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

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

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

Optical Sectioning Nonlinear Confocal Detector

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

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

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)

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

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

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)

A photosynthetic unit

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

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

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

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