Thomas H. Chia

Microprisms for In Vivo Multilayer Cortical Imaging

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 <500 microm. In addition, simultaneous imaging of multiple cortical layers is difficult due to the stratified organization of the cortex. We demonstrate the use of 1-mm microprisms for in vivo, two-photon neocortical imaging. These prisms enable simultaneous imaging of multiple cortical layers, including layer V, at an angle typical of slice preparations. Images were collected from the mouse motor and somatosensory cortex and show a nearly 900-microm-wide field of view. At high-magnification imaging using an objective with 1-mm of coverglass correction, resolution is sufficient to resolve dendritic spines on layer V neurons. Images collected using the microprism are comparable to images collected from a traditional slice preparation. Functional imaging of blood flow at various neocortical depths is also presented, allowing for quantification of red blood cell flux and velocity. H&E staining shows the surrounding tissue remains in its native, stratified organization. Estimation of neuronal damage using propidium iodide and a fluorescent Nissl stain reveals cell damage is limited to <100 microm from the tissue-glass interface. Microprisms are a straightforward tool offering numerous advantages for into neocortical tissue.

Multiphoton fluorescence lifetime imaging of intrinsic fluorescence in human and rat brain tissue reveals spatially distinct NADH binding

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.

Multiphoton microscopy of cleared mouse organs

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.

Detection of counterfeit U.S. paper money using intrinsic fluorescence lifetime

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.

In vivo imaging of deep cortical layers using a microprism.

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.

Multi-layer in vivo imaging of neocortex using a microprism.

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 μm 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.

Multiphoton fluorescence imaging of NADH to quantify metabolic changes in epileptic tissue in vitro

A powerful advantage of multiphoton microscopy is its ability to image endogenous fluorophores such as the ubiquitous coenzyme NADH in discrete cellular populations. NADH is integral in both oxidative and non-oxidative cellular metabolism. NADH loses fluorescence upon oxidation to NAD+; thus changes in NADH fluorescence can be used to monitor metabolism. Recent studies have suggested that hypo metabolic astrocytes play an important role in cases of temporal lobe epilepsy (TLE). Current theories suggest this may be due to defective and/or a reduced number of mitochondria or dysfunction of the neuronal-astrocytic metabolic coupling. Measuring NADH fluorescence changes following chemical stimulation enables the quantification of the cellular distribution of metabolic anomalies in epileptic brain tissue compared to healthy tissue. We present what we believe to be the first multiphoton microscopy images of NADH from the human brain. We also present images of NADH fluorescence from the hippocampus of the kainate-treated rat TLE model. In some experiments, human and rat astrocytes were selectively labeled with the fluorescent dye sulforhodamine 101 (SR101). Our results demonstrate that multiphoton microscopy is a powerful tool for assaying the metabolic pathologies associated with temporal lobe epilepsy in humans and in rodent models.

Counterfeit money detection by intrinsic fluorescence lifetime

Genuine U.S. Federal Reserve Notes have a consistent, two-component intrinsic fluorescence lifetime. We used scanning two-photon laser excitation and the time-correlated single photon counting method to identify three different types of counterfeit U.S. paper money.

Microprisms for In Vivo Multiphoton Microscopy of Mouse Cortex

Fluorescence microscopy of cortical slices, yielding ready access to all six layers of cortex, has proven to be a powerful technique in neurophysiology, however it lacks the context of in vivo experiments. In vivo microscopy, primarily multiphoton microscopy, provides this context but without ready access to deeper layers and typically involves imaging of a field-of-view that is roughly parallel to the cortical layers. Needle-like gradient index (GRIN) lenses have been used as invasive relay lenses to access deeper brain structures, however these lenses damage the apical dendrites of the neurons of interest during insertion into the cortex, and are therefore of limited use for functional cortical imaging.We present here the use of micro-prisms for performing in vivo multiphoton microscopy of mouse cortex. Small (∼1 mm ) prisms with a reflective coating on the hypotenuse act as a miniature periscope, rotating the image plane from one parallel to the cortical layers to one that is perpendicular to the layers. This enables simultaneous imaging of the entire thickness of cortex, much as is done it cortical slice preparations, while maintaining a large degree of the in vivo context.

Microprisms for in vivo Multiphoton Microscopy of Cortex

We demonstrate the use of microprisms for in vivo multiphoton microscopy of mouse cortex. These prisms enable a point-of-view more typical of ex vivo, cortical slice preparations, but in an in vivo context.