ABSTRACT
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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.
Subject(s)
Amacrine Cells/physiology , Dendrites/physiology , Models, Neurological , Motion Perception/physiology , Orientation/physiology , Retina/cytology , Synapses/physiology , Acetylcholinesterase/genetics , Acetylcholinesterase/metabolism , Amacrine Cells/drug effects , Animals , Animals, Newborn , Calcium/metabolism , Computer Simulation , Disks Large Homolog 4 Protein , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Glutamic Acid/pharmacology , Guanylate Kinases/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motion Perception/drug effects , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Synapses/drug effects , Synapses/ultrastructure , Visual Pathways/physiologyABSTRACT
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.
Subject(s)
Fluorescent Dyes , Image Enhancement/instrumentation , Microscopy, Fluorescence/instrumentation , Microsurgery/instrumentation , Subtraction Technique/instrumentation , Surgery, Computer-Assisted/instrumentation , Computer Systems , Equipment Design , Equipment Failure Analysis , Infrared Rays , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
While upconverting lanthanide nanoparticles have numerous advantages over other exogenous contrast agents used in scanned multiphoton imaging, their long luminescence lifetimes cause images collected with non-descanned detection to be greatly blurred. We demonstrate herein the use of Richardson-Lucy deconvolution to deblur luminescence images obtained via multiphoton scanning microscopy. Images were taken of three dimensional models of colon and ovarian cancer following incubation with NaYF4:Yb,Er nanoparticles functionalized with an antibody for EGFR and folic acid respectively. Following deconvolution, images had a lateral resolution on par with the optimal performance of the imaging system used, ~1.2 µm, and an axial resolution of ~5 µm. Due to the relatively high multiphoton excitation efficiency of these nanoparticles, it is possible to follow binding of individual particles in tissue. In addition, their extreme photostability allows for prolonged imaging without significant loss in luminescence signal. With these advantageous properties in mind, we also discuss the potential application of upconverting lanthanide nanoparticles for tracking of specific, cancer relevant receptors in tissue.
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, â¼1.2 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 three-dimensional functional tissue imaging at exceptionally low excitation power densities.
Subject(s)
Ear/anatomy & histology , Image Interpretation, Computer-Assisted/methods , Lanthanoid Series Elements , Liposomes/chemical synthesis , Microscopy, Confocal/methods , Microscopy, Fluorescence, Multiphoton/methods , Nanoparticles , Animals , Contrast Media/chemical synthesis , Image Enhancement/methods , Lanthanoid Series Elements/chemistry , Mice , Nanoparticles/chemistry , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
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.
ABSTRACT
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.
