Noninvasive imaging of visual system components in vivo is critical for understanding the causal mechanisms of retinal diseases and for developing therapies for their treatment. However, ultraviolet light needed to excite endogenous fluorophores that participate in metabolic processes of the retina is highly attenuated by the anterior segment of the human eye. In contrast, 2-photon excitation fluorescence imaging with pulsed infrared light overcomes this obstacle. Reducing retinal exposure to laser radiation remains a major barrier in advancing this technology to studies in humans. To increase fluorescence intensity and reduce the requisite laser power, we modulated ultrashort laser pulses with high-order dispersion compensation and applied sensorless adaptive optics and custom image recovery software and observed an over 300% increase in fluorescence of endogenous retinal fluorophores when laser pulses were shortened from 75 fs to 20 fs. No functional or structural changes to the retina were detected after exposure to 2-photon excitation imaging light with 20-fs pulses. Moreover, wide bandwidth associated with short pulses enables excitation of multiple fluorophores with different absorption spectra and thus can provide information about their relative changes and intracellular distribution. These data constitute a substantial advancement for safe 2-photon fluorescence imaging of the human eye.

We compare four optical coherence tomography techniques for noninvasive visualization of microcapillary network in the human retina and murine cortex. We perform phantom studies to investigate contrast-to-noise ratio for angiographic images obtained with each of the algorithm. We show that the computationally simplest absolute intensity difference angiographic OCT algorithm that bases only on two cross-sectional intensity images may be successfully used in clinical study of healthy eyes and eyes with diabetic maculopathy and branch retinal vein occlusion.

Microperimetry is a subjective ophthalmologic test used to assess retinal function at various specific and focal locations of the visual field. Historically, visible light has been described as ranging from 400 to 720 nm. However, we previously demonstrated that infra-red light can initiate visual transduction in rod photoreceptors by a mechanism of two-photon absorption by visual pigments. Here we introduce a newly designed and constructed two-photon microperimeter. We provide for the first time evidence of the presence of a nonlinear process occurring in the human retina based on psychophysical tests using newly developed instrumentation. Since infra-red light penetrates the aged front of the eye better than visible light, it has the potential for improved functional diagnostics in patients with age-related visual disorders.

We demonstrate in vivo three-dimensional (3-D) visualization of crystalline lens sutures in healthy eyes using swept source optical coherence tomography (SS-OCT). Volumetric data sets of the crystalline lenses were acquired and processed to obtain enhanced contrast projection images and to extract suture patterns in both anterior and posterior lens. The results presented different types of the sutures including Y-sutures, simple and complex star sutures. Age-related changes in suture arrangement were characterized quantitatively. Crystalline lens suture imaging with SS-OCT might be a useful tool in fundamental studies on development and ageing of human lens.

A three-dimensional optical coherence tomography (OCT) crystalline lens imaging method based on oblique scanning and image stitching is presented. The method was designed to increase OCT imaging volume of crystalline lens in vivo. A long-range swept-source (SS)-OCT imaging system, which can measure the entire anterior segment of eye in a single acquisition, is used to acquire one central volume and 4 extra volumes with different angles between optical axis of OCT instrument and the pupillary axis. The volumes are then stitched automatically by developed software. To show its effectiveness and verify its validity, we scanned the subjects before and after pupil dilation drops and compared the experimental results. By determining the number of voxels representing the signal from the crystalline lens in 3-D OCT images, our method can provide around 17% additional volumetric lens coverage compared with a regular imaging procedure. The proposed approach could be used clinically in early diagnosis of cortical cataract. Wider field of view offered by this method may facilitate more accurate lens biometry in its peripheral zones, which potentially contributes to understanding of lens shape modifications of the accommodating eye.

Examination of brain functions in small animal models may help improve the diagnosis and treatment of neurological conditions. Transcranial imaging of small rodents’ brains poses a major challenge for optical microscopy. Another challenge is to reduce the measurement time. We describe methods and algorithms for three-dimensional assessment of blood flow in the brains of small animals, through the intact skull, using spectral and time domain optical coherence tomography. By introducing a resonant scanner to the optical setup of the optical coherence tomography (OCT) system, we have developed and applied a high-speed spectral OCT technique that allows us to vary the imaging range of flow and to shorten measurement time. Multi-parameter signal analysis enables us to obtain both qualitative and quantitative information about flow velocity from the same set of data.