a Department of Physics and Institute for Optical Sciences, University of Toronto, Toronto, Canada

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. Confocal Optical Sectioning Nonlinear Detector

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

8. Third Harmonic Generation from a Focused Laser Beam • Enhanced by interfaces and multilayer structures1 • Orientation dependent Dn, Dc(3) [1] Saeta, P.N. and Miller, N.A. (2001). Applied Physics Letters 79 (17)

9. Nonlinear Multimodal Microscope M1 M6 ~25fs, 830nm, 27MHz laser L4 M5 Dichroic Mirror MPF PMT L1 L3 Pinhole Sample L2 M4 THG PMT M2 M7 Scanning Mirrors M3 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. Chloroplast1 Plant Leaf Mesophyll Cells1 Grana2 Computerized model of Grana2 Chloroplast1 Chloroplasts • Plant cell organelle responsible for light harvesting [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. MPF SHG THG SHG THG MPF 10 μm 10 μm 10 μm Isolated LHCII • Major light harvesting chlorophyll a/b pigment-protein complex of photosystem II (LHCII) was imaged with MPF, SHG and 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 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