Intravital imaging of the mouse popliteal lymph node.

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network. Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Role of surface lysine residues of adipocyte fatty acid-binding protein in fatty acid transfer to phospholipid vesicles.

The tertiary structure of murine adipocyte fatty acid-binding protein (AFABP) is a flattened 10-stranded beta-barrel capped by a helix-turn-helix segment. This helical domain is hypothesized to behave as a "lid" or portal for ligand entry into and exit from the binding cavity. Previously, we demonstrated that anthroyloxy-labeled fatty acid (AOFA) transfer from AFABP to phospholipid membranes occurs by a collisional process, in which ionic interactions between positively charged lysine residues on the protein surface and negatively charged phospholipid headgroups are involved. In the present study, the role of specific lysine residues located in the portal and other regions of AFABP was directly examined using site-directed mutagenesis. The results showed that isoleucine replacement for lysine in the portal region, including the alphaI- and alphaII-helices and the beta C-D turn, resulted in much slower 2-(9-anthroyloxy)palmitate (2AP) transfer rates to acidic membranes than those of native AFABP. An additive effect was found for mutant K22,59I, displaying the slowest rates of FA transfer. Rates of 2AP transfer from "nonportal" mutants on the beta-G and I strands were affected only moderately; however, a lysine --> isoleucine mutation in the nonportal beta-A strand decreased the 2AP transfer rate. These studies suggest that lysines in the helical cap domain are important for governing ionic interactions between AFABP and membranes. Furthermore, it appears that more than one distinct region, including the alphaI-helix, alphaII-helix, beta C-D turn, and the beta-A strand, is involved in these charge-charge interactions.

Self-tuning fuzzy logic control for ultrasound hyperthermia with reference temperature based on objective functions.

The purpose of this paper is to develop and evaluate a self-tuning fuzzy logic controller for a scanned focused ultrasound hyperthermia system with the reference temperature (Tr) determined from objective functions. This work employs simulation programs to develop the power deposition for the scanned focused ultrasound system and to solve the responses of temperature profiles based on the transient bioheat transfer equation. A fuzzy logic control algorithm is employed to determine the output power level for the heating system and an observer for blood perfusion variation is used to enhance the capability of the controller to adjust the required output power level for the treatment due to the drastic change of the blood perfusion. The reference temperature (Tr) for the controller is based on objective functions to tune its value during the heating process, while a control temperature (Tc) from the thermosensors located in the tumor region is used as the input for the controller. The objective function based on the entire temperature profile is used to evaluate the appropriateness of the heating temperature distribution for a time-variational blood perfusion. Simulation results demonstrate that the tumor region can be rapidly heated to the desired temperature level and maintained at that level despite blood perfusion variation. The resulting temperature profile, the objective function, and the output power level are related to the magnitude of blood perfusion, but are almost independent of the Tc location and the initial setting value of Tr. The fuzzy logic control algorithm with Tr determined from objective functions can be used for controlling the entire temperature distribution through a single control temperature, and the combination of control and optimization allows appropriate temperature fields to be created during the entire heating process. The control algorithm does not require the accurate prior knowledge of the locations of the thermosensors and the appropriate setting value for Tr.

Prevalence and predictors of undercorrected refractive errors in the Victorian population.

PURPOSE: To investigate the prevalence and predictors of undercorrected refractive errors in the Victorian population. METHODS: In this prospective study, a population-based sample of residents was recruited. The improvement in visual acuity with subjective refraction was assessed. Several individual characteristics were investigated as predictors of undercorrected refractive error. RESULTS: There were 5,615 eligible residents, of which 4,735 (84%) participated in the study (53% were women). In all, 466 participants (10%) had significant undercorrected refractive error leading to an improvement of 1 or more lines of visual acuity with refraction. Age was the most important predisposing factor. The risk of undercorrected refractive error increased by 1.8 times for every decade of life starting at 40 years of age. The next most important factor was the absence of distance refractive correction. These individuals were 6.8 times more at risk compared with those who wore distance spectacles. Other significant predictors of undercorrected refractive error were the presence of cataract and European or Middle Eastern languages spoken at home. People with tertiary education or hypermetropia were less likely to need refractive error improvement. Gender, country of birth, and employment status did not have any statistically significant effect after controlling for confounders. CONCLUSIONS: The results of this study disclose people in the community who are more at risk of compromising their vision because of undercorrected refractive errors. A campaign is warranted to alert people that it may be possible to improve their vision.

Letter contrast sensitivity function of the eye.

PURPOSE: To provide empirical data of letter CSF for various levels of defocus under controlled conditions of luminance and age. METHODS: Corrected distance visual acuities were tested at different levels of contrast and defocus. An experiment was conducted using the Medmont visual acuity tester on 10 young subjects and under normal room lighting. RESULTS: Empirical data of visual acuity were obtained for 7 levels of contrast (5, 10, 15, 25, 40, 60, 80%) and defocus levels of 0, +1 and +2D. A mathematical model was derived (R2=0.995) and this can be used to estimate visual acuity at various contrast levels for defocus of < or =+2D. CONCLUSION: This information is useful for the clinician as normative data and for further development of optical models to predict visual performance of the eye.

Anatomically accurate, finite model eye for optical modeling.

There is a need for a schematic eye that models vision accurately under various conditions such as refractive surgical procedures, contact lens and spectacle wear, and near vision. Here we propose a new model eye close to anatomical, biometric, and optical realities. This is a finite model with four aspheric refracting surfaces and a gradient-index lens. It has an equivalent power of 60.35 D and an axial length of 23.95 mm. The new model eye provides spherical aberration values within the limits of empirical results and predicts chromatic aberration for wavelengths between 380 and 750 nm. It provides a model for calculating optical transfer functions and predicting optical performance of the eye.

The prediction of spherical aberration with schematic eyes.

Many model eyes have been proposed; they differ in optical characteristics and therefore have different aberrations and image quality. In predicting the visual performance of the eye, we are most concerned with the central foveal vision. Spherical aberration is the only on-axis monochromatic aberration and can be used as a criterion to assess the degree of resemblance of eye models to the human eye. We reviewed and compiled experimental values of the spherical aberration of the eye, calculated the spherical aberration of several different categories of model eyes and compared the calculated results to the experimental data. Results show an over-estimation of spherical aberration by all models, the finite schematic eyes predicting values of spherical aberration closest to the experimental data. Current model eyes do not predict the average experimental values of the spherical aberration of the eye. A new model eye satisfying this assessment criterion is required for investigations of the visual performance of the eye.