Homodyne en face optical coherence tomography

We demonstrate, for what we believe to be the first time, the use of a 3 x 3 fiber-optic coupler to realize a homodyne optical coherence tomography (OCT) system for en face imaging of highly scattering tissues and turbid media. The homodyne OCT setup exploits the inherent phase shifts between different output ports of a 3 x 3 fiber-optic coupler to extract amplitude information of a sample. Our homodyne en face OCT system features a measured resolution of 14 microm axially and 9.4 microm laterally with a 90 dB signal-to-noise ratio at 10 micros integration time. En face OCT imaging of a stage 52 Xenopus laevis was successfully demonstrated at a depth of 600 microm within the sample.     

Combined confocal/en face T-scan-based ultrahigh-resolution optical coherence tomography in vivo retinal imaging

Combined confocal scanning ophthalmoscopy/en face T-scan-based ultrahigh-resolution optical coherence tomography (OCT) of the human retina in vivo is reported for the first time to our knowledge. The system uses a superluminescent diode-based broadband source, which gives an axial resolution of 3.2 microm in the retina. We demonstrate acquisition of T-scan-based OCT B-scan and simultaneous confocal/C-scan images of the human retina of large lateral size (covering a field of up to 20 degrees ) at a frame rate of 2Hz.     

Multiscale multimodal imaging with multiphoton microscopy and optical coherence tomography

A multiscale multiphoton microscopy (MPM) and optical coherence tomography (OCT) system has been developed using a sub-10 fs Ti:sapphire laser. The system performs cross-sectional OCT imaging over millimeter field-of-view and en-face high-resolution MPM imaging with submicrometer resolution from the same sample location. With fish cornea, we have demonstrated cross-sectional imaging of cornea tissue layers using OCT, and the zoom-in imaging of cells and collagen fibers in each layer using MPM. The multiscale MPM/OCT system shows the potential of a rapid coarse scan to search for abnormal regions and the subsequent fine zoom-in imaging for diagnosis.     

Effect of multimodal coupling in imaging micro-endoscopic fiber bundle on optical coherence tomography

The effect of higher-order modes in fiber bundle imager-based optical coherence tomography (OCT) has been theoretically modeled using coupled fiber mode analysis ignoring the polarization and core size variation in order to visualize the pure effect of multimodal coupling of the imaging bundle. In this model, the optical imaging fiber couples several higher-order modes in addition to the fundamental one due to its high numerical aperture for achieving light confinement to the single core pixel. Those modes become evident in a distance domain using A-mode (depth) OCT based on a mirror sample experiment where multiple peaks are generated by the spatial convolution and coherence function of the light source. The distance between the peaks corresponding to each mode can be estimated by considering the effective indices of coupled (guided) modes obtained from numerically solving the fiber mode characteristics equations and the fiber length. The results have been compared for various types (fiber dimensions and wavelengths) and lengths of fibers, which have mode separation of 715 μm (1404 μm) and 764 μm (1527 μm) for the measurement and analysis, respectively in a 152.5 mm (305 mm)-long imaging fiber.     

Real time en face Fourier-domain optical coherence tomography with direct hardware frequency demodulation

We demonstrate en face swept source optical coherence tomography (ss-OCT) without requiring a Fourier transformation step. The electronic optical coherence tomography (OCT) interference signal from a k-space linear Fourier domain mode-locked laser is mixed with an adjustable local oscillator, yielding the analytic reflectance signal from one image depth for each frequency sweep of the laser. Furthermore, a method for arbitrarily shaping the spectral intensity profile of the laser is presented, without requiring the step of numerical apodization. In combination, these two techniques enable sampling of the in-phase and quadrature signal with a slow analog-to-digital converter and allow for real-time display of en face projections even for highest axial scan rates. Image data generated with this technique is compared to en face images extracted from a three-dimensional OCT data set. This technique can allow for real-time visualization of arbitrarily oriented en face planes for the purpose of alignment, registration, or operator-guided survey scans while simultaneously maintaining the full capability of high-speed volumetric ss-OCT functionality.     

Fractal analysis of en face tomographic images obtained with full field optical coherence tomography

The quantitative modeling of the imaging signal of pathological areas and healthy areas is necessary to improve the specificity of diagnosis with tomographic en face images obtained with full field optical coherence tomography (FFOCT). In this work, we propose to use the depth‐resolved change in the fractal parameter as a quantitative specific biomarker of the stages of disease. The idea is based on the fact that tissue is a random medium and only statistical parameters that characterize tissue structure are appropriate. We successfully relate the imaging signal in FFOCT to the tissue structure in terms of the scattering function and the coherent transfer function of the system. The formula is then used to analyze the ratio of the Fourier transforms of the cancerous tissue to the normal tissue. We found that when the tissue changes from the normal to cancerous the ratio of the spectrum of the index inhomogeneities takes the form of an inverse power law and the changes in the fractal parameter can be determined by estimating slopes of the spectra of the ratio plotted on a log‐log scale. The fresh normal and cancer liver tissues were imaged to demonstrate the potential diagnostic value of the method at early stages when there are no significant changes in tissue microstructures.     

Zebrafish imaging and two-photon fluorescence imaging using ZnSe quantum dots

This study is to report a ZnSe quantum dot with a large two-photon absorption cross section and good biocompatibility,which can be used in bioimaging.Fluorescence emission at 410 nm is observed in the quantum dot under 760-nm laser excitation.These biocompatible quantum dots exhibit a two-photon cross-section of 9.1×10~5 GM(1 GM=10~(-50) cm~4·s/photon).Two-photon excited laser scanning microscopic images show that cells co-cultured with ZnSe quantum dots are found in the blue channel at a fluorescence intensity that is 14.5 times that of control cells not cocultured with quantum dots.After incubating zebrafish larvae with ZnSe quantum dots for 24 h,the fluorescence intensity of the yolk sac stimulated by ultraviolet light is 2.9 times that of the control group.The proposed material shows a great potential application in biological imaging.     

Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous Raman imaging. The distribution of the UV-absorbing fluorophore Hoechst 33342 in the apoptotic HeLa cells is measured in the combined cw two-photon-excited fluorescence and Raman microscopy modes. The 647-nm line of a Kr-ion laser is used to excite both the Raman scattering and the two-photon-excited fluorescence emission. The lateral and axial resolutions in the two imaging modes are compared by use of the Gaussian beam approximation and backprojection of the focal volume through the confocal pinhole.     

En face imaging of single cell layers by differential phase-contrast optical coherence microscopy

Optical coherence microscopy (OCM) is capable of imaging the backscattering potential of a sample with high transversal and axial resolution. We report on a combination of OCM with a differential phase-contrast technique that permits imaging of the subwavelength optical path differences that occur between a narrow beam probing a sample and its surrounding. This technique allows small transversal refractive-index variations close to a selected interface to be seen. We report on the method and present first images of a test sample and a single cell layer. The cells act as phase objects; imaging the phase properties improves the contrast compared with that of intensity images.     

Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy

We demonstrate that simultaneous second-harmonic generation (SHG) and two-photon-excited fluorescence (TPEF) can be used to rapidly image biological membranes labeled with a styryl dye. The SHG power is made compatible with the TPEF power by use of near-resonance excitation, in accord with a model based on the theory of phased-array antennas, which shows that the SHG radiation is highly structured. Because of its sensitivity to local asymmetry, SHG microscopy promises to be a powerful tool for the study of membrane dynamics.     

Attenuation compensation for optical coherence tomography imaging

Optical coherence tomography (OCT) is a noninvasive technique that provides micrometer-scale imaging of tissue. As most biological tissues are considered turbid, it causes attenuation of the OCT signal and limits the depth penetration. Although a few algorithms had been developed to compensate the attenuation, almost all of them need to extract the scattering parameters before doing the compensation procedure. Because the real biological samples are anisotropic and multilayer-like structure, it is not time-efficient to model and solve these scattering parameters. This paper introduces a new method to compensate the OCT signal attenuation in depth. By analyzing the input signal, a compensation function is adaptively derived for each A-scan line, which can be used effectively to compensate the energy loss in the large sections and enhance the details in the deep, dark-like areas. Three bio-samples, a piece of onion, a Poecilia Wingei fish and a piece of rabbit abdominal aorta, were used to test our method. OCT images obtained by a swept-source OCT system were processed by the proposed method. Results show the visualization of structures in OCT images has been evidently improved, especially in deep region.     

Simultaneous optical coherence tomography--Indocyanine Green dye fluorescence imaging system for investigations of the eye's fundus

We have developed a dual-channel optical coherence tomography-Indocyanine Green dye (OCT-ICG) fluorescence system based on a previously reported ophthalmic OCT confocal imaging system. The confocal channel is tuned to the fluorescence wavelength range of the ICG, and light from the same optical source is used to generate the OCT image and to excite the ICG fluorescence. The system enables the clinician to visualize simultaneously en face OCT slices and corresponding ICG angiograms of the ocular fundus, displayed side by side. C-scan (constant depth) and B-scan (cross section) images are collected by a fast en face scan (T scan). The pixel-to-pixel correspondence between the OCT and angiography images allows the user to capture OCT B scans precisely at selected points on the ICG confocal images.     

Combined scanning optical coherence and two-photon-excited fluorescence microscopy

We demonstrate simultaneous imaging by optical coherence microscopy (OCM) and two-photon-excited (TPE) fluorescence microscopy. A mode-locked Ti:sapphire laser is focused and scanned in three dimensions through a fixed sample, generating both backscattered light and fluorescence light, which are independently detected. Both imaging modes provide rapid en-face imaging with submicrometer resolution. High-power delivery into the sample yields an OCM sensitivity in excess of 130 dB at 100-kHz pixel rates. Simultaneous imaging of cell nuclei with OCM and TPE is demonstrated in live drosophila embryos.     

Adaptive optics optical coherence tomography for retina imaging

When optical coherence tomography (OCT) is used for human retina imaging, its transverse resolution is limited by the aberrations of human eyes. To overcome this disadvantage, a high resolution imaging system for living human retina, which consists of a time domain OCT system and a 37-elements adaptive optics (AO) system, has been developed. The AO closed loop rate is 20 frames per second, and the OCT has a 6.7-μm axial resolution. In this paper, this system is introduced and the high resolution imaging results for retina are presented.     

Sequential optical coherence tomography and confocal imaging

We report a system capable of sequentially acquiring two en-face images of different depth resolutions. The two images are generated by use of different principles, optical coherence tomography (OCT) and confocal microscopy, and have depth resolutions, at present, of better than 20 microm and over 0.12 mm, respectively. The lower-depth-resolution image is ideal for target positioning before collection of stacks of en-face OCT images. Switching between the two types of image by flipping an opaque screen in the reference arm, coupled with self-adjusting gain operation of avalanche photodiodes in the receiver. We illustrate the usefulness of the system by imaging a leaf and an optic nerve in vivo.     

Spectral-domain optical coherence phase microscopy for quantitative phase-contrast imaging

We describe a novel microscopy technique for quantitative phase-contrast imaging of a transparent specimen. The technique is based on depth-resolved phase information provided by common path spectral-domain optical coherence tomography and can measure minute phase variations caused by changes in refractive index and thickness inside the specimen. We demonstrate subnanometer level path-length sensitivity and present images obtained on reflection from a known phase object and human epithelial cheek cells.     

Pulsed magneto-motive optical coherence tomography for remote cellular imaging

We developed pulsed magneto-motive optical coherence tomography (PMM-OCT) to reduce environmental temperature in the measurement volume and to expand the effective magnetic field distance from a pulse source. The proposed PMM-OCT system consisted of a spectral-domain OCT system and a customarily designed electrical pulse generator. The enhanced magnetic field allowed the proposed system to be able to image magnetically labeled cells in a distance as far as 30?mm away from the pulse generator. As an easy and sensitive approach, our PMM-OCT may be beneficially applied to a molecular-level imaging systems.     

Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence

A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.