Phase-gradient microscopy in thick tissue with oblique back-illumination.

Phase-contrast techniques, such as differential interference contrast microscopy, are widely used to obtain morphological images of unstained biological samples. The transillumination geometry required for these techniques restricts their application to thin samples. We introduce oblique back-illumination microscopy, a method of collecting en face phase-gradient images of thick scattering samples, enabling near-video-rate in vivo phase imaging with a miniaturized probe suitable for endoscopy.

Fast volumetric phase-gradient imaging in thick samples.

Oblique back-illumination microscopy (OBM) provides high resolution, sub-surface phase-gradient images from arbitrarily thick samples. We present an image formation theory for OBM and demonstrate that OBM lends itself to volumetric imaging because of its capacity for optical sectioning. In particular, OBM can provide extended depth of field (EDOF) images from single exposures, by rapidly scanning the focal plane with an electrically tunable lens. These EDOF images can be further enhanced by deconvolution. We corroborate our theory with experimental volumetric images obtained from transparent bead samples and mouse cortical brain slices.

Quantitative phase imaging using a partitioned detection aperture.

We present a technique to quantitatively image the phase of thin quasi-transparent samples using extended source incoherent illumination and off-axis detection apertures. Our technique is achromatic and polarization independent, requires no active elements, and can be readily adapted to standard bright-field microscopes. We demonstrate our technique by quantitatively reconstructing the phase of cheek cells and a microlens. The light efficient, single-shot nature of our technique enables phase imaging at frame rates that are camera limited.

Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle.

We present an endomicroscope apparatus that exhibits out-of-focus background rejection based on wide-field illumination through a flexible imaging fiber bundle. Our technique, called HiLo microscopy, involves acquiring two images, one with grid-pattern illumination and another with standard uniform illumination. An evaluation of the image contrast with grid-pattern illumination provides an optically sectioned image with low resolution. This is complemented with high-resolution information from the uniform illumination image, leading to a full-resolution image that is optically sectioned. HiLo endomicroscope movies are presented of fluorescently labeled rat colonic mucosa.

Development of a Primary Human Co-Culture Model of Inflamed Airway Mucosa.

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.

In vivo imaging of airway cilia and mucus clearance with micro-optical coherence tomography.

We have designed and fabricated a 4 mm diameter rigid endoscopic probe to obtain high resolution micro-optical coherence tomography (µOCT) images from the tracheal epithelium of living swine. Our common-path fiber-optic probe used gradient-index focusing optics, a selectively coated prism reflector to implement a circular-obscuration apodization for depth-of-focus enhancement, and a common-path reference arm and an ultra-broadbrand supercontinuum laser to achieve high axial resolution. Benchtop characterization demonstrated lateral and axial resolutions of 3.4 µm and 1.7 µm, respectively (in tissue). Mechanical standoff rails flanking the imaging window allowed the epithelial surface to be maintained in focus without disrupting mucus flow. During in vivo imaging, relative motion was mitigated by inflating an airway balloon to hold the standoff rails on the epithelium. Software implemented image stabilization was also implemented during post-processing. The resulting image sequences yielded co-registered quantitative outputs of airway surface liquid and periciliary liquid layer thicknesses, ciliary beat frequency, and mucociliary transport rate, metrics that directly indicate airway epithelial function that have dominated in vitro research in diseases such as cystic fibrosis, but have not been available in vivo.

Video-rate imaging of microcirculation with single-exposure oblique back-illumination microscopy.

Oblique back-illumination microscopy (OBM) is a new technique for simultaneous, independent measurements of phase gradients and absorption in thick scattering tissues based on widefield imaging. To date, OBM has been used with sequential camera exposures, which reduces temporal resolution, and can produce motion artifacts in dynamic samples. Here, a variation of OBM that allows single-exposure operation with wavelength multiplexing and image splitting with a Wollaston prism is introduced. Asymmetric anamorphic distortion induced by the prism is characterized and corrected in real time using a graphics-processing unit. To demonstrate the capacity of single-exposure OBM to perform artifact-free imaging of blood flow, video-rate movies of microcirculation in ovo in the chorioallantoic membrane of the developing chick are presented. Imaging is performed with a high-resolution rigid Hopkins lens suitable for endoscopy.

Fast optically sectioned fluorescence HiLo endomicroscopy.

We describe a nonscanning, fiber bundle endomicroscope that performs optically sectioned fluorescence imaging with fast frame rates and real-time processing. Our sectioning technique is based on HiLo imaging, wherein two widefield images are acquired under uniform and structured illumination and numerically processed to reject out-of-focus background. This work is an improvement upon an earlier demonstration of widefield optical sectioning through a flexible fiber bundle. The improved device features lateral and axial resolutions of 2.6 and 17 µm, respectively, a net frame rate of 9.5 Hz obtained by real-time image processing with a graphics processing unit (GPU) and significantly reduced motion artifacts obtained by the use of a double-shutter camera. We demonstrate the performance of our system with optically sectioned images and videos of a fluorescently labeled chorioallantoic membrane (CAM) in the developing G. gallus embryo. HiLo endomicroscopy is a candidate technique for low-cost, high-speed clinical optical biopsies.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy.

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

Microvascular oxygen quantification using two-photon microscopy.

An instrument is demonstrated that is capable of three-dimensional (3D) vasculature imaging and pO(2) quantification with high spatial resolution. The instrument combines two-photon (2P) microscopy with phosphorescence quenching to measure pO(2). The instrument was demonstrated by performing depth-resolved microvascular pO(2) measurements of rat cortical vessels down to 120 microm below the surface. 2P excitation of porphyrin was confirmed, and measured pO(2) values were consistent with previously published data for normoxic and hyperoxic conditions. The ability to perform 3D pO(2) measurements using optical techniques will allow researchers to overcome existing limitations imposed by polarographic electrodes, magnetic resonance techniques, and surface-only pO(2) measurement techniques.