Summary The starburst amacrine cell in the mouse retina presents an opportunity to examine the precise role of sensory input location on neuronal computations. Using visual receptive field mapping, glutamate uncaging, two-photon Ca2+ imaging, and genetic labeling of putative synapses, we identify a unique arrangement of excitatory inputs and neurotransmitter release sites on starburst amacrine cell dendrites: the excitatory input distribution is skewed away from the release sites. By comparing computational simulations with Ca2+ transients recorded near release sites, we show that this anatomical arrangement of inputs and outputs supports a dendritic mechanism for computing motion direction. Direction selective Ca2+ transients persist in the presence of a GABA-A receptor antagonist, though the directional tuning is reduced. These results indicate a synergistic interaction between dendritic and circuit mechanisms for generating direction selectivity in the starburst amacrine cell.

Control of the two strongest upconversion emission lines in NaYF4:Yb3+, Er3+ nanoparticles is demonstrated by varying the excitation repetition rate. This technique may enable new multiplexed sensing modalities based on multicolor luminescent nanoparticles, currently contemplated for biomedical imaging and diagnostics.

Abstract. The use of upconverting lanthanide nanoparticles in fast-scanning microscopy is hindered by a long luminescence decay time, which greatly blurs images acquired in a nondescanned mode. We demonstrate herein an image processing method based on Richardson-Lucy deconvolution that mitigates the detrimental effects of their luminescence lifetime. This technique generates images with lateral resolution on par with the system's performance, ∼1.2 μm, while maintaining an axial resolution of 5 μm or better at a scan rate comparable with traditional two-photon microscopy. Remarkably, this can be accomplished with near infrared excitation power densities of 850 W∕cm 2 , several orders of magnitude below those used in two-photon imaging with molecular fluorophores. By way of illustration, we introduce the use of lipids to coat and functionalize these nanoparticles, rendering them water dispersible and readily conjugated to biologically relevant ligands, in this case epidermal growth factor receptor antibody. This deconvolution technique combined with the functionalized nanoparticles will enable threedimensional functional tissue imaging at exceptionally low excitation power densities.

SUMMARY Visually-guided behavior can depend critically on detecting the direction of object movement. This computation is first performed in the retina where direction is encoded by direction-selective ganglion cells (DSGCs) that respond strongly to an object moving in the preferred direction and weakly to an object moving in the opposite, or null, direction (reviewed in [1]). These DSGCs come in multiple types that are classified based on their morphologies, response properties and targets in the brain. This study focuses on two types – ON and ON-OFF DSGCs. Though animals can sense motion in all directions, the preferred directions of DSGCs in adult retina cluster along distinct directions that we refer to as the cardinal axes. ON DSGCs have three cardinal axes – temporal, ventral and dorsonasal – while ON-OFF DSGCs have four – nasal, temporal, dorsal, and ventral. How these preferred directions emerge during development is still not understood. Several studies have demonstrated that ON [2] and ON-OFF DSGCs are well tuned at eye-opening, and even a few days prior to eye-opening, in rabbits [3], rats [4] and mice [5–8], suggesting that visual experience is not required to produce direction selective tuning. However, here we show that at eye-opening the preferred directions of both ON and ON-OFF DSGCs are diffusely distributed and that visual deprivation prevents the preferred directions from clustering along the cardinal axes. Our findings indicate a critical role for visual experience in shaping responses in the retina.

Abstract. Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

SUMMARYVisually-guided behavior can depend critically on detecting the direction of object movement. This computation is first performed in the retina where direction is encoded by direction-selective ganglion cells (DSGCs) that respond strongly to an object moving in the preferred direction and weakly to an object moving in the opposite, or null, direction (reviewed in [1]). These DSGCs come in multiple types that are classified based on their morphologies, response properties and targets in the brain. This study focuses on two types -ON and ON-OFF DSGCs. Though animals can sense motion in all directions, the preferred directions of DSGCs in adult retina cluster along distinct directions that we refer to as the cardinal axes. ON DSGCs have three cardinal axestemporal, ventral and dorsonasal -while ON-OFF DSGCs have four -nasal, temporal, dorsal, and ventral. How these preferred directions emerge during development is still not understood. Several studies have demonstrated that ON [2] and ON-OFF DSGCs are well tuned at eye-opening, and even a few days prior to eye-opening, in rabbits [3], rats [4] and mice [5][6][7][8], suggesting that visual experience is not required to produce direction selective tuning. However, here we show that at eye-opening the preferred directions of both ON and ON-OFF DSGCs are diffusely distributed and that visual deprivation prevents the preferred directions from clustering along the cardinal axes. Our findings indicate a critical role for visual experience in shaping responses in the retina. HHS Public Access RESULTS Clustering of DSGC preferred directions along cardinal axes after eye-opening requires visual experienceWe used two-photon calcium imaging to study the development of DSGC populations in mouse retina. This imaging technique has proven to be quite powerful in characterizing the receptive field properties of retinal ganglion cells [9][10][11][12]. We loaded retinas of WT mice near eye-opening (P13-P14) and in adulthood (>P30) with the calcium dye Oregon green 488 BAPTA-1 hexapotassium salt (OGB-1) via electroporation, thus uniformly labeling the ganglion cell layer ( Figure 1A; see Supplemental Information). Ventral portions of the retina were stimulated with light centered at 385 nm to maximally activate UV-cones [13,14] while minimizing cross talk with the imaging detectors ( Figure S1). This approach allowed us to record from all DSGC subtypes within a single field of view (~40000 μm 2 ).To identify DSGCs, we first used full field UV-light flashes to classify cells as ON, OFF or ON-OFF retinal ganglion cells (RGCs) ( Figure S1; Table S1). We found that around 80% of the cells in the ganglion cell layer of young and adult retinas responded to light flashes with changes in fluorescence (Table S1), similar to the RGC percentages reported in previous studies [9,11,12]. In addition, over development we observed a decrease in the percentage of ON-OFF RGCs (28.03% at P13-14 to 20.84% in adult) (Table S1) [15].Second, we used light bars on a dark background drifting in 8 dir...

Upconverting lanthanide nanoparticles overcome many of the problems associated with more traditionally used luminescent contrast agents, such as photobleaching, auto°uorescence, cytotoxicity and phototoxicity. For this reason, they are an attractive choice for biomedical imaging applications, particularly for imaging in living tissues. The last decade has seen numerous improvements to these nanocrystals, but a comprehensive guide to the synthesis of upconverting lanthanide nanoparticles has not yet been written. Methods vary from paper to paper and from group to group, and results vary between research groups for each method. For this reason, development of these nanoparticles remains a signi¯cant endeavor for any research group interested in joining the¯eld. In this review, we look at the varying synthetic methods employed over the last decade and detail methodology for a select few that have been favored in the¯eld.

In the interest of generating new biomedical sensing techniques as well as improving those that currently exist, a great deal of attention has been given to upconverting lanthanide nanoparticles in recent years. In order to develop these nanoparticles for use in multiplexed and ratiometric sensing techniques, many recent studies have focused on experimental control of their emission wavelengths. Here we describe a new method for controlling the relative intensity of green and red emission bands in NaYF4:Yb3+, Er3+ nanoparticles via control of the excitation pulse repetition rate. Using this method, particles of the same composition may be tuned to produce red and green light in user-defined ratios. We discuss the mechanism behind this control as well as potential applications that could make use of this property, specifically in super resolution imaging techniques.