CARS Microscopy: Chemical Imaging & Spectroscopy in Biophotonics, Lab Reports of Biology

Download CARS Microscopy: Chemical Imaging & Spectroscopy in Biophotonics and more Lab Reports Biology in PDF only on Docsity! Work at LLNL was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory, under contract No. W-7405-Eng-48. This work was also supported by funding from the National Science Foundation. The Center for Biophotonics, an NSF Science and Technology Center, is managed by the University of California, Davis, under Cooperative Agreement No. PHY 0120999. Application of Coherent anti-Stokes Raman Scattering (CARS) Microscopy for Chemical Imaging and Spectroscopy Special Topics in Biophotonics (EAD289) James Chan, Ph.D. Lawrence Livermore National Laboratory NSF Center for Biophotonics Science and Technology (CBST) January 18, 2007 Outline • Motivation • Background theory on Raman spectroscopy • Spontaneous Raman imaging • Background theory on Coherent Anti-Stokes Raman Scattering (CARS) • CARS Instrumentation • Brief Introductions to F-CARS, E-CARS, M-CARS • Application of CARS to cell imaging • Future directions • Summary Raman scattering is the interaction of photons and intrinsic molecular bonds C.V. Raman 1930 Nobel Prize Incident light Molecular vibration in sample Ei = hωi anti-Stokes ωas Eas = hωas ωi ωas Wavelength ωs Scattered light ν0 ν1 Stokes ωi Es = hωs ωi ωs Boltzmann distribution Ground state Virtual state Excited state Polarizability induced dipole equation Classical picture of Raman and Rayleigh scattering with a diatomic molecule Electric field of incident light oscillating at frequency Induced dipole from this E-field Molecular polarizability changes with bond length The bond length oscillates at vibrational frequency Polarizability oscillates at vibrational frequency Rayleigh Anti-Stokes Stokes E = Eo cos (ωi t) µind = αE = α Eo cos (ωi t) α = αo + (r – req) (dα / dr) r – req = rmax cos (ωvib t) α = αo + (dα / dr)rmaxcos(ωvib t) µind = αoEocos(ωit) + (1/2)Eormax(dα / dr)[cos((ωi+ωvib) t) + cos((ωi-ωvib)t)] Raman spectra of cells provide a wealth of biological information Wavenumber units Single live human T cell k = 1/λ (cm-1) Raman shift = (1/λincident) – (1/λscattered) Raman mapping combined with fluorescence microscopy is a useful dual modality for studying cells 30 min per image Huang et. al., Biochemistry, V44, 10009-10019 (2005) Live S. pombe cell 632 nm laser GFP mitochondria image Researchers studying S. pombe cells discovered a yet to be assigned 1602 cm-1 Raman band 1602 cm-1 signal colocalizes with GFP signal Advantages and limitations of spontaneous Raman imaging Limitations • Fluorescence interference • Limited spatial resolution • Weak signal – long integration times Raman scattering is extremely inefficient (10-30 cm2 cross sections) 1 in 108 incident photons are Raman scattered Advantages • Minimally invasive technique • Non-photobleaching signal for live cell studies • Works under different conditions (temperatures and pressures) • Chemical imaging without exogenous tags • Works with different wavelengths Why develop CARS? • Contrast signal based on vibrational characteristics, no need for fluorescent tagging. • CARS signal is at high frequency (lower wavelength) – no fluorescence interference • Higher resolution • More sensitive (stronger signals) than spontaneous Raman microscopy – faster, more efficient imaging for real-time analysis Major improvements developed in 1999 • First CARS studies in 1965 • Advancement of laser technology • Tight focusing conditions relax phase matching conditions • Collinear geometry makes it much easier to implement • 3-D sectioning, through cells, tissue • Near IR light reduces potential laser damage to cells, tissue CARS is a third order nonlinear optical process, requiring high intensity laser pulses Requires high intensity, pulsed laser sources (ps, fs) Higher order terms becomes important when peak powers are high Polarization Phase matching conditions For CARS, kS kP kP kAS P(t) = χ(1) E(t) + χ(2) E(t)2 + χ(3) E(t)3 + … PAS = χ(3) Ep2 Es IAS = Ip2 IS [ sin (∆kz/2) / (∆kz/2) ] 2 Tight focusing using a high NA objective is key for CARS microscopic imaging Microscope coverslip 100X, 1.3 NA oil immersion objective Translation stage Trapped particle • Phase matching condition relaxed • Tight focus generates highest intensity at laser focus • CARS signal generated within focal volume • 3-D sectioning capability Intensity distribution of an optical field focused by a 1.4 NA objective Cheng et. al., J. Phys. Chem. B. V108, 827-840 (2004) Picosecond or femtosecond pulses, which is better? There are several tradeoffs Femtosecond pulses (80 fs ~ 70 cm-1) Picosecond pulses (5 ps ~ 3.6 cm-1) Raman bands typically 10 cm-1 Wavenumber Ps pulses focus all energy to a single Raman band to maximize coherent vibration, at expense of losing peak intensity and multiplex advantage with fs pulses Key components in a CARS microscope setup 100X Microscope objective Dichroic beamsplitter Scan stage or scanning mirrors Filter No spatial filter APD CARS signalωsωp t Telescopes Forward CARS Epi CARS First demonstration on 910 nm polystyrene beads Zumbusch et. al., Phys. Rev. Lett. V82, 4142 (1999) Example : CARS image Laser powers - 2 and 1 mW, tuned to 1570 cm-1 (protein, nucleic acid) image acquired in 8 min, smallest feature <300 nm Unstained live human epithelial cell Cheng et. al., J. Phys. Chem. B. V105,1277 (2001) Example : CARS imaging of bacterial spores 5 µm Raman spectrum of bacterial spore CARS lasers tuned to 1013 cm-1 vibration CARS signal at 697 nm CARS image of spores on glass substrate ω0 = 750 nm ωS = 812 nm ωAS Long-term dynamic cell processes can be monitored with CARS microscopy Conversion of 3T3-L1 fibroblast cells to adipocyte (fat) cells 0 hr 48 hr 60 hr 192 hr Imaging of triglyceride droplets at 2845 cm-1 (lipid vibration) Nan et. al., J. Lipid Res. V44, 2202 (2003) Comparison of F-CARS and E-CARS image NIH 3T3 cells C-H 2870 cm-1 lipid membrane Nuclear membrane edge visible in F-CARS, large axial length Dark image due to destructive interference in E-CARS Cytoplasm overwhelmed by solvent signal Cheng et. al., Biophys. Journal, V83, 502-509 (2002) Small scatterers in cytoplasm visible in E-CARS Nonresonant background is a major issue in CARS microscopy Polarization CARS (P-CARS) – Cheng et. al, Optics Letters, V26 1341 (2001) Epi-CARS (E-CARS) – suppression of bulk background solvent Dual pump CARS microscopy can be used to subtract nonresonant background Burkacky et. al., Optics Letters, V31, 3656 (2006) On resonance Off resonance Difference ωStokesωStokes ωPump We have been applying Raman spectroscopy for single cell cancer detection Coverslip 100X, 1.3 NA oil immersion objective Translation stage Trapped cell Presentation by D. Taylor on Laser Tweezers Raman Spectroscopy Normal T cell Cancer T cell Spontaneous Raman spectra takes 2 minutes per cell Chan et. al., Biophys. Journal. V90, 648 (2006) Future applications : CARS cytometry for rapid, label- less cancer cell detection and sorting Chan et. al., IEEE J. Sel. Topics. Quant. Elec. V11 858 (2005) Potential solution for faster chemical analysis of cells Trapped polystyrene bead using two CARS beams Microsecond temporal resolution CARS signal from a C=C bond We have demonstrated optical trapping combined with CARS for faster spectral analysis Future applications : CARS in-vivo imaging Stratum corneum Adipocytes of the dermis Adipocytes of subcutaneous layer 2845 cm-1 vibration C-H lipid Evans et. al., PNAS, V102 16807 (2005)