Upload
rodger-welch
View
216
Download
0
Tags:
Embed Size (px)
Citation preview
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