There is a critical need for a technology that can assist doctors in more accurately evaluating lung nodules at the time of biopsy. To address this need, a multispectral fluorescence line-scan confocal endomicroscope was developed that employs a fiber bundle probe to image tissue at the distal tip of the biopsy introducer needle. The multispectral nature of the instrument allows the simultaneous use of multiple FDA-approved dyes that stain different cellular/tissue compartments in different spectral regions to distinguish between lung cancer and benign conditions of the lung. The imaging system has been used to image normal rat lung as well as ex vivo human core-biopsy lung tissue.
KEYWORDS: Lung, Biopsy, Confocal microscopy, Tissues, Lung cancer, Real time imaging, Multispectral imaging, Luminescence, Line scan image sensors, Imaging systems
There is a critical need for a technology that can assist doctors in more accurately evaluating lung nodules at the time of biopsy. To address this need, a multispectral fluorescence line-scan confocal microendoscope was developed that employs a fiber bundle to image tissue at the distal tip of the biopsy needle. The multispectral nature of the instrument allows the simultaneous use of multiple FDA-approved dyes that stain different cellular/tissue compartments in different spectral regions to distinguish between lung cancer and benign lesions of the lung. The imaging system has been used to image ex vivo mouse and human lung tissue.
Recent evidence suggests that ovarian cancer can originate in the fallopian tube. Unlike many other cancers, poor access to the ovary and fallopian tubes has limited the ability to study the progression of this deadly disease and to diagnosis it during the early stage when it is most amenable to therapy. A rigid confocal microlaparoscope system designed to image the epithelial surface of the ovary in vivo was previously reported. A new confocal microlaparoscope with an articulating distal tip has been developed to enable in vivo access to human fallopian tubes. The new microlaparoscope is compatible with 5-mm trocars and includes a 2.2-mm-diameter articulating distal tip consisting of a bare fiber bundle and an automated dye delivery system for fluorescence confocal imaging. This small articulating device should enable the confocal microlaparoscope to image early stage ovarian cancer arising inside the fallopian tube. Ex vivo images of animal tissue and human fallopian tube using the new articulating device are presented along with in vivo imaging results using the rigid confocal microlaparoscope system.
Ultrathin flexible fiberscopes typically have separate illumination and imaging channels and are available in diameters ranging from 0.5 to 2.5 mm. Diameters can potentially be reduced by combining the illumination and imaging paths into a single fiberoptic channel. Single-channel fiberscopes must incorporate a system to minimize Fresnel reflections from air–glass interfaces within the common illumination and detection path. The Fresnel reflection at the proximal surface of the fiber bundle is particularly problematic. This paper describes and compares methods to reduce the background signal from the proximal surface of the fiber bundle. Three techniques are evaluated: (1) antireflective (AR)-coating the proximal face of the fiber, (2) incorporating crossed polarizers into the light path, and (3) a novel technique called numerical aperture sharing, whereby a portion of the image numerical aperture is devoted to illumination and a portion to detection.
Recent evidence suggests that epithelial ovarian cancer may originate in the fimbriated end of the fallopian tube1. Unlike
many other cancers, poor access to the ovary and fallopian tubes has limited the ability to study the progression of this
deadly disease and to diagnosis it during the early stage when it is most amenable to therapy. We have previously
reported on a rigid confocal microlaparoscope system that is currently undergoing a clinical trial to image the epithelial
surface of the ovary2. In order to gain in vivo access to the fallopian tubes we have developed a new confocal
microlaparoscope with an articulating distal tip. The new instrument builds upon the technology developed for the
existing confocal microlaparoscope. It has an ergonomic handle fabricated by a rapid prototyping printer. While
maintaining compatibility with a 5 mm trocar, the articulating distal tip of the instrument consists of a 2.2 mm diameter
bare fiber bundle catheter with automated dye delivery for fluorescence imaging. This small and flexible catheter design
should enable the confocal microlaparoscope to image early stage ovarian cancer arising inside the fallopian tube. Early
ex vivo mages of human fallopian tube and in vivo imaging results from recent open surgeries using the rigid confocal
microlaparoscope system are presented. Ex vivo images from animal models using the new articulating bare fiber system
are also presented. These high quality images collected by the new flexible system are similar in quality to those
obtained from the epithelial surface of ovaries with the rigid clinical confocal microlaparoscope.
Optical biopsies are aimed at providing fast and thorough screening of biological tissues in vivo. Disease diagnosis is
based on the morphological structures and biochemical features of tissues that can be sampled in situ with high
resolution. Some optical screening techniques, such as fluorescence confocal microendoscopy, provide a limited imaging
depth due to the shallow penetration of visible light. Despite confocal microendoscopy's high resolution and image
quality, morphological changes that occur deeper in the tissue cannot be detected. Other imaging techniques, such as
optical coherence tomography (OCT), are able to obtain information at greater depth into tissue. A combination of
fluorescence confocal and OCT into a single instrument capable of rapidly switching between these modalities, has the
potential of providing complementary en face confocal images showing the morphologic features of cells within a
surface layer, and cross-sectional OCT images showing tissue microarchitecture below the surface. The concept for this
dual system is to utilize the optical train of an existing multi-spectral confocal microendoscope as a spectral-domain
OCT system. Progress made on the implementation of this combined dual integrated imaging system is presented. A
performance analysis, discussion of the limitations inherent to the use of an imaging fiber bundle, and recent imaging
results are presented.
Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure,
but relies on exogenous fluorophores, has a relatively limited penetration depth (100 μm) and field of view (700 μm),
and produces a high rate of detailed information to the user. A new catheter based multi-modal system has been designed
that combines confocal imaging and oblique incidence reflectometry (OIR), which is a non-invasive method capable of
rapidly extracting tissue absorption, μa, and reduced scattering, μ's, spectra from tissue. The system builds on previous
developments of a custom slit-scan multi-spectral confocal microendoscope and is designed to rapidly switch between
diffuse spectroscopy and confocal fluorescence imaging modes of operation. An experimental proof-of-principle catheter
has been developed that consists of a fiber bundle for traditional confocal fluorescence imaging and a single OIR source
fiber which is manually redirected at +/- 26 degrees. Diffusely scattered light from each orientation of the source fiber is
collected via the fiber bundle, with a frame of data representing spectra collected at a range of distances from the OIR
source point. Initial results with intralipid phantoms show good agreement to published data over the 550-650 nm
spectral range. We successfully imaged and measured the optical properties of rodent cardiac muscle.
We demonstrate the implementation of a Fourier domain optical coherence tomography (OCT) imaging system
incorporated into the optical train of a fluorescence confocal microendoscope. The slit-scanning confocal system has
been presented previously and achieves 3μm lateral resolution and 25μm axial resolution over a field of view of 430μm.
Its multi-spectral mode of operation captures images with 6nm average spectral resolution. To incorporate OCT imaging,
a common-path interferometer is made with a super luminescent diode and a reference coverslip located at the distal end
of the fiber bundle catheter. The infrared diode spectral width allows a theoretical OCT axial resolution of 12.9μm. Light
from the reference and sample combine, and a diffraction grating produces a spectral interferogram on the same 2D CCD
camera used for confocal microendoscopic imaging. OCT depth information is recovered by a Fourier transform along
the spectral dispersion direction. Proper operation of the system scan mirrors allows rapid switching between confocal
and OCT imaging modes. The OCT extension takes advantage of the slit geometry, so that a 2D image is acquired
without scanning. Combining confocal and OCT imaging modalities provides a more comprehensive view of tissue and
the potential to improve disease diagnosis. A preliminary bench-top system design and imaging results are presented.
Slit-scanning geometries for confocal microendoscopy represent a compromise between acquisition rate and optical
performance. Such systems provide high frame rates that freeze motion but recent Monte Carlo simulations show that
scattered light severely limits the practical imaging depth for in vivo applications. A new multi-point scanning
architecture for confocal microendoscopy has been developed. The new scanner is based on a relatively simple
modification to the slit-scanning geometry that results in a parallelized point-scanning confocal microendoscope that
maintains the high frame rate of a slit-scanning system while providing optical performance close to that of a single point
scanning system. The multi-point scanner has been incorporated into an existing multi-spectral slit-scanning confocal
microendoscope. The new confocal aperture consists of a slit and a rotating low duty cycle binary transmission grating,
which effectively produces a set of continuously moving widely spaced illumination points along the slit. The design
maintains the ability to rapidly switch between grayscale and multi-spectral imaging modes. The improved axial
resolution of the multi-point scanning confocal microendoscope leads to significantly better confocal sectioning and
deeper imaging, which greatly improves the diagnostic potential of the instrument.
Successful treatment of cancer is highly dependent on the stage at which it is diagnosed. Early diagnosis, when the disease is still localized at its origin, results in very high cure rates-even for cancers that typically have poor prognosis. Biopsies are often used for diagnosis of disease. However, because biopsies are destructive, only a limited number can be taken. This leads to reduced sensitivity for detection due to sampling error. A real-time fluorescence confocal microlaparoscope has been developed that provides instant in vivo cellular images, comparable to those provided by histology, through a nondestructive procedure. The device includes an integrated contrast agent delivery mechanism and a computerized depth scan system. The instrument uses a fiber bundle to relay the image plane of a slit-scan confocal microlaparoscope into tissue. It has a 3-µm lateral resolution and a 25-µm axial resolution. Initial in vivo clinical testing using the device to image human ovaries has been done in 21 patients. Results indicate that the device can successfully image organs in vivo without complications. Results with excised tissue demonstrate that the instrument can resolve sufficient cellular detail to visualize the cellular changes associated with the onset of cancer.
We characterize and compare the axial and lateral performance of fluorescence confocal systems imaging in turbid media. The aperture configurations studied are a single pinhole, a slit, a Nipkow disk, and a linear array of pinholes. Systems with parallelized apertures are used clinically because they enable high-speed and real-time imaging. Understanding how they perform in highly scattering tissue is important. A Monte Carlo model was developed to characterize parallelized system performance in a scattering media representative of human tissues. The results indicate that a slit aperture has degraded performance, both laterally and axially. In contrast, the analysis reveals that multipinhole apertures such as a Nipkow disk or a linear pinhole array can achieve performance nearly equivalent to a single pinhole aperture. The optimal aperture spacing for the multipinhole apertures was determined for a specific tissue model. In addition to comparing aperture configurations, the effects of tissue nonradiative absorption, scattering anisotropy, and fluorophore concentration on lateral and axial performance of confocal systems were studied.
KEYWORDS: Laparoscopy, Confocal microscopy, Control systems, In vivo imaging, Ovary, Surgery, Imaging systems, Tissues, Clinical trials, Ovarian cancer
We previously reported on the development of a multi-spectral confocal laparoscope for clinical imaging. In this
paper we present current results using the system to image ovaries with a new laparoscope design using the
contrast agent acridine orange. This new laparoscope integrates computer controlled systems for focus, depth
scans, and localized contrast agent delivery. Precise axial position control is accomplished with tiny stepper
motors integrated inside the laparoscope handle. Ergonomic handle controls allow for data acquisition, deliver
of contrast agents, and adjustment of imaging depth during procedures by the surgeon. We have approval to use
acridine orange in our clinical trials to image ovaries in vivo during oophorectomies. We present in vivo results
using both acridine orange and fluorescein as the topically administered contrast agent.
We describe the design and operation of a multispectral confocal microendoscope. This fiber-based fluorescence imaging system consists of a slit-scan confocal microscope coupled to an imaging catheter that is designed to be minimally invasive and allow for cellular level imaging in vivo. The system can operate in two imaging modes. The grayscale mode of operation provides high resolution real-time in vivo images showing the intensity of fluorescent signal from the specimen. The multispectral mode of operation uses a prism as a dispersive element to collect a full multispectral image of the fluorescence emission. The instrument can switch back and forth nearly instantaneously between the two imaging modes (less than half a second). In the current configuration, the multispectral confocal microendoscope achieves 3-µm lateral resolution and 30-µm axial resolution. The system records light from 500 to 750 nm, and the minimum resolvable wavelength difference varies from 2.9 to 8.3 nm over this spectral range. Grayscale and multispectral imaging results from ex-vivo human tissues and small animal tissues are presented.
The confocal microendoscope is an instrument for imaging the surface of the human ovary. Images taken with this instrument from normal and diseased tissue show significant differences in cellular distribution. A real-time computer-aided system to facilitate the identification of ovarian cancer is introduced. The cellular-level structure present in ex vivo confocal microendoscope images is modeled as texture. Features are extracted based on first-order statistics, spatial gray-level-dependence matrices, and spatial-frequency content. Selection of the features is performed using stepwise discriminant analysis, forward sequential search, a nonparametric method, principal component analysis, and a heuristic technique that combines the results of these other methods. The selected features are used for classification, and the performance of various machine classifiers is compared by analyzing areas under their receiver operating characteristic curves. The machine classifiers studied included linear discriminant analysis, quadratic discriminant analysis, and the k-nearest-neighbor algorithm. The results suggest it is possible to automatically identify pathology based on texture features extracted from confocal microendoscope images and that the machine performance is superior to that of a human observer.
We have developed a mobile confocal microendoscope system that provides live cellular imaging during surgery
to aid in diagnosing microscopic abnormalities including cancer. We present initial clinical trial results using the
device to image ovaries in-vivo using fluorescein and ex-vivo results using acridine orange. The imaging catheter
has improved depth control and localized dye delivery mechanisms than previously presented. A manual control
now provides a simple way for the surgeon to adjust and optimize imaging depth during the procedure while a
tiny piezo valve in the imaging catheter controls the dye delivery.
A mobile confocal microendoscope for use in a clinical setting has been developed. This system
employs an endoscope consisting of a custom designed objective lens with a fiber optic imaging bundle to
collect in-vivo images of patients. Some highlights and features of this mobile system include frame rates
of up to 30 frames per second, an automated focus mechanism, automated dye delivery, clinician control,
and the ability to be used in an area where there is a single 110V outlet. All optics are self-contained and
the entire enclosure and catheter can be moved between surgical suites, sterilized and brought online in
under 15 minutes. At this time, all data have been collected with a 488 nm laser, but the system is able to
have a second laser line added to provide additional imaging capability. Preliminary in vivo results of
images from the ovaries using topical fluorescein as a contrast agent are shown. Future plans for the system include use of acridine orange (AO) or SYTO-16 as a nucleic acid stain.
We present a modified multi-spectral configuration of a slit-scanning confocal microendoscope that provides higher
spectral resolution in a fully automated interface. Tissue fluorescence signal is directed through a dispersive element that
spreads the spectral information across the CCD camera mapping spectral information perpendicular to the confocal slit.
The dispersive element may be chosen to meet the specific requirements defined by the user. Our current system uses a
BK7 prism with a 45o wedge angle and a 20mm diameter clear aperture. The prism is shifted from the optical axis
allowing automated switching from grayscale (beam on-axis) to multi-spectral (beam off-axis) imaging by tilting a
computer controlled mirror. The system records over a spectral range of 450nm to 750nm. The minimum resolvable
wavelength difference varies from 2.1nm to 8.3nm over the spectral range. The lateral and axial resolution of the system
is approximately 3&mgr;m by 30&mgr;m, respectively, and is the same for both grayscale and multi-spectral imaging modes.
Multi-spectral imaging results from ex-vivo tissues are presented.
We present a laparoscope for fluorescence confocal microendoscopy specifically designed for microscopic imaging during diagnostic laparoscopic surgery. The catheter consists of a disposable rigid distal tip which houses a flexible microendoscope and dye channel. The laparoscopic tip is a small disposable polycarbonate sheath
containing two inner lumens with a glass window on the distal end. The sheath outer diameter suitable for use in a 5mm trocar. The smaller inner lumen provides a channel for delivering fluorescent contrast agents to the tissue through a 200um hole in the glass window. On the proximal end, the smaller lumen is coupled to a computer controlled fluid delivery system that controls the amount of contrast agent dispensed onto the tissue down to a fraction of a micro liter. The main lumen houses the microendoscope. The microendoscope incorporates a computer-controlled focus mechanism that can quickly and accurately focus while correcting for
hysteresis. This fluorescence confocal micro-laparoscope will be tested in a small-scale clinical trial on women undergoing oophorectomy in the near future.
We previously reported on the development and testing of a multi-spectral confocal microendoscope. Here we present a
new system that will be used during an early stage clinical trial. The new microendoscope is significantly smaller, uses
fewer optical elements, and is structurally more robust. The slit-scanning confocal system employs two synchronized
single-axes scan mirrors and an externally coupled imaging catheter with automated focus control and dye delivery
systems. In grayscale collection mode the confocal microendoscope operates at 30 frames-per-second with 3μm lateral
resolution and 25μm axial resolution. The multi-spectral collection mode operates at 0.5 frames-per-second when
acquiring 32 spectral channels with an average minimum resolvable wavelength difference of 12nm. The system will be
used, in grayscale mode, to image ovaries during a small scale clinical trial on women undergoing oophorectomy.
Recent grayscale and multi-spectral imaging results from ex-vivo human tissues are presented.
The fluorescence confocal microendoscope provides high-resolution, in-vivo imaging of cellular pathology during optical biopsy. There are indications that the examination of human ovaries with this instrument has diagnostic implications for the early detection of ovarian cancer. The purpose of this study was to develop a computer-aided system to facilitate the identification of ovarian cancer from digital images captured with the confocal microendoscope system. To achieve this goal, we modeled the cellular-level structure present in these images as texture and extracted features based on first-order statistics, spatial gray-level dependence matrices, and spatial-frequency content. Selection of the best features for classification was performed using traditional feature selection techniques including stepwise discriminant analysis, forward sequential search, a non-parametric method, principal component analysis, and a heuristic technique that combines the results of these methods. The best set of features selected was used for classification, and performance of various machine classifiers was compared by analyzing the areas under their receiver operating characteristic curves. The results show that it is possible to automatically identify patients with ovarian cancer based on texture features extracted from confocal microendoscope images and that the machine performance is superior to that of the human observer.
A multi-spectral confocal microendoscope (MCME) for in-vivo imaging has been developed. The MCME employs a flexible fiber-optic catheter coupled to a slit-scan confocal microscope with an imaging spectrometer. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The focus mechanism allows for imaging to a maximum tissue depth of 200 microns. The 3mm diameter catheter may be used on its own or routed though the instrument channel of a commercial endoscope. The confocal nature of the system provides optical sectioning with 3 micron lateral resolution and 30 micron axial resolution. The system incorporates two laser sources and is therefore capable of simultaneous acquisition of spectra from multiple dyes using dual excitation. The prism based multi-spectral detection assembly is typically configured to collect 30 spectral samples over the visible range. The spectral sampling rate varies from 4nm/pixel at 490nm to 8nm/pixel at 660nm and the minimum resolvable wavelength difference varies from 8nm to 16nm over the same spectral range. Each of these characteristics are primarily dictated by the dispersion characteristics of the prism. The MCME is designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. The primary applications for the system include diagnosis of disease in the gastro-intestinal tract and female reproductive system. In-vitro, and ex-vivo multi-spectral results are presented.
Patients diagnosed with pancreatic cancer have a 5-year survival rate of only 3%. Endoscopic imaging of the pancreas is limited by the small size of the pancreatic duct, which has an average size of 3 mm. To improve imaging capabilities for the pancreatic duct, two small catheter-based imaging systems have been developed that will fit through the therapeutic channel of a clinical endoscope and into the pancreatic duct. One is a miniature endoscope designed to provide macro-imaging of tissue with both white light reflectance and fluorescence imaging modes. The 1.75 mm diameter catheter consists of separate illumination and imaging channels. At a nominal focal distance of 10 mm, the field of view of the system is ~ 10 mm, and the corresponding in-plane resolution is 60 microns. To complement the broadfield view of the tissue, a confocal microendoscope with 2 micron lateral resolution over a field of view of 450 microns and 25 micron axial resolution has been developed. With an outer diameter of 3 mm, the catheter in this system will also fit through the therapeutic channel and into the pancreatic duct. Images of tissue with both the miniature endoscope and confocal microendoscope are presented.
KEYWORDS: Confocal microscopy, Tissues, Objectives, Fiber optics, Chemical elements, Endoscopy, Natural surfaces, Modulation transfer functions, In vivo imaging, Tissue optics
A fluorescence confocal microendoscope has been developed to provide high resolution, in-vivo imaging of cellular pathology. The microendoscope employs a fiber-optic imaging bundle, a miniature objective, and a miniature focusing mechanism to allow imaging in remote locations of the body. The system uses a 2mm diameter flexible catheter that is capped by a rigid opto-mechanical system measuring 3mm in diameter and 12mm in length. The small size of the confocal microendoscope was chosen so that it may be routed through the therapeutic channel of a clinical endoscope, adding microscopic functionality to conventional endoscopy procedures. The confocal nature of the microendoscope provides optical sectioning with 2 micron lateral resolution and 25 micron axial resolution. The pneumatic focusing mechanism located in the distal opto-mechanical assembly allows for imaging to a maximum depth of 200 micron in the tissue. The system is capable of providing conventional grayscale fluorescence images at 10 frames-per-second as well as spatially resolved multi-spectral fluorescence images at several seconds a frame. Preliminary in-vivo results are be presented.
A confocal micro endoscope has been developed to examine cellular pathology during optical biopsy. The system employs a flexible fiber optic catheter coupled to a slit-scan confocal microscope to image tissue at remote locations in the body. The catheter of the confocal micro endoscope consists of a fiber-optic imaging bundle, a miniature objective, and a miniature focusing mechanism. The system has a lateral resolution of 1.8 micrometers and an axial resolution of 25 micrometers . The confocal micro endoscope can operate in a grayscale or multi-spectral imaging mode. Extensive work has been done to design a new miniature objective and focusing mechanism that will allow the catheter to be routed through the therapeutic channel of a clinical endoscope. We present the design for a miniature F/1 achromatic objective with nearly diffraction limited performance. The objective will be coupled to a pneumatic focusing mechanism to provide focus control to 200 micrometers below the surface of the tissue. The new catheter has an overall diameter of 3mm with a rigid tip of only 20mm in length.
A confocal microendoscope for in vivo imaging at the cellular level has been developed. The system consists of a slit-scanning confocal microscope coupled to a fiber optic imaging bundle with a miniature objective lens and focusing mechanism at the distal tip of the catheter. The system is used primarily for imaging tissue fluorescence--either autofluorescence or fluorescence from an exogenous compound. The microendoscope has a lateral resolution of 2 micrometers allowing visualization of structures at the cellular level, and because it is confocal, it can image at selected depths below the tissue surface. Excellent in vivo imaging results have been obtained in animal models and encouraging ex vivo results have been achieved with human tissue.
We describe fluorescence spectral-imaging results with the computed-tomography imaging spectrometer (CTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, the CTIS can be used to image dynamic phenomena involving multiple, spectrally overlapping fluorescence probes. This system is also optimal for simultaneously monitoring changes in spectral characteristics of multiple probes from different locations within the same sample. This advantage will provide additional information about the physiological changes in function form populations of cells which respond in a heterogeneous manner. The results presented in this paper consist of proof-of-concept imaging results from the CTIS in combination with two different systems of fore- optics. In the first configuration, raw image data were collected using the CTIS coupled to an inverted fluorescence microscope. The second configuration combined the CTIS with a confocal microscope equipped with a fiber-optic imaging bundle, previously for in vivo imaging. Image data were collected at frame rates of 15 frame per second and emission spectra were sample at 10-nm intervals with a minimum of 29 spectral bands. The smallest spatial sampling interval presented in this paper is 0.7 micrometers .
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