CARS Microscopy Third Annual Workshop

1
CARS Microscopy Third Annual Workshop
Caitriona Creely (Workshop Attendee)
Harvard University Summer 2006
2
Introduction

• I attended the 3rd Annual CARS Workshop in
  Harvard in June 2003
• Here are some of the basic ideas of the
  technique.

3
Workshop

• Sunney Xie Chemist
• Came to Harvard in 1999
• Pioneered CARS developments
• Third annual CARS workshop
• Physicists, Biologists, Industry people
• Lectures
• Lab practicals

IMPORTANT CARS Images, some text and schemes
taken directly from the group website
at http//bernstein.harvard.edu, or from the
references cited.
4
Outline

• What is CARS?
• Why use it?
• Non Resonant Background
• Sources
• Detection schemes
• Applications
• Problems?
• Discussion

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What is CARS?

• Non linear Raman process
• Generated at the focus of the beam
• ?AS 2?p - ?s

CARS Microscopy for Biology and Medicine E.
Potma X. S. Xie Optics and Photonics News 40
November (2004)
6
What is CARS?

• Two lasers at different frequencies
• ps or fs pulse trains with high peak Intensity
• Two beams spatially overlapped at focii
• Two beams temporally overlapped

CARS Microscopy for Biology and Medicine E.
Potma X. S. Xie Optics and Photonics News 40
November (2004)
7
Why use CARS?

• Intrinsic vibrational contrast
• Strong, directional signal gt Sensitive
• Requires moderate average powers good for
  biological samples
• Only generated at focus gt 3D sectioning
  capability
• Higher in frequency than one-photon fluorescence
  gt easily detected in presence of a strong
  fluorescent background.
• Near IR gt little scattering, deep penetration in
  tissues
• Near IR gt little absorption, Low photodamage

8
Non-resonant Background

• Non-resonant Background from the bulk!

Coherent Anti-Stokes Raman Scattering Microscopy
Instrumentation, Theory, and Applications J.
Cheng X. S. Xie J. Phys. Chem. B 108 827 (2004)
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Sources

• Two colours
• Difference between pump and Stokes span the
  vibrational spectrum
• Modelocked sources
• Picosecond pulses
• Time-bandwidth product
• Tunability for exciting different vibrations
• OPO pumped by ps Laser

Coherent Anti-Stokes Raman Scattering Microscopy
Instrumentation, Theory, and Applications J.
Cheng X. S. Xie J. Phys. Chem. B 108 827 (2004)
10
Detection Schemes

• F CARS Forward detected
• E CARS Epi detected
• P- CARS Polarisation dependent
• FM CARS Frequency modulated

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F CARS
Vibrational contrast when ?p ?s tuned to a
Raman active vibrational band
Three-Dimensional Vibrational Imaging by Coherent
Anti-Stokes Raman Scattering Andreas Zumbusch,
Gary R. Holtom, and X. Sunney Xie, Phys. Rev.
Lett. 82 (20), 4142 (1999)
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F CARS
CARS Microscopy for Biology and Medicine E. Potma
X. S. Xie Optics and Photonics News pg 40
November (2004)
13
Detection Schemes

• F CARS Forward detected
• E CARS Epi detected
• P- CARS Polarisation dependent
• FM CARS Frequency modulated

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E CARS

• Avoid non resonant background!
• Objects smaller than ?p/3 (too short for
  destuctive interference)
• Interfaces with different ?3 - to propagation
• Back-scattering of F CARS, reflactive/turbid
  media i.e. tissue

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E CARS
Chemical imaging of tissue in vivo with
video-rate coherent anti-Stokes Raman scattering
microscopy C. Evans et al. Proc. Natl. Acad. Sci.
102 (46) 16807 (2005)
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E CARS
Diffusion of baby oil through the skin
Chemical imaging of tissue in vivo with
video-rate coherent anti-Stokes Raman scattering
microscopy C. Evans et al. Proc. Natl. Acad. Sci.
102 (46) 16807 (2005)
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Detection Schemes

• F CARS Forward detected
• E CARS Epi detected
• P- CARS Polarisation dependent
• FM CARS Frequency modulated

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P CARS
Polarization coherent anti-Stokes Raman
scattering microscopy J. Cheng et al. Opt. Letts.
26 (17) 1341 (2001)
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P CARS
Lose power, have to have good signal!
Ordering of water molecules between phospholipid
bilayers visualized by coherent anti-Stokes Raman
scattering microscopy J. Cheng et al. Proc. Natl.
Acad. Sci. 100 (17) 9826. (2003)
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Detection Schemes

• F CARS Forward detected
• E CARS Epi detected
• P- CARS Polarisation dependent
• FM CARS Frequency modulated

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FM CARS
Source switched rapidly between two frequencies,
this FM modulation results in an AM of the CARS
signal ?I(d) I (?1) I (?2) gt Use lock in
detector Non Resonant background is not AM
High-sensitivity vibrational imaging with
frequency modulation coherent anti-Stokes Raman
scattering (FM CARS) microscopy F. Ghanikanov et
al. Opt. Letts. 31(12) 1872 (2006)
22
Applications

• Sensitive probe for lipids
• Lipid bilayer, thin objects, small objects
• Fast dynamic scanning of processes in living
  cells
• High damage threshold
• In vivo capabilites

23
Problems?

• Non resonant background term very strong
• Expensive laser sources
• Have to know beforehand the vibrational band of
  interest
• Currently limited tunability of sources
  improving in line with the laser sources.

24
Discussion?

• Good workshop!
• Only covered small part of the information on
  CARS

IMPORTANT CARS Images, some text and schemes
taken directly from the group website
at http//bernstein.harvard.edu, or from the
references cited.