The invention relates to the field of microscopy, and particularly related 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 typically uses two laser fields, a pump electromagnetic field with a center frequency at ωp and a Stokes electromagnetic field with a center frequency at ωs. The pump and stokes fields interact with a sample and generate a coherent anti-Stokes field having a frequency of ωAS=2ωp−ωs in the phase matched direction. When the Raman shift of ωp−ωs is tuned to be resonant at a given vibrational mode, an enhanced CARS signal is observed at the anti-Stokes frequency ωAS.
Unlike fluorescence microscopy, CARS microscopy does not require the use of fluorophores (which may undergo photobleaching), since the imaging relies on the 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 ωAS>ωp, ωs allows the signal to be detected in the presence of one-photon background fluorescence. CARS microscopy provides information about the intrinsic vibrational resonances of a sample with high sensitivity, allowing for label-free, chemically-specific imaging.
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 '237 patent discloses a non-collinear geometry of the two laser beams and a detection of the signal beam in the phase matching direction with a two-dimensional detector.
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.
There is also a nonresonant contribution to the CARS signal, however, that does not carry chemically-specific information that can distort and even overwhelm the resonant signal of interest. This nonresonant contribution provides background with no vibrational contrast from which the desired signal must be filtered or somehow distinguished. For example, a conventional lateral CARS intensity profile of a 535 nm polystyrene bead embedded in water includes a substantial amount of CARS background from water in addition to the characteristic CARS signal from the bead. 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 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, ωp–ωS.
One approach to reducing the non-resonant background field in CARS spectroscopy is to take advantage of the fact that the non-resonant background has different polarization properties than the resonant signal. For example, see Polarization-Sensitive Coherent Anti-Stokes Raman Spectroscopy, by Oudar, Smith and Shen, Applied Physics Letters, June 1979, pp. 758–760 (1979); and Coherent ellipsometry of Raman Scattering of Light, by Akhmanov, Bunkin, Ivanov and Koroteev, JETP Letters, Vol. 25, pp. 416–420 (1977), which employ non-collinear excitation beams with different polarization directions.
U.S. Pat. No. 6,798,507 discloses a system in which the pump and Stokes beams are polarized, and a polarization sensitive detector is employed. In high resolution CARS microscopy, however, tightly focused collinear excitation beams are sometimes necessary. It is known that tightly focusing polarized beams will result in polarization scrambling. See Principles of Optics, Born and Wolf, Pergaman Press, 1989, pp. 435–449.
U.S. Pat. No. 6,809,814 discloses a system in which a CARS signal is received in the reverse direction (epi-direction) from the sample. The epi directed signal, however, is significantly smaller than the forward directed signal, and a stronger signal may be desired for certain applications.
There is a need, therefore, for a system and method for providing improved sensitivity of CAS microscopy for certain applications, and in particular, to provide a CARS detection scheme that reduces the non-resonant background hence yields a higher signal-to-background ratio.
In accordance with an embodiment, the invention provides a system for detecting a nonlinear coherent field induced in a sample, said system. The system includes optics, a modulation system, and a detector system. The optics are for directing a first electromagnetic field at a first frequency ω1 and a second electromagnetic field at a second frequency ω2 towards a focal volume such that a difference frequency ω1–ω2 is resonant with a vibrational frequency of a sample in the focal volume. The modulation system is for modulating the difference frequency ω1–ω2 such that the difference frequency ω1–ω2 is tuned in and out of the vibrational frequency of the sample at a modulation frequency. The detector system is for detecting an optical field that is generated through non-linear interaction of ω1 and ω2 and the sample responsive to the modulation frequency.
In accordance with another embodiment of the invention, the system includes a source system, a modulation system, optics, and a detector system. The source system is for generating a first electromagnetic field at a first frequency, a second electromagnetic field at a second frequency that is different from said first frequency, and a third electromagnetic field at a third frequency that is different from the first frequency and different from the second frequency. The modulation system is for providing a modulated electromagnetic field that is switched between the second and third frequencies at a modulation frequency. The optics are for directing the first electromagnetic field and the modulated electromagnetic field toward a common focal volume. The detector system is for detecting a nonlinear coherent field that is generated responsive to the first and modulated electromagnetic fields in the focal volume.
In accordance with a further embodiment, the invention provides a method of detecting a nonlinear coherent field induced in a sample. The method includes the steps of directing a first electromagnetic field at a first frequency ω1 and a second electromagnetic field at a second frequency ω2 toward a focal volume such that a difference frequency ω1–ω2 is resonant with a vibrational frequency of a sample in the focal volume, modulating the different frequency ω1–ω2 such that the difference frequency ω1–ω2 is tuned in and out of the vibrational frequency of the sample at a modulation frequency, and detecting an optical field that is generated through non-linear interaction of ω1 and ω2 and the sample responsive to the modulation frequency.
The following description may be further understood with reference to the accompanying drawings in which:
The drawings are shown for illustrative purposes only.
The invention involves performing CARS microscopy such that the frequency difference ω1–ω2 is rapidly changed to and from the frequency of a desired molecular vibration. In accordance with an embodiment, the Stokes beam is maintained at a fixed optical frequency and the optical frequency of the pump beam is rapidly switched to modulate the frequency difference. In another embodiment, the optical frequency of the Stokes beam may be rapidly modulated while the pump beam is maintained at an optical fixed frequency. In further embodiments, the optical frequency of both the Stokes beam and the pump beam may be rapidly switched to produce a modulated frequency difference.
CARS microscopy systems of various embodiments of the invention, therefore, provide significant increases in the detection sensitivity because non-resonant background information is suppressed by locking into the switching periodicity of the modulated frequency difference. CARS signals are generated by collinearly overlapped, tightly focused, and raster scanned pump and Stokes laser beams, whose difference in frequency is rapidly modulated. The resulting CARS signal is detected by a detector system that is response to the modulation frequency. This scheme efficiently suppresses the nonresonant background and allows for the detection of far fewer vibrational oscillators than possible though existing CARS microscopy methods.
If the anti-Stokes signal that is obtained changes when the frequency difference is modulated, then it is known that the signal is due to a vibrational resonance. The nonresonant background, which will not change when the frequency difference is modulated, is therefore easily subtracted from the received anti-Stokes signal. With reference to
The output signal may be passed through a lock-in amplifier to provide that only changes at the time scale of the modulation period are provided in the final output. In accordance with other embodiments, an RF modulator/demodulator may be employed. For example, as shown at 14 in
The CARS response originates from the third order nonlinear susceptibility, which is the sum of a resonant contribution,
and a nonresonant electronic component,
The total detected CARS signal is given by:
The frequency dependence of the three terms
and
are shown at 20, 22 and 24 respectively in
The dashed line 24 represents the heterodyne component of Equation (1). The curves are calculated with an assumption that
The nonresonant term can often obscure the resonant CARS signal of interest, making it difficult to identify the chemically selective contributions to an image. This is especially true when imaging biological materials as the aqueous environment gives rise to a substantial nonresonant response that often overwhelms the resonant signal.
Consider an isolated resonance centered at vibrational frequency ΩR with FWHM linewidth Γ (as shown in
When the concentration of resonant species in a sample is high under this suppression conditions, the quadratic term
in Equation 1 is the greatest contribution to the detected signal. At much lower concentrations, however, the linear term
in Equation 1 becomes dominant. This heterodyne term contains a factor of
which implies that the component can be effectively enhanced by the nonresonant response of the solvent. The above approach is implemented by modulating the optical frequency of the pump beam ωp, at a high enough rate (>500 kHz) to separate the modulated signal from the lower frequency laser noise. The modulation frequency may depend on the particular laser source that is employed. A value may be set to be far from the noise spectrum peak of the laser (relaxation resonance frequency) as well as characteristic mechanical resonance frequencies of the beam steering and laser resonator optics.
As shown in
As shown in
As shown in
As shown in
In accordance with an embodiment of the invention, three pulsed lasers are coupled into a modified laser-scanning microscope (Olympus, FV300). The Stokes beam is about 10% of the output from a passively mode-locked, fixed-frequency Nd:YVO4 laser (High-Q, picoTRAIN, 7 ps, 1064 nm, 76 MHz rep. rate). The 90% output of the Nd:YVO4 source is used to synchronously pump an intracavity doubled optical parametric oscillator (OPO) producing tunable 5 ps near-IR radiation for use as a pump beam (Pump-1). The second pump beam (Pump-2) is provided by a mode-locked Ti:Al2O3 oscillator delivering tunable 3 ps pulses that are electronically synchronized to the Nd:YVO4 source. A half-wave plate inserted into the Pump-1 beam path is used to rotate the polarization so that Pump-1 and Pump-2 are perpendicularly polarized. The two pump beams are then combined in a two-port Glan-Taylor prism and sent collinearly into a Pockel's cell. Square waveforms with a 50% duty cycle, derived from a pulse delay generator synchronized to the laser pulse train, supply a modulation signal at a frequency of ˜500 kHz to the Pockel's cell. When the waveform is in the low state, Pump-1 is allowed to pass through the exit analyzer. When the waveform is in the high state, the polarization of both beams is rotated by π/2, such that Pump-2 now passes unattenuated though the analyzer while Pump-1 is blocked. This arrangement provides the rapid wavelength modulation needed for the experiment. The modulated pump beams are spatially combined with the Stokes beam on a dichroic mirror and the combined beams are directed into the scanning microscope. The CARS signal from the sample is detected by a PMT and fed into a lock-in amplifier. The lock-in reference is provided by the external signal supplied from the pulse generator driving the Pockel's cell. The half-wave plate introduced into the Pump-1 beam path can be used in conjunction with the Glan-Taylor prism to balance the intensity of the two beams for maximum nonresonant signal suppression.
As shown in
In addition to truly resonant imaging, FM-CARS also allows for increased detection sensitivity over conventional CARS microscopy. To quantify the increased sensitivity, solutions of methanol dissolved in water were used. Methanol is well characterized by Raman spectroscopy and contains only a single CH3 moiety that gives rise to two relatively narrow (ΓFWHM equal to about 25 cm−1), well-spaced Lorentzian-like peaks in the CH stretching region. For this experiment, Pump-1 was tuned to target 2928 cm−1, which corresponds to the symmetric CH3 stretch of methanol, while Pump-2 was tuned to target 3048 cm−1 were there is no vibrational resonance. As considered earlier, the FM-CARS intensity in terms of detuning (Δ1,2=ωp1,p2−ωs−Ω) is equal to ΔI(δ)=I(Δ1)−I(Δ2). At relatively low concentrations, I(Δ1,2) can be expressed in terms of the fraction of maximum solute concentration, n, by the following equation.
where
is the nonresonant CARS intensity from pure water, and R is the ratio of peak CARS signal from pure method to
The FM-CARS signal is maximized at
The R parameter can be readily measured experimentally at the resonance maximum, which is R=24 for this experiment.
Since FM-CARS makes use of a lock-in amplifier, the noise floor for the detected resonant signal may be reduced by narrowing the detector bandwidth to achieve better resonant signal detection sensitivity, with the ultimate sensitivity reached at an infinitely narrow bandwidth. While a detection bandwidth of f1=25 kHz (filled circles) achieves significantly better sensitivity than seen in normal CARS, the ultimate sensitivity of our configuration is reached at a bandwidth of f2=1.6Hz (open circles). The equation (2) relationship (solid line) shown at 140 provides a direct fit to the data. The efficient removal of nonresonant background originating from water with a narrow detection bandwidth (f2) allowed the detection of resonant signal from methanol with only 5×105 oscillators in the probed volume of 100 attoliters, as opposed to approximately 4×108 oscillators achieved with normal forward-CARS in the same experiment. The minimum detectable signal for both bandwidths differs from the expected value since they should scale linearly with the bandwidth (f). This suggests that the CARS signal noise spectrum has significant components in sub-Hz region, which most likely originate from beam pointing instability as well as laser intensity and spatial mode fluctuations.
Improvements to this technique, including dual-wavelength laser sources, OPOs with fast electro-optic tuning, and acousto-optic tunable filters for rapid wavelength modulation will very likely improve the detection limit by eliminating sources of noise. Systems of the invention may also be used to detect small changes in a vibrational band through appropriate choice of the modulated wavelengths.
In various embodiments, therefore, the invention provides a method for CARS microscopy that involves the efficient suppression of nonresonant signals based on rapid modulation of the difference frequency between the pump and Stokes beams. This approach vastly enhances the ability to distinguish resonant features from the nonresonant background, providing resonant images with an improvement of nearly three orders of magnitude in sensitivity for chemical species at low concentrations.
The present application claims priority to U.S. Provisional Patent Application Ser. No. 60/730,558 filed Oct. 26, 2005 as well as U.S. Provisional Patent Application Ser. Ser. No. 60/760,189 filed Jan. 19, 2006.
This invention was sponsored by NIH grants OD000277 and GM062536 and the government has certain rights to this invention.
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