Objective: To demonstrate optical coherence tomography for high-resolution, noninvasive imaging of the human retina. Optical coherence tomography is a new imaging technique analogous to ultrasound B scan that can provide cross-sectional images of the retina with micrometer-scale resolution. Design: Survey optical coherence tomographic examination of the retina, including the macula and optic nerve head in normal human subjects. Settings Research laboratory. Participants: Convenience sample of normal human subjects. Main Outcome Measures: Correlation of optical coherence retinal tomographs with known normal retinal anatomy. Results: Optical coherence tomographs can discriminate the cross-sectional morphologic features of the fovea and optic disc, the layered structure of the retina, and normal anatomic variations in retinal and retinal nerve fiber layer thicknesses with 10- μm depth resolution. Conclusion: Optical coherence tomography is a potentially useful technique for high depth resolution, cross-sectional examination of the fundus.

Optical Coherence Tomography of the Human Retina

We review recent advances in laser cell surgery, and investigate the working mechanisms of femtosecond laser nanoprocessing in biomaterials with oscillator pulses of 80-MHz repetition rate and with amplified pulses of 1-kHz repetition rate. Plasma formation in water, the evolution of the temperature distribution, thermoelastic stress generation, and stress-induced bubble formation are numerically simulated for NA=1.3, and the outcome is compared to experimental results. Mechanisms and the spatial resolution of femtosecond laser surgery are then compared to the features of continuous-wave (cw) microbeams. We find that free electrons are produced in a fairly large irradiance range below the optical breakdown threshold, with a deterministic relationship between free-electron density and irradiance. This provides a large ‘tuning range’ for the creation of spatially extremely confined chemical, thermal, and mechanical effects via free-electron generation. Dissection at 80-MHz repetition rate is performed in the low-density plasma regime at pulse energies well below the optical breakdown threshold and only slightly higher than used for nonlinear imaging. It is mediated by free-electron-induced chemical decomposition (bond breaking) in conjunction with multiphoton-induced chemistry, and hardly related to heating or thermoelastic stresses. When the energy is raised, accumulative heating occurs and long-lasting bubbles are produced by tissue dissociation into volatile fragments, which is usually unwanted. By contrast, dissection at 1-kHz repetition rate is performed using more than 10-fold larger pulse energies and relies on thermoelastically induced formation of minute transient cavities with lifetimes <100 ns. Both modes of femtosecond laser nanoprocessing can achieve a 2–3 fold better precision than cell surgery using cw irradiation, and enable manipulation at arbitrary locations.

Mechanisms of femtosecond laser nanosurgery of cells and tissues

We present what is to our knowledge the first in vivo tomograms of human retina obtained by Fourier domain optical coherence tomography. We would like to show that this technique might be as powerful as other optical coherence tomography techniques in the ophthalmologic imaging field. The method, experimental setup, data processing, and images are discussed.

https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics/volume-7/issue-03/0000/In-vivo-human-retinal-imaging-by-Fourier-domain-optical-coherence/10.1117/1.1482379.pdf

In vivo human retinal imaging by Fourier domain optical coherence tomography.

Optical Coherence Tomography: An Emerging Technology for Biomedical Imaging and Optical Biopsy

Here we present new technology for optical coherence tomography (OCT) that enables ultrahigh-resolution, non-invasive in vivo ophthalmologic imaging of retinal and corneal morphology with an axial resolution of 2–3 μm. This resolution represents a significant advance in performance over the 10–15-μm resolution currently available in ophthalmic OCT systems and, to our knowledge, is the highest resolution for in vivo ophthalmologic imaging achieved to date. This resolution enables in vivo visualization of intraretinal and intra-corneal architectural morphology that had previously only been possible with histopathology. We demonstrate image processing and segmentation techniques for automatic identification and quantification of retinal morphology. Ultrahigh-resolution OCT promises to enhance early diagnosis and objective measurement for tracking progression of ocular diseases, as well as monitoring the efficacy of therapy.

Current clinical practice emphasizes the development of techniques to diagnose disease in its early stages, when treatment is most effective and irreversible damage can be prevented or delayed. In ophthalmology, the precise visualization of pathology is especially critical for the diagnosis and staging of ocular diseases. Therefore, new imaging techniques have been developed to augment conventional fundoscopy and slit-lamp biomicroscopy. Ultrasonography is routinely used in ophthalmology, but requires physical contact with the eye and has axial resolutions of approximately 200 μm (ref. 1). High-frequency ultrasound enables approximately 20 μm axial resolutions, but due to limited penetration, only anterior eye structures can be imaged2. Confocal microscopy has been used to image the cornea with sub-micrometer transverse resolution3. Scanning laser ophthalmoscopy enables en face fundus imaging with micron-scale transverse and approximately 300-μm axial resolution4,5. None of these techniques, however, permits high-resolution, cross-sectional imaging of the retina in vivo.

Recently, optical coherence tomography (OCT) has emerged as a promising new technique for high-resolution, cross-sectional imaging6,7. OCT is attractive for ophthalmic imaging because image resolutions are 1–2 orders of magnitude higher than conventional ultrasound, imaging can be performed non-invasively and in real time, and quantitative morphometric information can be obtained. OCT is somewhat analogous to ultrasound imaging except that it uses light instead of sound. High-resolution, cross-sectional images are obtained by measuring the echo time delay of reflected infrared light using a technique known as low coherence interferometry8,9. OCT imaging was first demonstrated in the human retina in vitro6 and in vivo10,11. Recently, it has been extended to a wide range of other non-transparent tissues to function as a type of optical biopsy12–15. To date, however, the most important clinical applications of OCT have been retinal imaging in ophthalmic diagnosis7,16–22. Current ophthalmic OCT systems have 10–15-μm axial resolution and provide more detailed structural information than any other non-invasive ophthalmic imaging technique6,7,10,11. However, the resolution of current clinical ophthalmic OCT technology is significantly below what is theoretically possible. Improving the resolution of OCT ophthalmic imaging would enable structural imaging of retinal pathology at an intraretinal level, as well as improve the accuracy of morphometric quantification.

The axial resolution of conventional ophthalmic OCT systems is limited to 10–15 μm by the bandwidth of light sources used for imaging. Short-pulse, solid-state lasers can generate ultrabroad bandwidth, low-coherence light13. A broadband Cr:Forsterite laser operating in the near infrared (1,300 nm) has permitted cellular-level OCT imaging in developmental biology specimens with 6-μm axial resolution13. For ophthalmic imaging, light sources operating at 800 nm are necessary to avoid absorption in the ocular media. Recently, a state-of-the-art broadband Ti:Al2O3 laser has been developed for ultrahigh (~1 μm) axial resolution, spectroscopic OCT imaging in non-transparent tissue at 800-nm center wavelength23,24. We describe here ultrahigh-resolution ophthalmic OCT based on this state-of-the-art optical technology and demonstrate its potential to provide enhanced structural and quantitative information for ophthalmologic imaging.

/pdf/ultrahigh-resolution-ophthalmic-optical-coherence-tomography-3uqbz2mu8p.pdf

Ultrahigh-resolution ophthalmic optical coherence tomography

Objective: To compare the cross-sectional images of primate retinal morphology obtained by optical coherence tomography (OCT) with light microscopy to determine the retinal components represented in OCT images. Methods: Laser pulses were delivered to the retina to create small marker lesions in a Macaca mulatta . These lesions were used to align in vivo OCT scans and ex vivum histologic cross sections for image comparison. Results: The OCT images demonstrated reproducible patterns of retinal morphology that corresponded to the location of retinal layers seen on light microscopic overlays. Layers of relative high reflectivity corresponded to horizontally aligned retinal components such as the nerve fiber layer and plexiform layers, as well as to the retinal pigment epithelium and choroid. In contrast, the nuclear layers and the photoreceptor inner and outer segments demonstrated relative low reflectivity by OCT. Conclusions: Retinal morphology and macular OCT imaging correlate well, with alignment of areas of high and low reflectivity to specific retinal and choroidal elements. Resolution of retinal structures by OCT depends on the contrast in relative reflectivity of adjacent structures. Use of this tool will enable expanded study of retinal morphology, both normal and pathologic, as it evolves in vivo.

A comparison of retinal morphology viewed by optical coherence tomography and by light microscopy

Argon laser retinal lesions evaluated in vivo by optical coherence tomography

Purpose. To demonstrate how current theories regarding ultrashort laser pulse effects may apply to ocular tissue, a prospective clinicopathologic study of macular lesions from ultrashort laser pulses compared the pathologic effects with the clinical and fluorescein angiographic appearance of the laser lesions. Methods. Ninety-femtosecond, 3-picosecond, and 60-picosecond laser pulses, throughout a range of energies, were delivered to the retina of Macaca mulatto,. Clinical examination and fluorescein angiography were performed at 1 hour in all eyes and 24 hours after exposure in selected eyes. Eyes were enucleated at 1 or 24 hours after lesion placement. The structure and extent of retinal lesions were scored for comparison with the clinical findings. Results. Focal retinal pathologic appearance correlated well with a clinically visible lesion observed 24 hours after laser delivery. Retinal lesions were small foci of retinal pigment epithelium (RPE) and retinal disruption, without choriocapillaris involvement. Lesions that contained focal RPE vacuoles or lifting of the RPE also demonstrated leakage, in fluorescein angiographic studies. Supratlireshold laser delivery frequendy caused focal columns of retinal injury and intraretinal hemorrhages from retinal vessel bleeding, with no rupture of choroidal blood vessels. Conclusions. The retinal response to ultrashort laser pulses at moderate energy followed a pattern of focal damage from laser-induced breakdown without significant thermal spread. Invest Ophthalmol Vis Sci. 1997;38:2204-2213. Hivaluation of a new laser system for medical application, in diagnosis or in treatment, has two components: the safety of the laser for the proposed application and the potential for the laser to improve medical care in a cost-effective manner. Satisfaction of these requirements depends on the interaction of the laser with biologic tissues and the consequent information

Pathology of macular lesions from subnanosecond pulses of visible laser energy.

We investigate the relationship between the laser beam at the retina (spot size) and the extent of retinal injury from single ul- trashort laser pulses. From previous studies it is believed that the reti- nal effect of single 3-ps laser pulses should vary in extent and loca- tion, 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 mu- latta. The retinal response is captured sequentially with optical coher- ence tomography (OCT). The in vivo OCT images and the extent of pathology on final microscopic sections of the laser site are com- pared. 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 mm 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 ex- tent 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 pur- suing a specific retinal effect from ultrashort laser pulses. © 2004 Society

http://biophotonics.illinois.edu/pubs/biophotonics_current/retinalresponseofmacacamulatta.pdf

Retinal response of Macaca mulatta to picosecond laser pulses of varying energy and spot size

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.

Drew G. Narayan

Papers

A comparison of retinal morphology viewed by optical coherence tomography and by light microscopy

Argon laser retinal lesions evaluated in vivo by optical coherence tomography

Pathology of macular lesions from subnanosecond pulses of visible laser energy.

Retinal response of Macaca mulatta to picosecond laser pulses of varying energy and spot size

Histopathology of ultrashort-laser-pulse retinal damage