Action organizations within single dendritic spines in CA1 pyramidal neurons studies with two-photon photoactivation

The major cytoskeleton of dendritic spines is filamentous actin (F-actin).Organizations of F-actin within individual spines, however, have not beenelucidated.　I have here investigated sub-spine actin organizations usingtwo-photon photoactivation of PA-GFP fused with β-actin in rat CA1 pyramidalneurons in slice culture preparations.　I found segregated and discontinuousorganizations of two pools of F-actin, dynamic and stable pools, which tunedover with time constants of 1.2 min and 17 min, respectively.　The stable F-actinpool was localized at the base of spine head, often intruding into thick spine neck,while the dynamic pool occupied the rest of spine head.　Fractions of the stableF-actin pool were greater in larger spines, therefore, the entire F-actin poor wasmore stable in larger spines.　I succeeded in visualizing a retrograde flow ofF-actin in the dynamic pool from the apex to the base of spine, and found thatboth the speeds (0.2-1.2 μm/min) and lengths (0.2-0.7μm) of the F-actin flowwere greater in spines with larger head volumes.　Moreover, spine headsrapidly shrank when actin polymerization was blocked by latrunculin A,suggesting that the rate of actin polymerization in each spine actively andcontinuously determines the volume of spine head via the length of F-actin.Thus, I have revealed the sub-spine organizations of actin filaments that playkey role in spine structures and diversity

Intravital imaging-based analysis tools for vessel identification and assessment of concurrent dynamic vascular events

The vasculature undergoes changes in diameter, permeability and blood flow in response to specific stimuli. The dynamics and interdependence of these responses in different vessels are largely unknown. Here we report a non-invasive technique to study dynamic events in different vessel categories by multi-photon microscopy and an image analysis tool, RVDM (relative velocity, direction, and morphology) allowing the identification of vessel categories by their red blood cell (RBC) parameters. Moreover, Claudin5 promoter-driven green fluorescent protein (GFP) expression is used to distinguish capillary subtypes. Intradermal injection of vascular endothelial growth factor A (VEGFA) is shown to induce leakage of circulating dextran, with vessel-type-dependent kinetics, from capillaries and venules devoid of GFP expression. VEGFA-induced leakage in capillaries coincides with vessel dilation and reduced flow velocity. Thus, intravital imaging of non-invasive stimulation combined with RVDM analysis allows for recording and quantification of very rapid events in the vasculature

In vivo subcellular imaging of tumors in mouse models using a fluorophore-conjugated anti-carcinoembryonic antigen antibody in two-photon excitation microscopy

Recently, there has been growing interest in applying fluorescence imaging techniques to the study of various disease processes and complex biological phenomena in vivo. To apply these methods to clinical settings, several groups have developed protocols for fluorescence imaging using antibodies against tumor markers conjugated to fluorescent substances. Although these probes have been useful in macroscopic imaging, the specificity and sensitivity of these methods must be improved to enable them to detect micro-lesions in the early phases of cancer, resulting in better treatment outcomes. To establish a sensitive and highly specific imaging method, we used a fluorophore-conjugated anti-carcinoembryonic antigen (CEA) antibody to perform macroscopic and microscopic in vivo imaging of inoculated cancer cells expressing GFP with or without CEA. Macroscopic imaging by fluorescence zoom microscopy revealed that bio-conjugation of Alexa Fluor 594 to the anti-CEA antibody allowed visualization of tumor mass consisting of CEA-expressing human cancer cells, but the background levels were unacceptably high. In contrast, microscopic imaging using a two-photon excitation microscope and the same fluorescent antibody resulted in subcellular-resolution imaging that was more specific and sensitive than conventional imaging using a fluorescence zoom microscope. These results suggest that two-photon excitation microscopy in conjunction with fluorophore-conjugated antibodies could be widely adapted to detection of cancer-specific cell-surface molecules, both in cancer research and in clinical applications