11

Applications of nonlinear microscopy f or studying time dynamics and structural correlation in biological

systems

Dr. R.W.Sparrow

Date 4/7/2006

Applications of nonlinear Applications of nonlinear microscopy for studying microscopy for studying

time dynamics and time dynamics and structural correlation in structural correlation in

biological systemsbiological systems

Nicole Prenta, Richard Ciseka, Catherine Greenhalgha, Raymond Sparrowb, Neeresh Rohitlallb, Maike-Svenja

Milkereita, Chantal Greena and Virginijus Barzdaa

aDepartment of Physics and Institute for Optical Sciences, University of Toronto, Toronto, Canada

bNational Laser Centre, Council for Scientific and Industrial Research, Pretoria, Republic of South Africa

OutlineOutline

Introduction to nonlinear microscopy Nonlinear multimodal microscope set-up Applications of nonlinear microscopy for

studying biological specimens

→ Structural analysis→ Dynamic investigations→ Functional understanding

Research MotivationResearch Motivation

Live 3D imaging of biological structures Observe complex cellular dynamics of

biological specimens Obtain a physical understanding of the

functional activities of biological specimens

Minimal invasion into the natural state of the biological sample

55

Optical SectioningOptical Sectioning

Detector

ConfocalNonlinear

66

Nonlinear Excitation Nonlinear Excitation ProcessesProcesses

ω1

ω1

ω2

ω1

ω1 ω3

ω1

Two-photon excitation

fluorescence

Three-photon excitation

fluorescence

Energyω

ω

ω 3ωω

ω

2ω

Second HarmonicGeneration (SHG)

Third HarmonicGeneration (THG)

Boyd, R.W. (2003) Nonlinear optics, Academic Press

(3) (3) 2 * (3) 3 3( ) (3 c.c.) ( c.c.)i t i tP t E E e E e

(2) (2) * (2) 2 2( ) 2 ( c.c.)i tP t EE E e

P(t) → polarization E(t) → electromagnetic field → nth – order nonlinear susceptibility

Harmonic GenerationHarmonic Generation

(1) (2) 2 (3) 3( ) ( ) ( ) ( )P t E t E t E t

SHG from noncentrosymmetric media

( )n

Boyd, R.W. (2003) Nonlinear optics, Academic Press

Third Harmonic Third Harmonic Generation from a Generation from a

Focused Laser BeamFocused Laser Beam Enhanced by interfaces and

multilayer structures1

Orientation dependent

n, (3)

[1] Saeta, P.N. and Miller, N.A. (2001). Applied Physics Letters 79 (17)

Nonlinear Multimodal Nonlinear Multimodal MicroscopeMicroscope

~25fs, 830nm,27MHz laser

Sample

M4

M1

M2

M3

Dichroic Mirror

L1

L2

Pinhole

M5

L3

L4M6

M7

ScanningMirrors

MPFPMT

SHG PMT

THGPMT

Imaging Biological Imaging Biological StructuresStructures

Second harmonic generation from chiral macrostructures and microcrystalline arrangments

Third harmonic generation from multilamellar structures

Multimodal nonlinear microscopy for observing and understanding cellular dynamics

ChloroplastsChloroplasts

Plant cell organelle responsible for light harvesting

Chloroplast1 Computerized model of Grana2

Chloroplast1

Mesophyll Cells1

Grana2

Plant Leaf

[1] Addison Wesley Longman, 1999 “Structures of the Plant Cell” [2] Mustardy, L. and Garab, G. (2003)Trends in Plant Science 8 (3)

1212

1313

A photosynthetic unit

1414

Circular Dichroism of Circular Dichroism of LHCIILHCII

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

400 450 500 550 600 650 700 750 800

Wavelength (nm)

Cir

cula

r D

ich

rois

m, x

10-3

(ΔA

)

Isolated LHCIIIsolated LHCII

Major light harvesting chlorophyll a/b pigment-protein complex of photosystem II (LHCII) was imaged with MPF, SHG and THG

MPF SHG THG

MPF

10 μm10 μm

SHG

10 μm

THG

ConclusionConclusion

Nonlinear microscopy is a useful tool for 3D imaging of biological samples

Nonlinear signals act as different contrast mechanisms

Observation of cellular dynamics Beneficial for a fundamental understanding of

cellular communication and bioenergetics

AcknowledgementsAcknowledgements

Arkady Majora

Steve Elmoreb

Johannes HGM van Beekb

Juerg Aus der Auc

Jeff Squierc

Virgis Barzdaa

aDepartment of Physics and Institute for Optical Sciences, University of Toronto, Toronto, Canada

bDepartment of Molecular Cell Physiology, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands

cDepartment of Physics, Colorado School of Mines, 1523 Illinois Street Golden, CO 80401, USA

University of TorontoNational Science and

Engineering Research CouncilCanada Foundation for

InnovationOntario Innovation Trust

National Laser Centre