Intro to Super Resolution: When and why you need it - PKU · 2016-11-28 · Tubulin and...

Imagination at work

Intro to Super Resolution: When and why you need itStructured Illumination Microscopy

Leanna Ferrand, Product Support LeaderCellular Imaging & Analysis, GE Healthcare15 November 2016

Back to Basics

Functions of a microscope

Magnification

Increase the size of an

object

Resolution

Distinguish one object

from another

Contrast

Define the boundaries of

an object

311/15/2016

Resolution limit of a light microscope

d = λ / 2NA

λ = wavelength of light

NA = numerical aperture of

objective

We can’t change wavelength and there is a practical limit to

how high we can push NA

This is the diffraction limit!Molecules closer than

~200nm apart will appear to be the same structure

Distance (D)

Inte

nsi

ty

Distance (D)

Inte

nsi

ty

Distance (D)

Inte

nsi

ty

411/15/2016200𝑛𝑚 =

568

2 ∗ 1.42

What is Super Resolution?

Conventional microscope

250 x 250 x 600 nm

Vs

3D-SIM

100 x 100 x 300

=

8 fold volume resolution improvement!!

5

What is Super Resolution?

The diffraction limit exists because we can’t change

wavelength and there is a physical limit to how far we

can push NA. Why not?

PHYSICS! We can’t break the laws of physics, so what

can we do?

• Super Resolution technologies use physics to enable imaging below the diffraction limit

• Diffraction limit in commercially available methods:

– Confocal or Widefield: 250nm in x, y and 550nm in z

– Structured Illumination: 120 nm in x, y and 320nm in z (488nm wavelength)

– Localization Microscopy ~30-50nm in x, y

– Stimulated Emission Depletion (STED): ~50-70nm in x,y and ~300nm in z

6

Super Resolution TechnologiesLocalization

How it works

• Collect series of many images, activating a few molecules at a time

• Use Gaussian model to fit and localize

• Plot all found points; create an image

7

What does it get you?

• 30-50nm resolution

Advantages

• 30-50nm resolution

• Complimentary to immuno-EM

• Simple hardware design

Disadvantages

• No live cell capability for Fluors

• Limited to biology at the coverslip

• Challenging sample preparation

• Requires time-sensitive buffers

Super Resolution TechnologiesStimulated Emission Depletion - STED

How it works

• Two excitation sources, 1 traditional laser spot + 1 doughnut shaped beam

• Fluorescence w/in doughnut is quenched, leaves smaller PSF sample

8


• 60-70nm in lateral resolution

• 560-700nm resolution axially

Advantages

• Tunable lateral resolution; Resolution depends on power of STED beam

• Only direct visualization super resolution imaging technique; No math required

Disadvantages

• Special probes

• Multi color is limited

• Photobleaching due to laser power

• Lack of increased z resolution

Super Resolution TechnologiesStructured Illumination

How it works

• Illuminate sample with known pattern

• Measure Optical Transform Function

• Utilize maths to extract higher resolution information (frequencies)

9


• Two-fold increase in resolution in x, y, and z

• Significant increase in contrast

Advantages

• Multiple imaging modes

• 3D SIM mode provides two fold increase in resolution in x, y, and z

• Live cell time-lapse imaging!!

Disadvantages

• Only two-fold increase in resolution in x, y, and z

• Artifacts

• Limited z range due to widefield mode

Super Resolution Technologies

SIM Scientific Questions

When do you need SIM?

Structural and diffraction limited

• Where does a protein localize within a centrosome (or synaptonemalcomplex or structure of interest)?

• Desmosome structure

12

Colocalization

• Two proteins which appear to colocalize by high resolution microscopy

Temporal and diffraction limited

• Resolving localization of two proteins through the cell cycle

• Bacterial cell dynamics

ImmunoEM fails or suggests additional information

• Does EM suggest additional information is to be gained?

• Do you need 3D data?

SIM Scientific QuestionsWhen do you need SIM?

Structural and diffraction limited: Imaging centrosomes

13

A-M. Gabryjonczyk, A. Ferrand – University of Basel

Sonnen et al., JCS, 2013

Distal Proximal Walls Merged

Wide-field Deconvolved 3D-SIM

SIM Scientific QuestionsWhen do you need SIM? Temporal and diffraction limited-FtsZ dynamics in E. coli

OMX 3D-SIM – Max ProjectionWidefield – Max Projection

FtsZ-mNeonGreen

Z

X

Resolve

ring

structure in

living cells

Desmond Moore, Erickson Lab Duke

Max projections

SIM applicationsFixed samples

Wid

efi

eld

3D

-SIM

Lothar Schermelleh, Juergen Neumann, LMI Munich

16

OMX 3D-Structured Illumination MicroscopyNuclear Pore Complexes

OMX 3D-Structured Illumination MicroscopyResolve ring structure at base of cilia

17

Wid

efi

eld

OM

X 3

D-S

IM

1 µm

Green OverlayRed

1 µm Max projections

Shah Lab, Harvard

DeLuca Lab, Colorado State University 18

OMX 3D-Structured Illumination MicroscopyTubulin and kinetochores in PTK1 cells

Structured Illumination How does it work?

Courtesy Stephen Cody Ph.D, Monash Micro Imaging

Structured Illumination Microscopy (SIM)

20


21


22

23

Fiber optic

cable

Widefield light path

ObjectiveOptics

Structured Illumination Microscopy (SIM)How is interference created?

24

Fiber optic

cable

Diffraction

grating


+1

0

-1

-2

+2

Collimating

lens

Objective

Structured Illumination Microscopy (3D-SIM)Imaging Sequence

Phase 1 Phase 2 Phase 3 Phase 4 Phase 5A

ng

le 1

An

gle

2A

ng

le 3

25


26

3D-SIM 2D-SIM TIRF-SIM

27

Fiber optic

cable

+1

0

-1

-2

+2

Collimating

lensDiffraction

grating


Objective

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Line spacing for each wavelength

fixed. Resolution compromised!

Back aperture

0

+1

-1

0

+1

-1

Line spacing for each wavelength

adjustable. Resolution optimized!

0

+1

-1

0

+1

-1

0

+1

-1

DeltaVision™ OMX Blaze ModuleAchieve the best resolution for each wavelength

Does it really work in live samples?

All microscopy requires compromise…

30

SpatialResolution

Phototoxicity Speed

Multi-D

Z-depth, l, t

Fixed cell imaging Photon Budget

SpatialResolution

Phototoxicity Speed

Multi-D

Z-depth, l, t

Live cell imaging Photon Budget

OMX Live Cell 3D-SIM ImagingResolve details of ER structure in living yeast

Snapp Lab, Albert Einstein

ER marker

OMX 3D-SIM Widefield

OMX Live Cell 3D-SIM Imaging2-color imaging for 200 time points at 96 fps

Widefield Image OMX 3D-SIM Image

OMX Seminar | January 2015

Mito-GFPER-mCherry

Vaughan Lab, UW32

DeltaVision™ OMX Live Cell 3D-SIM ImagingLLC-PK1 cells going through mitosis

Widefield Image OMX 3D-SIM Image

Heidi Hehnly, Scott Lab UW & HHMI

EB3-GFPMax Projections

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2D SIM TIRF Applications

mCherry-LifeAct-Actin in HeLa cells; Michael Porter, AQLM 201561 time points @ 1 2D SI TIRF frame every 5 seconds for 5 minutes!

TIRF Image 2D TIRF SIM Image

DeltaVision™ OMX SR Live Cell 2D TIRF SIMActin dynamics and “Nest” formation

mCherry-LifeAct-Actin in HeLa; Michael Porter, AQLM 2015241 time points @ 1 2D-SIM TIRF frame every 15 secs for 60 mins!


DeltaVision™ OMX SR Live Cell 2D TIRF SIMActin dynamics & Cell Viability over 60 minutes!

GFP-tagged caveolin movement

visualized with 2D-SIM TIRF

DeltaVision™ OMX SR Live Cell 2D TIRF SIMVisualize individual Caveolin to enable tracking


GFP-Caveolin in HeLa; Michael Porter, AQLM 2015151 time points @ 1 2D-SIM TIRF frame every 2 seconds for 5 minutes!

mCherry-Tubulin, GFP-Clathrin; Michael Porter, AQLM 2015360 time points @ 1 2D-SIM TIRF frame every 30 seconds for 3 hours!

DeltaVision™ OMX SR Live Cell 2D TIRF SIMClathrin plaques and microtubule dynamics


Solutions from GEHCStructured Illumination Microscopy

DeltaVision OMX SR

• Compact single enclosure system

• SIM only or Ring-TIRF only configurations available

• 2D/3D SIM light pattern generator (New Blaze module)

• 48 x 24 mm stage travel stage

• Multi-camera mounting and alignment system

• Compact Laser illumination system

• Multifunction environmental control system

Traditional SIM Light Paths: Rotary Stage or Cube Rotation Compromises

• Need to control 3 parameters within one device:

– Beam positioning for SIM

– Phase stepping

– Angular rotation

• Only one wavelength can get optimal resolution in any experiment

• Slow!

41

OMX BLAZE Structured Illumination Light Path

How did we get fast enough for live cell imaging?

Separate out the parameters!

Diffraction Grating splits the laser light into orders or beams

Phase Control Module positions the orders/beams to: Create the desired SI interference pattern Optimize resolution for each wavelength Create the phase shifts during experiments

Angle Control Module rotates the orders/beams to the 3 angles

Title or Job Number | XX Month 201X 42

What are the features of a great live cell super resolution system?

43

Optimal resolution

for all wavelengths

Localization

microscopy

Ring TIRF

Environmental

control3D SIM

Ultra-fast

widefield imaging

2D SIM

2D TIRF SIM

Acquisition speed

DeltaVision OMX SR: Improve your science, don’t reinvent it!

Super Resolution Technologies

“The more resolution you ask for the more pain you can expect . You want to use the method that gives you the poorest resolution but still gets you the biological answer you are looking for.”

– Eric Betzig, Science webinar July 2015

• OMX users published 115 peer-reviewed papers in 2015 alone!

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Sample Prep Recommendations for SIM and for conventional microscopy

47GE Title or job number

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SIM Sample Prep RecommendationsMount sample on #1.5 coverslips

Objective

#1.5 Coverslips are 170 µm thick

Microscopes are optimized for #1.5 cover glass!

High precision #1.5 coverslips

require less optimization!


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SIM Sample Prep RecommendationsDo NOT mount sample on glass slide

Objective

Glass Slide

#1.5 Coverslipx

Mounting on the slide introduces an

uncontrollable variable!

The working range of a high NA objective is very small <200µm!


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SIM Sample Prep RecommendationsMount sample on #1.5 coverslips

Objective

Glass Slide

#1.5 Coverslip

Mounting on the coverslip helps keep the sample in the working range of the

objective!


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SIM Sample Prep RecommendationsDo NOT mount tissue sections on glass slide

Glass Slide

#1.5 CoverslipxTissue Section

• Tissue sections are not flat• Uneven surface can add additional distance from objective• Makes matching RI difficult

Mounting on slide introduces additional

uncontrollable distances

Objective


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SIM Sample Prep RecommendationsMount tissue section on #1.5 coverslip

Glass Slide

#1.5 CoverslipTissue Section

Objective

Keeps sample within working range of objective & can help with imaging deeper structures


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SIM Sample Prep RecommendationsDo not use DAPI in the mounting media

Objective

Glass Slide

Coverslip

DAPI can cause high levels of background! To

image with DAPI, perform a separate labeling step &

wash well!

Use glycerol based mounting media with an anti-fade agent


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Fixed Sample Preparation


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Mounting and Antifades.

• Probably the most critical step from the imaging point of view.

• Mounting agents are designed to stabilize the sample but more importantly provide controlled(known?) refractive index to match optics.

• May protect against photobleaching if antifade agent included in formulation

• Many different formulations available.– Organic solvent based resins.

– Glycerol:Polyalcohol:Water mixes.


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Considerations for Mounting Agents

• Hardening mounting agents may give inconsistent refractive index values in sample depending on effectiveness of curing

• Non-hardening mounting agents may not preserve samples long term but typically have more stable RI

• Antifade agents generally a good idea for repeated imaging situations (3D-SIM)

• Most effective antifade reagents are not live cell compatible


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Considerations for Mounting Agents

• Hardening agents may also cause significant specimen compression if they shrink during curing, e.g. PVA/Mowiol based formulas.

• To avoid compression artifacts consider mounting sample with spacer or gasket to prevent coverslip being sucked down onto slide.

• Do not use any mounting agent that has a counterstain in it (like DAPI) as it will increase background significantly and usually gets worse over time.


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OMX Sample Prep RecommendationsMount with non-hardening mounting media

Non-hardening varieties (Vectashield) help preserve 3D structure


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Optical Considerations

• Coverslips

• Spherical Aberration – yes you have it too!

• Chromatic aberration/alignment

• Sub-resolution alignment

• Clean your lenses


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Use the right coverslips…

• Most microscope objectives are designed for #1.5 or 170 nm thick coverslips

• If you use something different you will not be able to properly correct the spherical aberration and your ability to image at depth will be compromised


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Sample Induced Spherical Aberration

X

Z

SphericalAberrationIdeal

C


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Spherical Aberration Is Real

Axial Stretching and Signal Degradation due to Spherical aberration.

100x Oil Immersion objective , 1.4 NA. 25 micron depth, 0.25 nm steps

RI 1.5 mounting media RI 1.38 mounting media

Coverslip Position


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Why do I care, I only look in 2D?

X

Y


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Axial Chromatic Aberration

UncorrectedAchromat

CorrectedApochromat

Objective

Type

Spherical

Aberration

Chromatic

Aberration

Field

Curvature

Achromat 1 Color 2 Colors No

Plan Achromat 1 Color 2 Colors Yes

Fluorite 2-3 Colors 2-3 Colors No

Plan Fluorite 3-4 Colors 2-4 Colors Yes

Plan Apochromat 3-4 Colors 4-5 Colors Yes


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SIM Sample Prep RecommendationsSummary

• Least strict requirements of all the super resolution methods

• Get sample as close as possible to #1.5 coverslip

• Use photostable dyes

• Consider using antifade agents in mounting media

• Minimize refractive index mismatch

AppendixSelected Publications in Structured Illumination Microscopy3D-SIM1. Wynne, et al. Kinetochore function is controlled by a phospho-

dependent coexpansion of inner and outer components. (http://jcb.rupress.org/content/210/6/899.long)

2. Crawley, et al. Intestinal brush border assembly driven by protocadherin-based intermicrovillar adhesion. (http://www.cell.com/cell/abstract/S0092-8674(14)00215-3)

3. Zhao, et al. The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis. (http://www.nature.com/ncb/journal/v15/n12/full/ncb2880.html)

4. Das, et al. Apical abscission alters cell polarity and dismantles the primary cilium during neurogenesis. (https://www.sciencemag.org/content/343/6167/200.short)


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AppendixSelected Publications in Structured Illumination MicroscopyLive Cell1. Strauss, et al. 3D-SIM Super resolution microscopy reveals a bead-

like arrangement for FtsZ and the division machinery: implications for triggering cytokinesis. (http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1001389)

2. Conduit, et al. A molecular mechanism of mitotic centrosome assembly in Drosophila. (http://elifesciences.org/content/early/2014/08/22/eLife.03399)

3. Liu, et al. Novel thioredoxin-like proteins are components of a protein complex coating the cortical microtubules of Toxoplasma gondii. (http://ec.asm.org/content/12/12/1588.long)


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Intro to Super Resolution: When and why you need it - PKU · 2016-11-28 · Tubulin and...

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