Optics: measuring and testing – By dispersed light spectroscopy – With raman type light scattering
Reexamination Certificate
2003-01-16
2004-10-26
Font, Frank G. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With raman type light scattering
C356S317000, C356S318000
Reexamination Certificate
active
06809814
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to the field of microscopy, and particularly relates to the field of coherent anti-stokes Raman scattering microscopy.
Coherent anti-stokes Raman scattering (CARS) microscopy provides for the imaging of chemical and biological samples by using molecular vibrations as a contrast mechanism. In particular, CARS microscopy uses at least two laser fields, a pump electromagnetic field with a center frequency at &ohgr;
p
and a Stokes electromagnetic field with a center frequency at &ohgr;
s
. The pump and Stokes fields interact with a sample and generate a coherent anti-Stokes field having a frequency of &ohgr;
AS
=2&ohgr;
p
−&ohgr;
s
in the phase matched direction. When the Raman shift of &ohgr;
p
−&ohgr;
s
is tuned to be resonant at a given vibrational mode, an enhanced CARS signal is observed at the anti-Stokes frequency &ohgr;
AS
.
Unlike fluorescence microscopy, CARS microscopy does not require the use of fluorophores (which may undergo photobleaching), since the imaging relies on vibrational contrast of biological and chemical materials. Further, the coherent nature of CARS microscopy offers significantly higher sensitivity than spontaneous Raman microscopy. This permits the use of lower average excitation powers (which is tolerable for biological samples). The fact that &ohgr;
AS
>&ohgr;
p
, &ohgr;
s
allows the signal to be detected in the presence of background fluorescence.
For example, U.S. Pat. No. 4,405,237 discloses a coherent anti-Stokes Raman spectroscopic imaging device in which two laser pulse trains of different wavelengths, temporally and spatially overlapped, are used to simultaneously illuminate a sample. The signal beam in the phase matching direction with a two-dimensional detector, which gives the spatial resolution.
U.S. Pat. No. 6,108,081 discloses a different method and apparatus for microscopic vibrational imaging using coherent anti-Stokes Raman scattering. In the apparatus of the '081 patent, collinear pump and Stokes beams were focused by a high numerical aperture (NA) objective lens. The nonlinear dependence of the signal on the excitation intensity ensures a small probe volume of the foci, allowing three-dimensional sectioning across a thick sample. The signal beam is detected in the forward direction.
A prior art CARS imaging system (based on the '081 patent)
10
is shown diagrammatically in
FIG. 1
, in which collinear pump and Stokes beams
12
at frequencies of &ohgr;
p
and &ohgr;
s
respectively, are directed to a microscope objective lens
16
, and onto a sample
18
. The CARS signal is detected in the forward direction, and is received by collecting optics
20
, filtered by one or more filters
22
, and detected by a detector
26
.
The signal beam that is created in CARS imaging, however, includes a substantial amount of background with no vibrational contrast from which the signal must be filtered or somehow distinguished. For example, as shown in
FIG. 2
, a conventional (forward-detected) lateral CARS intensity profile of a 535 nm polystyrene bead embedded in water includes a substantial amount of CARS background from water
30
in addition to the characteristic CARS signal from the bead
32
. The horizontal axis in
FIG. 2
represents the lateral dimension (in &mgr;m) across the scan area, and the vertical axis represents the strength of the CARS signal (in cts). The presence of this background from the isotropic bulk water has hindered efforts to increase the sensitivity of CARS imaging, particularly in biological applications. The CARS background is caused by a variety of circumstances. For example, because of electronic contributions to the third order nonlinear susceptibility, there exists a non-resonant contribution to the CARS signal of the sample of interest as well as of the surrounding isotropic bulk medium (i.e., solvent), which is independent of the Raman shift, surrounding isotropic bulk medium (i.e., solvent), which is independent of the Raman shift, &ohgr;
p
−&ohgr;
S
. In addition, in biological applications the common solvent water has strong resonant signals with broad spectral widths that may overwhelm the weak signal of the sample.
As shown in
FIG. 3
, a combined CARS image
40
and intensity profile
42
taken along line
44
—
44
of epithelial cells shows that the signal includes CARS background (as generally indicated at
46
) that may not be easily distinguished from the microscopic sample signal (as generally indicated at
48
). The lateral dimension (in &mgr;m) is shown along the horizontal axis, and signal strength (in cts) is shown along the vertical axis. In certain embodiments, these bulk solvent background contributions to the detected CARS signal may overwhelm the CARS sample signals.
There is a need, therefore, for a system and method for providing improved sensitivity of CARS microscopy, and in particular, to provide a CARS system that reduces the background from the bulk medium, and hence provides a higher signal-to-background ratio.
SUMMARY OF THE INVENTION
The invention provides systems and methods for detecting a coherent anti-Stokes Raman scattering signal from a microscopic sample. In one embodiment, the system includes at least two laser sources, a pump source for generating an electromagnetic field at the pump frequency, a Stokes source for generating an electromagnetic field at the Stokes frequency that is different from the pump frequency, optics to direct collinearly the pump and Stokes beams toward an objective lens, which provides a common focal spot, and a detector for measuring a coherent anti-Stokes signal in the backward (epi) direction that is generated by the interaction of pump and Stokes fields with the sample, and collected by the same lens focusing the pump and Stokes beams.
REFERENCES:
patent: 4284354 (1981-08-01), Liao
patent: 4405237 (1983-09-01), Manuccia et al.
patent: 5286970 (1994-02-01), Betzig et al.
patent: 5418797 (1995-05-01), Bashkansky et al.
patent: 5617206 (1997-04-01), Fay
patent: 5674698 (1997-10-01), Zarling et al.
patent: 5847394 (1998-12-01), Alfano et al.
patent: 6108081 (2000-08-01), Holtom et al.
patent: 6151522 (2000-11-01), Alfano et al.
patent: 6166385 (2000-12-01), Webb et al.
patent: 03042553 (1991-02-01), None
“Polarization- sensitive Coherent Anti-Stokes Raman Spectroscopy,” Oudar et al.Applied Physics Letters.Jun. 1979. vol. 34 (11).
“Time-Dependent Fluorescence Depolarization Analysis in Three-Dimensional Microspectroscopy,” Koshioka et al.Applied Spectroscopy.1995. vol. 49, No. 2.
“Electromagnetic Theory of Propagation, Interference and Diffraction of Light,” Born et al.Principles of Optics. Sixth Edition. 1989. p. 435-449.
“Polarization Cars Spectroscopy,” Brakel et al.Advances in Non-linear Spectroscopy. 1988. John Wiley & Sons, Ltd.
“Coherent Ellipsometry of Raman Scattering of Light,” Akhmanov et al.JFTP Letters, American Institute of Physics. 1977. vol. 25, No. 9.
“Optical Determination of Crystal Axis Orientation in Silicon Fragments or Devices,”IBM Technical Disclosure Bulletin.Dec. 1984.
Cheng Ji-Xin
Volkmer Andreas
Xie Xiaoling Sunney
Font Frank G.
Gauthier & Connors LLP
Lauchman Layla
President and Fellows of Harvard College
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