Microscope-integrated optical coherence tomography (MIOCT) is an emerging multimodal imaging technology in which live volumetric OCT (“4D-OCT”) is displayed simultaneous with standard stereo color microscopy. 4D-OCT provides ophthalmic surgeons with many visual cues not available in microscopy, but it cannot serve as a replacement due to lack of color features. In this work, we demonstrate progress toward a unified solution by fusion of data from both modalities, guided by segmented 3D features, yielding a more efficient visualization combining important cues from both modalities.
Microscope-integrated optical coherence tomography (MIOCT) is an emerging multimodal imaging technology in which live volumetric OCT (“4D-OCT”) is displayed simultaneously with standard stereo color microscopy. 4D-OCT provides ophthalmic surgeons with many visual cues not available in microscopy, but it cannot serve as a replacement due to lack of color features. In this work, we demonstrate progress toward a unified solution by fusion of data from both modalities, guided by segmented 3D features, yielding a more efficient visualization combining important cues from both modalities.
Ophthalmic microsurgery involves the manipulation of thin, semi-transparent structures and has traditionally been performed using stereoscopic microscopes that provide an en face view of the surgical field. However, new therapeutic interventions such as subretinal injections require precise tool placement and dosing that are difficult to determine from the traditional microscope view. Optical coherence tomography (OCT) provides micron scale cross-sectional imaging and has become a gold standard in clinical ophthalmology settings, but its use in surgery has been more limited. The high-speed 400 kHz intraoperative system presented here provides valuable image guidance and quantitative metrics for a variety of human surgeries.
Microscope-integrated optical coherence tomography (MIOCT) systems allow for 4D visualization of thin,
semi-transparent structures during ophthalmic microsurgery. While these visualizations have greatly
increased the information available to surgeons, new surgical techniques, such as subretinal injections of
gene therapies, would benefit from quantitative measurements of structures imaged using OCT. Calibration
of true scan dimensions in OCT is complicated by inter-patient ocular variability as well as poor control of
scanner-patient alignment. We introduce novel measurement techniques for calibration and de-warping of
OCT imagery which allows for accurate measurement of intraocular structures including subretinal
microinjection bleb volumes in the ocular posterior segment.
Ophthalmic surgery is typically performed through an en-face only surgical microscope that provides limited depth information. This work introduces a high speed (400 kHz) microscope integrated optical coherence tomography (MIOCT) system which provides real time volumetric “4D” visualization via a heads-up stereoscopic display. The MIOCT system provides sub retinal visualization of tools and enables surgeons to perform delicate manipulation of retinal structures during mock surgical procedures. Following these mock surgical procedures in porcine eyes, this system will be readily translated into human ophthalmic microsurgery.
Through biomedical engineering partnerships, our group has been able to develop and translate optical coherence tomography (OCT), an imaging tool conventionally used in the “eye imaging suite”, for bedside use in unstable infants or for intraoperative guidance for the ophthalmic surgeon. In both arenas, image-guidance is transforming patient care. In the nursery, this has enabled assessment of retinal development, markers of injury and a determination of their relationship to brain function. In surgery, efficient application of an investigational high-speed swept-source OCT into our surgical armamentarium provides unique guidance in simple and complex cases, is improving our understanding of microsurgical pathologies, and is enabling new and improved surgical techniques.
Optical coherence tomography angiography (OCTA) is an extension of OCTA that allows for non-invasive imaging of the retinal microvasculature. OCTA imaging of adult retinal diseases is area of active research in ophthalmology as OCTA can provide insight into the pathogenesis of many retinal diseases. Like these adult diseases, pediatric diseases such as retinopathy of prematurity (ROP) have a primarily vascular pathogenesis. However, table top OCTA systems require compliant, seated subjects and cannot be used on infants and young children. In this manuscript we describe the development of a non-contact handheld OCTA (HH-OCTA) probe for imaging of young children and infants in the operating room. The probe utilizes a novel, diverging light on the scanner optical design that provides improved performance over a traditional OCT scanner design. While most handheld OCT probes are designed to be held by the side of the case or by a handle, our operators tend to prefer to grip probes by the tip of the probe for supine imagine. The ergonomics of the HH-OCTA probe were designed to match this grip. The HH-OCTA probe used a 200 kHz OCT engine, has a motorized stage that provides +10 to -10 D refractive error correction, and weighs 700g. Initial OCTA imaging was performed in 9 children or infants during exam under anesthesia. The HH-OCTA images provide visualization of the retinal microvasculature in both normal and pathological eyes.
OCT is the gold standard for clinical diagnosis and treatment of many retinal diseases. Most clinical OCT systems are table top systems that can only image seated, compliant patients that can fixate. These systems are incapable of imaging several important patient populations including bedridden patients and infants. In this work we describe the use of a custom, light weight, handheld OCT probe based on a high speed swept source engine for imaging in the intensive care nursery. The probe uses custom optics, optomechanics, and a MEMS mirror to achieve a weight of only 211g. The portability and imaging speed of this probe facilitates repeat, volumetric, bedside imaging in a challenging imaging environment. To date we have imaged over 43 pre-term and full-term infants in the intensive care nursery, with some patients having up to 15 imaging sessions starting at 30 weeks post menstrual age. Volumetric OCT enables visualization of the complex 3D structures associated with retinal pathology that is unavailable to slower, B-scan based probes. Repeat imaging shows the development of both normal and diseased retinal structures. We believe that OCT imaging of these infants will reveal retinal abnormalities, enable further study of pediatric retinal diseases, and allow for better management and prediction of future visual outcomes.
Optical coherence tomography (OCT) allows for micron scale imaging of the human retina and cornea. Previous research and commercial intraoperative OCT prototypes have been limited to live B-scan imaging because they were based on previous-generation spectral domain OCT systems. Our group has developed and reported on an intraoperative microscope integrated OCT system based on a 100 kHz commercial swept source laser. This system is capable of live 4D imaging, and with a heads up display allows for dynamic intraoperative visualization of retinal structures, tool tissue interaction, and surgical maneuvers. OCT angiography (OCTA) is an emerging OCT technology that allows for imaging of retinal vasculature without the use of potentially harmful contrast agents. This structural information can provide insights into the state and development of a wide range of ophthalmic pathologies. The addition of OCTA into intraoperative OCT could allow for monitoring of changes in retinal vasculature during surgery and imaging of traditionally non-compliant patients. In this work we provide a brief update of intraoperative 4D MIOCT across a range of pathologies, and demonstrate intraoperative OCTA for the first time. To the best of knowledge, this is the first report of intraoperative OCTA, as well as the first OCTA images ever acquired in an infant.
The en face operating stereomicroscope offers limited depth perception and ophthalmic surgeons must often rely on stereopsis and instrument shadowing to estimate motion in the axial dimension. Recent research and commercial microscope-integrated optical coherence tomography (MIOCT) systems have allowed OCT of live surgery, but these were restricted to real-time cross-sectional (B-scan) imaging which captures limited information about maneuvers that extend over 3D space. We recently reported on a four dimensional (4D: 3D imaging over time) MIOCT and HUD system with real-time volumetric rendering for human ophthalmic surgery, but this 100 kHz OCT system was restricted to 3.3 volumes/sec to achieve sufficient lateral sampling over a 5x5 mm field of view (FOV). In this work, we present a high-speed 4D MIOCT (HS 4D MIOCT) system for volumetric imaging at 800 kHz A-scan rate. The proposed system employs a temporal spectral splitting (TSS) technique in which the spectrum of a buffered 400 kHz OCT system is windowed into sub-spectra to yield A-scans with reduced axial resolution but at a doubled A-scan rate of 800 kHz. The trade-offs of TSS for B-scan and volumetric retinal imaging were characterized in healthy adult volunteers. In addition, porcine eye surgical manipulations were imaged with HS 4D MIOCT imaging at 10.85 volumes/sec with 400x96x340 (X,Y,Z) usable voxels over a 5x5 mm lateral FOV. HS 4D MIOCT was capable of imaging subtle volumetric tissue manipulations with high temporal and spatial resolution using ANSI-limited optical power and is readily translatable to the human operating suite.
Optical coherence tomography (OCT) allows for micron scale imaging of the human retina and cornea. Current generation research and commercial intrasurgical OCT prototypes are limited to live B-scan imaging. Our group has developed an intraoperative microscope integrated OCT system capable of live 4D imaging. With a heads up display (HUD) 4D imaging allows for dynamic intrasurgical visualization of tool tissue interaction and surgical maneuvers. Currently our system relies on operator based manual tracking to correct for patient motion and motion caused by the surgeon, to track the surgical tool, and to display the correct B-scan to display on the HUD. Even when tracking only bulk motion, the operator sometimes lags behind and the surgical region of interest can drift out of the OCT field of view. To facilitate imaging we report on the development of a fast volume based tool segmentation algorithm. The algorithm is based on a previously reported volume rendering algorithm and can identify both the tool and retinal surface. The algorithm requires 45 ms per volume for segmentation and can be used to actively place the B-scan across the tool tissue interface. Alternatively, real-time tool segmentation can be used to allow the surgeon to use the surgical tool as an interactive B-scan pointer.
In vivo photoreceptor imaging has enhanced the way vision scientists and ophthalmologists understand the retinal structure, function, and etiology of numerous retinal pathologies. However, the complexity and large footprint of current systems capable of resolving photoreceptors has limited imaging to patients who are able to sit in an upright position and fixate for several minutes. Unfortunately, this excludes an important fraction of patients including bedridden patients, small children, and infants. Here, we show that our dual-modality, high-resolution handheld probe with a weight of only 94 g is capable of visualizing photoreceptors in supine children. Our device utilizes a microelectromechanical systems (MEMS) scanner and a novel telescope design to achieve over an order of magnitude reduction in size compared to similar systems. The probe has a 7° field of view and a lateral resolution of 8 µm. The optical coherence tomography (OCT) system has an axial resolution of 7 µm and a sensitivity of 101 dB. High definition scanning laser ophthalmoscopy (SLO) and OCT images were acquired from children ranging from 14 months to 12 years of age with and without pathology during examination under anesthesia in the operating room. Parafoveal cone imaging was shown using the SLO arm of this device without adaptive optics using a 3° FOV for the first time in children under 4 years old. This work lays the foundation for pediatric research, which will improve understanding of retinal development, maldevelopment and early onset of diseases at the cellular level during the beginning stages of human growth.
Conventional optical coherence tomography (OCT) systems have working distances of about 25 mm, and require cooperative subjects to immobilize and fixate on a target. Handheld OCT probes have also been demonstrated for successful imaging of pre-term infants and neonates up to ~1 year old. However, no technology yet exists for OCT in young children due to their lack of attention and inherent fear of large objects close to their face. In this work, we demonstrate a prototype retinal swept-source OCT system with a long working distance (from the last optical element to the subject’s eye) to facilitate pediatric imaging. To reduce the footprint and weight of the system compared to the conventional 4f scheme, a novel 2f scanning configuration was implemented to achieve a working distance of 348mm with a +/- 8° scanning angle prior to cornea. Employing two custom-designed lenses, the system design resolution was nearly diffraction limited throughout a -8D to +5D refractive corrections. A fixation target displayed on a LCD monitor and an iris camera were used to facilitate alignment and imaging. Our prototype was tested in consented adult subjects and has the potential to facilitate imaging of young children. With this apparatus, young children could potentially sit comfortably in caretaker’s lap while viewing entertainment on the fixation screen designed to direct their gaze into the imaging apparatus.
Ophthalmic surgeons manipulate micron-scale tissues using stereopsis through an operating microscope and instrument
shadowing for depth perception. While ophthalmic microsurgery has benefitted from rapid advances in instrumentation
and techniques, the basic principles of the stereo operating microscope have not changed since the 1930’s. Optical
Coherence Tomography (OCT) has revolutionized ophthalmic imaging and is now the gold standard for preoperative and
postoperative evaluation of most retinal and many corneal procedures. We and others have developed initial microscope-integrated
OCT (MIOCT) systems for concurrent OCT and operating microscope imaging, but these are limited to 2D
real-time imaging and require offline post-processing for 3D rendering and visualization. Our previously presented 4D
MIOCT system can record and display the 3D surgical field stereoscopically through the microscope oculars using a
dual-channel heads-up display (HUD) at up to 10 micron-scale volumes per second. In this work, we show that 4D
MIOCT guidance improves the accuracy of depth-based microsurgical maneuvers (with statistical significance) in mock
surgery trials in a wet lab environment. Additionally, 4D MIOCT was successfully performed in 38/45 (84%) posterior
and 14/14 (100%) anterior eye human surgeries, and revealed previously unrecognized lesions that were invisible
through the operating microscope. These lesions, such as residual and potentially damaging retinal deformation during
pathologic membrane peeling, were visualized in real-time by the surgeon. Our integrated system provides an enhanced
4D surgical visualization platform that can improve current ophthalmic surgical practice and may help develop and
refine future microsurgical techniques.
The first generation of intrasurgical optical coherence tomography (OCT) systems displayed OCT data onto a separate computer monitor, requiring surgeons to look away from the surgical microscope. In order to provide real-time OCT feedback without requiring surgeons to look away during surgeries, recent prototype research and commercial intrasurgical OCT systems have integrated heads-up display (HUD) systems into the surgical microscopes to allow the surgeons to access the OCT data and the surgical field through the oculars concurrently. However, all current intrasurgical OCT systems with a HUD are only capable of imaging through one ocular limiting the surgeon’s depth perception of OCT volumes. Stereoscopy is an effective technology to dramatically increase depth perception by presenting an image from slightly different angles to each eye. Conventional stereoscopic HUD use a pair of micro displays which require bulky optics. Several new approaches for HUDs are reported to use only one micro display at the expense of image brightness or increased footprint. Therefore, these techniques for HUD are not suitable to be integrated into microscopes. We have developed a novel stereoscopic HUD which uses spatial multiplexing to project stereo views into both oculars simultaneously with only one micro-display and three optical elements for our microscope-integrated OCT system. Simultaneous stereoscopic views of OCT volumes are computed in real time by GPU-enabled OCT system software. We present, to our knowledge, the first microscope integrated stereoscopic HUD used for intrasurgical OCT with a novel optical design for stereoscopic viewing devices and report on its preliminary use in human vitreoretinal surgeries.
We assessed the reproducibility of lateral and axial measurements performed with spectral-domain optical coherence tomography (SDOCT) instruments from a single manufacturer and across several manufacturers. One human retina phantom was imaged on two instruments each from four SDOCT platforms: Zeiss Cirrus, Heidelberg Spectralis, Bioptigen SDOIS, and hand-held Bioptigen Envisu. Built-in software calipers were used to perform manual measurements of a fixed lateral width (LW), central foveal thickness (CFT), and parafoveal thickness (PFT) 1 mm from foveal center. Inter- and intraplatform reproducibilities were assessed with analysis of variance and Tukey-Kramer tests. The range of measurements between platforms was 5171 to 5290 μm for mean LW (p<0.001), 162 to 196 μm for mean CFT (p<0.001), and 267 to 316 μm for mean PFT (p<0.001). All SDOCT platforms had significant differences between each other for all measurements, except LW between Bioptigen SDOIS and Envisu (p=0.27). Intraplatform differences were significantly smaller than interplatform differences for LW (p=0.020), CFT (p=0.045), and PFT (p=0.004). Conversion factors were generated for lateral and axial scaling between SDOCT platforms. Lateral and axial manual measurements have greater variance across different SDOCT platforms than between instruments from the same platform. Conversion factors for measurements from different platforms can produce normalized values for patient care and clinical studies.
Vitreoretinal surgical visualization by ophthalmic microscopy is limited in its ability to distinguish
thin translucent tissues from other retinal substructures. Conventional methods for supplementing
poor contrast, such as with increased illumination and application of exogenous contrast agents, have
been limited by the risks of toxicity at the retina. Spectral domain optical coherence tomography
(SDOCT) has demonstrated strong clinical success in retinal imaging, enabling high-resolution,
motion-artifact-free cross-sectional imaging and rapid accumulation of volumetric macular datasets.
Current generation SDOCT systems achieve <5 μm axial resolutions in tissue, and have been used to
obtain high resolution datasets from patients with neovascular AMD, high risk drusen, and
geographic atrophy. Recently, an intraoperative microscope-mounted OCT system (MMOCT) was
presented as a method of augmenting a surgical microscope to concurrently acquire high-resolution,
high-contrast SDOCT volumetric datasets. Here, we demonstrated the utility of intraoperative
MMOCT for the visualization of vitreoretinal surgical procedures. Vitreoretinal surgery was
simulated by performing procedures, through an ophthalmic surgical microscope, on cadaveric
porcine eyes. The datasets acquired with the MMOCT show both instrument-tissue interaction as
well as the ability of OCT to image certain surgical tools, which would directly translate to better
surgical visualization and impact the treatment of ocular diseases.
We describe an efficient approach for the automated segmentation of pathological/morphological structures in
ophthalmic Spectral Domain Optical Coherence Tomography (SDOCT) images. In this algorithm, image pixels
are treated as nodes of a graph with edge weights assigned to associate pairs of pixels. The weights vary according
to the distances, brightness differences, and feature variations between pixel pairs. Cuts through the graph with
minimum accumulated weights correspond to morphological layer boundaries. This approach has been applied
to SDOCT images with encouraging results and thus forms an adaptable framework for the segmentation of
many different ophthalmic structures.
Vitreoretinal surgery visualization is inherently limited by the ability to distinguish between tissues
with subtle contrast, and to judge the location of an object relative to other retinal substructures.
Inherent issues in visualizing thin translucent tissues, in contrast to underlying semitransparent ones,
require the use of stains such as indocyanine green, which is toxic to retinal tissue. Spectral domain
optical coherence tomography (SDOCT) has demonstrated strong clinical success in retinal imaging,
enabling high-resolution, motion-artifact-free cross-sectional imaging and rapid accumulation of
volumetric macular datasets. Current generation SDOCT systems achieve <5 μm axial resolutions in
tissue, and have been used to obtain high resolution datasets from patients with various retinopathies.
While OCT imaging has been considered for various non-ophthalmic intrasurgical applications, it is
uniquely suited for vitreoretinal surgery where multiple layers of the retinal structure are readily
accessible, and where high resolution cross-sectional viewing can have an impact on surgery as it is
performed today. Real-time cross-sectional OCT imaging would also provide critical information
relevant to the location and deformation of structures that may shift during surgery. Here, we
demonstrate an opto-mechanical design for an intraoperative microscope-mounted OCT system
(MMOCT) and preliminary in vivo human retinal imaging using this system in a test subject. By
adapting an Oculus Binocular Indirect Ophthalmo-Microscope (BIOM3) suspended from a Leica
microscope with SDOCT scanning and relay optics, we have demonstrated real-time cross-sectional
imaging of multiple layers of the retinal structure, allowing for SDOCT augmented intrasurgical
microscopy for intraocular visualization.
Accurate detection-characterization of drusen is an important imaging biomarker of age-related macular degeneration (AMD) progression. We report on the development of an automatic method for detection and segmentation of drusen in retinal images captured via high speed spectral domain coherence tomography (SDOCT) systems. The proposed algorithm takes advantage of a priori knowledge about the retina shape and structure in the AMD and normal eyes. In the first step, the location of the retinal nerve fiber layer (RNFL) is estimated by searching for the locally connected segments with high radiometric vertical gradients appearing in the upper section of the SDOCT scans. The high reflective and locally connected pixels that are spatially located below the RNFL layer are taken as the initial estimate of the retinal pigment epithelium (RPE) layer location. Such rough estimates are smoothed and improved by using a slightly modified implementation of the Xu-Prince gradient vector flow based deformable snake method. Further steps, including a two-pass scan of the image, remove outliers and improve the accuracy of the estimates. Unlike healthy eyes commonly exhibiting a convex RPE shape, the shape of the RPE layer in AMD eyes might include abnormalities due to the presence of drusen. Therefore by enforcing local convexity condition and fitting second or fourth order polynomials to the possibly unhealthy (abnormal) RPE curve, the health (normal) shape of the RPE layer is estimated. The area between the estimated normal and the segmented RPE outlines is marked as possible drusen location. Moreover, fine-tuning steps are incorporated to improve the accuracy of the proposed technique. All methods are implemented in a graphical user interface (GUI) software package based on MATLAB platform. Minor errors in estimating drusen volume can be easily manually corrected using the user-friendly software interface and the program is constantly refined to correct for the repeating errors. This semi-supervised approach significantly reduces the time and resources needed to conduct a large-scale AMD study. The computational complexity of the core automated segmentation technique is attractive as it only takes about 6.5 seconds on a conventional PC to segment, display, and record drusen locations in an image of size (512 × 1000) pixels. Experimental results on segmenting drusen in SDOCT images of different subjects are included, which attest to the effectiveness of the proposed technique.
KEYWORDS: Optical coherence tomography, Data acquisition, Diagnostics, Volume rendering, 3D image processing, Image segmentation, Retinal scanning, 3D acquisition, Data centers, Signal detection
We report on the development of quantitative, reproducible diagnostic observables for age-related macular degeneration
(AMD) based on high speed spectral domain optical coherence tomography (SDOCT). 3D SDOCT volumetric data sets
(512 x 1000 x 100 voxels) were collected (5.7 seconds acquisition time) in over 50 patients with age-related macular
degeneration and geographic atrophy using a state-of-the-art SDOCT scanner. Commercial and custom software utilities
were used for manual and semi-automated segmentation of photoreceptor layer thickness, total drusen volume, and
geographic atrophy cross-sectional area. In a preliminary test of reproducibility in segmentation of total drusen volume
and geographic atrophy surface area, inter-observer error was less than 5%. Extracted volume and surface area of AMD-related
drusen and geographic atrophy, respectively, may serve as useful observables for tracking disease state that were
not accessible without the rapid 3D volumetric imaging capability unique to retinal SDOCT.
Precise targeting of retinal structures including retinal pigment epithelial cells, feeder vessels, ganglion cells, photoreceptors, and other cells important for light transduction may enable earlier disease intervention with laser therapies and advanced methods for vision studies. A novel imaging system based upon scanning laser ophthalmoscopy (SLO) with adaptive optics (AO) and active image stabilization was designed, developed, and tested in humans and animals. An additional port allows delivery of aberration-corrected therapeutic/stimulus laser sources. The system design includes simultaneous presentation of non-AO, wide-field (~40 deg) and AO, high-magnification (1-2 deg) retinal scans easily positioned anywhere on the retina in a drag-and-drop manner. The AO optical design achieves an error of <0.45 waves (at 800 nm) over ±6 deg on the retina. A MEMS-based deformable mirror (Boston Micromachines Inc.) is used for wave-front correction. The third generation retinal tracking system achieves a bandwidth of greater than 1 kHz allowing acquisition of stabilized AO images with an accuracy of ~10 μm. Normal adult human volunteers and animals with previously-placed lesions (cynomolgus monkeys) were tested to optimize the tracking instrumentation and to characterize AO imaging performance. Ultrafast laser pulses were delivered to monkeys to characterize the ability to precisely place lesions and stimulus beams. Other advanced features such as real-time image averaging, automatic highresolution mosaic generation, and automatic blink detection and tracking re-lock were also tested. The system has the potential to become an important tool to clinicians and researchers for early detection and treatment of retinal diseases.
We investigate the relationship between the laser beam at the retina (spot size) and the extent of retinal injury from single ultrashort laser pulses. From previous studies it is believed that the retinal effect of single 3-ps laser pulses should vary in extent and location, depending on the occurrence of laser-induced breakdown (LIB) at the site of laser delivery. Single 3-ps pulses of 580-nm laser energy are delivered over a range of spot sizes to the retina of Macaca mulatta. The retinal response is captured sequentially with optical coherence tomography (OCT). The in vivo OCT images and the extent of pathology on final microscopic sections of the laser site are compared. With delivery of a laser pulse with peak irradiance greater than that required for LIB, OCT and light micrographs demonstrate inner retinal injury with many intraretinal and/or vitreous hemorrhages. In contrast, broad outer retinal injury with minimal to no choriocapillaris effect is seen after delivery of laser pulses to a larger retinal area (60 to 300 µm diam) when peak irradiance is less than that required for LIB. The broader lesions extend into the inner retina when higher energy delivery produces intraretinal injury. Microscopic examination of stained fixed tissues provide better resolution of retinal morphology than OCT. OCT provides less resolution but could be guided over an in vivo, visible retinal lesion for repeated sampling over time during the evolution of the lesion formation. For 3-ps visible wavelength laser pulses, varying the spot size and laser energy directly affects the extent of retinal injury. This again is believed to be partly due to the onset of LIB, as seen in previous studies. Spot-size dependence should be considered when comparing studies of retinal effects or when pursuing a specific retinal effect from ultrashort laser pulses.
We have shown in previous work that the threshold for laser- induced breakdown is higher than the threshold for ophthalmoscopically visible retinal damage, but they approach each other as pulse duration decreased form several nanoseconds to 100 femtoseconds. We discuss the most recent data collected for sub-50 fs laser induced breakdown thresholds and retinal damage thresholds. With these short pulse durations, the chromatic dispersion effect on the pulse should be considered to gain a full understanding of the mechanisms for damage. We discuss the most likely damage mechanisms operative in this pulse width regime and discuss relevance to laser safety.
Purpose: The direct comparison of in-vivo OCT images with fixed tissues sections assumes the fixation of tissue has no effect on the size and configuration of final pathology images such as light micrographs. Fixation artifact has been a concern in numerous studies of the pathology of retinal laser lesions. We tested this hypothesis. Methods: The Humphrey OCT model 2000 with a custom mirror and lens assembly was used to scan tissue phantoms and both fresh and fixed ex-vivum tissue samples. The optical configuration was determined by optimization of the contrast and signal strength on tissue phantoms. Fresh porcine retinas were scanned using this optimal configuration, then fixed using either glutaraldehyde or formalin. OCT images were taken of the tissue at various stages during the fixation process. Additionally, we examined fixed retinal tissue containing retinal laser lesions as a part of our study of ultrashort-pulsed laser effects on the macacca mulatta retina. Histologic sections were prepared and evaluated. Results: In this presentation, we describe our optical setup and image optimization process and assess the effects of glutaraldehyde and formalin processing on OCT image quality. The OCT images of glutaraldehyde-fixed laser lesions are compared with similar images of laser lesions in-vivo. Fixation artifacts appeared on OCT at 2 to 24 hours. Opacification of the lumen of large vessels was seen at two hours with both glutaraldehyde and formalin, while fixation induced retinal detachment appeared at 24 hours. Overall, there was a grater delineation of the laser lesions by OCT at 24 hours when compared to at 1 or 2 hours of fixation. Conclusions: Fixations induced changes in OCT scans of retinal tissue are present as early as 2 hours after immersion in fixative. Although both glutaraldehyde and formalin fixation preserve much of the tissue structure, these method of fixation have s significant effect on OCT imaging of both normal retinal tissue and laser lesions.
In order to provide a direct comparison of the damage thresholds for mode-locked systems to those with continuous-wave (CW) or non-pulsed output, we have performed an experiment with lasers possessing otherwise identical output characteristics. Our work presents an in-vivo minimal visible lesion (MVL) study. Titanium:Sapphire lasers produced 800-nm output for either mode-locked (76 MHz repetition rate, 120 femtosecond) or continuous-wave exposures. Alternating laser exposures were delivered to the paramacular retinal region of rhesus subjects. Laser exposure duration was set to one-quarter second for both types of exposures. Through ophthalmoscopic examination of the fundus, an MVL threshold for damage is established with probit analysis. Approximately 75 data points for each type of exposure were collected. The laser dosage thresholds and confidence intervals for minimal visible damage at twenty-four hours postexposure are reported for mode-locked and CW exposures. Results are compared with published studies conducted at similar pulse duration and similar CW wavelengths.
The Air Force has led a research effort to investigate the thresholds and mechanisms for retinal damage from ultrashort laser pulses. The results suggest that nonlinear optical phenomena mitigate the eventual damage threshold of the retina, while the fundamental mechanisms for damage remain unchanged from 100 fs to 10 microsecond(s) . The result of this research is a recommendation for the establishment of maximum permissible exposure limits in the visible and near IR that reflect the results of the nonlinear interaction. We review the progress made in determine trends in retinal damage from laser pulses from one nanosecond to one hundred femtoseconds for visible and near-IR wavelengths including variations in spot size and number of pulses. We discuss the most likely damage mechanisms, including nonlinear optical interactions pertinent in this pulse width regime and discuss relevance to laser safety.
Purpose: We analyzed the effect of energy and rate of cutting on the in vivo ocular response to 2.94 μm wavelength Free Electron Laser incision of the cornea. We were interested in the difference between our clinical observations of the initial laser lesion and the ocular response using the biomicroscope versus optical coherence tomographs. We were also interested in the difference between these clinical in vivo data and our findings from light micrographs of fixed tissue.
Methods: Corneas were incised with FEL at 2.94 μm wavelength and either 2.5 or 3.5 mJ/1.4 μsec. the rate of movement of the laser beam across tissue ranged from 0.2 mm/sec to 1.2 mm/sec. Eyes were examined for two hours postoperatively using optical coherence tomography (OCT) and compared to the clinical slit lamp examination and to light microscopic examination of fixed tissue sections.
Results: OCT revealed a dramatic fibrin response directly correlated to the slow sweep of the FEL beam across the tissue (longer duration of tissue exposure to the laser beam). The OCt was better than examination at the slit lamp at demonstrating sites of fibrin attachments.
We wish to identify the change in extent of retinal tissue injury due to varying the spot size at the retina of ultrashort laser pulses. We compared the effects of delivery of near infrared (1060 nm) single laser pulses to an 800 micron diameter retinal spot to previously reported laser retinal effects. We examined macular lesions 24 hours after delivery of near-infrared (1060 nm wavelength) ultrashort laser to 804 micron spot-size, using fundus examination, fundus photographs and fluorescein angiograms. Using light microscopy, we examined sections of these lesions obtained 24 hours after laser delivery. The degree of retinal damage was compared to our data published previously by using a modified version of our previous grading scale. The 150 fs near infrared, large spot laser lesions were remarkable in their clinical and pathological appearance. The lesions, rather than centering on a single focal spot of pallor as typically seen in pulsed laser lesions of the retina, demonstrated a spotted pattern of multiple focal lesions across the area of laser delivery. There was also choroidal damage in several eyes but the Bruch's membrane remained intact. Although there was choroidal damage in the 150 fs near infrared wavelength small spot laser lesions there was not significant thermal spread. The small spot ultrashort visible wavelength showed no significant thermal spread and no choroidal damage. Larger spot-size demonstrated a broader area of damage than that of the smaller spot-size and different choroidal effect when compared to smaller sized lesions.
For the past several years the US Air Force has led a research effort to investigate the thresholds and mechanisms for retinal damage from ultrashort laser pulses [i.e. nanosecond (10-9 sec) to femtosecond (10-15 sec) pulse widths]. The goal was to expand the biological database into the ultrashort pulse regime and thus to allow establishment of maximum permissible exposure limits for these lasers. We review the progress made in determining trends in retial damage by ultrashort laser pulses in the visible and near infrared, including variations in spot size and number of pulses. We also discuss the most likely damage mechanisms operative in this pulse width regime and discuss relevance to laser safety.
KEYWORDS: Tissues, Transmission electron microscopy, 3D image processing, Confocal microscopy, Microscopy, 3D modeling, Laser tissue interaction, Electron microscopy, Microscopes, 3D image reconstruction
In predicting and measuring laser effect on retinal tissue for most of the visible to near infrared spectrum, one is concerned with the melanosome as the major absorber of incident energy. Differences in the location and density of melanosomes in the retinal pigment epithelium may have an impact on the effect of laser energy delivered to those tissues. Current models use estimates of numbers of melanosomes usually in an even distribution across a 5 - 8 micrometer deep volume. The goal of our study is to identify the three-dimensional distribution of melanosomes within the retinal pigment epithelium (RPE) for the use of those modeling laser tissue effects. We examined normal retinal pigment epithelium using three-dimensional (3-D) reconstruction from images obtained by transmission electron microscopy (TEM), light microscopy (LM) and confocal microscopy. Images were captured on a digital camera system attached to the microscope for both the transmission electron and light microscopy. Three-dimensional reconstruction was performed after digital deconvolution of microscopic images (Vaytek, Inc.). Three- dimensional images were then utilized for analysis of distribution of melanosomes and organelles within the pigment epithelial block. The distribution of melanosomes will be useful for accurate mathematical modeling of laser impact on the retina.
Damage thresholds using multiple laser pulses to produce minimum visible lesions (MVL) in rhesus monkey eyes are reported for near-infrared (800 nm) at 130 femtoseconds. Previous studies by our research group using single pulses in the near-infrared (1060 nm) have determined damage thresholds and retinal spot size dependence. We report the first multiple pulse damage thresholds using femtosecond pulses. MVL thresholds at 1 hour and 24 hours postexposure were determined for 1, 100 and 1,000 pulses and we compare these with other reported multiple pulse thresholds. These new data will be added to the databank for retinal MVL's as a function of pulse repetition rate for this pulsewidth and a comparison will be made with the ANSI standard for multiple pulse exposures. Our measurements show that the retinal ED50 threshold/pulse in the paramacula decreases for increasing number of pulses. The MVL-ED50 at the threshold/pulse decreased by a factor of 4 (0.55 (mu) J to 0.13 (mu) J/pulse) for an increase from 1 to 100 pulses.
Single pulses in the near-infrared (800 nanometers) were used to measure retinal minimum visible lesion (MVL) thresholds in rhesus monkey eyes at a pulse width of 130 femtoseconds (fs) within both the macula and paramacula regions. We report the MVL thresholds, determined at 1 hour and 24 hours post exposure, which were obtained within the macula and adjacent paramacula. This data will provide a direct comparison of the sensitivities of different retinal areas to laser injury and provide additional insight to laser damage. These new data points will be added to the databank for MVLs for single pulses. The MVL-ED50 threshold for the macula was measured to be 0.35 (mu) J at 24 hours postexposure, which compares with 0.43 (mu) J measured at 580 nm and the 0.17 (mu) J measured at 532 nm in our laboratory. Our measurements show that the retinal ED50 threshold in the paramacula was larger by a factor of 1.6 than in the macula. This factor of 1.6 is in good agreement with the factor of 1.1 to 2.5 reported in previous studies.
Retinal lesions produced by ultrashort laser pulses in the pico- and femtosecond range were examined by electron microscopy. Retinal pigment epithelial (RPE) cells that contained fractured and striated melanosomes typically exhibited severe damage to the other components of the cell. However, having observed RPE cell damage without coincident fractured melanosomes, it is thought that melanosome fracture itself is not responsible for the damage that occurs within the RPE cell. Nevertheless, the percentage of melanosomes fractured per lesion seems to parallel the severity of damage within that lesion site. No trend existed between percentage of melanosomes fractured and the peak power of laser delivery. However, with decreasing laser pulsewidth, there was a decrease in the percentage of melanosomes showing fracture.
Extensive research of ultrashort ocular damage mechanisms has shown that less energy is required for retinal damage for pulses shorter than one nanosecond. Laser minimum visible lesion thresholds for retinal damage from ultrashort (i.e. < 1 ns) laser pulses occur at lower energies than in the nanosecond to microsecond laser pulse regime. WE review the progress made in determining the trends in retinal damage from laser pulses of one nanosecond to one hundred femtoseconds in the visible and near-infrared wavelength regimes. We discuss the most likely damage mechanism(s) operative in this pulse width regime and discuss implications on laser safety standards.
Ultrashort pulsed laser retinal effects vary widely depending on the configuration of the laser energy as it reaches the retina and surrounding structures. Tissue response is determined by: wavelength, pulsewidth, energy per pulse, peak irradiance, linear optics of the beam path, and non linear optics of the ultrashort beam. In vivo, we have reported a range of lesions from visible and from near infrared ultrashort laser pulses. New data from additional infrared studies in vivo is combined with our previous data to present an overview of retinal effects and how these might be selected for retinal surgical use.
Single pulses in the near-infrared (1060 nanometers) were used to measure retinal spot size dependence of minimum visible lesion (MVL) thresholds in rhesus monkey eyes at a pulsewidth of 150 femtoseconds. We report the MVL thresholds determined at 1 hour and 24 hours post exposure which were obtained with 2 different lenses placed in front of the eye to vary the retinal spot size. Also we report the fluorescein angiography thresholds (FAVL) for the above measurements. These new data points will be added to the databank for Retinal Maximum Permissible Exposure (MPE) as a function of spot size for this pulsewidth and a comparison will be made with previous spot size dependency studies. Our measurements show that the retinal ED50 threshold fluence decreases for increasing retinal spot sizes. The fluence at the MVL threshold decreased by a factor of 3 for an increase in retinal image diameter by a factor of 4.5 times from the smallest to largest spot size.
Single pulses in the near-infrared (1060 and 1064 nanometers) were used to measure ophthalmoscopically minimum visible lesion (MVL) thresholds in the rhesus monkey eyes for pulsewidths of 7 nanoseconds (ns), 20 picoseconds (ps), and 150 femtoseconds (fs). MVL thresholds for 1 hour reading and 24 hour reading are reported as the 50% probability for damage (ED50) together with their fiducial limits. These measured thresholds are compared with previously reported thresholds for near-IR and visible wavelengths for the complete range of pulsewidths (ns, ps, and fs). Threshold doses were lower at the 24 hour reading than at the 1 hour reading and both ED50 for the fs pulsewidths were less than 25% of those for ns pulsewidths. MVL thresholds ranged from 19 (mu) J at 7 ns down to 1 (mu) J at 150 fs. Thresholds measured for the nanosecond and picosecond pulsewidths using infrared laser pulses were an order of magnitude larger than for the visible wavelengths at similar pulsewidths while the 150 fs threshold was only about double the value for the 580 nm visible wavelength at 90 fs.
Minimum visible lesions (MVL) are reported for picosecond and nanosecond laser pulses at near-IR wavelengths in the primate eye, Macaca Mulatta. The 50 percent probability for damage (ED50) dosages are reported for the 24 hour for both MVL and fluorescein angiography visible lesion thresholds at the 95 percent confidence level. The thresholds decreased by as much as 48 percent between the 1- hour reading and were lower in all cases at 24 hours. MVL- (ED50) threshold doses were 19.1 uJ at 7 ns and 4.2 uJ and 4.6 uJ at 80 ps and 20 ps respectively. Our thresholds measured for the near-IR laser pulses were lower by a factor of 4 to 8 lower than previously reported values but almost an order in magnitude higher than visible MVL thresholds for similar pulsewidth in the visible wavelengths.
Purpose: to assess the early in vivo evolution of tissue response and wound healing from ultrashort pulsed laser retinal lesions by correlating the cross sectional morphology from sequential optical coherence tomography with histopathologic sectioning. Methods: single ultrashort laser pulses were placed in the Macacca mulatta retina and evaluated by cross-section optical coherence tomography (OCT). These images were compared at selected time-points with corresponding histological sections. Results: OCT was able to detect the acute tissue injury from laser delivery and the evolution of the healing response over 8 days after laser delivery. These OCT images correlated well with histopathologic findings. Conclusion: analysis of the extent of initial laser lesions and the type of healing response can be performed in serial sequence with OCT providing new insight into the healing response form laser injury. This information correlates well with microscopic data.
Recent studies of retinal damage due to ultrashort laser pulses have shown that less energy is required for retinal damage for pulses shorter than one nanosecond. Laser minimum visible lesion thresholds for retinal damage from ultrashort laser pulses are produced at lower energies than in the nanosecond to microsecond laser pulse regime. We review the progress made in determining the trends in retinal damage from laser pulses of one nanosecond to one hundred femtoseconds in the visible and near-infrared wavelength regimes. We have determined the most likely damage mechanism operative in this pulse width regime and discuss implications on laser safety standards.
One concern during IR-laser ablation of tissue under water is the mechanical injury that may be induced in tissue due to rapid bubble expansion and collapse or due to strong laser-induced pressure waves. The objective of this study was to evaluate the feasibility of using a liquid which is transparent to the IR-region of the spectrum in order to minimize these undesired mechanical side-effects. As transmitting medium perfluorocarbon liquid was used. Free- running Er:YAG and Ho:YAG laser pulses were delivered into the liquid via a 400 micrometers fiber. Bubble formation during the ablation process was recorded with fast flash photography while pressure transients were measured with a needle hydrophone. The effect of the surrounding material (air, water, perfluorooctane) on the tissue response of chicken breast was evaluated in vitro using histology. It was observed that a large bubble (up to 6 mm in diameter) was formed under perfluorooctane driven by the ablation products. This bubble, however, does not generate a pressure wave when collapsing. Although perfluorooctane only shows a weak absorption for infrared radiation, laser-induced thermal lensing in the liquid strongly decreases the radiant exposure and therefore the ablation efficiency.
Recent studies of retinal damage due to ultrashort laser pulses have shown interesting behavior. Laser induced retinal damage for ultrashort (i.e. less than 1 ns) laser pulses is produced at lower energies than in the nanosecond to microsecond laser pulse regime and the energy required for hemorrhagic lesions is much greater times greater for the nanosecond regime. We investigated the tissue effects exhibited in histopathology of retinal tissues exposed to ultrashort laser pulses.
Optical Coherence Tomography (OCT) is a new, non-invasive diagnostic technique for high resolution optical 3D imaging, which was developed and applied to several different biological materials during the lasi; five years [1, 2, 3]. A unique application ofthis technique is the microscopical cross-sectional imaging ofpostenor structures ofthe eye which are not accessable with other high resolution techniques in-vivo neither with x-ray-imaging nor with high frequency ultrasound scanning. The superior spatial resolution on the order ofabout lOtm laterally and axially, the high signal-to-noise ratio ofmore than 100 db and the fast acquisition-time of one second for a two dimensional scan provides a technique for cross-sectional in-vivo-momtoring ofintraocular structures and therefore the possibility to study the time course of anatomical and pathological developments in the eye. The acute morphological changes of ocular structures and their biological healing response after shortterm impacts such as high-intensity laser exposures are ofparticular interest for the understanding of the mechanisms responsible for therapeutic laser-application in ophthal-mology as well as for laser injury to the eye. A correlation between cross-sectional OCT-images and structural findings using classical histopathological techniques facilitates a better interpretation ofthe characteristic patterns seen in OCTimages and defines the sensitivity ofthe OCT-technique to image morphological details. On the other hand preparational artefacts not avoidable in all histological procedures can be identified and analyzed by comparing histological micrographs with OCT-images of exactly the same structure. First results of an experimental study where retinal effects were produced in monkey eyes using laser pulses from 200 ms to 130 fs in duration are presented in this article. The applied energies from 5tJ to 50 mJ were able to induce the whole spectrum of biological effects possible in the eye, ranging from intraretinal microruptures to extensive thermal denaturation and massive preretinal hemorrhages [4, 5, 6].
Threshold measurements at 90 femtoseconds (fs) and 600 fs have been made for minimum visible lesions (MVLs) using Dutch Belted rabbit and Rhesus monkey eyes. Laser induced breakdown (LIB) thresholds on biological materials including vitreous, normal saline, tap water, and ultrapure water are reported along with irradiance calculations utilizing nonlinear transmission properties including self-focusing. At both pulsewidths the ED50 dose required for the Rhesus monkey eye was less than half the value determined for the Dutch Belted rabbit eye, all thresholds being 1 microjoule ((mu) J) or less. Measurements on the Rhesus eye at 600 fs found the ED50 dose (0.26 (mu) J) to be much lower than the ED50 dose at 90 fs (0.43 (mu) J). But for these two pulsewidths, almost the same energy level was determined for the Dutch Belted rabbit eye (0.94 (mu) J vs. 1.0 (mu) J). LIB threshold measurements at 100 fs and 300 fs using a simulated eye with isolated vitreous found the ED50 dosages to be 3.5 and 6.0 (mu) J respectively. We found in all cases that the ED50 dosages required to produce MVLs in 24 hours for rabbit and monkey eyes were less than the ED50 values measured for LIB in vitreous or saline or any other breakdown values reported. Also observed was the fact that many of the threshold lesions did not appear in the 1-hour postexposure check but clearly showed up at the 24-hour reading which provided for a much lower threshold dose after 24 hours. We discuss the energy levels and peak powers at which nonlinear effects can begin to occur.
Threshold measurements for Minimum Visible Lesions (MVL) at the retina are reported for femtosecond (fs) and picosecond (ps) laser pulses in Rhesus monkey eyes using visible wavelengths. The 50% probability for damage (ED50) dosages are calculated for 1 hour and 24 hour post-exposures at the 95% confidence level. The ED50 values are found to decrease with pulsewidth down to 600 fs. At 90 fs the ED50 dosages were noted to increase slightly when compared with the 3 ps and 600 fs values. Fluorescein angiography (FA) was accomplished at both 1 hour and 24 hour post-exposure and did not demonstrate lower threshold for damage, which has been the case for MVL's created with longer pulse durations (>= nanoseconds). At the 90 fs pulse duration, MVLs were not observed below 0.1 (mu) J. At energies greater than 0.1 (mu) J, both MVL and the absence of MVL's were observed up to 1.4 (mu) J. Above 1.4 (mu) J all energies delivered showed MVL development. Out of 138 data points taken at 90 fs, 94 were between 0.1 and 14 (mu) J, and the observed lesions are distributed with approximately 50% probability throughout this energy rate.
We present our clinical evaluation of hemorrhagic and non-hemorrhagic 90 fs single pulses in rabbits and primates. The rabbit and primate eye present unique in vivo models for evaluation of retinal and choroidal laser induced hemorrhages with distinct differences in their retinal anatomy. We found two different hemorrhagic events to occur in the posterior pole with delivery of 90 fs pulses. First, in the Dutch Belted rabbit, we found large amounts of energy per pulse (from 20 to 60 times ED50) were required for formation of subretinal hemorrhages. Second, in the Rhesus monkey, we found significant numbers of small intraretinal hemorrhages from relatively low energy 90 fs pulses. Both the Dutch Belted rabbit and the Rhesus monkey failed to consistently show subretinal hemorrhagic lesions form very high pulse energies. Our findings suggest more energy absorption at the level of the retinal circulation than the choroidal circulation with our pulse parameters. The effects of the laser on the retinal circulation may be due to the use of a wavelength of 580 nm. At this wavelength the oxyhemoglobin to melanin absorption ratio is nearly at its peak (approximately 0.40), perhaps allowing improved absorption in the retinal vasculature. One precaution with this finding, however, are the distinct differences between primate and non-primate ocular systems. Further studies are required to resolve the differences in damage at the level of the RPE and choroid between rabbits and primates.
A model laser surgical probe was designed and built to employ laser induced breakdown (LIB) in cutting fibrovascular membranes within the vitreous cavity of the eye. The probe is a simple design of a gradient index (GRIN) lens attached to the tip of a multimode fiber. It is designed to fit through a sclerotomy incision and enter the vitreaous cavity for work anterior to the retina. The laser light is focused close to the tip of the probe without causing GRIN lens damage. Thus a widely divergent beam behind the focus will diminish potential laser damage posterior to the target tissue. A Nd:YAG 1064-nm 10-ns pulsed laser was used with the probe inserted into a cell of tap water. Threshold data for laser induced breakdown was taken ad fit to a probit curve. The data was compared with past LIB threshold data. The radiant exposure versus distance from the probe was plotted via spot size measurements. This measurement gave a rough indication of the distance the probe must remain from the retina to be well below retinal maximum permissible exposure (MPE) levels. In-vitro threshold measurements of bovine vitreous were taken and compared to the water threshold. Finally, collagen membranes were lased with the probe to demonstrate its functional application.
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