Harnessing the Power of Super‐Resolution Microscopy
Manasa V. Gudheti, Ph.D., Applications Scientist
Outline
• Brief Intro to Super‐Resolution Microscopy• Principle of Single Molecule Localization (SML)• Photoswitching Mechanisms• Sample Prep Optimization• Biological Examples
Evolution of Super Resolution Microscopy
Z
X
100 nm
Resolutionxy‐20 nm; z‐50 nm
Single molecule Localization (SML)
Confocal SIM STED
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
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(f)PALM, STORM, dSTORM, GSDIM
Super resolutionConventional
Alexa 647 labeled microtubules in a BSC1 cell
Comparison
2 um
Photoswitching Mechanism for Blinking
Van de Linde et al. Photochem. Photobiol. Sci., 8, 465–469 (2009)
Photoswitching Mechanism for Blinking
Henriques et al. Biopolymers 95, 499–506 (2011)
Photoswitching Mechanism for Blinking
Blinking buffer: Reducing and oxidizing system (ROXS)
Shi et al. Anal. Chem. 82, 6132–6138 (2010)
Van de Linde et al. Photochem. Photobiol. Sci. 10, 499–506 (2011)
β-mercaptoethanol, (BME) β-mercaptoethylamine (MEA) and glucose/oxidase catalase (GLOX)
Sample Prep Questions to Ask
• What type of sample do you want to image? Cells, tissues, bacteria, virus
• What is your current sample prep technique? • What kind of dyes do you currently use? • How do you mount your sample?• What are your expectations?
Single Molecule Localization ProbesPreferred Organic Dyes
Excitation Laser Line
(nm)
Dye ExcitationMaximum
(nm)
Emission Maximum
(nm)488 ATTO 488 501 523
Alexa 488 495 519561 Cy3B 559 570
Alexa 568 578 603Alexa 555 555 580
640 Alexa 647 650 665Cy5 649 670DyLight 650 652 672
750 DyLight 755 754 776Alexa 750 749 775
Choice of Organic Dyes
Dempsey et al. Nat. Methods. 6, 1027-1036 (2011)
Photoswitchable Fluorescent Proteins (Genetically-Encoded)
Probe Type λPA (nm) λX (nm) λEM (nm) VariantsPSCFP2 0→A (Irrev) Violet (~400) 490 511 PSCFPPA-GFP 0→A (Irrev) Violet 504 517
Dronpa 0→A (Rev*) *activ. w violetquench w blue
503 518 Fastlime, Dronpa3
Dendra2 A→B (Irrev) Violet-Blue 553 573 Dendra
EosFP A→B (Irrev) Violet 569 581 mEos3.2, tdEos
Kaede A→B (Irrev) Violet 572 580
KikGR A→B (Irrev) Violet 583 593
PAmCherry 0→A (Irrev) Violet 564 595 1&2
• Dendra2‐HA • PAmCherry‐actin
Fast 2‐Color Live Super‐Resolution Imaging Reveals Dynamic Associations
Gudheti et al. Manuscript in preparation
HA Moves Along Actin‐Rich Membrane Regions in Fibroblasts at 37C
● Dendra2-HA (2,788 mol)● PAmCherry-Actin (7,316 mol)● PAmKate-TfR (2,967 mol)
1 μm
3‐Color FPALM of Dendra2‐HA, PAmCherry‐Actin, and PAmKate‐Transferrin Receptor
Gunewardene et al. Super‐resolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells (2011) Biophysical J 101
Three color super-resolution imaging of multiple fluorescent proteins in living cells
Gunewardene et al. Super‐resolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells (2011) Biophysical J 101
Live‐cell Imaging using mEos3.2
• Biological System: Live HeLa Cell• Label: mEos3.2‐clathrin light chain• Imaged at 600 fps for 58 s• 2 seconds per SR image• Imaged in PBS
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 )
• Cells were imaged DMEM (high glucose, phenol red–free) supplemented with 2‐beta mercaptoethanol, glucose oxidase and catalase at room temperature.
Adapted from Huang et al. Nat. Meth. 10, 653‐658 (2013)
Live Cell Imaging of BSC1 cells Labeled with AF647 Transferrin (3000 frames total imaged)
Live‐Cell Imaging of Direct‐Labeled Cellular DNA
Benke & Manley. ChemBiochem. 13, 298-301 (2012)
• 1 mM ascorbic acid, 10% glucose, glucose oxidase and catalase in Leibowitz medium, pH 7.2
• 8000 frames• 30 ms acquisition time
• Biological System: Vero Cell• Green: Cage fluorescent dye 505
clathrin• Red: Rhodamine spiroamide 565
Tubulin• Imaged in PBS
Imaging Without Switching Buffer
Biological Applications
Infectious Diseases Reproduction
Developmental Biology Neuroscience
Cardiology
Cell Biology
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)
Labeled RNA viruses using multiply labeled tetravalent RNA imaging probes (MTRIPs)
Biological System: Canine cardiomyocyteRed: Alexa 647 alpha‐actinin
Arrows point to longitudinal depositions spanning between transverse sheets in the cell interior
Decrease in the spatial regularity of the α‐actinin distribution in heart failureversus control cells observed
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
Biological System: DrosophilaGreen: Alexa 488 Frizzled ReceptorRed: DyLight 649 Lamin C
Developmental Biology‐Thick Samples
•Light scattering reduced
•Increased tissue transparency to achieve refractive uniformity throughout the specimen which allows imaging at greater depth
August 2011
Scale BufferTypical composition of Scale buffer
pH 7.7
Refractive index of 1.38
4M Urea
30% glycerol
0.1% Triton X-100
Comparison of whole fixed embryos incubated in PBS and Scale for two weeks
Hama et al. “Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain”, Nature Neuroscience 14, 1481-1488 (2011)
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. JudesResearch Hospital
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
Mitochondria Imaging
Sample courtesy of Dr. Cliff Guy, St. Judes Research Hospital
Biological System: Cos 7 CellGreen: Alexa 568 Complex IVRed: Alexa 647 TOM 207000 frames for each color
Multi‐color Imaging in Neurons
Biological System: Rat Hippocampal NeuronsGreen: Alexa 647 phalloidin( labeling actin)Magenta: Cy3B βII‐Spectrin or Adducin
Xu et al. “Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons”, Science 339, 452‐456( (2013)
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)
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
Summary
• Sample prep optimization is crucial for obtaining good resolution with single molecule localization
• Choose the right dyes • Choose the right fixation reagents• Optimize primary and secondary antibody concentrations• Post‐fixing after secondary labeling is recommended• Choose the right imaging conditions (imaging buffer
compatible with dyes, laser powers, acquisition time, and number of frames)
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