In the last quarter century, enface OCT and adaptive optics SLO imaging have exploded the ophthalmoscopic perspective, transporting clinicians through keyholes to new dimensions of retinal visualization, inviting revolutionary advances in diagnosis and therapy. To bridge the clinic - laboratory divide we have developed custom instruments to further survey and probe pathologies captured with commercial instruments, expanding our clinical understanding along with our visualization. These vistas offer a glimpse of microworlds yet to be navigated.
Hyalocytes are resident macrophages residing in the vitreous cortex of the eye. They help maintain optical clarity, manage immunological threats, and respond to vascular insults. Recently, Castanos et al 2020 reported the ability to image these cells using clinical OCT and Hammer et al 2020 demonstrated their appearance using AO OCT. In this study we demonstrate the use of quad detection AO SLO imaging combined with clinical en face OCT in healthy human subjects to observe the dynamic morphological changes and the variable motility of these cells above the retinal surface over extended time intervals.
As techniques of retinal imaging have evolved, anatomic features that were only assessable in the laboratory have become available in the clinic for patient care. The retinal capillaries were initially described on microscope sections in the pathology laboratory. As optical methods have advanced these features have become part of the routine clinical landscape inspected daily by physicians. This paper briefly traces the evolution of these techniques and shows how they fit into the modern diagnostic armamentarium of ophthalmic retinal care.
Two factors are of importance to optical coherence tomography (OCT), resolution and sensitivity. Adaptive optics
improves the resolution of a system by correcting for aberrations causing distortions in the wave-front. Balanced
detection has been used in time domain OCT systems by removing excess photon noise, however it has not been used in
Fourier domain systems, as the cameras used in the spectrometers saturated before excess photon noise becomes a
problem. Advances in camera technology mean that this is no longer the case and balanced detection can now be used to
improve the signal to noise ratio in a Fourier domain (FD) OCT system. An FD-OCT system, enhanced with adaptive
optics, is presented and is used to show the improvement that balanced detection can provide. The signal to noise ratios
of single camera detection and balanced detection are assessed and in-vivo retinal images are acquired to demonstrate
better image quality when using balance detection.
We reported recently an active tracking device based on white light coherence ranging using a spectrally interrogated
Michelson interferometer, which was used to monitor and correct for the axial displacement of the eye and head of the
imaged subject in a confocal scanning ophthalmoscope/ en face OCT system (SLO/OCT) by tracking the axial position
of the eye fundus. Both the tracking and imaging interferometers share the eye interface optics and the patient eye and
also an optical path difference (OPD) changing device in the reference (fast voice coil mounted retroreflector), that keeps
them locked at constant OPD values. As a consequence, the sensitivity of the tracking interferometer is not affected by
the spectrometer sensitivity roll-off with increased OPD and mirror term ambiguity tracking errors close to OPD = 0 are
eliminated. Moreover, the axial tracking range is only limited by the voice coil stage travel range and the tracking system
has an update time better than 5 ms. We investigate the potential of the new configuration for acquiring volumetric data
free of axial eye motion artifacts for two different lateral field sizes. Sets of SLO and en face OCT images at
progressively deeper locations in the retina are simultaneously acquired for two lateral sizes, 15°x15° and 3.5°x3.5°. The
large lateral field size provides a means of navigating the retina, while the high magnification small lateral size imaging
reveals interesting microscopic details of the retinal morphology.
We report an active tracking device based on white light coherence ranging using a spectrally interrogated Michelson
interferometer, which is used to monitor and correct for the axial displacement of the eye and head of the subject in a
confocal scanning ophthalmoscope/ en face OCT system (SLO/OCT). The Nyquist limit range of the spectrometer in the
tracking interferometer is ~5.4 mm, which is adequate for monitoring the axial position of axially extended layered
objects like the human eye fundus. Both the tracking and imaging interferometers share the eye interface optics and the
sample and also an optical path (OPD) changing device in the reference (fast voice coil mounted retroreflector), that
keeps them locked at constant OPD values. As a consequence, the sensitivity of the tracking interferometer is not
affected by the spectrometer sensitivity roll-off with increased OPD and mirror term ambiguity tracking errors close to
OPD = 0 are eliminated. Moreover, the axial tracking range is only limited by the voice coil stage travel range. A real
time data acquisition processor board is used to digitize the spectrometer signal and calculate the correction signal
applied to the voice coil with an update time better than 5 ms. We demonstrate axial motion corrected combined
confocal/ en face OCT imaging of the human eye fundus in vivo.
KEYWORDS: Optical coherence tomography, Spectroscopy, Signal detection, Photodetectors, Signal to noise ratio, Sensors, Diffraction gratings, Signal processing, Optical spectroscopy, Photodiodes
A detection method based on 7 spectral windows at the photodetection stage in an optical coherence tomography system was tested. We investigated its utility in two directions: spectroscopic optical coherence tomography and signal to noise ratio improvement. A diffraction grating was used in the photodetection unit to diffract light over a 16 photodetector array. Currently, this array has been configured to deliver 7 channels only by binning two adjacent photodetectors. The improvement of the signal to noise ratio has been investigated. Preliminary results of spectroscopy analysis which will be shown are B-scan OCT imaging of paint layers. The ultimate aim of the project is to perform spectroscopic analysis of the retina.
A new approach of acquiring quasi-simultaneous optical coherence tomography (OCT) and confocal images is presented. The two images are generated using different principles, OCT and confocal microscopy. When the system is used to image the retina, the two images have depth resolutions, at present, of <20 µm and ~1 mm, respectively. The acquisition and display of en face OCT and confocal images are quasi-simultaneous, without the need of a beamsplitter. By using a chopper to periodically obstruct the reference beam in the OCT interferometer, synchronized with the XY-transversal scanner, much higher acquisition speed is obtained than in a previous report where we flipped an opaque screen in the reference arm of the interferometer. Successful operation of the novel configuration was achieved by: (1) stable synchronization of the chopper's movement with the horizontal line scanner and (2) fast self-adjusting of the gain value of avalanche photodiodes, depending on the optical power. Images from coin, leaves, and retina in vivo have been collected to demonstrate the functionality of the system.
A new approach of acquiring quasi-simultaneous OCT and confocal images is presented. The two images are generated
using different principles, optical coherence tomography (OCT) and confocal microscopy (CM). When the system is
used to image the retina, the two images have depth resolutions, at present, of less than 20 μm and approximately 1 mm
respectively. The acquisition and display of en-face OCT and confocal images are quasi-simultaneous, without the need
of a beam splitter. By using a chopper to periodically obstruct the reference beam in the OCT interferometer,
synchronized with the XY-transversal scanner, much higher acquisition speed is obtained than in a previous report where
we flipped an opaque screen in the reference arm of the interferometer. Successful operation of the novel configuration
was achieved by: (1) stable synchronization of the chopper's movement with the horizontal line scanner and (2) fast self-adjusting
of the gain value of avalanche photodiodes depending on the optical power. Images from coin, leafs and retina in vivo have been collected to demonstrate the functionality of the system.
KEYWORDS: Laser tissue interaction, Laser welding, Tissues, In vivo imaging, Skin, Animal model studies, Near infrared, Laser energy, Collagen, Absorption
Near infrared laser tissue welding (LTW) is achieved by subjecting the closely approximated surgically incised tissues to
a laser beam at a wavelength that is absorbed by water in the tissue. Full thickness welds are accomplished with
optimum laser power and penetration depths appropriate for the thickness of welded tissues. No extrinsic cross-linking
or bonding materials are used. The absorbed laser energy increases the entropy of collagen in the tissue. In LTW, tissue
water temperatures transiently rises to approximately 60° C, causing partial denaturing of collagen and other structural
proteins due to breaking of hydrogen bonds, electrostatic interactions and some interchain covalent bonds for a short
duration of time. This is followed by cross linking of proteins on either side of weld line, with reformation of the above
mentioned bonds as the tissue cools, resulting in the formation of water tight full thickness welds. In this study, a cw
fiber laser emitting at 1455 nm, corresponding to absorption by a water vibrational overtone, is used for in vivo LTW of
surgical incisions made in the skin of guinea pigs under general anesthesia. The tensile strength and healing rates of the
welded incisions are compared to suturing of similar incisions. Laser parameters, including power, scanning rates,
exposure area, and exposure duration, are optimized to reduce thermal damage while maintaining tensile strength.
A comparative analysis on the performance of different scanning regimes in time domain optical coherence tomography
is presented in terms of image size. Safety thresholds due to the different continuous irradiation time per transverse pixel
in different scanning regimes are also considered. We present the maximum exposure level for a variety of scanning
procedures, employing either A scanning (depth priority) or T scanning (transverse priority) when generating cross
section images, en-face images or collecting 3D volumes. We present a comparison between such B-scan images, and
different criteria to allow the user to choose the right mode of operation. Mainly, two criteria are detailed, a scanning
criterion and a safety criterion. The scanning criterion depends on the number of pixels along the lateral and axial
directions. The analysis shows that en-face scanning allows wider images while the longitudinal scanning is more
suitable to deep cross sections. The safety criterion refers to safety levels to be observed in each scanning mode. We
show that the flying spot OCT imaging has different safety limits for T- and A- based imaging modes. The analysis leads
to maximum permissible optical power levels that favors T-scan imaging of wide objects. We then apply the analysis
considering as object the eye.
A review is presented of the research on high resolution imaging of the eye based on en-face OCT. This can provide a
dual display of images with different depth resolutions, where the two images are OCT and the other confocal. Two
applications are presented: (i) OCT/ICG systems where the confocal channel is tuned to the fluorescence of indocyanine
green and (ii) aberration corrections in both OCT and confocal channels using closed loop adaptive optics for enhanced
contrast and transversal resolution.
The 1020-1080 nm spectral region appears as a viable alternative to the 700-900 nm spectral band for in vivo eye fundus
OCT imaging due to a local absorption minimum of water (main constituent of the eye aqueous and vitreous). Light at
these wavelengths also experiences less attenuation due to lower scattering and absorption by melanin in the retinal
pigment epithelium and choroid, which results in deeper penetration of the probe beam in the choroid. T-scan based en
face OCT is a modification of the OCT technique that has the unique capability of acquiring both longitudinal (B-scans)
and tranversal (C-scans) OCT images of the eye fundus in real time and allows the addition of a confocal scanning
ophthalmoscope channel to the OCT instrument. We report for the first time a combined T-scan based en face OCT and
confocal scanning opthalmoscopy system for imaging the human eye fundus in vivo in the 1050 nm region. The
instrument allows the visualization of choroidal blood vessels in both the confocal and OCT channels without the use of
contrast agents such as indocyanine green (ICG) dye and could prove an alternative tool for diagnosing eye conditions
like age related macular degeneration that are preceded by choroidal neovascularisation.
We develop a dual-channel optical coherence tomography/indocyanine green (OCT/ICG) fluorescence system based on our previously reported ophthalmic OCT/confocal imaging system. The confocal channel is tuned to the fluorescence wavelength range of the ICG dye and light from the same optical source is used to generate the OCT image and to excite the ICG fluorescence. The system enables the clinician to visualize simultaneously en face OCT slices and corresponding ICG angiograms of the ocular fundus, displayed side by side. C-scan (constant depth) and B-scan (cross section) images are collected by fast en face scanning (T-scan). The pixel-to-pixel correspondence between the OCT and angiography images enables the user to precisely capture OCT B-scans at selected points on the ICG confocal images.
In this study, a NIR erbium fiber laser tuned to a water vibrational overtone absorption band at 1455 nm was used to weld directly, in vitro, seventy-six porcine aorta tissues without the need for extrinsic solder materials. The tissues were divided into eleven groups based on the multiple and variable parameters that were used to weld the tissues. The effectiveness of the parameters used in each of the weld groups was evaluated directly at the time of the weld and also by tensile strength measurements done at the termination of the weld. Management of heat produced in tissues is of critical importance for good laser tissue welding (LTW). To address heat
management issues, we report LTW using a transparent cover over the tissue specimen as a heat sink. Multiple scanning helps distribute the laser-generated heat and allows the tissue to cool between scans, reducing thermal damage. Better heat management using a transparent cover slide enhances the welding success. It reduces collateral damage and limits water evaporation and control the buckling of tissue around the line of apposition so that the two pieces that are welded do not move apart along the line of apposition due to buckling pressure and ensure a full-length weld.
The authors report preliminary clinical results using an unique instrument which acquires and displays simultaneously an OCT image, a confocal image similar to that of a scanning laser ophthalmoscope and an indocyanine green fluorescence image. The three images are produced by three channels, an OCT and a confocal channel operating at 793 nm and a confocal channel tuned on the ICG fluorescence spectrum, which peaks at 835 nm. The system is based on our previously described ophthalmic Optical Coherence Tomography (OCT)/confocal imaging system, where the same source is used to produce the OCT image and excite fluorescence in the ICG dye. The system is compact and assembled on a chin rest and it enables the clinician to visualise the same area of the eye fundus in terms of both en-face OCT slices and ICG angiograms, displayed at the same time. The images are collected by fast T-scanning (en-face) which are then used to build B-scan or C-scan images.
We report a versatile imaging system combining scanning laser ophthalmoscopy (SLO) and T-scan based en face ultrahigh resolution optical coherence tomography (OCT). The image carrier is generated using the optical path difference modulation introduced by the X-Y galvo-scanner mirrors specific to en face OCT (without optical modulators in the reference arm). The light source is a compact superluminescent diode based source with 150 nm FWHM spectrum, centered at 890 nm. We demonstrate en face B-scan and C-scan ultrahigh resolution OCT imaging of the human retina in vivo, with an axial resolution of 3.2 μm in tissue. The system is capable of acquiring large lateral size ultrahigh resolution OCT scans of a maximum field size of 20°. The acquisition speed is up to 2 frames/s for both OCT B-scans and C-scans. The measured system sensitivity is more than 98 dB, for a power level to the target of 1 mW and maximum lateral scan size. The C-scans are, to the best of our knowledge, the first and the largest size reported ultrahigh resolution C-scans of the human retina in vivo. The instrument is assembled on a chin rest and ready to be used for clinical imaging. SLO and ultrahigh resolution OCT C-scans are acquired simultaneously and displayed side by side. This allows users in a clinical environment to correlate details of the same feature in the area of interest in both images and also choose precisely in the SLO image the location where to perform the ultrahigh resolution en face B-scan.
Monte Carlo simulations were performed to delineate the role of local fluence rates and absorption in histologic success and tensile strength analysis of laser welding of ocular corneal tissue using an erbium fiber laser system operating at 1455nm wavelength. Porcine cornea was used for in vitro welding, while varying power, scan time, and irradiance. Immediate histologic analysis was performed, as well as tensile strength studies. Simulations were performed using MCML code, with a total of 109 photons started. CONV code was used to convolve the output from MCML for a flat photon beam of 80-800 μ focal spot size and power specified by the experiment. The absorption coefficient, μa, was assumed to reflect that of water, 28.6 cm-1. The scattering coefficient, μs, and anisotropy factor, g, were both neglected due to the poor scattering capabilities of water in the wavelength of the laser beam. Fluence rates were determined and were within 0.3%-4% of surface dose calculations for a beam diameter of 80 μ. Interactive Data Language (IDL) was used to sum the dose for one convolved beam to an experiment with multiple scans across the porcine cornea. Achieving optimal usage of the laser system requires maximal use of the variables (power, scan patterns, scan time, irradiance) available to use, and the correlation between Monte Carlo-aided dosimetry and the histopathological and tensile strength studies was performed. Optimal parameters for use in this 1455 nm laser system can be studied, and will allow users the ability to predict histology scores of welding success and tissue injury based on absorption values. These results can refine our experience with laser tissue welding of porcine cornea and aid in determining optimal delivered dose for successful tissue apposition and minimal adverse thermal heating.
Laser skin welding (LSW) is being pursued for scarless wound healing. We present a new LSW approach using a contact glass slide over the sample and rapid scanning of the laser beam around the area to be welded. This led to dramatic improvement in welding efficacy. A 400 mW beam at 1455 nm with a focused spot diameter of 80 μm in air was scanned at a rate of 5mm/second over a 5mm line of incision in 5 mm x 20 mm human skin samples. Histological analysis of the welded samples using hematoxyline and eosin under unpolarized light showed full-thickness full-length weld, and that with picrosirius red F3BA stain under polarized light revealed that there was no appreciable damage. Measured tensile strength of 2.1 kg/cm2 is markedly greater than our previous LSW results of 1.05 ± 0.19 kg/cm2, which is greater than the typical values of 0.4 kg/cm2 obtained using sutures.
In this study, 72 different combinations of laser welding parameters were compared for their effectiveness in welding ocular tissue. The laser employed in the welding system was a near infrared (NIR) erbium fiber laser with a wavelength of 1.455 μm . The laser system used a motorized translational stage and shutter to control the laser exposure of the tissue being welded. The emission wavelength of the laser in the NIR range corresponds to one of the lesser absorption bands of water. Parameters of the laser welding system that could be changed to allow a more effective distribution of the laser energy and therefore management of thermal energy included: the number and kinds of intricate offset patterns of light on or around the incision, the number of lines per pattern, the power level, the speed of the laser beam movement over the tissues, the spot size, dwell time and the focus plane of the light beam in the tissue. Histopathology was used as an endpoint indication of the effects that the various sets of welding parameters had on the welded tissues. Standard Hematoxylin and Eosin stain and Sirius Red F3B (Direct Red 80) in combination with polarization microscopy were used to stain and visualize the welded ocular tissue. Paradoxically, the best cornea welds quantified using histopathology occurred with fluence of 4,500 mJ/cm2 or less while the corneal welds exhibiting the strongest tensile strengths, but most tissue damage had a delivered fluence above 7,000 mJ/cm2. The best histological representatives of welded corneas had an average delivered fluence of 2,687 mJ/cm2 and an irradiance of 14 W/cm2. Using the properly determined parameters, the NIR erbium fiber welding system provided full thickness welds without the requirement of extrinsic dyes, chromophores, or solders. The NIR laser system with the appropriately developed parameters can be used effectively to weld ocular tissues.
The authors report preliminary clinical results using a unique instrument which acquires and displays simultaneously an Optical Coherence Tomography (OCT) and an indocyanine green fluorescence image (ICGF). The two images are produced by two channels, an OCT and a confocal channel tuned to the ICG fluorescence spectrum. The system is based on our previously described ophthalmic OCT/confocal imaging system, where the same source is used to produce the OCT image and excite fluorescence in the ICG dye. The system is compact and assembled on a chin rest and it enables the clinician to visualise the same area of the eye fundus in terms of both en-face OCT slices and ICG angiograms, displayed side by side. The images are collected by fast T-scanning (en-face) which are then used to build B-scan or C-scan images. We present images of a variety of choroidal neovascular membranes, and lesions suspected of harboring choroidal neovascular membranes.
Two and three SLD diodes are grouped together in order to obtain a compounded source of wide band for OCT investigations. One SLD has a two-lobe spectrum around 840 nm and a compounded spectrum is achieved by using SLDs of smaller wavelengths. This has two advantages: (1) the smaller the wavelength the lower the loss of power through the vitreous due to water absorption; (2) medium band, standard SLDs if used bring a more significant reduction to the coherence length than their counterparts, of medium band but centered at longer wavelength. We show that it is possible to obtain high resolution OCT images with an inexpensive, compact, and easy to operate source.
By dividing both the object and reference beam in an OCT interferometer, two independent OCT imaging channels are assembled. The depth scanning proceeds simultaneously in the two OCT channels and from the same range, however a differential optical path difference can be introduced between the two channels. In this way, two simultaneous images are generated where the depth differs in each pixel by the differential optical path difference. A dual OCT system working at 850 nm was devised and we demonstrate the capability of the method by simultaneously acquiring images from the optic nerve and fovea of a volunteer. The configuration devised insures a strict pixel to pixel correspondence between the two images irrespective of the axial eye movements while the depth difference between the corresponding pixels is exactly the differential optical path difference. The images are collected by fast en-face scanning (T-scan) which allows both B-scan and C-scan acquisition.
We report a novel imaging procedure and a system implementing it, capable of acquiring two quasi-simultaneous en-face images from a multi-layer target using a single mode fiber optic interferometer. Two optical sources are employed, which are toggled on and off simultaneously with the ramp signal applied to the scanner whose movement determines the line in the raster image. In this way, half of the line in the final frame is generated by a particular source only. Imaging with two different depth resolutions or with two different wavelengths is made possible in this way. Because the switching is much faster than the observer eye can follow, the two images can be interpreted and compared simultaneously. The paper discusses the different functionality of the system depending whether the OCT image is made out of A or T-scans.
En-face OCT acquired simultaneously with paired confocal ophthalmoscopic (CO) images provides unprecedented point-to-point correlation between surface and subsurface anatomy of the retina. An advanced prototype of a dual channel OCT/CO instrument was developed in terms of signal to noise ratio and image size. The system can operate in A, B and C-scan regimes. The design is such that there is a strict pixel to pixel correspondence between the OCT and confocal images. An extensive array of clinic pathologies were studied including macular degeneration, central serous retinopathy (CSR), macular hole, macular pucker, cystoid macular edema (CME), diabetic maculopathy, and macular trauma. We report observation of reoccurring patterns in the en-face OCT images which could be identified with different diseases. The system can also simultaneously produce en-face OCT and indocyanine green (ICG) fluorescence images where the same source is used to produce the OCT image and excite the ICG. The system is compact and assembled on a chin rest and it enables the clinician to visualise the same area of the eye fundus in terms of both en face OCT slices and ICG angiograms, displayed side by side. The images are collected by fast en-face scanning (T-scan) followed by slower scanning along a transverse direction and depth scanning. The system is capable of providing chosen OCT B-scans at selected points from the ICG image.
KEYWORDS: Polarization, Optical coherence tomography, Birefringence, Single mode fibers, Tissue optics, Reflectivity, In vivo imaging, Signal detection, Mirrors, Tissues
Polarization sensitive optical coherence tomography (PS-OCT) takes into account the vector nature of light waves (state of polarization). The most complete information about the polarization properties of a biological target is given by the depth resolved Mueller matrix elements, however, it is difficult to construct such a system for in-vivo examination. We designed and assembled a simpler system, with two incoherent channels to provide limited information, but essential on the polarization properties of the tissue. The interferometer is a hybrid configuration of bulk optic and single mode optical fiber components. No polarization maintaining fiber is used. The reference and sample beams interfere in single mode optical couplers. The low coherence light source is a superluminescent diode of 850 nm center wavelength and 25 nm spectrum FWHM (which corresponds to a depth resolution of 12 microns in tissue). The system can display either a pair of two polarisation sensitive OCT images, corresponding to linear orthogonal polarisation directions or a pair of images, a polarisation insensitive (pure reflectivity) image and a birefringence retardation map. The 12 bit grayscale images are collected by fast en-face scanning (T-scan) at 2 frames/s. We demonstrate in vivo en face images of the retinal nerve fiber layer, lamina cribrosa, cornea and teeth. A rotation angle of 0.3 degrees per micron was evaluated from the retinal nerve fiber layer and lamina cribrosa.
KEYWORDS: Tissues, Laser tissue interaction, Cornea, Sclera, Laser welding, Near infrared, Eye, In vitro testing, Laser systems engineering, Control systems
In this study, an NIR fiber laser with an eye safe wavelength of 1.455 μm was used to successfully weld in vitro porcine cornea and sclera tissue. The emission wavelength overlaps an absorption band of water. The laser system was used in combination with a motorized translational system and shutter to control the laser exposure on the tissue being welded. Different welding conditions were analyzed for the porcine cornea and sclera. The welded tissues were examined using histopathology and tensile strength analysis. The NIR welding technique provides strong, full thickness welds and does not require the use of extrinsic dyes, chromophores, or solders. The NIR laser system used in this study can effectively weld cornea and sclera tissue, and this laser tissue welding (LTW) methodology typically causes minimal disruption of tissue, and thus, avoids opacities and irregularities in the tissue which may result in decreased visual acuity.
The authors report the construction of a dual channel OCT/indocyanine green (ICG) fluorescence system with the optical source common to both the OCT and fluorescence channels based on our previously described ophthalmic Optical Coherence Tomography (OCT)/confocal imaging system. The confocal channel is tuned to the fluorescence wavelength range of ICG dye. The system is compact and assembled on a chin rest and it enables the clinician to visualise the same area of the eye fundus in terms of both en face OCT slices and ICG angiograms, displayed side by side. The images are collected by fast en-face scanning (C-scan) followed by slower scanning along a transverse direction and depth scanning. We demonstrate the first such dual OCT/ICG-fluorescence images from healthy eyes. The system is still capable of providing chosen OCT B-scans at selected points from the ICG confocal image, in the same way the OCT/confocal configuration was used.
We have constructed a compact set-up based on incoherent detection in two optical channels to provide real time polarisation sensitive OCT imaging. The system can display either a pair of two polarisation sensitive OCT images, corresponding to linear orthogonal polarisation directions or a pair of images, a polarisation insensitive (pure reflectivity) image and a birefringence retardation map. The images are collected by fast en-face scanning (T-scan) followed by slower scanning along a rectangular transverse direction and depth scanning. B-scan and C-scan images from in-vivo retina and optic nerve are presented.
Using an advanced prototype of en-face OCT/cSLO instrument, an extensive array of clinic pathologies were studied including macular degeneration, central serous retinopathy (CSR), macular hole, macular pucker, cystoid macular edema (CME), diabetic maculopathy, and macular trauma. We report observation of reoccurring patterns in the en-face OCT images which could be identified with different diseases. Uniquely specific and reoccurring patterns could be characterized for macular hole ("Chrysanthemum flower"), CME ("Swiss cheese wheel"), Macular Pucker ("Star"), CSR ("Target") and RPE Detachment ("Ring of Light"). Other entities such as polypoidal choroidopathy and diabetic edema residues had easily recognizable features but were variable enough to defy specific descriptive comparison. To facilitate the interpretation of the en-face OCT images, a three dimensional interactive simulation was designed which allows the demonstration of characteristic features and artifacts encountered in the acquisition of transverse images.
The time-resolved and steady-state changes in fluorescence were investigated from one spore-forming (Bacillus subtilis) and four non-spore forming (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa) bacteria subjected to different bactericidal agents. The bactericidal agents were sodium hypochlorite (bleach) hydrogen peroxide, formaldehyde, and UV light exposure. Application of sodium hypochlorite resulted in an almost total lose of fluorescence signal and large decrease in the optical density of the bacterial suspension. Addition of hydrogen peroxide resulted in a 35% decrease in emission intensity fom the Sa and an 85-95% decrease for the other bacteria. Ultraviolet light exposure resulted in a 5-35% decrease in the emission intensity of the tryptophan band. The addition of formaldehyde to the bacteria did not result in significant changes in the steady-state emission intensity, but did shift the tryptophan emission peak position to shorter wavelengths by 3 to 5 nm. Time-resolved fluorescence measurements showed that the fluorescence lifetime of tryptophan in the bacteria could not be described by a single exponential decay, and was similar to that of tryptophan in neutral aqueous solution. Upon addition of formaldehyde to the Gram positive bacteria (Bs and Sa) the strength of the short lifetime component increased dramatically, while for the Gram negative bacteria, a smaller increase was observed. These fluorescence changes reflect the different mechanisms of the bactericidal agents and may provide a useful tool to monitor the effectiveness of disinfectants.
A review is presented of the developments in Kent in the field of optical coherence tomography (OCT) based instrumentation. Original versatile imaging systems have been devised which allow operation in different regimes under software control. Using such systems, B-scan and C-scan images are demonstrated from retina, anterior chamber, skin and teeth. The systems developed in Kent employ the flying spot concept, i.e. they use en-face scanning of the beam across the target. This has opened the possibility of providing simultaneous en-face OCT and confocal images (C-scans). Application of a standalone OCT/confocal system for investigating the retina in eyes with pathology, the anterior chamber, skin and teeth is demonstrated.
We report the first (to the best of out knowledge) en face polarization sensitive optical coherence tomography (PS-OCT) system. The transverse raster scanning of the target is achieved using a pair of galvo-scanner mirrors. The set-up is based on incoherent detection in two optical and electronic channels and employs balanced detection to reduce the excess photon noise generated by the low coherence source (superluminescent diode). The outputs of the two channels are processed using software to provide a polarisation insensitive (pure reflectivity) image and a birefringence retardation map. Images from ex vivo (human tooth) and in vivo targets (human retina) have been acquired. Particulars of en face optical coherence tomography imaging of birefringent tissue are discussed.
Native fluorescence emission and excitation spectra were used to monitor changes in Bacillus subtilis (Bs) and Staphylococcus aureus (Sa) subjected to starvation conditions. Initially, the fluorescence spectra from the Bs and Sa was dominated by tryptophan emission. After the second day, a fluorescence band with an emission peak at 410 nm and an excitation peak at 345 nm appeared in the Bs. This emission is from dipicolinic acid, a major constituent of bacterial endospores. The dipicolinic acid intensity increased steadily during the next 2 to 4 days as the number of Bs forming spores increased. No dipicolinic acid signal was observed in the (non-spore forming) Sa. The addition of β-hydroxybutyric acid to either the Bs or Sa
resulted in the emergence of a third band with very strong fluorescence emission maximum at 460 nm and with excitation maxima at 250, 270 and 400 nm. This 460 nm emission was quenched with the addition of Fe2+, indicating that the source of this emission is a siderophore produced by the bacteria.
Optical transmission and light scattering measurements were performed on three species of bacteria with different sizes and shapes (Pseudomonas aeruginosa. Staphylococcus aureus and Bacillus subtilis). The average bacteria size was determined from transmission measurements by using the Gaussian Ray Approximation of Anomalous Diffraction Theory. A rescaled spectra combining multiple angular data was analyzed in the framework of the Rayleigh-Gans theory of light scattering in order to determine particle shape and size distribution. Particle size and shape as determined by both
methods are in good agreement with size and shape measured by scanning
electron microscopy. These results demonstrate that light scattering may be able to detect and identify microbial contamination in the environment.
This paper demonstrates the clinical application of a multiplanar imaging system, which simultaneously acquires en-face (C-scan) OCT and corresponding confocal ophthalmoscopic images along with cross-sectional (B-scan) OCT at cursor designated locations on the confocal image. Advantages of the simultaneous OCT/confocal acquisition as well as the challenges of interpreting the C-scan OCT images are discussed. Variations in tissue inclination with respect to th coherence wave surface alters the sampling of structures within the depth in the retina, producing novel slice orientations which are often challenging to interpret. We evaluate for the first time the utility of C-scan OCT for a variety of pathologies including exudative ARMD, macular hole, central serous retinopathy, diabetic retinopathy, polypoidal choroidal vasculopathy and macular pucker. Several remarkable observations of new aspects of clinical anatomy were noted. The versatility of selective capture of C-scan OCT images and B-scan OCT images at precise points on the confocal image affords the clinician a more complete and interactive tool for 3D imaging of retinal pathology.
En-face optical coherence tomography (OCT) technology is employed to produce high resolution images from the retina, cornea, skin and teeth. Longitudinal (B-scan) and transversal (C-scan) images are demonstrated using en-face scanning method. The main advantage of the en-face imaging is that the C-scan images permit a straightforward comparison with the images produced by confocal microscopy. Other developments are also presented as the generation of 3D imaging of different tissue using stacks of en- face OCT images collected at different depths.
We investigate the effect of light source coherence length on longitudinal and en-face OCT images of the retina. The sources used in this comparative study are a superluminescent diode (SLD), a superliminescent diode fitted with an interference filter at its output and a tunable coherence length three-electrode laser device (3EL). We show that the use of sources of shortest coherence length is ideal for longitudinal OCT imaging. However, there are reasons for using adjustable coherence length sources for en-face OCT imaging. The effect of adjustable coherence length (and implicitly spectrum FWHM) on the achievable signal to noise ratio in the Oct is also presented. An increase in the coherence length enhances the excess photon noise but, at the same time, increases the signal collected from scattering tissue due to a larger thickness of the coherence gated backscattering layer in the target tissue. This suggests that the signal to noise ratio should not change with the light source coherence length. Nevertheless, the effect of light source coherence length change on the signal to noise ratio is more complex due to other noise sources in the system.
In this work, Raman spectroscopy was investigated as a tool for monitoring glutamate levels in the eye. Glutamate is a by product of nerve cell death, and is an indicator of macular degeneration. Raman spectra was from ex vivo porcine eyes was investigated, with glutamate injected into the eyes to simulate disease conditions. The Raman spectra from the native eye was dominated by the lens. However, an optical system was designed to optimize collection of signal from the vitreous and reduce the background lens signal. The molecular signature of glutamate was detectable in the Raman spectra by this system.
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