Plane 1 Plane 2
Model: Live A431 CellsLabel: QDot655‐EGFRSpeed: 50 frames/s
3D Particle Tracking
Model: Live BSC1 cellsLabel: Cy3B labeled mRNA Speed: 200 frames/s
Membrane TargetIntracellular Target
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High Power Lasers
Jones et al. Nat. Meth. 8, 499‐505 (2011)
405
488
561
640
750
488 – 1000mw561 – 1000mw640 – 1000mw750 – 1000mw
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750 nm Excitation
Biological System: BSC1 cellsGreen: AF647 TOM20 mitochondriaRed: AF750 Tubulin
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Vutara’s Edge to Edge Illumination
Good data region
Bad data region Whole region of interest is usable
Image from Vutara Image from other SML scope
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Ease of Use‐System Demo
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Multi‐color Imaging
Biological System: Vero CellGreen: Cy3B TOM20 mitochondriaRed: AF647 Tubulin
Biological System: Vero CellGreen: Cage fluorescent dye 505clathrinRed: Rhodamine spiroamide 565tubulin
Biological System: BSC1 cellsGreen: AF647 TOM20 mitochondriaRed: AF750 Tubulin
*750 on Vutara 350 Video Rate Super‐Resolution Microscope
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Biological Applications
Infectious Diseases Reproduction
Developmental Biology Cell Biology and Cancer Neuroscience
Cardiology
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Coverslip
Biological System: E.ColiTarget: Outer MembraneDye: Cy5
Color coded for depth
Courtesy of Dr. Tomasz Zal – MD Anderson Cancer Center, Texas
Single E. Coli standing on coverslip
3D Image with Single Z Plane Acquisition
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Biological System: hRSVGreen: Cy3B RNA antisense probeRed: Alexa 647 F‐protein
Human Respiratory Syncytial Virus (hRSV)
Alonas et al. ACS Nano 8, 302‐315 (2014)
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Biological System: Canine cardiomyocyteRed: Alexa 647 alpha‐actinin
Imaging in Cardiomyocytes
Lichter et al. Journal of Cellular and Molecular Cardiology 72, 186‐195 (2014)
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3D Super‐Resolution z‐stack in Mouse Spermatocyte
AF 647 labeled synaptonemal complex protein 3 (SYCP3)
Sample courtesy of Mark Lessard, The Jackson Laboratory
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3.2 µm 600 nm
Z slice
3D Super‐Resolution Accuracy and Validation
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Biological System: DrosophilaGreen: Alexa 488 Frizzled ReceptorRed: DyLight 649 Lamin C
Developmental Biology‐Thick Samples
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Images courtesy of Dr. Chris German, University of Utah
Biological System: 30 µm Striatal Rat Brain SlicesGreen: Cy3B SNAP‐25Red: Alexa 647 Vesicular Monoamine Transporter‐2 (VMAT2)
Super‐Resolution Imaging in TissueSaline
Drug
‐Treated
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Multi‐color Imaging in Cell Biology
Biological System: Cos 7 CellGreen: Alexa 568 Complex IVRed: Alexa 647 TOM 20
Image courtesy of Dr. Cliff Guy, St. Judes
Biological System: HeLa cellGreen: ATTO 488 tubulinRed: Alexa 647 midbody proteinBlue: Cy3B midbody protein
Biological System: HeLa cellGreen: Alexa 488 tubulinRed: Alexa 647 midbody protein
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Multi‐color Imaging in Neurons
Images courtesy of Andrew Taibi, Dr. Jason Shepherd, University of
Utah
Biological System: Rat Cortical NeuronsGreen: Alexa 488 MAP2 (dendritic/axonal marker)Magenta: alexa555 Arc (postsynaptic ‐associates with endocytic machinery to get rid of surface AMPA Receptors)Cyan: Alexa 647 GluR1 (live and surface labeled only)
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Biological System: Vero CellGreen: Cage fluorescent dye 505clathrinRed: Rhodamine spiroamide 565tubulin
Imaging without Switching Buffer
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Summary
• Enhanced Resolution: x,y‐20 nm, z‐50 nm• Speed: enables live cell imaging• Versatility: various biological applications• Ease of use: results
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