Fiber bundle fluorescence endomicroscopy is an effective method for in vivo imaging of biological tissue samples. Line-scanning confocal laser endomicroscopy realizes confocal imaging at a much higher frame rate compared to the point scanning system, but with reduced optical sectioning. To address this problem, we describe a fiber bundle endomicroscopy system that utilizes the HiLo technique to enhance the optical sectioning while still maintaining high image acquisition rates. Confocal HiLo endomicroscopy is achieved by synchronizing the scanning hybrid-illumination laser line with the rolling shutter of a CMOS camera. An evident improvement of axial sectioning is achieved as compared to the line-scanning confocal endomicroscopy without the HiLo technique. Comparisons are also made with epifluorescence endomicroscopy with and without HiLo. The optical sectioning enhancement is demonstrated on lens tissue as well as porcine kidney tissue.
We report a compact rigid instrument capable of delivering en-face optical coherence tomography (OCT) images alongside (epi)-fluorescence endomicroscopy (FEM) images by means of a robotic scanning device. Two working imaging channels are included: one for a one-dimensional scanning, forward-viewing OCT probe and another for a fiber bundle used for the FEM system. The robotic scanning system provides the second axis of scanning for the OCT channel while allowing the field of view (FoV) of the FEM channel to be increased by mosaicking. The OCT channel has resolutions of 25 / 60 μm (axial/lateral) and can provide en-face images with an FoV of 1.6 × 2.7 mm2. The FEM channel has a lateral resolution of better than 8 μm and can generate an FoV of 0.53 × 3.25 mm2 through mosaicking. The reproducibility of the scanning was determined using phantoms to be better than the lateral resolution of the OCT channel. Combined OCT and FEM imaging were validated with ex-vivo ovine and porcine tissues, with the instrument mounted on an arm to ensure constant contact of the probe with the tissue. The OCT imaging system alone was validated for in-vivo human dermal imaging with the handheld instrument. In both cases, the instrument was capable of resolving fine features such as the sweat glands in human dermal tissue and the alveoli in porcine lung tissue.
HiLo technique provides an effective means of eliminating signals from out-of-focus in widefield fluorescence microscopy by synthesizing two images sequentially acquired with uniform and structured illumination. However, light scattering within the sample often deteriorates the optical sectioning effect. Here, we demonstrate that optical sectioning can be improved by combining HiLo technique and confocal slit detection. Light scattering is reduced by using the rolling shutter of a CMOS scanner as a virtual detector slit. Synchronizing the camera rolling shutter with a scanning hybrid-illumination laser line results in a HiLo endomicroscopy with confocal line detection at a high frame rate of 60 fps. In endomicroscopy, an expanded laser beam passes through a beam splitter and is reflected by a spatial light modulator which toggles between two illumination patterns, grid and uniform. The illumination patterns are focused by a cylindrical lens and then delivered through a fiber bundle probe that transfers the laser line to the tissue and collects emitted fluorescence. To check the axial depth sectioning strength, a thin, uniform fluorescent plane is illuminated. The fullwidth half maximum of the axial distance scanning is 19 μm for the proposed HiLo confocal as opposed to 28.5 μm for the line scanning confocal and 51 um for the widefield HiLo. Experiments on imaging phantoms and ex vivo tissues demonstrate that the optical sectioning ability of the HiLo confocal endomicroscopy is improved when compared to its counterparts.
Fiber-bundle based confocal laser endomicroscopy combined with fluorescent biomarkers has shown promise for high-resolution imaging of tissue microstructure in vivo and in situ. However, limited image acquisition speed and a restriction to single fluorescence agents (due to single channel excitation and fluorescence collection spectral bands) for most existing systems makes simultaneous visualization of multiple morphological and functional features difficult. In this paper, we report the development of a high-speed dual-wavelength line-scan confocal laser endomicroscopy system suitable for multiplexed molecular imaging applications using 488 nm and 660 nm laser sources. The fluorescent confocal images are captured by a rolling-shutter CMOS camera at a constant frame rate of 120 Hz, with the rolling shutter of the CMOS camera acting as a virtual detector slit. Dual-wavelength imaging is achieved by switching between the laser sources for alternate frames, avoiding bleed-through, and providing an effective frame rate of 60 Hz. The two channels are pseudo-coloured and combined, and large area dual-wavelength mosaics are created by registering and stitching the image frames as the probe moves across the tissue. Preliminary images with a resolution of 1.2 µm are presented from fluorescently stained phantoms and ex vivo tissue, demonstrating the clinical feasibility of the technique.
Breast conserving surgery allows complete tumor resection while maintaining acceptable cosmesis for patients. Safe and rapid intraoperative margin assessment during the procedure is important to establish the completeness of tumor excision and minimizes the need for reoperation. Confocal laser endomicroscopy has demonstrated promise for real-time intraoperative margin assessment using acriflavine staining, but it is not approved for routine in-human use. We describe a custom high-speed line-scan confocal laser endomicroscopy (LS-CLE) system at 660 nm that enables high-resolution histomorphological imaging of breast tissue stained with methylene-blue, an alternative fluorescent stain for localizing sentinel nodes during breast surgery. Preliminary imaging results on freshly excised human breast tissue specimens are presented, demonstrating the potential of methylene-blue aided rapid LS-CLE to determine the oncological status of surgical margins in-vivo.
Optical fiber bundle based endomicroscopy is a low-cost optical biopsy technique for in vivo cellular level imaging. A
limitation of such an imaging system, however, is its small field-of-view (FOV), typically less than 1 mm2. With such a
small FOV it is difficult to associate individual image frames with the larger scale anatomical structure. Video-sequence
mosaicking algorithms have been proposed as a solution for increasing the image FOV while maintaining cellular-level
resolution by stitching together the endomicroscopy images. Although extensive research has focused on image
processing and mosaicking algorithms, there has been limited work on localization of the probe to assist with building
high quality mosaics over large areas of tissue.
In this paper, we propose the use of electromagnetic (EM) navigation to assist with large-area mosaicking of hand-held
high-resolution endomicroscopy probes. A six degree-of-freedom EM sensor is used to track in real-time the position
and orientation of the tip of the imaging probe during free-hand scanning. We present a proof-of-principle system for
EM-video data co-calibration and registration and then describe a two-step image registration algorithm that assists
mosaic reconstruction. Preliminary experimental investigations are carried out on phantoms and ex vivo porcine tissue
for free-hand scanning. The results demonstrate that the proposed methodology significantly improves the quality and
accuracy of reconstructed mosaics compared to reconstructions based only on conventional pair-wise image registration.
In principle, this approach can be applied to other optical biopsy techniques such as confocal endomicroscopy and
endocytoscopy.
Micro-thin wires are of significant importance to academia, research laboratories as well as industries engaged in
micro-fabrication of products related to diverse fields like micromechanics, bio-instrumentation, optoelectronics etc.
Critical dimension metrology of such wires often demands diameter estimation with tight tolerances. Amongst other
measurement techniques, Optical Diffractometry under Fraunhofer approximation has emerged over years as a nondestructive,
robust and precise technique for on-line diameter estimation of thin wires. However, it is observed that
existing Fraunhofer models invariably result in experimental overestimation of wire diameter, leading to
unacceptable error performances particularly for diameters below 50 μm. In this paper, a novel diffraction model
based on Geometric theory is proposed and demonstrated to theoretically quantify this diameter overestimation. The
proposed model utilizes hitherto unused paths-ways for the two lateral rays that contribute to the first diffraction
minimum. Based the 3-D geometry of the suggested model, a new 'diffraction formulation' is proposed. The
theoretical analysis reveals the following. For diffraction experiment, the Actual diameter of the diffracting wire is a
function of four parameters: source wavelength 'λ', axial distance 'z', diffraction angle corresponding to first
diffraction minimum 'θd' and a newly defined characteristic parameter 'm'. The analysis reveals further that the
proposed characteristic parameter 'm' varies non-linearly with diameter and presents a dependence only on the
experimentally measured diffraction angle 'θd'. Based on the proposed model, the communication reports for the
first time, a novel diameter-inversion procedure which, not only corrects for the overestimated but also facilitates
wire diameter-inversion with high resolution. Micro-thin metallic wires having diameters spanning the range 1-50
μm are examined. Experimental results are obtained that corroborate the theoretical approach.
A novel optical method is proposed and demonstrated, for real-time dimension estimation of thin opaque cylindrical
objects. The methodology relies on free-space Fraunhofer diffraction principle. The central region, of such tailored
diffraction pattern obtained under suitable choice of illumination conditions, comprises of a pair of 'equal intensity
maxima', whose separation remains constant and independent of the diameter of the diffracting object. An analysis of
'the intensity distribution in this region' reveals the following. At a point symmetrically located between the said
maxima, the light intensity varies characteristically with diameter of the diffracting object, exhibiting a relatively
stronger intensity modulation under spherical wave illumination than under a plane wave illumination. The analysis
reveals further, that the said intensity variation with diameter is controllable by the illumination conditions. Exploiting
these 'hitherto unexplored' features, the present communication reports for the first time, a reliable method of estimating
diameter of thin opaque cylindrical objects in real-time, with nanometer resolution from single point intensity
measurement. Based on the proposed methodology, results of few simulation and experimental investigations carried-out
on metallic wires with diameters spanning the range of 5 to 50μm, are presented. The results show that proposed method is well-suited for high resolution on-line monitoring of ultrathin wire diameters, extensively used in micro-mechanics
and semiconductor industries, where the conventional diffraction-based methods fail to produce accurate results.
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