Profile for Michael J. Levene

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism. Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light. The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations. Images are collected with a large field-of-view (approximately 900 microm). In addition, we provide details on the non-survival surgical procedure and microscope setup. Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts. Resolution was sufficient to image dendritic spines near the soma of layer V neurons. A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex. Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

INTRODUCTION Two-photon fluorescence microscopy is an integral tool in the field of neuroscience research. Applications of two-photon microscopy to in vivo deep-brain imaging are limited due to substantial light scattering by dense neural tissue. Conventional in vivo imaging can only access the superficial 300-400 mum of neocortex and suffers from nonuniform intensity and poor signal-to-noise as the depth increases. This protocol presents a technique to produce high-quality images deep into the mouse neocortex by inserting a 1-mm right-angle glass microprism into the neocortex. The microprism serves to reflect the laser excitation light and the fluorescence emission off a high-reflective coating on the prism's hypotenuse to provide a side-on imaging perspective of multiple neocortical layers simultaneously. This "inverted periscope" technique allows for wide field-of-view imaging of layer V green fluorescent protein (GFP) cell bodies with their apical dendrites extending through the superficial layers. Microprisms maintain spatial resolutions capable of resolving dendritic spines. Furthermore, microprisms can image the network of blood vessels in the neocortex and blood flow through microcapillaries to obtain information such as red blood cell (RBC) flux and velocity.

Typical imaging depths with multiphoton microscopy (MPM) are limited to less than 300 mum in many tissues due to light scattering. Optical clearing significantly reduces light scattering by replacing water in the organ tissue with a fluid having a similar index of refraction to that of proteins. We demonstrate MPM of intact, fixed, cleared mouse organs with penetration depths and fields of view in excess of 2 mm. MPM enables the creation of large 3-D data sets with flexibility in pixel format and ready access to intrinsic fluorescence and second-harmonic generation. We present high-resolution images and 3-D image stacks of the brain, small intestine, large intestine, kidney, lung, and testicle with image sizes as large as 4,096 x 4,096 pixels.

Genuine U.S. Federal Reserve Notes have a consistent, two-component intrinsic fluorescence lifetime. This allows for detection of counterfeit paper money because of its significant differences in fluorescence lifetime when compared to genuine paper money. We used scanning two-photon laser excitation and the time-correlated single photon counting (TCSPC) method to sample a approximately 4 mm(2) region. Three types of counterfeit samples were tested. Four out of the nine counterfeit samples fit to a one-component decay. Five out of nine counterfeit samples fit to a two-component model, but are identified as counterfeit due to significant deviations in the longer lifetime component compared to genuine bills.

Cortical slices allow for simultaneous imaging of multiple cortical layers. However, slices lack native physiological inputs and outputs. Although in vivo, two-photon imaging preserves the native context, it is typically limited to a depth of <100 microm from the tissue-glass interface. Microprisms are a straightforward tool offering numerous advantages for into neocortical tissue.

Two-photon fluorescence lifetime imaging (FLIM) of molecules can reveal important information on the local microenvironment. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic co-enzyme, has a lifetime that depends strongly on enzymatic binding. We present a custom image-processing algorithm for raw fluorescence lifetime and amplitude data that produces an image showing spatially distinct NADH fluorescence lifetimes in slices of rat and human brain. NADH FLIM images were collected in control and epileptic rat tissue. Differences in spatial patterns of NADH lifetimes support the hypothesis that NADH binding, and thus metabolic capacity, is significantly different between groups. This type of analysis can provide information on metabolic states in pathological material.

Although fluorescence microscopy has proven to be one of the most powerful tools in biology, its application to the intact animal has been limited to imaging several hundred micrometers below the surface. The rest of the animal has eluded investigation at the microscopic level without excising tissue or performing extensive surgery. However, the ability to image with subcellular resolution in the intact animal enables a contextual setting that may be critical for understanding proper function. Clinical applications such as disease diagnosis and optical biopsy may benefit from minimally invasive in vivo approaches. Gradient index (GRIN) lenses with needle-like dimensions can transfer high-quality images many centimeters from the object plane. Here, we show that multiphoton microscopy through GRIN lenses enables minimally invasive, subcellular resolution several millimeters in the anesthetized, intact animal, and we present in vivo images of cortical layer V and hippocampus in the anesthetized Thy1-YFP line H mouse. Microangiographies from deep capillaries and blood vessels containing fluorescein-dextran and quantum dot-labeled serum in wild-type mouse brain are also demonstrated.