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.     

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.     

Phase-resolved optical coherence tomography and optical Doppler tomography for imaging blood flow in human skin with fast scanning speed and high velocity sensitivity

We have developed a novel phase-resolved optical coherence tomography (OCT) and optical Doppler tomography (ODT) system that uses phase information derived from a Hilbert transformation to image blood flow in human skin with fast scanning speed and high velocity sensitivity. Using the phase change between sequential scans to construct flow-velocity imaging, this technique decouples spatial resolution and velocity sensitivity in flow images and increases imaging speed by more than 2 orders of magnitude without compromising spatial resolution or velocity sensitivity. The minimum flow velocity that can be detected with an axial-line scanning speed of 400 Hz and an average phase change over eight sequential scans is as low as 10 microm/s, while a spatial resolution of 10 microm is maintained. Using this technique, we present what are to our knowledge the first phase-resolved OCT/ODT images of blood flow in human skin.     

Engineered microsphere contrast agents for optical coherence tomography

Contrast agents are utilized in virtually every imaging modality to enhance diagnostic capabilities. We introduce a novel class of optical contrast agent, namely, encapsulating microspheres, that are based not on fluorescence but on scattering nanoparticles within the shell or core. The agents are suitable for reflection- or scattering-based techniques such as optical coherence tomography, light microscopy, and reflectance confocal microscopy. We characterize the optical properties of gold-, melanin-, and carbon-shelled contrast agents and demonstrate enhancement of optical coherence tomography imaging after intravenous injection of such an agent into a mouse.     

Confocal enhanced optical coherence tomography for nondestructive evaluation of paints and coatings

We investigate confocal enhanced optical coherence tomography as a nondestructive evaluation tool for imaging with improved image contrast and depth resolution through a highly scattering paint layer. The depth responses provided by confocal microscopy, optical coherence tomography, and confocal enhanced optical coherence tomography for imaging through paint are investigated. We demonstrate that a new combination of confocal microscopy and optical coherence tomography provides significantly improved resolution and contrast for coherence gated imaging of substrate structures under the paint.     

Ultrahigh-resolution full-field optical coherence tomography using spatial coherence gating and quasi-monochromatic illumination

We developed an ultrahigh-resolution full-field optical coherence tomography (FF-OCT) microscope that is based on the spatial, rather than the temporal, coherence gating. The microscope is capable of observing three-dimensional microbiological structures as small as 0.4 μm × 0.4 μm × 1.0 μm (xyz) using quasi-monochromatic light and a liquid crystal retarder. Unlike traditional FF-OCT systems, this microscope can be operated in high resolution for any preferable wavelength with minimized defocusing and dispersion effects. High-resolution images of an onion cell are presented.     

Polarization-resolved second-harmonic-generation optical coherence tomography in collagen

We describe a novel imaging technique, second-harmonic-generation optical coherence tomography (SHOCT). This technique combines the spatial resolution and depth penetration of optical coherence tomography (OCT) with the molecular sensitivity of second-harmonic-generation spectroscopy. As a consequence of the coherent detection required for OCT, polarization-resolved images arise naturally. We demonstrate this new technique on a skin sample from the belly of Icelandic salmon, acquiring polarization-resolved SHOCT and OCT images simultaneously.     

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.     

The effects of longitudinal spatial coherence of light in interference experiments

The physical conditions needed to observe the effects of longitudinal spatial coherence of emission of an extended thermal source are considered. Results of experiments on observation of interference pulses of longitudinal spatial coherence, performed using a scanning longitudinal shearing Michelson interferometer, are presented. Distinctions between the effects of temporal and longitudinal spatial coherence of light are established. It is shown that the presence of a noncompensated optical layer in one of the arms of the interferometer makes the interference pulses of the temporal and spatial coherence of light shift relative to each other (“recession”).     

Multiple-scattering speckle in holographic optical coherence imaging

We investigate the effect of multiple scattering on the image quality of holographic optical coherence imaging, which is a full-field coherence-domain imaging form of optical coherence tomography. The speckle holograms from turbid media and from multicellular tumor spheroids are characterized by high-contrast speckle on a multiply-scattered background caused by channel cross-talk. We quantify the multiple-scattered light that is accepted by the holographic coherence gate, and identify a cross-over from single-scattered to multiple-scattered light beyond 15 to 20 optical thicknesses. Speckle reduction relies on vibrating diffusers and on fast adaptive holograms in photorefractive quantum well devices. The high anisotropy factor for tumor tissue reduces multiply-scattered light contributions for biomedical tumor imaging.     

MISTRAL: a transmission soft X‐ray microscopy beamline for cryo nano‐tomography of biological samples and magnetic domains imaging

The performance of MISTRAL is reported, the soft X‐ray transmission microscopy beamline at the ALBA light source (Barcelona, Spain) which is primarily dedicated to cryo soft X‐ray tomography (cryo‐SXT) for three‐dimensional visualization of whole unstained cells at spatial resolutions down to 30 nm (half pitch). Short acquisition times allowing for high‐throughput and correlative microscopy studies have promoted cryo‐SXT as an emerging cellular imaging tool for structural cell biologists bridging the gap between optical and electron microscopy. In addition, the beamline offers the possibility of imaging magnetic domains in thin magnetic films that are illustrated here with an example.     

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.     

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.     

Hybrid Rayleigh,Raman and two‐photon excited fluorescence spectral confocal microscopy of living cells

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Near-infrared dyes as contrast-enhancing agents for spectroscopic optical coherence tomography

Optical coherence tomography (OCT) images of biological tissues often have low contrast. Spectroscopic optical coherence tomography (SOCT) methods have been developed to enhance contrast but remain limited because most tissues are not spectrally active in the frequency bands of laser sources commonly used in OCT. Near-infrared (NIR) dyes with absorption spectra features within the OCT source spectrum can be used for enhancing contrast in this situation. We introduce and demonstrate the use of NIR dyes as contrast agents for SOCT. Contrast-enhanced images are compared with fluorescence microscopy, demonstrating a link between SOCT and fluorescence imaging.     

Linear optical coherence tomography system with a downconverted fringe pattern

Linear optical coherence tomography (LOCT) systems are a simple and robust alternative to time-domain optical coherence tomography systems, but a detector with approximately 10(4) pixels is needed for an imaging depth of 2 mm. We present a new system for LOCT with a special mask attached to the image sensor. The mask essentially performs a downconversion of the spatial frequencies by multiplication with a second spatial frequency. This reduces the fringe frequency of the optical coherence tomography signal so that the signal can be sampled with fewer pixels.     

Second-harmonic optical coherence tomography

Second-harmonic optical coherence tomography, which uses coherence gating of second-order nonlinear optical responses of biological tissues for imaging, is described and demonstrated. Femtosecond laser pulses were used to excite second-harmonic waves from collagen harvested from rat tail tendon and a reference non-linear crystal. Second-harmonic interference fringe signals were detected and used for image construction. Because of the strong dependence of second-harmonic generation on molecular and tissue structures, this technique imparts contrast and resolution enhancement to conventional optical coherence tomography.     

Real-time phase-resolved functional optical coherence tomography by use of optical Hilbert transformation

We have developed a novel real-time phase-resolved functional optical coherence tomography system that uses optical Hilbert transformation. When we use a resonant scanner in the reference arm of the interferometer, with an axial scanning speed of 4 kHz, the frame rate of both structural and Doppler blood-flow imaging with a size of 100 by 100 pixels is 10 Hz. The system has high sensitivity and a larger dynamic range for measuring the Doppler frequency shift that is due to moving red blood cells. Real-time images of in vivo blood flow in human skin obtained with this interferometer are presented.     

Statistics and reduction of speckle in optical coherence tomography

Studies have shown that optical coherence tomography (OCT) is useful in imaging microscopic structures through highly scattering media. Because spatially coherent light is used in OCT, speckle in the reconstructed image is unavoidable, resulting in degradation of the quality of the OCT images and impaired ability to differentiate subsurface structures. Therefore speckle reduction is an important issue in OCT imaging. We develop speckle statistics that are appropriate to the OCT measurements and demonstrate a simple and practical speckle-reduction technique.     

Simultaneous swept source optical coherence tomography of the anterior segment and retina using coherence revival

We report on an implementation of coherence revival-based heterodyne swept source optical coherence tomography that is capable of simultaneously imaging the anterior and posterior eye. A polarization-encoded sample arm was used to efficiently focus orthogonal polarizations on the anterior segment and retina. Depth encoding was achieved using coherence revival, which allows for multiple depths within a sample to be simultaneously imaged and frequency encoded by carefully controlling the optical pathlength of each sample path. This design is a significant step toward whole-eye optical coherence tomography (OCT), which would enable customized ray-traced modeling of patient eyes to improve refractive surgical interventions and eliminate optical artifacts in retinal OCT diagnostics. We demonstrated the feasibility of this system for in vivo imaging by simultaneously acquiring images of the anterior segments and retinas in healthy human volunteers.