mike kerber bio-applications manager june 2015 · tips for storm sample preparation • compare...
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Mike Kerber Bio-applications Manager
June 2015
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N-STORM Nikon - Stochastic Optical
Reconstruction Microscopy
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Resolution =
Wavelength
2 NA
So…about 200 nm
Ernst Abbe 1840-1905
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Resolution =
Wavelength
2 NA
So…about 200 nm
High NA
Objective
Excitation Light Waves
Z
X
200-300 nm
60
0-8
00
nm
Point
Spread
Function
How big is 200nm?
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McGuffee et al. PLoS Comput Biol. 2010.
5nm
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10X Resolution Increase (X, Y, Z)
Modalities: 2D, TIRF, 3D, Multi-Color
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Normally, all light sources are “on,” and difficult to distinguish:
How does STORM work?
1.Emitter isolation: Only some lights are on at a time
2.Localization: Pinpoint centroids of visible lights
3.Repeat!
Requires photoswitchable fluorescent probes!
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“Emitter Isolation” = Only a subpopulation
of fluorophores is on at once
Conventional
Fluorescence
Emitter
Isolation
Can’t see individuals in the crowd… Individuals distinguishable!
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Continuous STORM
Common small molecule dyes will “blink” in the
presence of “imaging buffers” and high laser powers.
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With some assumptions, we can accurately
find the center of a diffuse spot
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Single-Molecule Localization
320 nm
Image of one fluorescent molecule
FIONA
Fluorescence Imaging with One Nanometer Accuracy
Paul Selvin
Fitting to a gaussian model
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STORM =
Emitter Isolation and Localization
Raw images Conventional fluorescence STORM Image
Activation Localization Deactivation
Rust, Bates & Zhuang, Nat. Methods, 2006 Bates, Huang, Dempsey & Zhuang, Science, 2007
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5 μm 500 nm
40,000 frames, 1,502,569 localization points
B-SC-1 cell, anti-β tubulin, Alexa 647
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Dual Color STORM
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Dual Color STORM
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Huang, Zhuang et al, Science (2008)
3D STORM
Camera
2γ
(x, y, z)
400
200
0
-200
-400
z (nm)
In
Focus
Above
Focus
Below
Focus
Tube Lens
Cylindrical Lens
In
Focus
Above
Focus
Below
Focus
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5 μm
Huang, Wang, Bates and Zhuang, Science, 2008
Scale bar: 200 nm
3D Imaging of the Microtubule Network
z (nm)
300
0
600
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Bad Sample = Bad STORM Image
Clathrin-STORM
Widefield Clathrin
1 µm
Clathrin-STORM
BAD
Good
What makes a good STORM sample?
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Determinants of Sample Quality
1. Probe Choice Dyes that work for N-STORM
2. Labeling Strategies Fixation
Immunostaining
* Don’t forget to use glass-bottom (#1.5)
dishes to hold STORM imaging buffer!
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Few Dyes Work for N-STORM
(Despite what is published)
Dempsey et al., 2011
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Where to get secondary antibodies for STORM
cSTORM 2º Antibodies Sources
Alexa647 Life Technologies, Jackson
Cy5 Jackson
Alexa568 Life Technologies
Cy3B DIY (Dye from GE)
Atto488 Rockland, Sigma
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Linkage Error: Antibodies separate the
fluorophore from the structure of interest
Microtubule AF647
10 nm
25 nm
10 nm
AF647
60 nm
Image courtesy of Michael Davidson, FSU
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Our sample prep protocol: a good starting point
The following optimizations
are also necessary…
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Tips for STORM Sample Preparation
• Compare antibodies from multiple sources.
• Optimize fixation (fixative concentration, permeabilization, etc.)
to maximize structural preservation and antibody binding.
• Minimize background signal levels by titrating primary antibody.
• Block with heat-treated sterile filtered blocking serum.
• Don’t skip on the washing steps and use 1% blocking serum to
remove antibodies AT EVERY STEP.
• Lock secondary antibodies in place with post-staining fixation.
• Remove residues with Tween 80 wash.
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STORM Sample Prep Summary
• Use glass-bottom (#1.5) dishes
• Label with Alexa 647 and Atto 488
• Block and wash samples thoroughly
• Use MEA-containing imaging buffer and keep it
fresh
• Optimization is usually necessary for the best
results!
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Widefield is powerful, but has limitations
Out of focus light = high background
Low Z resolution
Light is harmful to live cells
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TIRF = Total Internal Reflection Fluorescence
Plus:
• 150nm Z-resolution
• Reduced phototoxicity to live cells
• Very high speeds possible
No out of focus fluorescence Single-molecule sensitivity
So…What’s the catch?
Can only image the 150nm next to the coverslip!
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What near-coverslip applications require
high sensitivity, high speed, and low background?
Focal adhesions Single molecules
Membrane transport
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How does TIRF work?
High angle light reflects off of interfaces
Low refractive index
High refractive index
Totally Internally Reflected!
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TIRF creates an “Evanescent Field” that excites
fluorophores within ~150nm of the coverslip
150 nm
`
Higher signal:noise!
Coverslip
Oil
Conventional Widefield:
• All fluorophores in the volume are excited
“Evanescent Field”
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How to do TIRF:
1. Focus on your sample interface
2. Aim beam directly through sample and onto ceiling
3. Focus beam
4. Adjust beam angle until TIR is achieved
Sample
This is why you must focus
the objective first!
Spot projected
onto ceiling
X/Y positioning knobs
Locking screw Focus adjustment slider
HIGH NA
Objective
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How do I know if I’m “in TIRF?”
Widefield TIRF
Image Plane
Aperture Plane
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D = 10 um
DiI-coated bead with
matching refractive index
What if you want to actually measure the
penetration depth of the evanescent field?
X
Z
R2 = X2 + (R – Z)2
R R – Z
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Why “High NA?”
“Critical angle” = Angle necessary for reflection = sin-1(n2/n1) = 63°
Media
Glass
“High” NA Objective
Maximum angle for an objective NA = n sin ɵ
NA ɵ Depth 1.4 67° ~150 nm
1.45 72° ~100 nm 1.49 79° ~50 nm
So, higher NA lenses allow for thinner evanescent fields!
ɵ Penetration Depth
“Higher” NA Objective
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Also remember: Laser beams have a width
Critical angle threshold
Total internal reflection!
This is the other reason to use “Higher NA” objectives
Some laser light at critical angle and some not
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What if I want to do TIRF with multiple ?
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Multichannel TIRF considerations
Remember: shorter wavelengths of light are refracted more at interfaces.
Some options:
• Use chromatically corrected optics in your TIRF illuminator
• Use a motorized TIRF illuminator
• Use a separate TIRF illuminator for each wavelength
Different wavelengths will not strike the back aperture at the same location or in the same focus
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What do you need to do TIRF?
• High NA objective – 60X 1.49 Apo – 100X 1.49 Apo – 100X 1.49 SR Apo – 100X 1.45 ʎ Plan Apo
• TIRF cubes
• Laser • Samples
– Need a glass-water interface! – Get as close to coverslip as possible, minimize ECM!
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Extra hardware for
single-molecule live-cell TIRF:
• Maintaining focus is a must!
– Perfect Focus System!
• Single molecules are dim
– EMCCDs
• Molecular motors move fast
– Triggering
• Live-cell fluorophores, like GFP, bleach very fast
– Minimize exposure to light when not imaging
– Find focus using some other imaging modality (transmitted, SRIC)
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Common Pitfalls of TIRF
• Very small imperfections in your optics will
cause an interference fringe
– DIC prism, etc. can worsen interference patterns
and cause ghosting
• Light scatter causes directional smearing
– Scattering exacerbated by extra coatings
– Thinner evanescent fields cause less scatter
– Take images at multiple orientations
• Different wavelengths will not strike the specimen
at the same incident angle or same focus
– Ti TIRF arms have chromatic correction for focus
– Use different angles for each channel (Motorized
or multiple arms)
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TIRF Summary
• An evanescent field is created by reflecting laser light off of a glass/water interface
• Penetration depth depends on wavelength, angle, and refractive index
• Penetration depth can be calculated or measured experimentally
• Objective lenses with at least 1.45 NA are recommended
• TIRF advantages: high speed, high signal:noise, thin z-sectioning, and low phototoxicity
• TIRF disadvantages: can only see ~150nm adjacent to coverslip
TIRF
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43
Thank you!
Acknowledgements
Xiaowei Zhuang, Harvard University
Mike Davidson, FSU
microscopyu.com
Your local Nikon representatives:
Edwin DeFeijter
Jim Winkelmann