Spectral-domain optical coherence phase and multiphoton microscopy

We describe simultaneous quantitative phase contrast and multiphoton fluorescence imaging by combined spectral-domain optical coherence phase and multiphoton microscopy. The instrument employs two light sources for efficient optical coherence microscopic and multiphoton imaging and can generate structural and functional images of transparent specimens in the epidirection. Phase contrast imaging exhibits spatial and temporal phase stability in the subnanometer range. We also demonstrate the visualization of actin filaments in a fixed cell specimen, which is confirmed by simultaneous multiphoton fluorescence imaging.     

Quantitative phase-contrast imaging of cells with phase-sensitive optical coherence microscopy

We describe a method for en face phase-contrast imaging of cells with a fiber-based differential phase-contrast optical coherence microscopy system. Recorded en face images are quantitative phase-contrast maps of cells due to spatial variation of the refractive index and (or) thickness of various cellular components. Quantitative phase-contrast images of human epithelial cheek cells obtained with the fiber-based differential phase-contrast optical coherence microscopy system are presented.     

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.     

Spectral-domain phase microscopy

Broadband interferometry is an attractive technique for the detection of cellular motions because it provides depth-resolved phase information via coherence gating. We present a phase-sensitive technique called spectral-domain phase microscopy (SDPM). SDPM is a functional extension of spectral-domain optical coherence tomography that allows for the detection of nanometer-scale motions in living cells. The sensitivity of the technique is demonstrated, and its calibration is verified. A shot-noise limit to the displacement sensitivity of this technique is derived. Measurement of cellular dynamics was performed on spontaneously beating cardiomyocytes isolated from chick embryos.     

Digital holography for quantitative phase-contrast imaging

We present a new application of digital holography for phase-contrast imaging and optical metrology. This holographic imaging technique uses a CCD camera for recording of a digital Fresnel off-axis hologram and a numerical method for hologram reconstruction. The method simultaneously provides an amplitude-contrast image and a quantitative phase-contrast image. An application to surface profilometry is presented and shows excellent agreement with contact-stylus probe measurements.     

Spectral-domain optical coherence tomography with a Fresnel spectrometer

We propose a novel spectral-domain optical coherence tomography (SD-OCT) equipped with a Fresnel spectrometer, which utilizes a Fresnel zone plate (FZP) as both dispersion and focusing optics and thus spreads the spectral interferogram evenly in wavenumber domain because of the proportional relation between the focal length of the FZP and the wavenumber. With no need of the conversion calculation from wavelength to wavenumber in conventional SD-OCT, this new design is favorable for fast imaging with high resolution. As only a FZP and CCD are used, the Fresnel spectrometer is simple and compact. It is experimentally shown that its performance is as good as that of numerical interpolation in conventional SD-OCT. Imaging of bio-tissue by Fresnel SD-OCT is also demonstrated.     

Long-term quantitative phase-contrast imaging of living cells by digital holographic microscopy

The dynamic analysis of biological living samples is one of the particular interests in life sciences. An improved digital holographic microscope for long-term quantitative phase-contrast imaging of living cells is presented in this paper. The optical configuration is optimized in the form of a free-space-fiber hybrid system which promotes the flexibility of imaging in complex or semi-enclosed experimental environment. Aberrations compensation is implemented taking into account the additional phase aberration induced by liquid culture medium in long-term observation. The proposed approach is applied to investigate living samples of MC3T3-E1 and MLO-Y4 cells. The experimental results demonstrate its availability in the analysis of cellular changes.     

Spectral-domain optical coherence reflectometric sensor for highly sensitive molecular detection

We describe what we believe to be a novel use of spectral-domain optical coherence reflectometry (SD-OCR) for highly sensitive molecular detection in real time. The SD-OCR sensor allows identification of a sensor surface of interest in an OCR depth scan and monitoring the phase alteration due to molecular interaction at that surface with subnanometer optical thickness sensitivity. We present subfemtomole detection sensitivity for etching of SiO(2) molecules and demonstrate its application as a biosensor by measuring biotin-streptavidin binding in a microfluidic device.     

High-resolution optical coherence microscopy for high-speed, in vivo cellular imaging

Optical coherence microscopy (OCM) is demonstrated with a high-speed, broadband, reflective-grating phase modulator and a femtosecond Ti:Al2O3 laser. The novel system design permits high-resolution OCM imaging in a new operating regime in which a short coherence gate is used to relax the requirement for high-numerical-aperture confocal axial sectioning. In vivo cellular imaging is demonstrated in the Xenopus laevis tadpole and in human skin with a 3-microm coherence gate and a 30-microm confocal gate. The ability to achieve cellular imaging with a lower numerical aperture should facilitate the development of miniaturized probes for in vivo imaging applications.     

Dark-field optical coherence microscopy

Dark-field illumination is known to enhance scattering contrast in optical microscopy. We combined this concept with Fourier domain optical coherence microscopy (OCM). The detection and illumination paths are decoupled, and only the scattered light originating from the sample generates the tomogram signal, whereas any specular reflection is highly suppressed. We analyze and discuss this dark-field OCM concept and present its superior imaging quality on live cell samples.     

Full-field optical coherence microscopy

We present a new microscopy system for imaging in turbid media that is based on the spatial coherence gate principle and generates in parallel a complete two-dimensional head-on image without scanning. This system has been implemented in a commercial microscope and preserves the lateral resolution of the optics used. With a spatially incoherent source, speckle-free images with diffraction-limited resolution are recorded at successive depths with shot-noise-limited detection. The setup comprises a photoelastic modulator for path difference modulation and a two-dimensional CCD array and uses a multiplexed lock-in detection scheme.     

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.     

Spectroscopic spectral-domain optical coherence microscopy

The spectroscopic content within optical coherence tomography (OCT) data can provide a wealth of information. Spectroscopic OCT methods are frequently limited by time-frequency trade-offs that limit high spectral and spatial resolution simultaneously. We present spectroscopic spectral-domain optical coherence microscopy performed with a multimodality microscope. Restricting the spatial extent of the signal by using high-numerical-aperture optics makes high-resolution spectroscopic information accessible, facilitated with spectral-domain detection. Simultaneous acquisition of multiphoton microscopy images is used to validate tissue structure and localization of nuclei within individual cells.     

High-resolution line-scanning optical coherence microscopy

An optical coherence microscopy system based on line illumination and detection is demonstrated. The system uses a Linnik-type interferometer illuminated by a broadband Ti:sapphire laser and detected by a high-speed, line-scan CCD camera. This approach is less sensitive to incoherent scattering and sample motion than full-field imaging. Spatial resolutions of approximately 2 microm x approximately 3 microm(transverse x axial) are achieved. The sensitivity of the system is 93 dB with averaging over 30 line scans. En face real time, cellular-level imaging of biological tissues is demonstrated at approximately 2 frames/s.     

Visibility in differential phase-contrast imaging with partial coherence source

This paper gives theoretical analysis of visibility of fringes, which is influenced by distances, temporal and spatial coherence of source, in hard x-ray differential phase-contrast imaging with microfocus x-ray source. According to the character of longitudinal periodicity of the interferogram, the setup is insensitive to mechanical drift and vibrations. The effect of temporal coherence of x-ray source is investigated and its related bandwidth is derived. Based on the theory of partially coherent light, it shows that the requirement for the spatial coherence of x-ray source is not strict and can be met by the general microfocus x-ray tube for x-ray differential phase-contrast imaging.     

Double-reflection polygon mirror for high-speed optical coherence microscopy

We report on a high-speed, high-efficiency, high-duty-cycle, path-length-maintaining and linear beam scanner suitable for en face scanning optical coherence microscopy. Fast transverse beam scanning is achieved by use of a double-reflection polygon mirror (DRPM) rotating at a constant speed. With a motor speed of 18,000 rpm and a scanner diameter of 50 mm, the DRPM provides a line rate up to 3 kHz, +/-1.8 degrees scanning range, and 90% duty cycle. A much higher scanning speed and much larger scanning range can be readily achieved by increasing the scanner diameter.     

Cellular resolution optical coherence microscopy with high acquisition speed for in-vivo human skin volumetric imaging

In this Letter, we report for the first time (to our knowledge) in-vivo volumetric optical coherence microscopy images of skin epidermal cells. We achieved micrometer-class resolution, 2 μm laterally and axially, with an acquisition speed of 23 K A-scans/s and over 90 dB sensitivity to a depth of 1 mm by employing a custom, liquid-lens-based, dynamic-focusing objective, a broadband light source, and a custom, astigmatism-corrected Czerny-Turner spectrometer with a high-speed complementary metal-oxide-semiconductor camera.     

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.     

Phase-shifting Zernike phase contrast microscopy for quantitative phase measurement

Zernike phase contrast microscopy is extended and combined with a phase-shifting mechanism to perform quantitative phase measurements of microscopic objects. Dozens of discrete point light sources on a ring are constructed for illumination. For each point light source, three different levels of point-like phase steps are designed, which are alternatively located along a ring on a silica plate to perform phase retardation on the undiffracted (dc) component of the object waves. These three levels of the phase steps are respectively selected by rotating the silica plate. Thus, quantitative evaluation of phase specimens can be performed via phase-shifting mechanism. The proposed method has low "halo" and "shade-off" effects, low coherent noise level, and high lateral resolution due to the improved illumination scheme.