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

11/3/2015 12Carl Zeiss Microscopy

LSM 880 and Airyscan Light path

Inside the LSM 880Coupling of the Airyscan Module

11/3/2015 13Carl Zeiss Microscopy

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

11/3/2015 14Carl Zeiss Microscopy

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

11/3/2015 15Carl Zeiss Microscopy

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

11/3/2015 16Carl Zeiss Microscopy

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

11/3/2015 17Carl Zeiss Microscopy

Airyscan Detection

11/3/2015 18Carl Zeiss Microscopy

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

11/3/2015 19Carl Zeiss Microscopy

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

11/3/2015 20Carl Zeiss Microscopy

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)

11/3/2015 21Carl Zeiss Microscopy

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

11/3/2015 22Carl Zeiss Microscopy

Advanced Concepts of the AiryscanReducing the Effective PSF

11/3/2015 23Carl Zeiss Microscopy

Airyscan ProcessingImmediate Pixel Reassignment

11/3/2015 24Carl Zeiss Microscopy

Airyscan ProcessingIsotropic 1.7x Resolution Improvementdetector wise deconvolution

11/3/2015 25Carl Zeiss Microscopy

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

11/3/2015 26Carl Zeiss Microscopy

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

11/3/2015 27Carl Zeiss Microscopy

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

11/3/2015 29Carl Zeiss Microscopy

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?

11/3/2015 30Carl Zeiss Microscopy

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

11/3/2015 33Carl Zeiss Microscopy

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.,

11/3/2015 48Carl Zeiss Microscopy

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