We report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage.

A need exists to prolong the release of rapidly metabolized peptides of a low molecular weight, while delivering this peptide without environmental interference. Previous studies have used bovine serum albumin (BSA) as a model peptide to study release characteristics from alginate microcapsules. BSA is 66 kDa in size, while the peptide of interest here, connexin-43 carboxyl-terminus mimetic peptide (αCT1), is only 3.4 kDa. Such a change in size results in a much different set of release parameters. Our overall goal is a sustained release over a 24+ h period. Prolonged application of the peptide to a wound site to investigate therapeutic effects is ideal. As a result, a diffusion method using alginate microcapsules, along with the addition of poly-l-lysine and poly-l-ornithine, has been explored. We first aimed to establish and characterize our parameters through a set of parametric tests. Variations in polymer coating, change in pH, and changes in loading ratio have previously been shown to effect release using model compounds. Here we test specific changes in these parameters to show effects on the release of αCT1. Additionally, the microcapsules were attached to several biomaterials and surgical implants by ultraviolet cross-linking to study the effectiveness of attachment and delivery. Analysis and measurements using phase contrast microscopy, scanning electron microscopy, and atomic force microscopy were used to characterize changes in microcapsule morphology.

This study investigated the inflammatory effect of intraoperative mitomycin C (MMC) on adhesion reformation in human rectus muscles. Ten consecutive patients who underwent medial rectus resection had their postoperative rectus muscles divided into two groups: control group (n = 10) and MMC group (n = 10). In the MMC group, the muscle was soaked for 2 min with MMC, prepared as a 0.2 mg/mL (0.02%) solution. The 0.02% MMC reactions were examined using histological analysis with hematoxylin-eosin (inflammatory response) and Masson's trichrome (collagen fibrils), immunoreactivities of cyclooxygenase-II (inflammatory response), and collagen type I and III, scanning electron microscopy analysis to quantify the diameter and D-periodicity of collagen fibrils, and atomic force microscopy analysis to quantify the diameter, D-periodicity, and adhesion force of collagen fibrils. The rectus muscles treated with 0.02% MMC showed a significantly increased inflammatory response (p < 0.05), increased collagen density (p < 0.0001), increased fibril diameter (p < 0.001 or p < 0.05), and decreased fibril adhesion force (p < 0.005) compared to the rectus muscles in the control group. MMC simultaneously caused an inflammatory response as well as nanostructural and biomechanical property changes in the collagen fibril network.

Dermal bone is an important component of the teleost fins, and its ability to regenerate after fin amputation appears to be unlimited. The organic bone matrix contain type I collagen fibers, proteoglycans enriched in chondroitin sulfate, and noncollagenous matrix protein such as osteocalcin, osteopontin, and osteonectin. These molecules are synthesized by fin osteoblasts. Inorganic components chiefly consist of calcium and phosphate that form crystals of hydroxyapatite. Fin rays are described as models to study ossification. Due to this, the identification of the components involved in the synthesis of the organic and inorganic components of lepidotrichial bone are of great interest for the analysis of skeletal disorders in fish ossification. The present study investigates expression of alkaline phosphatase, osteopontin, osteocalcin, and chondroitin sulfate during pectoral fin regeneration in Carassius auratus gibelio. Alkaline phosphatase reaction has been found in the epidermis covering the wound, proximal blastema, near the cells that surround newly-formed lepidotrichia matrix and the tips of regenerating fin rays. Osteopontin has been observed throughout the regeneration blastema but excluded from the scleroblasts lining the inner side of the lepidotrichia. Osteocalcin and chondroitin sulfate expression coincides with the onset of mineralization of lepidotrichial matrix, suggesting its involvement in bone mineralization.

Transmission electron microscopy (TEM), fluorescence microscopy, and confocal Raman microscopy can be used to characterize ultrastructural and compositional heterogeneity of plant cell walls. In this study, TEM observations revealed the ultrastructural characterization of Cornus alba L. fiber, vessel, axial parenchyma, ray parenchyma, and pit membrane between cells, notably with the ray parenchyma consisting of two well-defined layers. Fluorescence microscopy evidenced that cell corner middle lamella was more lignified than adjacent compound middle lamella and secondary wall with variation in lignification level from cell to cell. In situ Raman images showed that the inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber cell wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose were visualized in the pit membrane areas. These complementary microanalytical methods provide more accurate and complete information with regard to ultrastructural and compositional characterization of plant cell walls.

Mesua ferrea is traditionally used for treating bleeding piles, fever, and renal diseases. It has been reported to have antimircobial activity. In the present study, antibacterial efficacy of leaf and fruit extracts on the growth and morphology of Staphylococcus aureus is evaluated. Both extracts display good antibacterial activity against S. aureus with a minimum inhibition concentration of 0.048 mg/mL. Both extracts are bacteriostatic at a minimum bacteriostatic concentration of 0.39 mg/mL. The bacteriostatic activity lasts for 24 h, and then cells start to grow as normal as shown in time-kill analysis. Scanning electron microscopy study indicated potential detrimental effect of the extracts of leaf and fruits of M. ferrea on the morphology of S. aureus. The treatment with the extracts caused extensive lysis of the cells, leakage of intracellular constituents, and aggregation of cytoplasmic contents forming an open meshwork of the matrix.