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Super‐Resolution Microscopy and its Applications in Fast and Complex Biological SystemsManasa V. Gudheti, Ph.D., Applications Scientist
Outline
• Introduction to Super‐Resolution Microscopy• Single Molecule Localization (SML)• Vutara 350 Design & Features• Biological Applications
Evolution of Super Resolution Microscopy
Z
X
100 nm
Resolutionxy‐20 nm; z‐50 nm
Single molecule Localization (SML)
Confocal SIM STED
250 nm
Principle of Single Molecule Localization (SML)
Principle of Single Molecule Localization (SML)
Images are captured from two focal planes simultaneously on the Vutara microscope
Super resolutionConventional
Alexa 647 labeled microtubules in a BSC1 cell
Comparison
2 um
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
• ResEnhanced Technology• 3D w/ Biplane• Quadfield• Parallel Processing
Innovative Design
Vutara 350
sCMOS
ResEnhanced
Pixel Specific Noise
ResEnhanced Technology for sCMOS
Adapted from Huang et al. Nat. Meth. 10, 653‐658 (2013)
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
x
z
camera camera
plane 2plane 1
0 nm
+125 nm
+250nm
‐125nm
‐250nm
1 µm
Joerg Bewersdorf
Biplane Approach
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))
Quadfield
Detector
Color1
Color2
Customized:• 2 Colors / 2 Planes• 4 colors
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
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
Speed
800fps 800fps 2500fps
Alexafluor‐647 labeled alpha‐tubulin (Red) and Cy3B labeled TOM20 (Green)in BSC1 cells
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)
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)
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)
Live Cell Imaging of BSC1 cells Labeled with AF647 Transferrin
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
High Power Lasers
Jones et al. Nat. Meth. 8, 499‐505 (2011)
405
488
561
640
750
488 – 1000mw561 – 1000mw640 – 1000mw750 – 1000mw
750 nm Excitation
Biological System: BSC1 cellsGreen: AF647 TOM20 mitochondriaRed: AF750 Tubulin
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
Ease of Use‐System Demo
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
Biological Applications
Infectious Diseases Reproduction
Developmental Biology Cell Biology and Cancer Neuroscience
Cardiology
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
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)
Biological System: Canine cardiomyocyteRed: Alexa 647 alpha‐actinin
Imaging in Cardiomyocytes
Lichter et al. Journal of Cellular and Molecular Cardiology 72, 186‐195 (2014)
3D Super‐Resolution z‐stack in Mouse Spermatocyte
AF 647 labeled synaptonemal complex protein 3 (SYCP3)
Sample courtesy of Mark Lessard, The Jackson Laboratory
3.2 µm 600 nm
Z slice
3D Super‐Resolution Accuracy and Validation
Biological System: DrosophilaGreen: Alexa 488 Frizzled ReceptorRed: DyLight 649 Lamin C
Developmental Biology‐Thick Samples
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
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
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)
Biological System: Vero CellGreen: Cage fluorescent dye 505clathrinRed: Rhodamine spiroamide 565tubulin
Imaging without Switching Buffer
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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