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Oblique back-illumination microscopy

Phase contrast microscopy provides exquisite high-resolution images of sample morphology, without the use of sample labeling. However standard phase contrast techniques, like differential interference contrast (DIC), only work in the transmission direction and thus cannot be used when imaging thick samples. For this reason, standard phase contrast techniques have not made a great impact in in-vivo biomedical or clinical imaging applications.

We have developed a new technique, called Oblique Back-illumination Microscopy (OBM) that provides microscopic resolution DIC-like images of sub-surface sample morphology even in very thick tissue. In addition, OBM provides simultaneous amplitude imaging by a method of shadowcasting. Because OBM works in a reflected light geometry, it is amenable to endoscopy applications.

OBM uses standard widefield detection optics. That is, light is projected from an object plane to an image plane with a series of lenses, and it is then detected with a camera (CCD or CMOS). In the case of an endoscope, some extra relay optics is introduced, such as an imaging fiber bundle. This is no different from standard widefield endoscopy. Where OBM differs is in the illumination path. A schematic of an OBM endoscope based on a flexible imaging fiber bundle is shown in Fig. 1a. Illumination from two LEDs is sequentially launched into a thick sample by diametrically opposed off-axis optical fibers attached to an endoscope probe housing. Multiple scattering in the sample redirects the light so that it trans-illuminates the focal plane of the probe micro-objective (magenta dashed line). An image from the focal plane is then relayed by a flexible fiber bundle and projected onto a digital camera. (b) Close-up of the probe distal end, onto which is superposed a density map obtained by Monte Carlo simulation of the light energy in the sample that was injected by a single fiber (from left) and collected by the micro-objective (this density map is often called a “photon banana”). Note the obliqueness of the light distribution through the focal plane. (c) Oblique trans-illumination is partially blocked by the micro-objective back aperture. Index of refraction variations at the focal plane refract the light causing changes in the image intensity that are proportional to the slope of the variations, thus leading to phase gradient contrast.

Publications Related to this Research Area

Fast hyperspectral phase and amplitude imaging in scattering tissue

C. Ba, J.-M. Tsang, J. Mertz,

Optics Letters

Hyperspectral imaging in scattering tissue generally suffers from low light collection efficiency. In this Letter, we propose a microscope based on Fourier transform spectroscopy and oblique back-illumination microscopy that provides hyperspectral phase and amplitude images of thick, scattering samples with high throughput. Images can be acquired at >0.1  Hz rates with spectral resolution better than 200  cm−1, over a wide spectral range of 450–1700 nm. Proof-of-principle demonstrations are presented with chorioallantoic membrane of a chick embryo, illustrating the possibility of high-resolution hemodynamics imaging in thick tissue, based on transmission contrast.

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Widefield fluorescence microscopy with sensor-based conjugate adaptive optics using oblique back illumination

Jiang Li, Thomas G. Bifano, Jerome Mertz,

Journal of Biomedical Optics

We describe a wavefront sensor strategy for the implementation of adaptive optics (AO) in microscope applications involving thick, scattering media. The strategy is based on the exploitation of multiple scattering to provide oblique back illumination of the wavefront-sensor focal plane, enabling a simple and direct measurement of the flux-density tilt angles caused by aberrations at this plane. Advantages of the sensor are that it provides a large measurement field of view (FOV) while requiring no guide star, making it particularly adapted to a type of AO called conjugate AO, which provides a large correction FOV in cases when sample-induced aberrations arise from a single dominant plane (e.g., the sample surface). We apply conjugate AO here to widefield (i.e., nonscanning) fluorescence microscopy for the first time and demonstrate dynamic wavefront correction in a closed-loop implementation.

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Dual-modality endomicroscopy with co-registered fluorescence and phase contrast

C. Ba, M. Palmiere, J. Ritt, J. Mertz,

Biomedical Optics Express

We describe a dual-modality laser scanning endomicroscope that provides simultaneous fluorescence contrast based on confocal laser endomicroscopy (CLE) and phase-gradient contrast based on scanning oblique back-scattering microscopy (sOBM). The probe consists of a 2.6mm-diameter micro-objective attached to a 30,000-core flexible fiber bundle. The dual contrasts are inherently co-registered, providing complementary information on labeled and unlabeled sample structure. Proof of principle demonstrations are presented with ex-vivo mouse colon tissue.

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Fast volumetric phase-gradient imaging in thick samples

J. D. Giese, T. N. Ford, J. Mertz,

Optics Express

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.

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Phase-gradient contrast in thick tissue with a scanning microscope

J. Mertz, A. Gasecka, A. Daradich, I. Davison, D. Cote ,

Biomedical Optics Express

It is well known that the principle of reciprocity is valid for light traveling even through scattering or absorptive media. This principle has been used to establish an equivalence between conventional widefield microscopes and scanning microscopes. We make use of this principle to introduce a scanning version of oblique back-illumination microscopy, or sOBM. This technique provides sub-surface phase-gradient and amplitude images from unlabeled tissue, in an epi-detection geometry. That is, it may be applied to arbitrarily thick tissue. sOBM may be implemented as a simple, cost-effective add-on with any scanning microscope, requiring only the availability of an extra input channel in the microscope electronics. We demonstrate here its implementation in combination with two-photon excited fluorescence (TPEF) microscopy and with coherent anti-Stokes Raman scattering (CARS) microscopy, applied to brain or spinal cord tissue imaging. In both cases, sOBM provides information on tissue morphology complementary to TPEF or CARS contrast. This information is obtained simultaneously and is automatically co-registered. Finally, we show that sOBM can be operated at video rate.

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

T. N. Ford, J. Mertz,

Journal of Biomedical Optics

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 highresolution rigid Hopkins lens suitable for endoscopy.

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

Tim N. Ford, Kengyeh K. Chu, Jerome Mertz,

Nature Methods

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.

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An offset fiber produces oblique illumination

Monte Carlo simulation of "photon banana" (i.e. density of photons in scattering sample that are injected by an off-axis fiber and detected by an on-axis contact-mode endomicroscope objective. Note that illumination through focal plane (dashed line) is oblique, leading to phase-gradient contrast.

OBM with extended field of view

OBM of 11-day old chick embryo vasculature. Extended field of view is achieved by mosaicing.

Single-shot OBM video of chick embryo vasculature

OBM produces phase-gradient (left) and amplitude (right) contrast simultaneously. Single-shot operation is achieved by using two different illumination colors. Acquisition is video rate.