Method and apparatus for performing second harmonic optical coherence tomography

The invention is an apparatus and method for second harmonic optical coherence tomography of a sample comprising a laser coupled to an interferometer which has a reference arm and in a sample arm. A nonlinear crystal in the reference arm generates a second harmonic reference signal. The sample typically backscatters some second harmonic light into the sample arm. A broadband beam splitter optically coupled to the reference arm and sample arm combines the signals from the reference arm and sample arm into interference fringes and a dichroic beam splitter splits the interference fringes into a fundamental and second harmonic interference signal. A detector is optically coupled to the dichroic beam splitter detects interference fringes from which both an OCT and second harmonic OCT image can be constructed using a conventional data processor.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of optical coherence tomography using optical second harmonic generation and nonlinear optical interferometry.

2. Description of the Prior Art

Optical coherence tomography (OCT) is an emerging imaging technology that provides in-vivo high-resolution, cross-sectional images of biological tissues. Using coherence gating technique, OCT is capable of detecting the backscattered light from highly scattering tissues at depths of 2-3 mm. OCT imaging contrast originates from the inhomogeneities of sample scattering properties that are linearly dependent on sample refractive indices. In many instances such as pathological processes in tissue, changes in sample linear scattering properties are small and difficult to measure. For example, many cancers originate in the epithelium that has a thickness suitable for OCT imaging, but in their early stages when these cancers are developing through cell dysplasia, changes in tissue morphology and refractive index between normal and diseased tissues are very small and difficult to detect. Therefore, to meet the challenges found in OCT clinical applications, imaging contrast enhancement is very important.

In recent years, many OCT contrast enhancement methods have been developed. These techniques include Doppler OCT, polarization sensitive OCT, spectroscopic OCT, pump-probe techniques, and using contrast agents for OCT. More recently, applying nonlinear optical effects of second harmonic generation (SHG) and coherent anti-Stokes Raman scattering for OCT contrast enhancement have also been demonstrated.

SHG is a powerful contrast mechanism in nonlinear optical microscopy. SHG signals provide unique information regarding sample structure symmetry because the signals strongly depend on the orientation, polarization and local symmetry properties of chiral molecules. SHG enables direct imaging of anisotropic biological structures, such as membranes, structure proteins, and microtubule ensembles. Besides successfully producing high-resolution and highly contrasting images of tissue morphology, recently SHG microscopy has also been applied to study dynamics in tissue physiology, such as monitoring collagen modification in tumors growing, and optically recording the action potentials change in neuron cells. SHG is emerging as a powerful nonlinear optical imaging modality for cell biology and biophysics.

BRIEF SUMMARY OF THE INVENTION

In the illustrated embodiment the invention is defined as an apparatus for second harmonic optical coherence tomography of a sample comprising a light source, such as a femtosecond pulsed laser, and an interferometer having an optical path in a reference arm and in a sample arm. A nonlinear crystal is disposed in the optical path of the reference arm. The sample in the optical path of the sample arm backscatters at least some second harmonic of the incident light. A broadband beam splitter is optically coupled to the reference arm and sample arm, combines the signals in the reference arm and sample arm into interference fringes. A dichroic beam splitter is optically coupled to the broadband beam splitter to splits the interference fringes into a fundamental and second harmonic interference signal. A detector is optically coupled to the dichroic beam splitter for detecting interference fringes, which include at least the second harmonic interference fringes, and a second detector preferably detects the fundamental harmonic interference fringes. The interferometer and detector simultaneously perform second harmonic OCT measurements at second harmonic frequency and conventional OCT measurements at a fundamental frequency.

A pair of prisms is disposed in the optical path of the signal arm or reference arm to compensate for the group-velocity dispersion of the fundamental and harmonic waves or group velocity mismatch in the signal arm and reference arm, thus enabling simultaneous observation of SH-OCT interference signals and conventional OCT interference signals.

The interferometer comprises means for independently axially or transversely scanning the sample in decoupled modes of operation to provide two dimensional tomographic imaging of the sample with only one dimensional movement of the light.

The apparatus further comprises optical elements, such as half wave plates and laser polarizers, for controlling input power into the interferometer.

In the embodiment where the light source comprises a mode-locked laser the apparatus further comprises an optical isolator for preventing back-scattered light from entering the light source and interfering with mode locking.

The apparatus further comprises optical elements, such as filters for filtering out second harmonic frequencies of light generated by the light source.

The apparatus further comprises optical elements coupled to the light source for determining a ratio of polarization modes of the light generated by the light source and for splitting the light from the light source into the reference arm and sample arm according to polarization mode of the light.

In the illustrated embodiment the nonlinear crystal is oriented for type I phase matching.

A dichroic mirror and translation stage coupled to the mirror serve as an optical terminus in the reference arm to act as an optical delay line. The dichroic mirror differentially reflects the fundamental and second harmonic frequency of the light signal in the reference arm to reduce the amount of reflected light at the fundamental frequency, which is transmitted toward the nonlinear crystal.

A bandpass filter centered at the second harmonic frequency and optically coupled to the dichroic beam splitter rejects background noise transmitted toward the detector.

A long pass filter and short pass filter differentiate between the fundamental and second harmonic frequency interference signals. The first detector which detects the fundamental frequency is optically filtered by the long pass filter and the second detector which detects the second harmonic frequency is optically filtered by the short pass filter.

A moving mirror is disposed in the reference arm and a lock-in amplifier is coupled to the detector. The lock-in amplifier is locked at f1,2=2v Δl/λ1,2, where v and Δl are the frequency and amplitude respectively of the moving mirror, and λ1,2are the wavelengths of the fundamental and second harmonic interference signals.

Additional optical elements can be disposed into the interferometer optical path for controlling the beam polarization of light in the sample arm and reference arm, oriented according to polarization characteristics of the sample.

The nonlinear crystal has a predetermined thickness according to the wavelength of the fundamental frequency for balanced SHG signal strength and spectral width. The predetermined thickness is approximately 0.1 mm when the wavelength of the fundamental frequency is approximately 800 nm.

The invention is also defined as a method of operating the forgoing apparatus to generate second harmonic OCT images and conventional OCT images. For example, the invention includes a method of performing optical tomography of a sample comprising the steps of providing a source of at least partially coherent broadband radiation through an interferometer having a sample arm for probing the sample and a reference arm; scanning the sample with the source of radiation through the interferometer; generating first and second harmonics from the sample and from a nonlinear thin crystal in the reference arm; detecting interference fringes of the first and second harmonics radiation backscattered from the sample into the interferometer; processing the detected interference fringes to determine first and second harmonics OCT signals of the detected backscattered interference fringes at each pixel in a data window; and generating a tomographic image of the sample at each pixel based on the first and second harmonics OCT interference fringes. In the same manner, the invention is an apparatus for performing optical tomography of a sample in which second harmonics of a radiation signal can be generated according to the above method.

The invention can be further defined as an improvement in an OCT tomographic imaging system having an interferometer with a reference arm and sample arm comprising means for generating a second harmonic frequency in the reference arm; means for combining the second harmonic frequency from the reference arm and a second harmonic frequency from the sample in the sample arm to produce a second harmonic interference fringe signal; and means for detecting the second harmonic interference fringe signal to enable the OCT tomographic imaging system to produce an image derived from the second harmonic interference fringe signal. Again, the invention can be defined as an improvement in a method of OCT tomographic imaging using an interferometer with a reference arm and sample arm in the foregoing improved system.

The invention can be further defined as an improvement in an OCT tomographic imaging system comprising means for generating third harmonic frequency in the reference arm, by replacing the nonlinear crystal with one optimized for third harmonic generation; means for combining the third harmonic frequency from a nonlinear crystal in the reference arm and a third harmonic frequency from the sample in the sample arm to produce a third harmonic interference fringe signal; and means for detecting the third harmonic interference fringe signal to enable the OCT tomographic imaging system to produce an image derived from the third harmonic interference fringe signal.

The invention can be further defined as an improvement in an OCT tomographic imaging system comprising means for generating a Raman frequency in the reference arm, by replacing the nonlinear crystal with a Raman reference; means for combining the Raman frequency from the Raman reference in the reference arm and a Raman frequency from the sample in the sample arm to produce a Raman interference fringe signal; and means for detecting the Raman interference fringe signal to enable the OCT tomographic imaging system to produce an image derived from the Raman interference fringe signal. The Raman reference may be any known sample or material which is capable of producing a known Raman frequency, typically in the range close to that expected in the unkown or imaged sample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An optical tomography technique of second harmonic optical coherence tomography is described. Femtosecond laser pulses at 800 nm wavelength are used to excite second harmonics at 400 nm from a rat tail tendon and a reference nonlinear thin crystal34. Second harmonic interference fringe signals were detected and used for image construction. A tomographic image shows the sample structure of two thin collagen layers sandwiched among glass slides as shown and described below in connection withFIG. 4c. Because of the strong dependence of second harmonic generation on molecular and tissue structures, this technique offers molecular contrast as well as resolution enhancement to the conventional optical coherence tomography.

The invention discloses a high-resolution SH-OCT system10as diagrammatically described below inFIG. 1b. Using broadband, pulsed laser illumination and nonlinear interferometry, the system10combines the molecular structure sensitivity of SHG with coherence gating of OCT. Since the axial and transverse scans are decoupled, two-dimensional cross-sectional imaging of anisotropic biological structures can be done with one-dimensional scanning of the sampling beam, which has the potential to be adapted to clinic endoscopic studies.

In the illustrated embodiment, a high numerical aperture single mode fiber17is used to broaden the spectrum of a femtosecond laser12. The sample46under illumination generates second harmonic generated (SHG) signal. The reference SHG signal is generated by a nonlinear crystal34. Coherence gating detection of these SHG signals produces interference fringes that can be used for image construction. The current system achieves an axial imaging resolution of 4.2 μm in free space, corresponding to 3.1 μm in tissue, which is a six-fold improvement over prior art systems. For the first time, a SH-OCT system is applied to image the biological tissue of a native, intact rat-tail tendon. Highly contrasting, high-resolution SH-OCT images showing collagen fibrils organization in tendon tissue have been recorded.

In the invention, we demonstrate an optical tomography technique, second harmonic optical coherence tomography (SH-OCT), which combines the molecular contrast of SHG with the coherence gating of OCT. The SH-OCT system is comprised of an interferometer20illuminated by a broadband light source12. If the sample46possesses certain structures lacking a center of symmetry, the illuminating light is converted into second harmonic waves at the sample site as well as in the reference arm32through a nonlinear crystal34. The temporal interference pattern of these second harmonic waves is then detected and used for image construction. Because the fundamental radiation is only partially converted into second harmonics, with proper optics, both SH-OCT and conventional OCT measurements can be simultaneously performed. Based on the high selectivity of SHG on tissue molecular structure, together with optical sectioning capability of coherence gating, SH-OCT is provides considerable imaging contrast and resolution enhancement to the conventional optical tomographic imaging techniques.

The experimental configuration of SH-OCT system10is shown inFIG. 1a. The light source is a Kerr-lens mode-locked Ti:sapphire laser12with the output power of about 600 mW at a wavelength of 800 nm. The pulse duration is about 100-170 femtoseconds at a repetition rate of 76 MHz. The output of laser12is coupled through a Faraday isolator16to a half-wave plate14to prevent back-scattered light from entering laser12and interfering with mode locking. Half-wave plate14is used in combination with a Glan prism18and second half-wave plate22to control the input power into the optic fiber input into interferometer20. A long-wave pass filter24is used to filter out the spurious second harmonic components produced by the laser12.

A fiber optic embodiment is depictedFIG. 1band will be described together with the free-space embodiment ofFIG. 1a. To generate a continuum centered at 800 nm, the femtosecond pulses from laser12are coupled through isolator16, polarizer23and half wave plate14into a section of 2.0-meter-long commercially available high numerical aperture single mode fiber17(Corning HI-780) by a microscope objective19as shown inFIG. 1b. The invention also contemplates a free space optical path. When the optical path is provided in a fiber17, the light output from the fiber17is re-collimated to a parallel beam with 1.5 mm diameter using an aspheric lens21inFIG. 1b. Glan laser polarizer18purifies the polarization of the continuum output. By rotating the half wave plate14in front of the coupling lens of the microscope objective in the high-resolution second harmonic OCT embodiment ofFIG. 1b, a linear polarized continuum is produced in either horizontal or vertical polarization with an average power exceeding 200 mW. The spectrum broadening of the laser pulses in the fiber17is shown inFIG. 5a, where the narrow curve is the original spectrum of the laser12and the broadened curve is the spectrum of the continuum generated in the fiber. Note that the input beam polarization and the length of fiber17can affect the spectrum shape and smoothness.

The filtered light is directed by mirrors26aand26binFIG. 1bor mirror26inFIG. 1ainto a polarizing beam splitter28which splits the input beam into the reference arm32and sample arm30of the interferometer10. The split ratio is controlled by the second half-wave plate22upstream in the optical path.

In the reference arm32, a 0.1 mm thick nonlinear BaB2O4(BBO) crystal34is employed, which is oriented for type I phase matching, to convert the input radiation to second harmonic wave at 400 nm. Type I phase-matching means that both waves at the fundamental frequency ω have the same polarization whereas the wave at the second harmonic frequency 2ω have orthogonal polarization. Both second harmonic and fundamental waves are then reflected by a dichroic metal mirror36mounted on a motorized or piezoelectric translation stage38, which acts as the delay line in OCT system10. The back-reflected radiation is partially reflected by a broadband nonpolarizing beam splitter40and propagated into the combining broadband, nonpolarizing beam splitter42. The dichroic mirror36reflects 90% of the second harmonic wave and 5% of the fundamental wave. A majority of the fundamental wave is dumped to avoid being reflected back to generate a second harmonic wave from the crystal34again, otherwise ghost lines may appear in the tomographic images.

In the signal arm after beam splitter28the fundamental radiation is transmitted through a half-wave plate44and is focused by a low numerical aperture lens48(N.A.=0.2, f=31.8 mm) onto sample46mounted on translation or scanning stage50. When the sample46has second order nonlinear properties, the fundamental radiation generates a second harmonic signal. Back reflected second harmonic and fundamental waves were collimated by the same lens48and directed by a dichroic beam splitter52in the embodiment ofFIG. 1atoward the combining beam splitter42. The dichroic beam splitter42reflects a maximum amount of second harmonic radiation and about 5% of the fundamental radiation. In the embodiment ofFIG. 1bthe returned signal from sample46is directed to beam splitter28and then toward dichroic beam splitter42where it is split between a bandpass filter57and PMT54on one hand and photodiode60on the other. The outputs of PMT54and photodiode60are then coupled to a preamplier77which serves as a data acquisition and processing circuit and thence to lock-in amplifier76and computer79.

In the embodiment ofFIG. 1athe radiation from signal arm30and reference arm32are recombined after passing through the combining beam splitter42. In the detection arm, a dichroic beam splitter42is used to separate the beam according to the wavelength. Fundamental and second harmonic interference fringes are detected by a photo diode60and a photomultiplier54, respectively. A band-pass filter58centered at 400 nm with 40 nm bandwidth is attached to the photomultiplier head to further reject background noise. By changing the optical path delay in the reference arm32, the pulses overlap temporally and interference fringes at fundamental and second harmonic wavelengths are generated.

The harmonic interference fringe signal is detected by a photomultiplier tube54after passing through a short-pass filter56which transmits light that is lower in wavelength than a predetermined value, which is chosen here to be below 800 nm but above 400 nm, and a 400 nm band-pass filter58. The fundamental interference fringe signal is detected by a photodiode60after passing through a long-pass filter62which transmits all the wavelengths longer than a predetermined wavelength number, which is set here below 800 nm but above 400 nm. A pair of prisms64made from BK7 glass are also inserted made from fused silica are inserted into the optical path of the signal arm30in the embodiment ofFIG. 1ato compensate for the group-velocity dispersion of the fundamental and harmonic waves or group velocity mismatch in the two arms30and32, thus enabling simultaneous observation of SH-OCT and conventional OCT signals. Because the material dispersion of the optical components is not uniform for all the wavelengths, the fundamental and second harmonic waves require different thicknesses of compensating material to generate optimized fringes at corresponding wavelengths. It is also within the scope of the invention that prism pair64could be inserted into the reference arm32between the crystal34and mirror36as shown inFIG. 1b.

To investigate the longitudinal resolutions for the fundamental and harmonic wavelengths in this hybrid OCT system10, a polished GaAs (111) crystal is used in place of sample46as a nonlinear optical mirror66to have the interference fringes generated at both the harmonic and fundamental wavelengths. The experiment setup inFIG. 1ais modified by replacing the module68in the dotted box with module70in the dotted box. The laser12is focused at 45° onto the surface of crystal66, and the reflected radiation (fundamental and second harmonic) is recollimated by another lens48′ identical to lens48and directed by mirrors72and74into beam splitter42.

Interference signals of fundamental and second harmonic waves are measured as shown in the graph ofFIG. 2, where second harmonic interference occurs at the double frequency of fundamental interference. The penetration depth of the 800 nm wavelength into the GaAs mirror66is less than one micron so the resolution of the system10is determined by the coherence length of laser radiation.

It is well known that the coherence length Icof a Gaussian pulse with spectral width Δλ and center wavelength λ0is lc=0.440 λ02/Δλ. Simple calculations show that for Gaussian pulses Δλ1/Δλ2=4/√2 and lc/lc2=√2, where Δλ1and Δλ2are the spectral width of fundamental and second harmonic waves, lcand lc2are the coherence lengths of the fundamental and second harmonic waves. The emission spectrum of the laser12in the illustrated embodiment is centered at 800 nm with a spectral width (full width at half-maximum) of 8.1 nm, as shown inFIG. 3a. The measured spectrum of SHG from the nonlinear crystal BBO34is shown inFIG. 3b, with a spectral width of 3.0 nm centered at 400 nm.FIG. 3candFIG. 3dare graphs which represent the measured interference fringes of fundamental and harmonic waves when the mirror36is scanned. The measured coherence lengths of the fundamental and harmonic waves are 33 μm and 24 μm in free space respectively, which agree well with the predicted values within the experimental accuracy.

In the high-resolution second harmonic OCT embodiment ofFIG. 1b, to measure the coherence length of the OCT system10, we remove the BBO crystal34from the reference arm32and place crystal34in front of the main beam splitter42, and replace the sample46with a mirror. Fundamental and second harmonic waves are present in both arms30and32and interfere to produce two sets of fringes, with the coherence point spread functions shown inFIG. 5candFIG. 5d. The fundamental wave has a coherence length of 6.0 μm and the second harmonic wave has a coherence length of 4.2 μm in free space, which determine the axial resolutions of the OCT system at corresponding wavelengths. The spectrum of the second harmonic wave from the 0.1-mm-thick BBO crystal34is shown inFIG. 5b.

In the fiber-based second harmonic OCT embodiment ofFIG. 1c, the free-space optical paths ofFIGS. 1aand1bare replaced with optic fibers, and the free-space optical components are replaced with fiber-optic counterparts according to well understood design principles. For example, the free-space beamsplitters/combiners28,40,42,52are replaced with 1×2 fiber-optic couplers100or 2×2 fiber-optic couplers102, and the free-space polarization optics of laser polarizers and waveplates14,22,23,44, are replaced with fiber-optic polarization controllers104, and the free-space optical detectors54,60are replaced with fiber-interfaced detectors106. Sample46is scanned using an fiber optic probe108.

In the fiber-based second harmonic OCT embodiment ofFIG. 1c, the 2×2 fiber-optic coupler102is for splitting and combining the light wave at second harmonic and fundamental frequency, one 1×2 fiber-optic coupler100ais for monitoring the light intensity fluctuation in the light source12, another 1×2 fiber-optic coupler100bis for splitting the interference signals onto two detectors106for detecting the second harmonic frequency and fundamental frequency respectively. The fiber-optic polarization controllers104are for controlling and matching the polarization of light in both arms30,32to produce maximum interference signals. The fiber-interfaced detectors106can be a fiber-interfaced photodiode, a avalanche photodiode or a photomultiplier tube.

In the fiber-based second harmonic OCT embodiment ofFIG. 1c, the optical fiber17is chosen to support pulsed light propagation and support wideband single mode operation. Pulsed light propagation is required because in the nonlinear process of second harmonic generation, sufficient optical peak power can occur only when the pulses are present. Wideband single mode operation is required because it is desirable to collect the second harmonic signals from a pre-determined optical path and with high efficiency. Large mode area photonics crystal fibers and photonics band-gap fibers are two candidates to construct the fiber-based system.

Consider now the use of system10first to measure an actual biological sample46. The sample46used in our study was Type I collagen harvested from rat tail tendon which is a well documented source of SHG in tissue. The sample consisted of two collagen layers82of about 30 μm thickness sandwiched among three 170 μm-thick glass slides84, with its structure shown inFIG. 4c. The average excitation power entering the sample arm was approximately 50 mW.

Rat-tail tendon was chosen for the imaging experiment since many of its important properties are known from other independent methods. Collagen is the most abundant protein in higher vertebrates, comprising over one-third of total body protein and 60-86 percent or more of the dry weight of the tendon. Other components of tendons include water, proteoglycans, cells, elastin, and other extracellular matrix components. All of these components are arranged in a fibrous structure, as shown in a 60× microscope image inFIG. 6b. It is known that collagen in rat-tail tendon consists of three parallel intertwined, polar helices. This non-central-symmetric structure makes it very efficient for second harmonic generation.

Using Gaussian beam approximation, estimated power density at the beam waist in the sample was about 3.19×109W/cm2, and the focusing lens48had a depth of focus of 0.52 mm, which was long enough to cover the two collagen layers. In this case, back-reflected SHG signals from the sample46can be easily detected. The measured SHG signal was the contribution of the SHG signal reflected from a thin layer at the collagen layer surface and the transmitted SHG waves generated at various planes along the light path. The latter can be back scattered by the non-uniformities within the sample or at the boundaries of the different sample layers.

FIG. 4cis a side cross-sectional view of a phantom manufactured with three glass layers80sandwiching collagen layers82. The tomography experiment was conducted by scanning the mirror36in the delay line of reference arm32and recording the fundamental and harmonic interference signals with a lock-in amplifier76coupled to PMT54and photodiode60. The lock-in amplifier76demodulated the interference fringe envelope signal with extremely high sensitivity and precision when its frequency was locked at f1,2=2v Δl/λ1,2, where v and Δl are the frequency and amplitude respectively of the moving mirror36, and λ1,2are the wavelengths of the fundamental and second harmonic waves. The output of amplifier76is coupled to an analog-to-digital converter77and thence to computer79where data reduction and storage is performed according to well understood principles to create the actual SH-OCT image.

The measured OCT signals of one typical axial scan are shown in the graph ofFIG. 4a. The conventional OCT signal inFIG. 4ashows the sandwich structure of the sample46shown inFIG. 4c. The strong reflectance occurring at the first air-glass interface78suppresses signals from following layers. The SH-OCT signal inFIG. 4bshows two peaks that correspond to the two-layer structure as second harmonic signals are only produced in the two collagen layers80and82. Comparison ofFIG. 4aandFIG. 4bshows that there is no SH-OCT signal come from the air-glass interface78, indicating that SH-OCT provides good contrast to linear reflections. The SH-OCT signal reveals information regarding the second-order nonlinear properties of the sample46that can not be provided by conventional OCT signals. Furthermore, it is evident that the resolution of SH-OCT is higher than that of conventional OCT.

In addition to molecular sensitivity, SHG also can serve as a unique contrasting mechanism for tissue structure since the second harmonic signal is highly dependent on the orientation, polarization, and local symmetry properties of tissue. Therefore, the SHG efficiency in collagen depends on orientation of collagen fibrils relative to the incident electric field polarizations. In the experiment ofFIGS. 4a-4c, the half-wave plate44for the fundamental wavelength was used to control the input beam polarization to the sample46. To maximize the second harmonic interference signal, another half-wave plate84, shown in dotted outline and optimized for the second harmonic wavelength, is inserted into the reference arm32after the reference crystal34. By rotating half-wave plates44and84in both arms30and32, collagen fibrils with different orientation can be preferentially highlighted to produce polarization dependent tomographic images.

In another measurement of rat tail tendon the images ofFIGS. 6aand6bwere obtained. In this study, rat-tail tendon was removed from thawed rat-tails and stored in phosphate-buffered saline solution for several minutes. A 10-mm-long section was cut from the tendon and embedded between two microscope cover slips spaced by 0.1 mm diameter wire ring. The edge of the sample was sealed with epoxy.

We used a microscope objective to focus the beam onto the specimen. The average laser power was 80 mW at the sample site. Typical energy per pulse was approximately 1 nJ with energy density of 0.05-0.07 J/cm2, which is much less than the tissue damage threshold in the range of 0.5-1.0 J/cm2. When the optical path length difference between the sample arm30and reference arm32is within the coherence length of the second harmonic wave, the second harmonic interference can be detected. The interference fringes signal was demodulated by lock-in amplifier76and used for image construction.FIG. 6ashows a high-resolution SH-OCT image in the rat-tail tendon obtained with a 0.25-μm scanning resolution. The image shows the collagen fibrils organization within an area of 100×50 μm. As the tension-bearing element in the tendon, collagen appears in clearly defined, parallel, cable-like and slightly wavy bundles. In this image, highly organized collagen fiber bundles (fascicles) oriented in the same direction can be clearly identified. Because of the cross-sectioning nature of OCT, collagen fiber bundles localized at different imaging planes parallel to the axial direction exhibit different thicknesses as projected into this image. The transverse and axial resolutions of this image are 1.9 μm and 4.2 μm, as determined by the Gaussian beam waist diameter at the focus and coherence length of second harmonic wave respectively.

Understanding the origin of the back-scattered SHG signal from the sample is important because in the coherent process of SHG, the majority of the second-harmonic wave co-propagates with the excitation laser beam. This phenomenon has been experimentally investigated in nearly transparent thin layers. The research results suggest that laterally oriented collagen fibrils scatter in both forward and backward directions, but axially oriented collagen fibrils scatter mostly forward with signal intensity orders of magnitude larger than lateral ones. In highly scattering thick tissues like tendons and muscles, essentially no SHG signals can be collected in transmission mode, and the SHG signals detected in the backward direction are mostly from the back scattering of the forward-generated SHG signals, since SHG signals are predominantly generated in forward direction, and immediately suffer from heavy scattering within the tissue until they either get absorbed or escape from the sample surface in the backward direction. Collected by the same excitation objective, these back-scattered SHG signals are particularly important for thick tissues and in-vivo clinical applications.

The BBO crystal34used in the reference arm has a thickness of 0.1 mm and is oriented for type-I phase matching. For second harmonic wavelength conversion of a broadband laser source12using a nonlinear crystal, spectrum narrowing effect induced by the crystal dispersion must be considered. Because the spectral width of SHG in the bulk of a nonlinear crystal is limited by the crystal thickness, the nonlinear crystal has to be made very thin to accommodate for a large bandwidth of the fundamental laser spectrum. Under the same excitation conditions, the coherence lengths measured when using different thickness crystals are shown inFIG. 7a, and the Fourier transforms of the measured fringes are shown inFIG. 7b. Although thicker crystal (0.5 mm) produces much greater SHG signals than thinner ones (0.1 and 0.05 mm), its coherence length is also much larger, which means thicker crystal limits the SHG spectrum more than thinner crystal. However, further reducing crystal thickness from 0.1 mm to 0.05 mm does not generate much more useful spectral components, but produces even weaker SHG signals. Therefore, a 0.1 mm is an optimum crystal thickness for balanced SHG signal strength and spectral width in current system.

The BBO crystal34can also work as the polarization selector for the second harmonic interference. With current experiment setup inFIG. 1a, when the crystal34is followed by another quarter wave plate designed for the second harmonic wavelength, the polarization plane of second harmonic wave in the reference arm can be rotated to match that from the sample to produce polarization selective SH-OCT images.

Collagen is the predominant structural protein in most biological tissues, as well as the major source of SHG. Modifications of the collagen fibrillar matrix structure are associated with various physiologic processes, such as wound healing, aging, diabetes, and cancer. Research results suggest morphologic changes in collagen structure produces predictable alterations in the SHG signal, and can be intrinsic indicators of disease states. Therefore, SHG is very promising as a sensitive probe in tissue morphology and physiology studies. With the development of novel microstructure fibers that support femtosecond laser pulses, it is possible to implement SH-OCT with fiber optics and adapt it for in-vivo endoscopic imaging inside bodies of living animals and human patients.

In summary, we have presented a noninvasive optical tomography technique of second harmonic optical coherence tomography and experimentally demonstrated the feasibility of using this technique to image biological samples. Compared with conventional OCT performed at fundamental wavelength, SH-OCT offers enhanced molecular contrast and spatial resolution. It is also an improvement over existing second harmonic scanning microscopy technology as the intrinsic coherence gating mechanism enables the detection and discrimination of second harmonic signals generated at deeper locations. The enhanced molecular contrast of SH-OCT extends conventional OCT's capability for detecting small changes in molecular structure. Second harmonic-OCT is promising for the diagnosis of cancers and other diseases at an early stage when changes in tissue and molecular structure are small.

Detailed structural information about collagen fibrils organization in rat-tail tendon has been revealed in the recorded images. This new technique may offer several distinct advantages for imaging ordered, or partially ordered, biological tissues. First, the SHG signal from tissue tends to be a very sensitive indicator of tissue molecular structure and symmetry changes. Second, coherence gating extends the capability of high-resolution detection of SHG signals at locations deep inside the sample. Third, SHG signals are produced intrinsically so imaging does not require staining the sample with dyes or fluorophores. Fourth, decoupled axial and transverse scans enable two dimensional tomographic imaging of sample with only one dimension moving of the probing beam, which is essential for in-vivo endoscopic applications.

Many alterations and modifications may be made by those having ordinary skill in the art without departing from the spirit and scope of the invention.