Infrared Spectroscopic Studies of Cells and Tissues: Triple Helix Proteins as a Potential Biomarker for Tumors

In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection. Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues. An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders. © 2013 Stelling et al

Quality assessment of corneal storage media and their components

Background: To keep the loss of endothelial cell density in donor corneas to a minimum, a storage medium which is adjusted to their nutritional needs is necessary. Different media, used either serum-supplemented or serum-free, are available. The quality of medium- and serum-batches as well as support of endothelial cell viability by the medium are to be tested with a quality assured screening system that allows routine examination. Methods: A screening system was developed which is based on cell-culture tests with the well-established human corneal endothelial cell line HCEC-12, and therefore can be performed without the need for donor corneas. The cells are plated at a defined density in cell-culture dishes, and are cultured for a defined period of time in the test media. Evaluation is carried out by assaying cell count, activity of cell metabolism (resazurin conversion), and determining the number of apoptotic and necrotic cells (combined vital staining with YO-PRO®-1/propidium iodide and subsequent flow cytometry). Results: Human corneal endothelial cells that are cultured in a medium which is adjusted to their nutritional needs achieve higher cell numbers and show a higher metabolic rate. Simultaneously, the percentage of apoptotic and necrotic cells is lower. The screening system developed in this study allows for easy and reliable detection of slightest differences between different media, different processing steps for same media, and different supplements, as well as different serum batches. Conclusions: The differentiated results show that the screening system is sensitive enough to show even minor quality differences. Therefore, it is more suitable than the hitherto commonly used growth assay with primary, mostly porcine, corneal endothelial cells

Comparison of two in vivo microscopy techniques to visualize alveolar mechanics

OBJECTIVE: In conventional in vivo microscopy, a three dimensional illustration of tissue is lacking. Concerning the microscopic analysis of the pulmonary alveolar network, surgical preparation of the thorax and fixation of the lung is required to place the microscope's objective. These effects may have influence on the mechanical behaviour of alveoli. Relatively new methods exist for in vivo microscopy being less invasive and enabling an observation without fixation of the lung. The aim of this study was to compare a fibered confocal laser scanning microscopy (FCLSM) with optical coherence tomography (OCT) in a mouse and a rabbit model. Moreover, FCLSM was also used endoscopically in the rabbit model. METHODS: Smallest possible thoracic windows were excised at the lower margin of the upper right lung lobe and an interpleural catheter inserted before re-coverage with a transparent membrane foil. The OCT-scanner was positioned by a motor driven translation stage. The imaging was gated to endinspiratory plateau. For CLSM, Fluorescein 0.1% was given into the central venous streak line. The confocal probe with a diameter of 650 microm was carefully positioned at the very same lung region. Images were directly recorded real-time and the observed region qualitatively compared with FD-OCT images. Additionally, in the rabbit model, CLSM was used endoscopically under bronchoscopic sight control. In a postprocessing analysis, images taken were analyzed and compared by using an "air index" (AI). RESULTS: In the mouse model, the very same region could be re-identified with both techniques. Concerning alveolar shape and size, qualitatively comparable images could be gained. The AI was 40.5% for the OCT and 40.1% for the CLSM images. In the rabbit, even an endoscopic view on alveoli was possible. Likewise AI was 43.2% for CLSM through the thoracic window and 43.6% from endoscopically. For the OCT an AI of 44.6% was analysed in the rabbit model. CONCLUSIONS: Both FD-OCT and CLSM provide high-resolution images of alveolar structure giving depth information that is beneficial to conventional microscopy. CLSM also facilitates endoscopic view on alveoli being well comparable to images gained through a thoracic window