Michael J. Levene

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.

High-resolution, 2- and 3-dimensional imaging of uncut, unembedded tissue biopsy samples.

CONTEXT Despite continuing advances in tissue processing automation, traditional embedding, cutting, and staining methods limit our ability for rapid, comprehensive visual examination. These limitations are particularly relevant to biopsies for which immediate therapeutic decisions are most necessary, faster feedback to the patient is desired, and preservation of tissue for ancillary studies is most important. The recent development of improved tissue clearing techniques has made it possible to consider use of multiphoton microscopy (MPM) tools in clinical settings, which could address difficulties of established methods. OBJECTIVE To demonstrate the potential of MPM of cleared tissue for the evaluation of unembedded and uncut pathology samples. DESIGN Human prostate, liver, breast, and kidney specimens were fixed and dehydrated by using traditional histologic techniques, with or without incorporation of nucleic acid fluorescent stains into dehydration steps. A benzyl alcohol/benzyl benzoate clearing protocol was substituted for xylene. Multiphoton microscopy was performed on a home-built system. RESULTS Excellent morphologic detail was achievable with MPM at depths greater than 500 μm. Pseudocoloring produced images analogous to hematoxylin-eosin-stained images. Concurrent second-harmonic generation detection allowed mapping of collagen. Subsequent traditional section staining with hematoxylin-eosin did not reveal any detrimental morphologic effects. Sample immunostains on renal tissue showed preservation of normal reactivity. Complete reconstructions of 1-mm cubic samples elucidated 3-dimensional architectural organization. CONCLUSIONS Multiphoton microscopy on cleared, unembedded, uncut biopsy specimens shows potential as a practical clinical tool with significant advantages over traditional histology while maintaining compatibility with gold standard techniques. Further investigation to address remaining implementation barriers is warranted.

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.

Multiphoton microscopy of cleared mouse brain expressing YFP.

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.(1,2) However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light(3) do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.(4) Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

In vivo multiphoton microscopy of deep tissue with gradient index lenses

Gradient index lenses enable multiphoton microscopy of deep tissues in the intact animal. In order to assess their applicability to clinical research, we present in vivo multiphoton microscopy with gradient index lenses in brain regions associated with Alzheimers disease and Parkinsons disease in both transgenic and wild-type mice. We also demonstrate microscopy of ovary in wild type mouse using only intrinsic fluorescence and second harmonic generation, signal sources which may prove useful for both the study and diagnosis of cancer.

Clinical Measurement of von Willebrand Factor by Fluorescence Correlation Spectroscopy

BACKGROUND Identification of von Willebrand factor (vWF) abnormalities in a variety of conditions is hampered by the limitations of currently available diagnostic tests. Although direct multimer visualization by immunoelectrophoresis is a commonly used method, it is impractical as a routine clinical test. In this study, we used a biophysical analysis tool, fluorescence correlation spectroscopy (FCS), to measure vWF distributions. The goals were to develop a method that is quicker and simpler than vWF gel electrophoresis and to evaluate the potential of FCS as a clinical diagnostic technique. METHODS We analyzed plasma from 12 patients with type 1 von Willebrand disease (vWD), 14 patients with type 2 vWD, and 10 healthy controls using a fluctuation-based immunoassay approach. RESULTS FCS enabled identification and proper classification of type 1 and type 2 vWD, producing quantitative results that correspond to qualitative gel multimer patterns. FCS required minimal sample preparation and only a 5-min analysis time. CONCLUSIONS This study represents the first implementation of FCS for clinical diagnostics directly on human plasma. The technique shows potential for further vWF studies and as a generally applicable laboratory test method.

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.

Microprisms for in vivo Multiphoton Microscopy of Mouse Cortex

Microprisms inserted into the cortex of mouse enable in vivo multiphoton microscopy, rotating the field-of-view from parallel to perpendicular to the surface of cortex and allowing imaging of the full cortical thickness.

GRIN lenses for deep in vivo multiphoton imaging

Needle-like gradient index lenses are shown to be effective for acquiring multiphoton images from millimeters to centimeters deep in anesthetized animals. We present in vivo two-photon images in mouse of ovaries, of neurons from cortical layer V and hippocampus, and of capillaries several millimeters deep in the brain.