The neural representation of taste quality at the periphery.

The mammalian taste system is responsible for sensing and responding to the five basic taste qualities: sweet, sour, bitter, salty and umami. Previously, we showed that each taste is detected by dedicated taste receptor cells (TRCs) on the tongue and palate epithelium. To understand how TRCs transmit information to higher neural centres, we examined the tuning properties of large ensembles of neurons in the first neural station of the gustatory system. Here, we generated and characterized a collection of transgenic mice expressing a genetically encoded calcium indicator in central and peripheral neurons, and used a gradient refractive index microendoscope combined with high-resolution two-photon microscopy to image taste responses from ganglion neurons buried deep at the base of the brain. Our results reveal fine selectivity in the taste preference of ganglion neurons; demonstrate a strong match between TRCs in the tongue and the principal neural afferents relaying taste information to the brain; and expose the highly specific transfer of taste information between taste cells and the central nervous system.

In vivo microendoscopy of the hippocampus.

Conventional intravital microscopy has generally been limited to superficial brain areas such as the olfactory bulb, the neocortex, or the cerebellar cortex. In vivo optical microendoscopy uses gradient refractive index (GRIN) microlenses that can be inserted into tissue to image cells in deeper areas. This protocol describes in vivo microendoscopy of the mouse hippocampus. The general methodology can be applied to many deep brain regions and other areas of the body.

In vivo optical microendoscopy for imaging cells lying deep within live tissue.

Although in vivo microscopy has been pivotal in enabling studies of neuronal structure and function in the intact mammalian brain, conventional intravital microscopy has generally been limited to superficial brain areas such as the olfactory bulb, the neocortex, or the cerebellar cortex. For imaging cells in deeper areas, this article discusses in vivo optical microendoscopy using gradient refractive index (GRIN) microlenses that can be inserted into tissue. Our general methodology is broadly applicable to many deep brain regions and areas of the body. Microendoscopes are available in a wide variety of optical designs, allowing imaging across a range of spatial scales and with spatial resolution that can now closely approach that offered by standard water-immersion microscope objectives. The incorporation of microendoscope probes into portable miniaturized microscopes allows imaging in freely behaving animals. When combined with the broad sets of available fluorescent markers, animal preparations, and genetically modified mice, microendoscopic methods enable sophisticated experimental designs for probing how cellular characteristics may underlie or reflect animal behavior and life experience, in healthy animals and animal models of disease.

Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy.

The combination of intravital microscopy and animal models of disease has propelled studies of disease mechanisms and treatments. However, many disorders afflict tissues inaccessible to light microscopy in live subjects. Here we introduce cellular-level time-lapse imaging deep within the live mammalian brain by one- and two-photon fluorescence microendoscopy over multiple weeks. Bilateral imaging sites allowed longitudinal comparisons within individual subjects, including of normal and diseased tissues. Using this approach, we tracked CA1 hippocampal pyramidal neuron dendrites in adult mice, revealing these dendrites' extreme stability and rare examples of their structural alterations. To illustrate disease studies, we tracked deep lying gliomas by observing tumor growth, visualizing three-dimensional vasculature structure and determining microcirculatory speeds. Average erythrocyte speeds in gliomas declined markedly as the disease advanced, notwithstanding significant increases in capillary diameters. Time-lapse microendoscopy will be applicable to studies of numerous disorders, including neurovascular, neurological, cancerous and trauma-induced conditions.

In vivo fluorescence imaging with high-resolution microlenses.

Micro-optics are increasingly used for minimally invasive in vivo imaging, in miniaturized microscopes and in lab-on-a-chip devices. Owing to optical aberrations and lower numerical apertures, a main class of microlens, gradient refractive index lenses, has not achieved resolution comparable to conventional microscopy. Here we describe high-resolution microlenses, and illustrate two-photon imaging of dendritic spines on hippocampal neurons and dual-color nonlinear optical imaging of neuromuscular junctions in live mice.

High-speed, miniaturized fluorescence microscopy in freely moving mice.

A central goal in biomedicine is to explain organismic behavior in terms of causal cellular processes. However, concurrent observation of mammalian behavior and underlying cellular dynamics has been a longstanding challenge. We describe a miniaturized (1.1 g mass) epifluorescence microscope for cellular-level brain imaging in freely moving mice, and its application to imaging microcirculation and neuronal Ca(2+) dynamics.

Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans.

Sarcomeres are the basic contractile units of striated muscle. Our knowledge about sarcomere dynamics has primarily come from in vitro studies of muscle fibres and analysis of optical diffraction patterns obtained from living muscles. Both approaches involve highly invasive procedures and neither allows examination of individual sarcomeres in live subjects. Here we report direct visualization of individual sarcomeres and their dynamical length variations using minimally invasive optical microendoscopy to observe second-harmonic frequencies of light generated in the muscle fibres of live mice and humans. Using microendoscopes as small as 350 microm in diameter, we imaged individual sarcomeres in both passive and activated muscle. Our measurements permit in vivo characterization of sarcomere length changes that occur with alterations in body posture and visualization of local variations in sarcomere length not apparent in aggregate length determinations. High-speed data acquisition enabled observation of sarcomere contractile dynamics with millisecond-scale resolution. These experiments point the way to in vivo imaging studies demonstrating how sarcomere performance varies with physical conditioning and physiological state, as well as imaging diagnostics revealing how neuromuscular diseases affect contractile dynamics.

Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two- dimensional scanning mirror.

Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce two-photon imaging based on microelectromechanical systems (MEMS) scanners. Single crystalline silicon scanning mirrors that are 0.75 mm x 0.75 mm in size and driven in two dimensions by microfabricated vertical comb electrostatic actuators can provide optical deflection angles through a range of approximately16 degrees . Using such scanners we demonstrated two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.52 kHz.