Functional mapping of brain activity is important in elucidating how neural networks operate in the living brain. The whisker sensory system of rodents is an excellent model to study peripherally evoked neural activity in the central nervous system. Each facial whisker is represented by discrete modules of neurons all along the pathway leading to the neocortex. These modules are called “barrels” in layer 4 of the primary somatosensory cortex. Their location (approximately 300–500 μm below cortical surface) allows for convenient imaging of whisker-evoked neural activity in vivo. Fluorescence laminar optical tomography (FLOT) provides depth-resolved fluorescence molecular information with an imaging depth of a few millimeters. Angled illumination and detection configurations can improve both resolution and penetration depth. We applied angled FLOT (aFLOT) to record 3D neural activities evoked in the whisker system of mice by deflection of a single whisker in vivo. A 100 μm capillary and a pair of microelectrodes were inserted to the mouse brain to test the capability of the imaging system. The results show that it is possible to obtain 3D functional maps of the sensory periphery in the brain. This approach can be broadly applicable to functional imaging of other brain structures.

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T m ), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (⌬⌬G d 50°C ‫ס‬ −4 kcal/mole; ⌬T m ‫ס‬ −22°C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (⌬⌬G d 50°C ‫ס‬ −3 kcal/mole; ⌬T m ‫ס‬ −11°C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys 44 -Asp 45 peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (⌬⌬G d 50°C ‫ס‬ −1 kcal/mole; ⌬T m ‫ס‬ +2°C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (⌬⌬G d 50°C ‫ס‬ +1 kcal/mole; ⌬T m ‫ס‬ +17°C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.Keywords: Disulfide-bond; cross-link; protein stability; differential scanning calorimetry; denaturation; folding Supplemental material: See www.proteinscience.org.The conformational stability of a protein is important to its function. Certain diseases such as Alzheimer's, prion, and cystic fibrosis, are associated with misfolded, unfolded, or aggregated proteins (Kelly 1996;Harper and Lansbury, Jr. 1997;Horwich and Weissman 1997, Qu et al. 1997). Much has been characterized regarding contributions to protein stability of hydrogen bonds, i...

We have previously shown that the male sex steroid testosterone inhibits slightly, but the female sex steroid 17␤-estradiol (E 2 ) potentiates dramatically, the capsaicin receptor-mediated current in rat dorsal root ganglion (DRG) neurons. Here, we used pharmacological methods and the nociceptive behavioral test to determine whether there is a sex difference in capsaicininduced acute pain in rats in vivo and what mechanism underlies this sex difference. Results revealed that intradermal injection of capsaicin induced a dose-dependent nocifensive response in males and females, with the dose required to produce a comparable level of nociception being approximately 3-to 4-fold higher in males than in females. In addition, females during the proestrus stage exhibited significantly greater capsaicin-induced nocifensive responses compared with the estrus stage. Moreover, the female's enhanced sensitivity to the capsaicin-induced nocifensive response was completely reversed by ovariectomy 6 weeks before capsaicin injection. It is noteworthy that intradermal coinjection of E 2 but not progesterone with capsaicin potentiated the capsaicininduced nocifensive response in ovariectomized rats. Likewise, intradermal E 2 injection dose-dependently potentiated the capsaicin-induced nocifensive response in male rats. Furthermore, potentiation by E 2 of the capsaicin-induced nocifensive response in male rats was not significantly reduced by a selective protein kinase C (PKC) inhibitor or by a selective protein kinase A (PKA) inhibitor, indicating that neither PKC nor PKA was involved in the effect of E 2 . These data demonstrate that E 2 mediates the female's enhanced sensitivity to capsaicin-induced acute pain, consistent with potentiation by E 2 of the capsaicin receptor-mediated current in rat DRG neurons.

Optical coherence tomography (OCT) provides high-resolution, cross-sectional imaging of tissue microstructure in situ and in real-time, while fluorescence molecular imaging (FMI) enables the visualization of basic molecular processes. There are great interests in combining these two modalities so that the tissue's structural and molecular information can be obtained simultaneously. This could greatly benefit biomedical applications such as detecting early diseases and monitoring therapeutic interventions. In this research, an optical system that combines OCT and FMI was developed. The system demonstrated that it could co-register en face OCT and FMI images with a 2.4 × 2.4 mm field of view. The transverse resolutions of OCT and FMI of the system are both ~10 μm. Capillary tubes filled with fluorescent dye Cy 5.5 in different concentrations under a scattering medium are used as the phantom. En face OCT images of the phantoms were obtained and successfully co-registered with FMI images that were acquired simultaneously. A linear relationship between FMI intensity and dye concentration was observed. The relationship between FMI intensity and target fluorescence tube depth measured by OCT images was also observed and compared with theoretical modeling. This relationship could help in correcting reconstructed dye concentration. Imaging of colon polyps of APC min mouse model is presented as an example of biological applications of this co-registered OCT/FMI system.

A simple porogen-leaching method to fabricate macroporous cyclic acetal hydrogel cell scaffolds is presented. Optical coherence tomography (OCT) was applied for nondestructive imaging and quantitative characterization of the scaffold structures. High-resolution OCT reveals the microstructures of the engineered tissue scaffolds in three dimensions. It also enables subsequent image processing to investigate quantitatively several key morphological design parameters for macroporous scaffolds, including the volume porosity, pore interconnectivity, and pore size. Two image-processing algorithms were adapted: three-dimensional labeling was applied to assess the interconnectivity, and erosion was applied to assess the pore size. Scaffolds with different design parameters were imaged, characterized, and compared. OCT imaging and image processing successfully discriminated scaffolds made from different formulations in terms of volume porosity, interconnectivity, and pore size. The cell viability and their spread across the scaffolds were confirmed by the fluorescence microscopy co-registered with OCT.

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Noise is usually undesired yet inevitable in science and engineering. However, by introducing the engineered noise to the precise solution of Jones matrix elements, we break the fundamental limit of polarization multiplexing capacity of metasurfaces that roots from the dimension constraints of the Jones matrix. We experimentally demonstrate up to 11 independent holographic images using a single metasurface illuminated by visible light with different polarizations. To the best of our knowledge, it is the highest capacity reported for polarization multiplexing. Combining the position multiplexing scheme, the metasurface can generate 36 distinct images, forming a holographic keyboard pattern. This discovery implies a new paradigm for high-capacity optical display, information encryption, and data storage.

Optimization of regenerative medicine strategies includes the design of biomaterials, development of cell-seeding methods, and control of cell-biomaterial interactions within the engineered tissues. Among these steps, one paramount challenge is to non-destructively image the engineered tissues in their entirety to assess structure, function, and molecular expression. It is especially important to be able to enable cell phenotyping and monitor the distribution and migration of cells throughout the bulk scaffold. Advanced fluorescence microscopic techniques are commonly employed to perform such tasks; however, they are limited to superficial examination of tissue constructs. Therefore, the field of tissue engineering and regenerative medicine would greatly benefit from the development of molecular imaging techniques which are capable of non-destructive imaging of three-dimensional cellular distribution and maturation within a tissue-engineered scaffold beyond the limited depth of current microscopic techniques. In this review, we focus on an emerging depth-resolved optical mesoscopic imaging technique, termed Laminar Optical Tomography (LOT) or Mesoscopic Fluorescence Molecular Tomography (MFMT), which enables longitudinal imaging of cellular distribution in thick tissue engineering constructs at depths of a few millimeters and with relatively high resolution. The physical principle, image formation, and instrumentation of LOT/MFMT systems are introduced. Representative applications in tissue engineering include imaging the distribution of human mesenchymal stem cells (hMSCs) embedded in hydrogels, imaging of bio-printed tissues, and in vivo applications.