2. Applications of nonlinear microscopy for studying time dynamics and structural correlation in biological 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

3. Outline Introduction to nonlinear microscopy
Nonlinear multimodal microscope set-up
Applications of nonlinear microscopy for studying biological specimens
Structural analysis
Dynamic investigations
Functional understanding

4. Research 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

5. Optical Sectioning
Nonlinear
Confocal
Detector

6. Nonlinear Excitation Processes
Energy  ω ω1  ω1 ω1 ω ω 3ω ω3 2ω ω2 ω1 ω1 ω ω
Two-photon excitation fluorescence
Three-photon excitation fluorescence
Second Harmonic
Generation (SHG)
Third Harmonic
Generation (THG)
Boyd, R.W. (2003) Nonlinear optics, Academic Press

7. Harmonic Generation SHG from noncentrosymmetric media
P(t) → polarization
E(t) → electromagnetic field
→ nth – order nonlinear susceptibility
SHG from noncentrosymmetric media
Boyd, R.W. (2003) Nonlinear optics, Academic Press

8. Third Harmonic Generation from a Focused Laser Beam
Dn, Dc(3)
Enhanced by interfaces and multilayer structures1
Orientation dependent
For a phase mismatch that is zero or negative, the energy in the TH peaks at the focus of the
fundamental beam, and, after the focus, destructive interference
causes all the TH to be reabsorbed by the fundamental.
For a phase mismatch that is zero or
negative, the energy in the TH peaks at the focus of the
Phase mismatch that is zero or negative the energy in the TH peaks at the focus of the fundamental beam, but after the focus destructive interference results in neglible far field THG
[1] Saeta, P.N. and Miller, N.A. (2001). Applied Physics Letters 79 (17)

9. Nonlinear Multimodal Microscope
~25fs, 830nm,
27MHz laser L4 M5
Dichroic Mirror
MPF
PMT L1 L3
Pinhole
Sample L2 M4 M2 M7
THG
PMT M3
Scanning
Mirrors
SHG
PMT

10. Imaging Biological Structures
Second harmonic generation from chiral macrostructures and microcrystalline arrangments
Third harmonic generation from multilamellar structures
Multimodal nonlinear microscopy for observing and understanding cellular dynamics

11. Chloroplasts Plant cell organelle responsible for light harvesting
Plant Leaf
Mesophyll Cells1
Chloroplast1
Plant cell organelle responsible for light harvesting
Grana2
Chloroplast1
Computerized model of Grana2
[1] Addison Wesley Longman, 1999 “Structures of the Plant Cell”
[2] Mustardy, L. and Garab, G. (2003)Trends in Plant Science 8 (3)

13. A photosynthetic unit

14. Circular Dichroism of LHCII
Circular Dichroism, x10-3 (ΔA)

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

16. Conclusion
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

17. Acknowledgements University of Toronto
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 Toronto
National Science and Engineering Research Council
Canada Foundation for Innovation
Ontario Innovation Trust
National Laser Centre