Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy.

The combination of intravital microscopy and animal models of disease has propelled studies of disease mechanisms and treatments. However, many disorders afflict tissues inaccessible to light microscopy in live subjects. Here we introduce cellular-level time-lapse imaging deep within the live mammalian brain by one- and two-photon fluorescence microendoscopy over multiple weeks. Bilateral imaging sites allowed longitudinal comparisons within individual subjects, including of normal and diseased tissues. Using this approach, we tracked CA1 hippocampal pyramidal neuron dendrites in adult mice, revealing these dendrites' extreme stability and rare examples of their structural alterations. To illustrate disease studies, we tracked deep lying gliomas by observing tumor growth, visualizing three-dimensional vasculature structure and determining microcirculatory speeds. Average erythrocyte speeds in gliomas declined markedly as the disease advanced, notwithstanding significant increases in capillary diameters. Time-lapse microendoscopy will be applicable to studies of numerous disorders, including neurovascular, neurological, cancerous and trauma-induced conditions.

High-speed, miniaturized fluorescence microscopy in freely moving mice.

A central goal in biomedicine is to explain organismic behavior in terms of causal cellular processes. However, concurrent observation of mammalian behavior and underlying cellular dynamics has been a longstanding challenge. We describe a miniaturized (1.1 g mass) epifluorescence microscope for cellular-level brain imaging in freely moving mice, and its application to imaging microcirculation and neuronal Ca(2+) dynamics.

Microstructural abnormalities in MEWDS demonstrated by ultrahigh resolution optical coherence tomography.

BACKGROUND: Histopathological studies of acute multiple evanescent white dot syndrome (MEWDS) have not been reported because of the transient and benign nature of the disease. Ultrahigh resolution optical coherence tomography (UHR-OCT), capable of high resolution in vivo imaging, offers a unique opportunity to visualize retinal microstructure in the disease. METHODS: UHR-OCT images of the maculae of five patients with MEWDS were obtained and analyzed. Diagnosis was based on clinical presentation, examination, visual field testing, and angiography. RESULTS: UHR-OCT revealed disturbances in the photoreceptor inner/outer segment junction (IS/OS) in each of the five patients (six eyes) with MEWDS. In addition, thinning of the outer nuclear layer was seen in the case of recurrent MEWDS, suggesting that repeated episodes of MEWDS may result in photoreceptor atrophy. CONCLUSIONS: Subtle disruptions of the photoreceptor IS/OS are demonstrated in all eyes affected by MEWDS. UHR-OCT may be a useful adjunct to diagnosis and monitoring of MEWDS.

Ultrahigh resolution optical coherence tomography of macular holes.

PURPOSE: To evaluate ultrahigh resolution optical coherence tomography (UHR OCT) for visualization of intraretinal layers, especially the photoreceptor inner segment and outer segment layers, in eyes with macular holes and after surgical intervention. METHODS: An UHR OCT system based on a titanium:sapphire laser was used, enabling in vivo cross-sectional retinal imaging with 3-micro m axial resolution. Typical, representative tomograms of 5 of 48 eyes from 36 patients demonstrated the potential of UHR OCT to detect morphologic changes in different stages of full-thickness macular holes and changes induced by surgical intervention. RESULTS: UHR OCT could detect subtle intraretinal changes in macular hole formation. Unprecedented visualization of photoreceptor impairment was achieved that appeared to be more extensive than the hole itself. Postoperatively, clinically closed holes showed restoration of the photoreceptor inner and outer segment layers of various extents, with residual disease in some eyes. CONCLUSION: In macular holes, UHR OCT allows for detection of even small morphologic changes of the retinal layers, especially the photoreceptor inner and outer segment layers. Therefore, it also represents a superior method to monitor the effect of surgical interventions. Preoperative photoreceptor impairment and the degree of postoperative restoration could possibly be associated with visual function. Hence, UHR OCT could lead to better understanding of macular hole pathogenesis and to more accurate disease prognosis.

Ultra-high resolution optical coherence tomography assessment of photoreceptors in retinitis pigmentosa and related diseases.

PURPOSE: To assess photoreceptor integrity in patients with retinitis pigmentosa (RP) and related diseases using ultra-high resolution optical coherence tomography (UHR-OCT) and to correlate foveal photoreceptor loss with visual acuity. DESIGN: Observational case series. METHODS: Nine eyes of nine patients with RP and related diseases were imaged with UHR-OCT at the ophthalmology clinic. Patients were diagnosed based on history, examination, fluorescein angiography, and electroretinography. Concurrently, 36 eyes of 36 normal subjects were imaged with UHR-OCT. Central foveal thickness (CFT) and foveal outer segment/pigment epithelium thickness (FOSPET) were defined and measured on UHR-OCT images in all subjects and were compared between the two groups using unpaired t tests. The two thickness measurements in RP patients were correlated with visual acuity using Pearson correlation and linear regression. RESULTS: UHR-OCT demonstrated macular photoreceptor thinning in all RP patients. The difference in CFT between RP patients and normal subjects was not statistically significant (P = .103), but the difference in FOSPET between the two groups was significant (P = .003). Visual acuity showed a fair correlation with CFT (Pearson r = -0.43, r(2) = 0.187, P = .245) and an excellent correlation with FOSPET (Pearson r = -0.942, r(2) = 0.887, P < .0001). CONCLUSIONS: In the current study using UHR-OCT, a new thickness measurement termed FOSPET is demonstrated to quantify photoreceptor loss. FOSPET was statistically thinner in patients with RP and related diseases than in normal eyes and showed correlation with logMAR visual acuity. FOSPET appears to be a probable predictor of visual acuity in RP.

High-definition and 3-dimensional imaging of macular pathologies with high-speed ultrahigh-resolution optical coherence tomography.

OBJECTIVE: To assess high-speed ultrahigh-resolution optical coherence tomography (OCT) image resolution, acquisition speed, image quality, and retinal coverage for the visualization of macular pathologies. DESIGN: Retrospective cross-sectional study. PARTICIPANTS: Five hundred eighty-eight eyes of 327 patients with various macular pathologies. METHODS: High-speed ultrahigh-resolution OCT images were obtained in 588 eyes of 327 patients with selected macular diseases. Ultrahigh-resolution OCT using Fourier/spectral domain detection achieves approximately 3-mum axial image resolutions, acquisition speeds of approximately 25 000 axial scans per second, and >3 times finer resolution and >50 times higher speed than standard OCT. Three scan protocols were investigated. The first acquires a small number of high-definition images through the fovea. The second acquires a raster series of high-transverse pixel density images. The third acquires 3-dimensional OCT data using a dense raster pattern. Three-dimensional OCT can generate OCT fundus images that enable precise registration of OCT images with the fundus. Using the OCT fundus images, OCT results were correlated with standard ophthalmoscopic examination techniques. MAIN OUTCOME MEASURES: High-definition macular pathologies. RESULTS: Macular holes, age-related macular degeneration, epiretinal membranes, diabetic retinopathy, retinal dystrophies, central serous chorioretinopathy, and other pathologies were imaged and correlated with ophthalmic examination, standard OCT, fundus photography, and fluorescein angiography, where applicable. High-speed ultrahigh-resolution OCT generates images of retinal pathologies with improved quality, more comprehensive retinal coverage, and more precise registration than standard OCT. The speed preserves retinal topography, thus enabling the visualization of subtle changes associated with disease. High-definition high-transverse pixel density OCT images improve visualization of photoreceptor and pigment epithelial morphology, as well as thin intraretinal and epiretinal structures. Three-dimensional OCT enables comprehensive retinal coverage, reduces sampling errors, and enables assessment of 3-dimensional pathology. CONCLUSIONS: High-definition 3-dimensional imaging using high-speed ultrahigh-resolution OCT improves image quality, retinal coverage, and registration. This new technology has the potential to become a useful tool for elucidating disease pathogenesis and improving disease diagnosis and management.

Noninvasive volumetric imaging and morphometry of the rodent retina with high-speed, ultrahigh-resolution optical coherence tomography.

PURPOSE: To demonstrate high-speed, ultrahigh-resolution optical coherence tomography (OCT) for noninvasive, in vivo, three-dimensional imaging of the retina in rat and mouse models. METHODS: A high-speed, ultrahigh-resolution OCT system using spectral, or Fourier domain, detection has been developed for small animal retinal imaging. Imaging is performed with a contact lens and postobjective scanning. An axial image resolution of 2.8 mum is achieved with a spectrally broadband superluminescent diode light source with a bandwidth of approximately 150 nm at approximately 900-nm center wavelength. Imaging can be performed at 24,000 axial scans per second, which is approximately 100 times faster than previous ultrahigh-resolution OCT systems. High-definition and three-dimensional retinal imaging is performed in vivo in mouse and rat models. RESULTS: High-speed, ultrahigh-resolution OCT enabled high-definition, high transverse pixel density imaging of the murine retina and visualization of all major intraretinal layers. Raster scan protocols enabled three-dimensional volumetric imagingand comprehensive retinal segmentation algorithms allowed measurement of retinal layers. An OCT fundus image, akin to a fundus photograph was generated by axial summation of three-dimensional OCT data, thus enabling precise registration of OCT measurements to retinal fundus features. CONCLUSIONS: High-speed, ultrahigh-resolution OCT enables imaging of retinal architectural morphology in small animal models. OCT fundus images allow precise registration of OCT images and repeated measurements with respect to retinal fundus features. Three-dimensional OCT imaging enables visualization and quantification of retinal structure, which promises to allow repeated, noninvasive measurements to track disease progression, thereby reducing the need for killing the animal for histology. This capability can accelerate basic research studies in rats and mice and their translation into clinical patient care.

Quantification of photoreceptor layer thickness in normal eyes using optical coherence tomography.

OBJECTIVE: To demonstrate the ability to segment and analyze individual intraretinal layers, including the outer retinal complex (ORC; outer nuclear layer and inner and outer segments of the photoreceptor cells), in healthy eyes using images acquired from the latest commercially available optical coherence tomography (OCT) system (StratusOCT; Carl Zeiss Meditec, Inc., Dublin, CA) and from the ultrahigh resolution OCT (UHR-OCT) prototype. METHODS: Thirty-seven eyes from 37 healthy subjects underwent complete ophthalmologic examination using StratusOCT and UHR-OCT. ORC was identified and measured using a segmentation algorithm. RESULTS: For StratusOCT, mean weighted ORC thickness +/- SD was 91.1 +/- 7.9 microm, and mean weighted total retinal thickness +/- SD was determined to be 258.9 +/- 10.1 microm. For UHR-OCT, mean weighted ORC thickness +/- SD was 96.4 +/- 6.3 microm, and mean weighted total retinal thickness +/- SD was determined to be 263.4 +/- 9.2 mum. There was a higher rate of algorithm failure with UHR-OCT images. CONCLUSIONS: Photoreceptor layer thickness can be calculated by measuring ORC on OCT images using a macular segmentation algorithm. ORC values may serve as a useful objective parameter in determining the efficacy of various therapeutic modalities that target the photoreceptor layer in various diseases.

Ultrahigh-resolution optical coherence tomography of surgically closed macular holes.

OBJECTIVE: To evaluate retinal anatomy using ultrahigh-resolution optical coherence tomography (OCT) in eyes after successful surgical repair of full-thickness macular hole. METHODS: Twenty-two eyes of 22 patients were diagnosed as having macular hole, underwent pars plana vitrectomy, and had flat/closed macular anatomy after surgery, as confirmed with biomicroscopic and OCT examination findings. An ultrahigh-resolution-OCT system developed for retinal imaging, with the capability to achieve approximately 3-microm axial resolution, was used to evaluate retinal anatomy after hole repair. RESULTS: Despite successful closure of the macular hole, all 22 eyes had macular abnormalities on ultrahigh-resolution-OCT images after surgery. These abnormalities were separated into the following 5 categories: (1) outer foveal defects in 14 eyes (64%), (2) persistent foveal detachment in 4 (18%), (3) moderately reflective foveal lesions in 12 (55%), (4) epiretinal membranes in 14 (64%), and (5) nerve fiber layer defects in 3 (14%). CONCLUSIONS: With improved visualization of fine retinal architectural features, ultrahigh-resolution OCT can visualize persistent retinal abnormalities despite anatomically successful macular hole surgery. Outer foveal hyporeflective disruptions of the junction between the inner and outer segments of the photoreceptors likely represent areas of foveal photoreceptor degeneration. Moderately reflective lesions likely represent glial cell proliferation at the site of hole reapproximation. Thin epiretinal membranes do not seem to decrease visual acuity and may play a role in reestablishing foveal anatomy after surgery.

Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two- dimensional scanning mirror.

Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce two-photon imaging based on microelectromechanical systems (MEMS) scanners. Single crystalline silicon scanning mirrors that are 0.75 mm x 0.75 mm in size and driven in two dimensions by microfabricated vertical comb electrostatic actuators can provide optical deflection angles through a range of approximately16 degrees . Using such scanners we demonstrated two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.52 kHz.

Ultrahigh-resolution optical coherence tomography of canthaxanthine retinal crystals.

A case of crystalline retinopathy caused by prolonged ingestion of an oral tanning agent containing canthaxanthine is described. Color fundus photography and ultrahigh-resolution optical coherence tomography were performed.

Ultrahigh-resolution optical coherence tomography in patients with decreased visual acuity after retinal detachment repair.

OBJECTIVE: To assess microstructural changes in the retina that may explain incomplete visual recovery after anatomically successful repair of rhegmatogenous retinal detachments (RD) using ultrahigh-resolution optical coherence tomography (UHR OCT). DESIGN: Retrospective observational case series. PARTICIPANTS: Seventeen patients with decreased visual acuity after RD repair. Twelve patients had macula-involving and 5 had macula-sparing RDs. METHODS: The UHR OCT prototype capable of approximately 3 mum axial resolution was developed for clinical use. The UHR OCT images through the center of the fovea in 17 patients with visual complaints after RD surgery were obtained. Patients were either postoperative patients from the New England Eye Center or tertiary referrals. Baseline visual acuity, preoperative lens status, location of retinal detachment, macular involvement, and postoperative visual acuity were recorded. MAIN OUTCOME MEASURES: The UHR OCT images after RD repair. RESULTS: The UHR OCT images were obtained 1 to 84 months (median, 5 months) postoperatively. The mean preoperative logarithm of the minimum angle of resolution (logMAR) visual acuity was 1.37 (Snellen equivalent, 20/390). The mean postoperative logMAR visual acuity was 0.48 (Snellen equivalent, 20/60). Anatomical abnormalities that were detected included distortion of the photoreceptor inner/outer segments (IS/OS) junction in 14 of 17 patients (82%), epiretinal membranes in 10 of 17 patients (59%), residual subretinal fluid in 3 of 17 patients (18%), and cystoid macular edema in 2 of 17 patients (12%). Of the 5 patients with preoperative macula-on detachments, 4 had distortion of the outer retina after RD repair. CONCLUSIONS: The higher resolution of UHR OCT facilitates imaging of the IS/OS junction. Therefore, UHR OCT is able to confirm prior histopathologic findings that damage to photoreceptor outer segments may occur as a consequence of retinal detachment. This may explain poor postoperative visual acuity in eyes with anatomically successful repair.

Redefining lamellar holes and the vitreomacular interface: an ultrahigh-resolution optical coherence tomography study.

OBJECTIVES: To define optical coherence tomographic (OCT) criteria for the diagnosis of a lamellar macular hole, and to increase understanding of lamellar hole pathogenesis by examining fine anatomic features using ultrahigh-resolution optical coherence tomography (UHR OCT). DESIGN: Retrospective observational case series. PARTICIPANTS: Nineteen eyes of 18 patients with lamellar holes were imaged with UHR OCT between 2002 and 2004. METHODS: A UHR OCT system was developed for use in the ophthalmology clinic. All 6 UHR OCT images for each eye imaged were examined. Lamellar holes were diagnosed based on a characteristic OCT appearance. Criteria for the OCT diagnosis of a lamellar hole were as follows: (1) irregular foveal contour; (2) break in the inner fovea; (3) intraretinal split; and (4) intact foveal photoreceptors. From 1205 eyes of 664 patients imaged with UHR OCT, and retrospectively reviewed, 19 eyes of 18 patients were diagnosed with a lamellar hole based on these criteria. All 19 eyes were also imaged with standard resolution OCT. Their charts were retrospectively reviewed. MAIN OUTCOME MEASURES: Standard and ultrahigh-resolution OCT images. RESULTS: On chart review, clinical diagnosis of a lamellar hole was made in only 7 of 19 eyes (37%). Twelve of 19 eyes (63%) had an epiretinal membrane (ERM) on clinical examination. Ten of 19 eyes (53%) had a posterior vitreous detachment. On UHR OCT, 17 of 19 eyes (89%) had ERMs. Eleven ERMs had an unusual thick appearance on UHR OCT. Due to poor visual acuity, 4 eyes underwent vitrectomy. Only 1 of 4 surgeries (25%) was visually and anatomically successful. Another eye improved visually, but a lamellar hole persisted. One eye progressed to a full-thickness macular hole preoperatively, which reopened after surgery. One eye developed a full-thickness hole postoperatively. CONCLUSIONS: The diagnosis of a lamellar hole can be made based on OCT criteria, which could be applied to both standard and ultrahigh-resolution OCT. The increased resolution of UHR OCT sheds light on the pathogenesis of the lamellar hole. Epiretinal membranes were visualized on UHR OCT in the majority of eyes. Many ERMs had an unusual thick appearance on UHR OCT, which may represent either trapped vitreous or posterior hyaloid, and may help stabilize retinal anatomy. Conversely, ERM contraction may play a role in lamellar hole formation. Vitrectomy surgery was anatomically and visually successful in only 1 of 4 patients, suggesting caution when performing vitrectomy on lamellar holes.

Normal macular thickness measurements in healthy eyes using Stratus optical coherence tomography.

OBJECTIVE: To report normal macular thickness measurements in healthy eyes using the latest commercially available optical coherence tomography (OCT) mapping software, version 3.0, from the Stratus OCT (OCT3). METHODS: Thirty-seven eyes from 37 healthy subjects underwent a complete ophthalmologic examination, including OCT. Six radial scans, 6 mm in length and centered on the fovea, were obtained using the OCT3. Retinal thickness was automatically calculated by OCT mapping software. Measurements were displayed as the mean and standard deviation for each of the 9 regions defined in the Early Treatment Diabetic Retinopathy Study. RESULTS: Foveal thickness (mean thickness in the central 1000-microm diameter area) and central foveal thickness (mean thickness at the point of intersection of 6 radial scans) on the OCT3 were 212 +/- 20 and 182 +/- 23 microm, respectively. Macular thickness measurements were thinnest at the center of the fovea, thickest within 3-mm diameter of the center, and diminished toward the periphery of the macula. The temporal quadrant was thinner than the nasal quadrant. Central foveal thickness was also manually determined as 170 +/- 18 microm, approximately 12 microm less than the value automatically obtained from the OCT3 software. There was no correlation between age and foveal thickness (P = .80). CONCLUSIONS: Mean foveal thickness measurements were 38 to 62 microm thicker than previously reported values, while mean central foveal thickness measurements were 20 to 49 microm thicker than previously published values. This discrepancy should be considered when interpreting OCT scans.

Idiopathic juxtafoveal retinal telangiectasis: new findings by ultrahigh-resolution optical coherence tomography.

OBJECTIVE: To investigate the capabilities of ultrahigh-resolution optical coherence tomography (UHR OCT); to compare with the commercially available OCT standard-resolution system, StratusOCT, for imaging of idiopathic juxtafoveal retinal telangiectasis (IJT); and to demonstrate that UHR OCT provides additional information on disease morphology, pathogenesis, and management. DESIGN: Retrospective, observational, interventional case series. PARTICIPANTS: Nineteen eyes of 10 patients diagnosed with IJT in at least one eye. METHOD: All patients were imaged with UHR OCT and StratusOCT at the same visit. A subset of patients was also imaged before and after treatment of IJT. MAIN OUTCOME MEASURES: Ultrahigh- and standard-resolution cross-sectional tomograms of IJT pathology. RESULTS: Using both standard- and ultrahigh-resolution OCT, we identified the following features of IJT: (1) a lack of correlation between retinal thickening on OCT and leakage on fluorescein angiography, (2) loss and disruption of the photoreceptor layer, (3) cystlike structures in the foveola and within internal retinal layers such as the inner nuclear or ganglion cell layers, (4) a unique internal limiting membrane draping across the foveola related to an underlying loss of tissue, (5) intraretinal neovascularization near the fovea, and (6) central intraretinal deposits and plaques. In 63% of cases, the presence of abnormal vessels and a discontinuity of the photoreceptor layer correlated with visual acuity. CONCLUSIONS: Ultrahigh-resolution OCT improves visualization of the retinal pathology associated with IJT and allows identification of new features associated with it. Some of these features, such as discontinuity of the photoreceptor layer, are revealed only by UHR OCT.

Ultrahigh resolution optical coherence tomography in macular dystrophy.

PURPOSE: To visualize and investigate intraretinal changes in macular dystrophies with ultrahigh resolution optical coherence tomography (UHR OCT). DESIGN: Prospective observational case series. METHODS: setting: Department of Ophthalmology and Center for Biomedical Engineering and Physics, Christian Doppler Laboratory, Medical University of Vienna, Vienna, Austria. patients: Thirteen patients (23 eyes) with adult-onset foveomacular vitelliform dystrophy (AOFVD) and 14 patients (27 eyes) with Stargardt's disease (SD) or fundus flavimaculatus (FF). OBSERVATIONS: Imaging using a compact, new generation UHR OCT system, achieving considerably improved visualization of intraretinal layers, especially the photoreceptor layer. main outcome measures: UHR OCT tomograms visualizing intraretinal differences in morphology of AOFVD and SD/FF as location and extension of deposits and loss of photoreceptors. Central foveal thickness defined as distance between internal limiting membrane and photoreceptors/retinal pigment epithelium interface. RESULTS: Patients with AOFVD had a mostly intact photoreceptor layer, a central foveal thickness of 142 +/- 23 microm as well as subretinal deposits. Patients with SD generally had a diffuse degenerative change with a visible reduction in thickness of all intraretinal layers, resulting in a corresponding reduction of central foveal thickness (94 +/- 38 microm) and central loss of photoreceptors (PRs). Comparative central foveal thickness of patients with AOFVD and SD/FF was significantly different (P < .001). Patients with FF had pigment epithelial deposits and paracentral focal photoreceptor loss. CONCLUSIONS: UHR OCT is a clinically feasible tool for examining intraretinal changes, in particular photoreceptor atrophy in macular dystrophies and, therefore, has the potential to be an adequate imaging system for monitoring the course of disease.