where is CARS headed Eric Potma University of
- Slides: 31
where is CARS headed? Eric Potma University of California, Irvine
target
Raman molecular signatures Bacteriophage P 22 (DNA and capsid proteins) O-P-O Amide I CH 3 CH 2 Thomas, Annu. Rev. Biophys. Struct. 28, 1 (1999) DNA @ 788 cm-1 protein @ 1451 cm-1 Fixed apoptotic He. La cell > 30 minutes Uzunbajakava et al. , Biophys. J 84, 3986 (2003)
towards building CARS C. V. Raman in 1928: “. . that the effect is a true scattering and not a fluorescence is indicated in the first place by its feebleness in comparison with the ordinary scattering. ” Nicolaas Bloembergen in 1978: “As spontaneous Raman spectroscopy has blossomed and grown during one half-century, it may be predicted with some confidence that coherent nonlinear Raman spectroscopy will yield many new results in the next half-century. ”
more photons: CARS wprobe wpump wvib w. Stokes w. CARS For a 1 µm polystyrene bead, same average power: CARS is > 104 times stronger than spontaneous Raman Detector
first CARS experiments
first CARS microscope Duncan, Reintjes, Manuccia, Optics Lett. 7, 350 (1982)
from slow to fast 1999 2003 30 minutes 1 sec/frame Zumbusch, Holtom, Xie, Phys. Rev. Lett. 82, 4142 (1999) Nan, Cheng, Xie, J. Lipid. Res. 44, 2202 (2003)
CARS and Microscopy ‘turnkey’ system 780 -930 nm OPO 1064 nm pump Confocal microscope -1500 – 3500 cm-1 range - ~ 5 ps pulses - up to 20 m. W @ 80 MHz - down to 1 sec/frame (512 x 512)
nonlinear coherent signals under tightly focused conditions L
Imaging modes det 2 µm 200 nm Polystyrene bead @ 3050 cm-1 det 2 µm
signal in epi-CARS
vibrational imaging in 3 D F-CARS Epithelial cells CH 2 stretch @ 2845 cm-1 E-CARS
Real-time hydrodynamics OH-stretch vibration of water (3200 -3600 cm-1) 60 ms/line scan
Real-time hydrodynamics Flushing cells with D 2 O buffer
Diffusion of lipid droplets 5 mm LDs in Y-1 cells. 1 s/frame Pump: 1 m. W Stokes: 0. 5 m. W @ (8 MHz)
high sensitivity: single bilayers H H F-CARS: lysed red blood cell H H C C C H y Ep, ES x C H H C C H H H
visualizing lipid phases 1: 1 DOPC: DSPC (70% D) 2090 cm-1 2140 cm-1
in vivo tissue imaging CH 2 vibration (fat) @ 2845 cm-1 Pump 816 nm (50 m. W), Stokes 1064 nm (50 m. W) 20 frames/s, 640 x 480 pixels
back-scattering in tissue 60 B 50 40 image plane Percent reflected back 30 20 10 0 0 50 100 150 Thickness (µm) 200 250
in vivo tissue imaging mouse ear Stratum corneum Fat cells Sebaceous glands Subcutaneous layer images are averaged
in vivo tissue imaging
tissue imaging 3 D rendering of mouse sebaceous gland courtesy of Conor Evans, Harvard
in vivo tissue imaging
Improving CARS Raman CARS
CARS: the next phase Es = Er + E nr Eref
Heterodyne CARS Raman CARS H-CARS
signal amplification CARS interferometry
heterodyne CARS imaging 2845 cm-1 | (3)|2 Im (3) Re (3) 2950 cm-1 | (3)|2
The future of CARS turnkey, hands-off system for routine imaging improving sensitivity through optimizing excitation and detection schemes video-rate imaging of structures in skin tissue towards biomedical applications of nonlinear vibrational microscopy
Thanks! Sunney Xie – Harvard University Conor Evans, Xiaolin Nan, Wei Yang – Xie Group Jun Ye – JILA, university of Colorado Charles Lin – Wellman, MGH Kenneth Kosik – Harvard Institute of Medicine Axel Nohturfft – Harvard Molecular Cellular Biology John Pezacki – Steacie Institute Ottawa Yiwei Jia – Olympus Microscopes Y. Pang – Coherent Lasers Daniel Kopf – High-Q Lasers
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