In vivo fluorescent imaging of the mouse retina using adaptive optics

In vivo imaging of the mouse retina using visible and near infrared wavelengths does not achieve diffraction-limited resolution due to wavefront aberrations induced by the eye. Considering the pupil size and axial dimension of the eye, it is expected that unaberrated imaging of the retina would have a transverse resolution of 2 microm. Higher-order aberrations in retinal imaging of human can be compensated for by using adaptive optics. We demonstrate an adaptive optics system for in vivo imaging of fluorescent structures in the retina of a mouse, using a microelectromechanical system membrane mirror and a Shack-Hartmann wavefront sensor that detects fluorescent wavefront.     

Contrast improvement of confocal retinal imaging by use of phase-correcting plates

We have developed a custom scanning laser ophthalmoscope that uses phase plates produced by photolithography to improve the contrast of human retinal images. We combined the scanning engine from a commercial real-time confocal microscope with custom optics to provide medium magnification imaging of the human retina (3 degrees field of view). Defocus and astigmatism were corrected with conventional trial lenses. Higher-order aberrations of the eye were corrected with a phase plate. A 633-nm laser was used for illuminating the retina. Inserting the phase plate into the optical system increased the contrast of a sample retinal vessel by 26%. Additionally, a number of small features of the retina, which were not visible with standard commercial imaging systems, became visible. There results illustrate that, with the rapid development of custom fabrication techniques for refractive corrections, improved diagnostic imaging with little added complexity to existing ophthalmic imaging systems may be realistic.     

Stokes vector analysis of adaptive optics images of the retina

A high-resolution Stokes vector imaging polarimeter was developed to measure the polarization properties at the cellular level in living human eyes. The application of this cellular level polarimetric technique to in vivo retinal imaging has allowed us to measure depolarization in the retina and to improve the retinal image contrast of retinal structures based on their polarization properties.     

Invivo depth-resolved birefringence measurements of the human retinal nerve fiber layer by polarization-sensitive optical coherence tomography

To our knowledge, this is the first demonstration of in vivo depth-resolved birefringence measurements of the human retinal nerve fiber layer (RNFL) by use of polarization-sensitive optical coherence tomography (PS-OCT). Because glaucoma causes nerve fiber layer damage, which may cause loss of retinal birefringence, PS-OCT is a potentially useful technique for the early detection of glaucoma. We built a fiber-based PS-OCT setup that produces quasi-real-time images of the human retina in vivo . Preliminary measurements of a healthy volunteer showed that the double-pass phase retardation per unit depth of the RNFL near the optic nerve head is 39+/-6( degrees )/100microm .     

人眼的高级像差对视功能的影响

准确测量和分析人眼的高级像差有助于更好地改善视觉,具有重要的实验和临床意义。测量了实际人眼的波前像差,分析了高级像差对人眼视功能的影响。像差数据由哈特曼-夏克（Hartmann-Shack）波前传感器分别对不同瞳孔的人眼进行测量而获得,视功能的评价方法采用了调制传递函数（MTF）、对比敏感度函数（CSF）、斯特雷尔（Strehl）比率。由人眼的波前像差数据和视网膜空间像调制度（AIM）曲线直接给出了视锐度值与对比敏感度值。矫正了离焦与像散后．被测者的平均视锐度值达到了1．0,对比敏感度在低频（20c／mm）处约为52,高频（80c／mm）处约为1;矫正了前4阶像差后,平均视锐度值可提高到1．2,对比敏感度在低频（20c／mm）处提高到96,高频（80c／mm）处提高到7。实际人眼各不相同,达到理想成像需矫正高级像差的阶数也不同。矫正了前九阶像差后,均可达到理想成像,斯特雷尔比率值大于0．8。     

Optically deviated focusing method based high-speed SD-OCT for in vivo retinal clinical applications

The aim of this study is to provide accurately focused, high-resolution in vivo human retinal depth images using an optically deviated focusing method with spectral-domain optical coherence tomography (SD-OCT) system. The proposed method was applied to increase the retinal diagnosing speed of patients with various values of retinal distances (i.e., the distance between the crystalline eye lens and the retina). The increased diagnosing speed was facilitated through an optical modification in the OCT sample arm configuration. Moreover, the optical path length matching process was compensated using the proposed optically deviated focusing method. The developed system was mounted on a bench-top cradle to overcome the motion artifacts. Further, we demonstrated the capability of the system by carrying out in vivo retinal imaging experiments. The clinical trials confirmed that the system was effective in diagnosing normal and abnormal retinal layers as several retinal abnormalities were identified using non-averaged single-shot OCT images, which demonstrate the feasibility of the method for clinical applications.     

Adaptive optics optical coherence tomography for retina imaging

When optical coherence tomography (OCT) is used for human retina imaging, its transverse resolution is limited by the aberrations of human eyes. To overcome this disadvantage, a high resolution imaging system for living human retina, which consists of a time domain OCT system and a 37-elements adaptive optics (AO) system, has been developed. The AO closed loop rate is 20 frames per second, and the OCT has a 6.7-μm axial resolution. In this paper, this system is introduced and the high resolution imaging results for retina are presented.     

In vivo Fourier-domain full-field OCT of the human retina with 1.5 million A-lines/s

In vivo full-field (FF) optical coherence tomography (OCT) images of human retina are presented by using a rapidly tunable laser source in combination with an ultra-high-speed camera. Fourier-domain FF-OCT provided a way to increase the speed of retinal imaging by parallel acquisition of A-scans. Reduced contrast caused by cross talk was observed only below the retinal pigment epithelium. With a 100Hz sweep rate, FF-OCT was fast enough to acquire OCT images with acceptable motion artifacts. FF-OCT allows ultrafast retinal imaging, boosting image speed by a lack of moving parts and a considerably higher irradiation power.     

High-speed, high-resolution Fourier-domain optical coherence tomography system for retinal imaging in the 1060 nm wavelength region

A high-speed (47,000 A-scans/s), ultrahigh axial resolution Fourier domain optical coherence tomography (OCT) system for retinal imaging at approximately 1060 nm, based on a 1024 pixel linear array, 47 kHz readout rate InGaAs camera is presented. When interfaced with a custom superluminescent diode (lambda(c) = 1020 nm, Deltalambda = 108 nm, Pout = 9 mW), the system provides 3.3 microm axial OCT resolution at the surface of biological tissue, approximately 4.5 microm in vivo in rat retina, approximately 5.7 microm in vivo in human retina, and 110 dB sensitivity for 870 microW incident power and 21 mus integration time. Retinal tomograms acquired in vivo from a human volunteer and a rat animal model show clear visualization of all intraretinal layer and increased penetration into the choroid.     

Real-time blind deconvolution of retinal images in adaptive optics scanning laser ophthalmoscopy

With the use of adaptive optics (AO), the ocular aberrations can be compensated to get high-resolution image of living human retina. However, the wavefront correction is not perfect due to the wavefront measure error and hardware restrictions. Thus, it is necessary to use a deconvolution algorithm to recover the retinal images. In this paper, a blind deconvolution technique called Incremental Wiener filter is used to restore the adaptive optics confocal scanning laser ophthalmoscope (AOSLO) images. The point-spread function (PSF) measured by wavefront sensor is only used as an initial value of our algorithm. We also realize the Incremental Wiener filter on graphics processing unit (GPU) in real-time. When the image size is 512 × 480 pixels, six iterations of our algorithm only spend about 10 ms. Retinal blood vessels as well as cells in retinal images are restored by our algorithm, and the PSFs are also revised. Retinal images with and without adaptive optics are both restored. The results show that Incremental Wiener filter reduces the noises and improve the image quality.     

Capability of a Liquid-Crystal Adaptive Optics System Based on Feedback Interferometry for Retinal Imaging

A modified arrangement of the adaptive optical retinal imaging system that we described previously is proposed to reduce the intensity loss in the system, so that it works properly even when the intensity of light incident on the eye is very weak. Experiments to verify the system performance were conducted using a conventional artificial eye with a specular reflector as a model retina. We observed that an image of a test target (mimicking a retina) blurred by an aberration plate (mimicking the ocular aberrations) was successfully restored in the adaptive optics fashion even when the intensity of the incident light probing the aberration of the eye became about 1.5% of that required in the previous system. Effect of a more realistic artificial eye with a scattering object as a model retina was also examined experimentally. We found that not only the ocular aberrations, but also the retinal scattering cause the wave-front deformations and that our adaptive optics system compensates for both of them simultaneously.     

High resolution mosaic image of capillaries in human retina by adaptive optics

Adaptive optics (AO) has been proved as a powerful means for high resolution imaging of human retina.Because of the pixel number of charge-coupled device (CCD) camera, the field of view is limited to 1°.In order to have image of capillaries around vivo human fovea, we use mosaic method to obtain high resolution image in area of 6°× 6°. Detailed structures of capillaries around fovea with resolution of 2.3μm are clearly shown. Comparison shows that this method has a much higher resolution than current clinic retina imaging methods.     

Retinal axial focusing and multi-layer imaging with a liquid crystal adaptive optics camera

With the help of adaptive optics(AO) technology, cellular level imaging of living human retina can be achieved.Aiming to reduce distressing feelings and to avoid potential drug induced diseases, we attempted to image retina with dilated pupil and froze accommodation without drugs. An optimized liquid crystal adaptive optics camera was adopted for retinal imaging. A novel eye stared system was used for stimulating accommodation and fixating imaging area. Illumination sources and imaging camera kept linkage for focusing and imaging different layers. Four subjects with diverse degree of myopia were imaged. Based on the optical properties of the human eye, the eye stared system reduced the defocus to less than the typical ocular depth of focus. In this way, the illumination light can be projected on certain retina layer precisely.Since that the defocus had been compensated by the eye stared system, the adopted 512 × 512 liquid crystal spatial light modulator(LC-SLM) corrector provided the crucial spatial fidelity to fully compensate high-order aberrations. The Strehl ratio of a subject with-8 diopter myopia was improved to 0.78, which was nearly close to diffraction-limited imaging. By finely adjusting the axial displacement of illumination sources and imaging camera, cone photoreceptors, blood vessels and nerve fiber layer were clearly imaged successfully.     

Label-free morphometry of retinal nerve fiber bundles by second-harmonic-generation microscopy

We present an application of second-harmonic generation (SHG) microscopy for label-free visualization and quantification of the morphology of nerve fibers in the retina. We show that SHG arises from the retinal nerve fiber layer and that it is specifically associated with uniformly oriented microtubules in the axons. The utility of axonal SHG is demonstrated for imaging the neuroanatomy of fresh ex vivo retina, and the three-dimensional structure of the axons of retinal ganglion cells is quantitatively analyzed.     

Contributions of the cornea and the lens to the aberrations of the human eye

The relative contributions of optical aberrations of the cornea and the crystalline lens to the final image quality of the human eye were studied. The aberrations of the entire eye were obtained from pairs of double-pass retinal images, and the aberrations of the cornea were obtained from videokeratographic data. Third-order spherical aberration and coma were significantly larger for the cornea than for the complete eye, indicating a significant role of the lens in compensating for corneal aberrations. In a second experiment retinal images were recorded in an eye before and after we neutralized the aberrations of the cornea by having the subjects wear swimming goggles filled with saline water, providing a direct estimate of the optical performance of the crystalline lens.     

Simultaneous swept source optical coherence tomography of the anterior segment and retina using coherence revival

We report on an implementation of coherence revival-based heterodyne swept source optical coherence tomography that is capable of simultaneously imaging the anterior and posterior eye. A polarization-encoded sample arm was used to efficiently focus orthogonal polarizations on the anterior segment and retina. Depth encoding was achieved using coherence revival, which allows for multiple depths within a sample to be simultaneously imaged and frequency encoded by carefully controlling the optical pathlength of each sample path. This design is a significant step toward whole-eye optical coherence tomography (OCT), which would enable customized ray-traced modeling of patient eyes to improve refractive surgical interventions and eliminate optical artifacts in retinal OCT diagnostics. We demonstrated the feasibility of this system for in vivo imaging by simultaneously acquiring images of the anterior segments and retinas in healthy human volunteers.     

In vivo imaging of intrinsic optical signals in chicken retina with functional optical coherence tomography

Visually evoked intrinsic optical signals (IOSs) were measured in vivo for the first time to our knowledge from all retina layers of the chicken retina with a combined functional optical coherence tomography and electroretinography (ERG) system. IOS traces were recorded from a small volume in the retina with 3.5 μm axial resolution and 7 ms time resolution. Comparison of the IOS and ERG traces shows a correlation between the positive and negative IOS measured from different retinal layers and the timing of the a and b waves in the ERG recording.     

Corticospinal interaction during isometric compensation for modulated forces with different frequencies

Background

The zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae.

Results

We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1 μg/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells.

Conclusions

The coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.     

Monte Carlo simulations on corneal and total aberrations before and after LASIK

Previous Monte Carlo simulations which manipulate each Zernike coefficient of total aberrations of human eyes indicate that interactions among wave-front aberrations can provide better visual quality for both pre-LASIK eyes and post-LASIK eyes. In this paper, we go a step further for Monte Carlo simulations which are not only on total aberrations but also on corneal aberrations, before and after LASIK, for a set of eyes. The corneal aberrations after LASIK are acquired through a new reliable method. Then a series of Monte Carlo simulations (including sign simulation, value simulation and meridional simulation) are performed by manipulating each Zernike coefficient (second through sixth-order) of total aberrations as well as corneal aberrations. The results are evaluated by modulation transfer function (MTF) ratio. Total aberrations for post-LASIK eyes still show MTF advantage over randomized aberrations, with slightly change as compared to that for pre-LASIK eyes. However, true corneal aberrations before and after LASIK have no MTF advantage over random aberrations. From this research, we draw conclusions: there is apparent advantage for the complete eye's true aberrations over random aberrations, whether pre-LASIK or post-LASIK, which does not exist for any biological optical surfaces in isolation, and the ability of adaptive mechanism of human eyes, increases after LASIK.