Confocal, Airyscan and Structured Illumination ... XXX Collaborators Bruce W. Fouke, Geology, UIUC 1...
Transcript of Confocal, Airyscan and Structured Illumination ... XXX Collaborators Bruce W. Fouke, Geology, UIUC 1...
Confocal, Airyscan and Structured Illumination
Superresolution microscopy– Mayandi Sivaguru
Why and When Choose Airyscan OR SR-SIM Superresolution
Speed, data size, and processing
Performance comparison
Summary
Theory, Light Path, Resolution Comparison
Specialized Applications and Live Cell Imaging
Why and When Choose Airyscan OR SR-SIM Superresolution
Speed, data size, and processing
Performance comparison
Summary
Theory, Light Path, Resolution Comparison
Specialized Applications and Live Cell Imaging
Diffraction limit and PSF of an Optical System
Excerpt from Point Spread Function by Jeff Lichtman iBiology.org
Lateral
0.61λ/NA
Axial
2nλ/NA2
~1.5mm Depth
Shermelleh et al.,
JCB 2010, a Review
Superresolution PSF Dimensions- Theoretical comparisons
-Courtesy NYTIMES
Airyscan
352
147
Confocal Microscopes- Light Path-Optical Sectioning, Resolution
Images are from Zeiss Campus
Resolution and Depth Performance Confocal Microscopy
Sivaguru et al., 2012, Plos One
DOI: 10.1371/journal.pone.0039129
Structured Illumination Microscopy (SIM)Concepts (Real Space)
“known“ pattern “Sample“ image
Carl Zeiss MicroImaging GmbH, Dr. Stephan Kuppig, Product Management BioSciences (Jena)
9
SR-SIM: Structured Illumination Microscopy Concepts (Frequency Space)
kx
ky
Frequency Space
k0
Object Image
Object Image
Carl Zeiss MicroImaging GmbH, Dr. Stephan Kuppig, Product Management BioSciences (Jena)
Gustafsson et al., Biophys J 2008
Matts Gustafsson 1960-2011Photo Courtesy of Paul Fetters, Nature Methods
SR-SIM: Structured Illumination Microscopy-TheoryConcepts (Frequency Space)
2fc
S(fx-2fc’)S(fx+2fc’)S(fx)
Klaus Weisshart, 2014 Zeiss White Paper
18.05.2011 11Carl Zeiss MicroImaging GmbH, Dr. Stephan Kuppig, Product Management BioSciences (Jena)
System ComponentsELYRA illumination module
Sample illumination in all camera-based
detection modes in ELYRA is made
possible by:
… a single illumination module
… the same laser module
… the same set of lasers
Solid-state lasers for a broad range
of fluorochromes:
405 nm (50 mW)
488 nm (100 mW)
561 nm (100 mW)
642 nm (100 mW)
Laser Port
(405 nm line)
Laser Port
(RGB lines)
Telescope
Field Rotator
Slider with
Phase Gratings
TIRF Mirror
Field adjustment
Port for
X-Cite 120
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LSM 880 and Airyscan Light path
Inside the LSM 880Coupling of the Airyscan Module
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Efficient handling at laser input
Apochromatic
pinhole optics
Fastest linear
scanning, tp.
controlled
Low incident angle
dichroics, high
laser rejection
QUASAR: single-shot spectral
detection; cooled and improved
electronics, higher data throughput
Airyscan detector
for superresolution
Hexagonal GaAsP
detection array
• As pinhole is reduced
below 1 AU, wave
optical properties
begin to dominate
• An infinitely small
pinhole yields identical
illumination and
detection PSFs
• Both lateral and axial
resolution criteria can
be reduced by a
factor of 1.4
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Resolution Limits of a Confocal LSMEffects of Smaller Pinhole Sizes
𝐝𝐳 =𝟎. 𝟔𝟒 𝛌
𝒏 − 𝒏𝟐− 𝐍𝐀𝟐
𝐝𝐱𝐲 =𝟎. 𝟑𝟕 𝛌
𝐍𝐀
𝐝𝐱𝐲 =𝟎. 𝟓𝟏 𝛌𝐞𝐱𝐍𝐀
𝐝𝐳 =𝟎. 𝟖𝟖 𝛌𝐞𝐱
𝒏 − 𝒏𝟐− 𝐍𝐀𝟐
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Resolution Limits of a Confocal LSMAt Small Pinholes, Signal Loss > Resolution Gains
Normalized to Peaks Normalized to 2 AU Intensity
Small pinhole diameters lead to improved
resolution steadily until about 0.2 AU, results
in deeper dips between two objects
However, constricting the pinhole
actually yields a drastic reduction in
signal below 1 AU
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Airyscan DetectionFarewell to the Pinhole
• 32 GaAsP detectors
in hexagonal lattice
• Each detector
approximately 0.2 AU
in diameter
• Total detection area
approximately 1.25
AU in diameter
• Simultaneous
improvement in
resolution and
signal
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Airyscan Detection
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Advanced Concepts of the AiryscanConventional Scanning Confocal (1 AU)
A point-like emitter generates a
diffraction limited pattern (~ PSF)
PH = 1 AU
scan
scan
Inte
nsity
excitation
At 1 AU the PSF is
mapped directly 1:1
Excitation and detection
are scanned in sync
~ 240 nm
detection
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Advanced Concepts of the AiryscanAiryscan – A Single Element Improves Resolution
detection
PH = 1.25 AU
excitation
scan
subunit
~ 0.2 AU
By collecting just the central element,
the PSF is weaker but narrower
A point-like emitter generates a
diffraction limited pattern (~ PSF)
Inte
nsity
scan
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Advanced Concepts of the AiryscanAiryscan – An Offset Element Improves Resolution
detection
PH = 1.25 AU
excitation
subunit
~ 0.2 AU
Inte
nsity
scan
scan
By an element offset from the center,
the resolution is still improved
A point-like emitter generates a
diffraction limited pattern (~ PSF)
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Advanced Concepts of the AiryscanAiryscan – Combining the Data
detection
PH = 1.25 AU
excitation
subunit
~ 0.2 AU
Inte
nsity
scan
scan
An Airyscan image is formed by:
1. Reassigning the offset signal
2. Summing the contributions
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Advanced Concepts of the AiryscanReducing the Effective PSF
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Airyscan ProcessingImmediate Pixel Reassignment
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Airyscan ProcessingIsotropic 1.7x Resolution Improvementdetector wise deconvolution
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Airyscan ProcessingDetector-Wise Deconvolution
Confocal microscope
Plan-Apochromat 63x/1.4
633nm illumination
Approx. resolution: 260 nm
Pixel reassignment
1.4x improved resolution
Airyscan processing
1.7x improved resolution
170 nm fluorescent beads
0.5 mm 0.5 mm 0.5 mm
Approx. resolution: 185 nm Approx. resolution: 153 nm
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Why Linear Scanners?Ensuring Versatility and Quantifiable Results
field of view
scan line (x)
pix
el ti
me
85% on
15% off
Linear Scanning (Zeiss)
scan line (x)
pix
el ti
me
field of view40% off
60% on
Duty CycleSine Scanning
requires
equalization
12% discarded
usable pixel time
natively linear
no discarding
of the signal;
time-efficient
Duty Cycle
*Compared at same imaging speed and FOV
**Compared at same S/N level
• Linear scans feature:- 29% higher S/N*
- 66% longer pixel time*
- Lower light dosage**
- Decreased
photodamage**
- Constant scan speed
- Uniform excitation
• Sine (and resonant)
scanners yield images
that are inherently not
quantitative
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Improved signal recording:
Crisp details, clear image data
Improved S/N ratio:
Black background
What Defines Sensitivity?Aspects of Signal and Noise
Detectors-QE: Confocal, Airyscan and SR-SIM
AiryscanSR-SIM
Confocal
Better image quality- Higher signal-to-noise
with detection of faint
signals; look deeper
Faster scanning- Shorter pixel dwell
times, reduced need for
averaging
Longer imaging- Lower laser power
prevents phototoxicity
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Cultured 2h8 cells labeled with extremely low expression of GFP and
mCherry. Courtesy A. Bruckbauer Cancer Research, London, UK
PMT GaAsP
PMT
What Defines Sensitivity?And What Does Increased Sensitivity Enable?
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What Defines Sensitivity?Aspects of Signal and Noise
Why and When Choose Airyscan OR SR-SIM Superresolution
Speed, data size, and processing
Performance comparison
Summary
Theory, Light Path, Resolution Comparison
Specialized Applications and Live Cell Imaging
01/30/2013 32Unpublished data
Experimental PSFs-3 Channels
WF SR-SIM
56
1 n
m4
88 n
m
Confocal Airyscan130-170 nm
FWHM
120-157 nm
FWHM
SENSITIVITY• No need for additional spatial sampling;
uses photons discarded in conventional
confocals with mechanical pinhole
SPEED• Higher inherent S/N ratio can be traded
for faster imaging as needed
RESOLUTION• A 1.7-fold higher resolution in all
three dimensions, yielding 5 times
smaller confocal volumes
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Concepts of the AiryscanMultifunctional Benefits for “Photon Budget”
For the same resolution,
a standard confocal pinhole would
have to be closed to 0.2 AU
(with consequent loss in S/N)
Why and When Choose Airyscan OR SR-SIM Superresolution
Performance comparison
Summary
Theory, Light Path, Resolution Comparison
Specialized Applications and Live Cell Imaging
Speed, data size, and processing
Thin Vs Thick samplesSR-SIM
Jingyi Fei, TJ Ha Lab
Urban and Barclay, Surangi Punyasena Lab
Airyscan
Jennifer Mitchell, Martha Gillette Lab
Unlabeled and Labeled Pollen Comparison_SR-SIM
Unlabeled Labeled-PAS
Comparison in 3D Performance
Confocal/AS/SR-SIM
Comparison
SEM data Mander et al., 2013, Royal Society Proceeding
SR-SIM Resolving the Areolae and Granulae of grass pollen
SR-SIM in Resolving Areolae and Granulae Contrast in Pollen
Sivaguru et al., 2015, MRT (in preparation)
SEM data Mander et al., 2013
Royal Society Proceeding
SR-SIM resolving the Granulae
and Areolae of grass pollen
Unlabeled pollen
Optical Transfer Function Comparison- Confocal, Airyscan and SR-SIM
Sivaguru et al., 2015, MRT (in preparation)
125 nm
Klaus Weisshart, 2014 Zeiss White Paper
Why and When Choose Airyscan OR SR-SIM Superresolution
Speed, data size, and processing
Performance comparison
Summary
Theory, Light Path, Resolution Comparison
Specialized Applications and Live Cell Imaging
SR-SIM
AiryscanAt 512 x 512 pixels 13 fps
At 512 x 16 pixels 430 fps
At max speeds 4x larger field of v
Image Size 16 times larger
1024x1024
Confocal Mode ()/Airyscan Mode/After Processing
Why and When Choose Airyscan OR SR-SIM Superresolution
Speed, data size, and processing
Performance comparison
Summary
Theory, Light Path, Resolution Comparison
Specialized Applications and Live Cell Imaging
Speed, data size, and processing
Live Cell Imaging Mitochondria- Ratiometric ROS indicator-SR-SIM
Vladimir and Jessica, Rex Gaskin‘s Lab
Airyscan Live Cell Imaging FRET Acceptor Photobleaching
in LSM 880 with GaAsP detector
Xinyu K, Debra Leckband Lab
Ryan Lake, Chemistry Dept.,
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Mitosis in HeLa EB3 EGFP-Histone 2B mCherry, Airyscan Superresolution_Jan Ellenberg, EMBL
Why and When Choose Airyscan OR SR-SIM Superresolution
Performance comparison
Summary
Theory, Light Path, Resolution Comparison
Specialized Applications and Live Cell Imaging
Speed, data size, and processing
Choosing the right optical system for your application*
XXX
XXX
http://www.igb.illinois.edu/core
CollaboratorsBruce W. Fouke, Geology, UIUC1
Kimani Toussaint, MEL, UIUC1
Manabu Nakamura, FSHN1
Elvira Demejia, FSHN1
Surangi Punyasena, Plant Biol. UIUC1
Michael Urban, Plant Biol. UIUC*
Luke Mander, The Open Univ.1
Richard Barclay, Smithsonian Institute*
M Taher A Saif. MEL, UIUC1
Hyun Joon Kong, Bioengineering, UIUC*
Logan Liu, ECE, UIUC1
Dean E. Riechers, Plant Biol. UIUC1
Aleel Grennan, Plant Biol. UIUC*
Don Ort, Plant Biol. UIUC*
Steven Huber, Plant Biol. UIUC1
Allison Stewart, Vetmed, UIUC1
Matthew Stewart, Vetmed, UIUC1
Aaron Johnson, Speech and Hearing, UIUC*
Sakthivel Sadayappan, Loyola Univ. Chicago1
Matthew Curtis, Carl Zeiss LLC
Scott Oleynch, Carl Zeiss LLC
Courtney Akitake, Carl Zeiss LLC
Core StaffGlenn A. Fried, Director1
Xudong Guan
Austin Cyphersmith*
Kingsley Boateng*
Joyce Koberlain