Multiphoton Imaging and Spectroscopy in Cell and Tissue Biophysics. J Moger and C P Winlove

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1 Multiphoton Imaging and Spectroscopy in Cell and Tissue Biophysics J Moger and C P Winlove

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3 Relating Structure to Function Biochemistry Raman microspectrometry Surface enhanced Raman spectrometry (SERS) Coherent antistokes Raman spectrometry (CARS) Structure Two photon fluorescence (TPF) Second harmonic generation (SHG)

4 Molecular Bases of Elastin Elasticity Raman Microspectrometer. Model is a Rennishaw RM1000 Microscope Body Notch filter & polariser / waveplate Laser light only Laser & Raman light Raman light only Diffraction Grating CCD Detector Mechanical Testing Apparatus Force transducer Clamps Stress Extension Transducer Stepper Motor Mirror Slit Sample Mirror Objective Lens Pinhole Laser in Trough Sample Heater Micrometer Elastin: Unique mammalian elastic protein 100% strain Lasts a lifetime Experiments: Ellen Green

5 Raman Spectrometry on Elastin Fibres 1500 Counts w a t e r A m i d e I Counts Raman Shift (cm-1) Raman Shift (cm-1) Amide I Effects of strain on: Peak position - standardisation? Peak height/area - baseline? Amide bands (secondary structure) decomposition algorithm? Hydration baseline & decomposition? Polarisation quantification? X X X

6 Surface Enhanced Raman Scattering (SERS) Raman spectrum excited by plasmons on metallic nanostructures (Fleischmann ) Enhancement of signal for adsorbed molecules Electromagnetic? Chemical? (1) Fleischmann, M, Chem. Phys. Lett (1974)

7 Surface Enhanced Raman Spectrometry Colloids: Metallic particles (1 100 nm) Aggregation => plasmons in gaps Resonance ~ particle diameter and cluster size Nanoshells: Do not require aggregation to support surface plasmons Enhancement without aggregation. Overcome colloid instability 10 7 x enhancement Antibody targetting? Experiments: Natalie Cornes Metal coat Polymer core

8 Coherent Anti-Stokes Raman Scattering (CARS) ω as = 2ω p - ω s Optically pump molecule into excited state: ω ω s ω ω as I as 2 2 ωasl = ε c n n 9 (3) 2 2 χ I I sin c 2 p s o as pns Δk. 2 L p p Optimal excitation is achieved by two temporally overlapped pico-second pulses (ωp, ωs ) Tune (ω p ω s ) to match vibrational levels (Active driving & coherent build up => 10 5 x amplification) Scan ω p and record intensity of anti-stokes signal =>spectrum. Surface enhanced CARS => enhancement (SERS 3 )

9 Multiphoton Microscopy & Spectroscopy System SHG/TPF Ti:Saph OPO PP 532nm 532/1064 nm OPO DM DM PP IX50 FV300 IX71 Optical parametric oscillator Pulse Picker Confocal headstage & spectrometer: Raman/CARS Confocal headstage: fluorescence, back & forward SHG, TPF Lasers

10 Second Harmonic Generation (SHG) and Two Photon Fluorescence (TPF) SHG Half incident wavelength Occurs in asymmetrical structures e.g: Rod shaped proteins: collagen, myosin and tubulin Membranes Polarization dependent Reflects molecular asymmetry and supramolecular organisation TPF > Half incident wavelength Reflects distribution of specific fluorophores 800nm laser light stimulates: - Elastin (ECM) - NAD(P)H (cells) - Flavoproteins (cells)

11 Advantages of Non-Linear Microscopy No staining No sectioning IR penetration images at depth > 100μm Resolution < 1μm 3D scans Minimal photo-bleaching in vivo technique?

12 Imaging Articular Cartilage Structure: Surface zone Transitional zone Radial zone Tidemark Collagen Fibre Organisation Calcified Cartilage Subchondral Bone Cellular Composition Questions: Microscopic composition Responses to e.g mechanical loads? Focal changes in e.g arthritis?

13 Backscattered Cartilage Surface Imaging SHG (λ = 400nm) TPF (460nm <λ < 550nm) Fibrous structure Site of chondrocyte Nucleus? Site of chondrocyte Pericellular matrix Field 125 X 125μm Experiments Jessica Mansfield

14 Polarisation Variations with Depth 6a. 6b. Polarization Angle Depth in Microns Normalised Intensity Polarization Angle Depth in Microns Normalised Intensity SHG TPF

15 Problems in Multiphoton Imaging of Tissue Sources of fluorescence Mechanisms of second (and higher) harmonic generation Effects of overlying tissue Scattering/absorbtion Birefringence Radiation effects on tissue Spectrum analysis/quantification RM Williams et al. Biophys. J. 88, (2005)