Fourier-domain digital holographic optical coherence imaging of living tissue

Digital holographic optical coherence imaging is a full-frame coherence-gated imaging approach that uses a CCD camera to record and reconstruct digital holograms from living tissue. Recording digital holograms at the optical Fourier plane has advantages for diffuse targets compared with Fresnel off-axis digital holography. A digital hologram captured at the Fourier plane requires only a 2D fast Fourier transform for numerical reconstruction. We have applied this technique for the depth-resolved imaging of rat osteogenic tumor multicellular spheroids and acquired cross-section images of the anterior segment and the retinal region of a mouse eye. A penetration depth of 1.4 mm for the tumor spheroids was achieved.     

Holographic optical coherence imaging of rat osteogenic sarcoma tumor spheroids

Holographic optical coherence imaging is a full-frame variant of coherence-domain imaging. An optoelectronic semiconductor holographic film functions as a coherence filter placed before a conventional digital video camera that passes coherent (structure-bearing) light to the camera during holographic readout while preferentially rejecting scattered light. The data are acquired as a succession of en face images at increasing depth inside the sample in a fly-through acquisition. The samples of living tissue were rat osteogenic sarcoma multicellular tumor spheroids that were grown from a single osteoblast cell line in a bioreactor. Tumor spheroids are nearly spherical and have radial symmetry, presenting a simple geometry for analysis. The tumors investigated ranged in diameter from several hundred micrometers to over 1 mm. Holographic features from the tumors were observed in reflection to depths of 500-600 microm with a total tissue path length of approximately 14 mean free paths. The volumetric data from the tumor spheroids reveal heterogeneous structure, presumably caused by necrosis and microcalcifications characteristic of some human avascular tumors.     

Demonstration of complex-conjugate-resolved harmonic Fourier-domain optical coherence tomography imaging of biological samples

Complex-conjugate-resolved Fourier-domain optical coherence tomography, where the quadrature components of the interferogram are obtained by simultaneous acquisition of the first and second harmonics of the phase-modulated interferogram, is applied to multisurface test targets and biological samples. The method provides efficient suppression of the complex-conjugate, dc, and autocorrelation artifacts. A complex-conjugate rejection ratio as high as 70 dB is achieved.     

Polarization contrast imaging of biological tissues by polarization-sensitive Fourier-domain optical coherence tomography

Jones matrix imaging of biological samples by a polarization-sensitive Fourier-domain optical coherence tomography has been demonstrated using a two-dimensional CCD camera to obtain two spectra corresponding to the orthogonal polarization components simultaneously. The measurement results of a quarter-wave plate are compared between the two incident polarization sets, H-V linear and R-L circular polarization. Jones matrix imaging of the bovine tendon is demonstrated. Measured Jones matrix images are converted to equivalent Müller matrix images. Local polarization properties are obtained by longitudinal differentiation of Jones matrix components. The layered structure of the bovine tendon and birefringence are revealed.     

Time-domain optical coherence tomography with digital holographic microscopy

We show that digital holography can be combined easily with optical coherence tomography approach. Varying the reference path length is the means used to acquire a series of holograms at different depths, providing after reconstruction images of slices at different depths in the specimen thanks to the short-coherence length of light source. A metallic object, covered by a 150-microm-thick onion cell, is imaged with high resolution. Applications in ophthalmology are shown: structures of the anterior eye, the cornea, and the iris, are studied on enucleated porcine eyes. Tomographic images of the iris border close to the pupil were obtained 165 microm underneath the eye surface.     

Influence and compensation of autocorrelation terms in depth-resolved spectroscopic Fourier-domain optical coherence tomography

We demonstrate depth-resolved spectral absorption measurements in the wavelength range from 750 to 850 nm using a broadband light source consisting of three spectrally shifted superluminescent light-emitting diode modules and a low-cost spectrometer-based Fourier-domain optical coherence tomography system. We present the theoretical model and experimental verification of interferences between autocorrelation terms and the signal carrying cross-correlation terms, strongly affecting the absorption measurements. A simple background subtraction, minimizing the artifacts caused by the interferences of autocorrelation and cross-correlation terms, is presented.     

Surface imaging using holographic optical tweezers

We present an imaging technique using an optically trapped cigar-shaped probe controlled using holographic optical tweezers. The probe is raster scanned over a surface, allowing an image to be taken in a manner analogous to scanning probe microscopy (SPM), with automatic closed loop feedback control provided by analysis of the probe position recorded using a high speed CMOS camera. The probe is held using two optical traps centred at least 10 μm from the ends, minimizing laser illumination of the tip, so reducing the chance of optical damage to delicate samples. The technique imparts less force on samples than contact SPM techniques, and allows highly curved and strongly scattering samples to be imaged, which present difficulties for imaging using photonic force microscopy. To calibrate our technique, we first image a known sample--the interface between two 8 μm polystyrene beads. We then demonstrate the advantages of this technique by imaging the surface of the soft alga Pseudopediastrum. The scattering force of our laser applied directly onto this sample is enough to remove it from the surface, but we can use our technique to image the algal surface with minimal disruption while it is alive, not adhered and in physiological conditions. The resolution is currently equivalent to confocal microscopy, but as our technique is not diffraction limited, there is scope for significant improvement by reducing the tip diameter and limiting the thermal motion of the probe.     

Optical sectioning by holographic coherence imaging: a generalized analysis

A theory of optical sectioning by image plane holography is developed, emphasizing the use of broad-spectrum holographic methods to enhance the process. It is shown that a broad-spectrum source in a grating interferometer imitates the behavior of a monochromatic broad source.     

Estimation of longitudinal resolution in optical coherence imaging

The spectral shape of a source is of prime importance in optical coherence imaging because it determines several aspects of image quality, especially longitudinal resolution. Wide spectral bandwidth, which provides short coherence length, is sought to obtain high-resolution imaging. To estimate longitudinal resolution, the spectral shape of a source is usually assumed to be Gaussian, although the spectra of real sources are typically non-Gaussian. We discuss the limit of this assumption regarding the estimation of longitudinal resolution. To this end, we also investigate how coherence length is related to longitudinal resolution through the evaluation of different definitions of the coherence length. To demonstrate our purpose, the coherence length for several theoretical and real spectral shapes of sources having the same spectral bandwidth and central wavelength is computed. The reliability of coherence length computations toward the estimation of longitudinal resolution is discussed.     

Dispersion control with a Fourier-domain optical delay line in a fiber-optic imaging interferometer

Recently, Fourier-domain (FD) optical delay lines (ODLs) were introduced for high-speed scanning and dispersion compensation in imaging interferometry. We investigate the effect of first- and second-order dispersion on the photocurrent signal associated with an optical coherence imaging system implemented with a single-mode fiber, a superluminescent diode centered at 950 nm +/- 35 nm, a FD ODL, a mirror, and a layered LiTAO3 that has suitable dispersion characteristics to model a skin specimen. We present a practical and useful method to minimize the effect of dispersion through the interferometer and the specimen combined, as well as to quantify the results using two general metrics for resolution. Theoretical and associated experimental results show that, under the optimum solution, the maximum broadening of the point-spread function through a 1-mm-deep specimen is limited to 57% of its original rms width value (i.e., 8.1 microm optimal, 12.7 microm at maximum broadening) compared with approximately 110% when compensation is performed without the specimen taken into account.     

Characterization of optical fiber imaging bundles for swept-source optical coherence tomography

Fiber imaging bundles have been investigated for use in endoscopic optical coherence tomography (OCT) systems, to obviate the requirement for scanning components within the endoscope probe section. Images have been acquired using several optical configurations, two of which are common path in design. Configurations have been selected as having potential for miniaturization and inclusion in endoscopic-type systems, since the advantages of employing imaging bundles are most clearly seen in this type of system. The various types of bundle available are described, and the properties of the leached bundles used here are discussed in detail, with reference to their effect upon the performance of OCT systems. Images are displayed from measurements made on a range of samples.     

Task-based optimization and performance assessment in optical coherence imaging

Optimization of an optical coherence imaging (OCI) system on the basis of task performance is a challenging undertaking. We present a mathematical framework based on task performance that uses statistical decision theory for the optimization and assessment of such a system. Specifically, we apply the framework to a relatively simple OCI system combined with a specimen model for a detection task and a resolution task. We consider three theoretical Gaussian sources of coherence lengths of 2, 20, and 40 microm. For each of these coherence lengths we establish a benchmark performance that specifies the smallest change in index of refraction that can be detected by the system. We also quantify the dependence of the resolution performance on the specimen model being imaged.     

Polarization-sensitive optical coherence tomography for imaging human atherosclerosis

Polarization-sensitive optical coherence tomography (PS-OCT) combines the advantages of OCT with image contrast enhancement, which is based on its ability to detect phase retardation and the fast-axis angle. Both PS-OCT images and histopathology have demonstrated similar features that allowed differentiation of atherosclerotic structures (i.e., plaques) from normal tissue. Moreover, the picrosirius polarization method was used to confirm PS-OCT assessment of collagen in the fibrous cap of atherosclerotic plaques, and high-frequency (40 MHz) ultrasound images were used to identify calcium in the vessel wall. Our preliminary ex vivo investigation of human aortic specimens indicated that PS-OCT might help to identify atherosclerotic lesions.     

Multiscan time-domain optical coherence tomography for retina imaging

A versatile time-domain optical coherence tomography system is presented that can generate cross-sectional images by using either transverse priority or depth priority scanning. This is made possible by using a transmissive scanning delay line compatible with balance detection operating at a speed similar to that of the transverse scanner used to scan the beam across the target. In vivo images from the retina are generated and shown using the same system switched to either transverse or depth priority scanning regime, by using the scanning delay line either in slow or fast scanning modes, respectively. A comparative analysis of different scanning regimes depending on image size to fit different areas to be imaged is presented. Safety thresholds due to the different continuous irradiation time per transverse pixel in different scanning regimes are also considered. We present the maximum exposure level for a variety of scanning procedures, employing either A scanning (depth priority) or T scanning (transverse priority) when generating cross-sectional images, en face images, or collecting 3D volumes.     

Wide-field optical coherence tomography: imaging of biological tissues

We describe a two-dimensional optical coherence tomography technique with which we were able to obtain multiple longitudinal slices of a biological sample directly in a single Z scan. The system is based on a femtosecond Cr4+:forsterite laser and an infrared camera for wide-field imaging of the sample with a depth resolution of 5 microm. With this imaging apparatus we were able to investigate human skin and mouse ear samples and to observe the different constitutive tissues.     

Cancellation of coherent artifacts in optical coherence tomography imaging

Coherent artifacts in optical coherence tomography (OCT) images can severely degrade image quality by introducing false targets if no targets are present at the artifact locations. Coherent artifacts can also add constructively or destructively to the targets that are present at the artifact locations. This constructive or destructive interference will result in cancellation of the true targets or in display of incorrect echo amplitudes of the targets. We introduce the use of a nonlinear deconvolution algorithm, CLEAN, to cancel coherent artifacts in OCT images of extracted human teeth. The results show that CLEAN can reduce the coherent artifacts to the noise background, sharpen the air-enamel and enamel-dentin interfaces, and improve the image contrast.     

Manipulation of live mouse embryonic stem cells using holographic optical tweezers

We report the ability to move and arrange patterns of live embryonic stem cells using holographic optical tweezers. Single cell suspensions of mouse embryonic stem cells were manipulated with holographic optical tweezers into a variety of patterns including lines, curves and circles. Individual cells were also lifted out of the sample plane highlighting the potential for 3D positional control. Trypan blue dye exclusion and Live/Dead? staining (CMFDA^?1, EthHD^?1) showed that the cells were still viable after manipulation with the optical tweezers. The ability to move individual stem cells into specific, pre-defined patterns provides a method to study how arrangement and associated small-scale interactions occur between neighbouring cells.     

Primate retina imaging with polarization-sensitive optical coherence tomography.

Polarization-sensitive optical coherence tomography (PSOCT) is applied to determine the depth-resolved polarization state of light backreflected from the eye. The birefringence of the retinal nerve fiber layer (RNFL) was observed and measured from PSOCT images recorded postmortem in a Rhesus monkey. An image-processing algorithm was developed to identify birefringent regions in acquired PSOCT retinal images and automatically determine the thickness of the RNFL. Values of the RNFL thickness determined from histology and PSOCT were compared. PSOCT may provide a new method to determine RNFL thickness and birefringence for glaucoma diagnostics.     

Reconstruction in interferometric synthetic aperture microscopy: comparison with optical coherence tomography and digital holographic microscopy

It is shown that the spatial frequencies recorded in interferometric synthetic aperture microscopy do not correspond to exact backscattering [as they do in unistatic synthetic aperture radar (SAR)] and that the reconstruction process based on SAR is therefore based on an approximation. The spatial frequency response is developed based on the three-dimensional coherent transfer function approach and compared with that in optical coherence tomography and digital holographic microscopy.     

Monte Carlo modeling of optical coherence tomography imaging through turbid media

We combine a Monte Carlo technique with Mie theory to develop a method for simulating optical coherence tomography (OCT) imaging through homogeneous turbid media. In our model the propagating light is represented by a plane wavelet; its line propagation direction and path length in the turbid medium are determined by the Monte Carlo technique, and the process of scattering by small particles is computed according to Mie theory. Incorporated into the model is the numerical phase function obtained with Mie theory. The effect of phase function on simulation is also illustrated. Based on this improved Monte Carlo technique, OCT imaging is directly simulated and phase information is recorded. Speckles, resolution, and coherence gating are discussed. The simulation results show that axial and transversal resolutions decrease as probing depth increases. Adapting a light source with a low coherence improves the resolution. The selection of an appropriate coherence length involves a trade-off between intensity and resolution.