Noncontact, depth-resolved, optical probing of retinal response to visual stimulation with a <10-m spatial resolution, achieved by using functional ultrahigh-resolution optical coherence tomography (fUHROCT), is demonstrated in isolated rabbit retinas. The method takes advantage of the fact that physiological changes in dark-adapted retinas caused by light stimulation can result in local variation of the tissue reflectivity. fUHROCT scans were acquired from isolated retinas synchronously with electrical recordings before, during, and after light stimulation. Pronounced stimulusrelated changes in the retinal reflectivity profile were observed in the inner͞outer segments of the photoreceptor layer and the plexiform layers. Control experiments (e.g., dark adaptation vs. light stimulation), pharmacological inhibition of photoreceptor function, and synaptic transmission to the inner retina confirmed that the origin of the observed optical changes is the altered physiological state of the retina evoked by the light stimulus. We have demonstrated that fUHROCT allows for simultaneous, noninvasive probing of both retinal morphology and function, which could significantly improve the early diagnosis of various ophthalmic pathologies and could lead to better understanding of pathogenesis.electroretinogram ͉ functional optical coherence tomography ͉ inner plexiform layer ͉ photoreceptors ͉ retinal imaging T he vertebrate retina consists of several distinct layers: nuclear layers containing cell bodies can be differentiated from plexiform layers with axons and dendrites forming the neuronal network that preprocesses light-evoked signals before transmission to the brain. Early stages of retinal disorders are often confined to one of these layers and are manifested by both morphological abnormalities and impaired physiological responses. Detection of such pathologies requires high-resolution imaging methods. Various imaging modalities such as fundus photography, ultrasound imaging, and optical coherence tomography (OCT) are clinically used for imaging retinal morphology. OCT is an emerging imaging technique that allows for noncontact, in vivo visualization of biological tissue morphology with a micrometer-scale resolution at imaging depths of 1-2 mm (1-3). Currently, electrophysiological tests such as electroretinography (ERG) (4) and multifocal ERG (5) are used for clinical assessment of retinal function.More then 25 years ago, it was observed that the isolated retina when stimulated with visible light changes the amount of transmitted near-infrared light (NIR) (6, 7). Photoreceptors (PRs) were determined to be the main source of this effect, and in the following years, this method was used for investigation and quantitative evaluation of the activation of the PR G protein transducin and the time course of transduction events (8-10 and reviewed in ref. 11). In the last few years, other physiological processes at the cellular and subcellular level such as membrane depolarization (12), cell swelling (13), and altered metabolism...

Visualizing deep-brain vasculature and hemodynamics is key to understanding brain physiology and pathology. Among the various adopted imaging modalities, multiphoton microscopy (MPM) is well-known for its deep-brain structural and hemodynamic imaging capability. However, the largest imaging depth in MPM is limited by signal depletion in the deep brain. Here we demonstrate that quantum dots are an enabling material for significantly deeper structural and hemodynamic MPM in mouse brain in vivo. We characterized both three-photon excitation and emission parameters for quantum dots: the measured three-photon cross sections of quantum dots are 4–5 orders of magnitude larger than those of conventional fluorescent dyes excited at the 1700 nm window, while the three-photon emission spectrum measured in the circulating blood in vivo shows a slight red shift and broadening compared with ex vivo measurement. On the basis of these measured results, we further demonstrate both structural and hemodynamic three-photon microscopy in the mouse brain in vivo labeled by quantum dots, at record depths among all MPM modalities at all demonstrated excitation wavelengths.

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1700-nm window has been demonstrated to be a promising excitation window for deep-tissue multiphoton microscopy (MPM). Long working-distance water immersion objective lenses are typically used for deep-tissue imaging. However, absorption due to immersion water at 1700 nm is still high and leads to dramatic decrease in signals. In this paper, we demonstrate measurement of absorption spectrum of deuterium oxide (D2O) from 1200 nm to 2600 nm, covering the three low water-absorption windows potentially applicable for deep-tissue imaging (1300 nm, 1700 nm, and 2200 nm). We apply this measured result to signal enhancement in MPM at the 1700-nm window. Compared with water immersion, D2O immersion enhances signal levels in second-harmonic generation imaging, 3-photon fluorescence imaging, and third-harmonic generation imaging by 8.1, 24.8, and 24.7 times with 1662-nm excitation, in good agreement with theoretical calculation based on our absorption measurement. This suggests D2O a promising immersion medium for deep-tissue imaging.