Investigations on structure-property-relationship of self-associating photosensitizers by means of time-resolved spectroscopy

Die Struktur-Eigenschafts-Beziehung von ausgewählten tetrapyrrolischen Molekülen (sogenannte Photosensibilisatoren) wurde untersucht in homogener Lösung und in vitro mittels zeitaufgelöster Laserspektroskopie und Mikroskopie. Es konnte gezeigt werden, dass das beobachtete Maximum der photodynamischen Effizienz von amphiphilen Photosensibilisatoren auf den unterschiedlichen Aggregationsgrad der Moleküle zurückzuführen ist. Aggregate der untersuchten lipophilen Photosensiblisatoren konnten erstmals eindeutig in dem Zellinneren nachgewiesen werden. Zur Charakterisierung der Energierelaxationsmechanismen in J-Aggregaten von meso-Tetra-(sulphonatophenyl)porphyrin (TPPS4) wurde ein Model basierend auf der Theorie der Singulett-Exciton-Exciton-Wechselwirkung entwickelt

Isotropic 3D Nuclear Morphometry of Normal, Fibrocystic and Malignant Breast Epithelial Cells Reveals New Structural Alterations

Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis

A versatile method for dynamically controlled patterning of small populations of epithelial cells on substrates via non-contact piezoelectric inkjet printing

<div><p>Intercellular interactions play a central role at the tissue and whole organism level modulating key cellular functions in normal and disease states. Studies of cell-cell communications are challenging due to ensemble averaging effects brought about by intrinsic heterogeneity in cellular function which requires such studies to be conducted with small populations of cells. Most of the current methods for producing and studying such small cell populations are complex to implement and require skilled personnel limiting their widespread utility in biomedical research labs. We present a simple and rapid method to produce small populations with varying size of epithelial cells (10–50 cells/population) with high-throughput (~ 1 population/second) on flat surfaces via patterning of extracellular matrix (ECM) proteins and random seeding of cells. We demonstrate that despite inherent limitations of non-contact, drop-on-demand piezoelectric inkjet printing for protein patterning, varying mixtures of ECM proteins can be deposited with high reproducibility and level of control on glass substrates using a set of dynamically adjustable optimized deposition parameters. We demonstrate high consistency for the number of cells per population (~1 cell standard error of mean), the population’s size (~0.2 coefficient of variation) and shape, as well as accurate spatial placement of and distance between colonies of a panel of metaplastic and dysplastic esophageal epithelial cells with differing adhesion and motility characteristics. The number of cells per colony, colony size and shape can be varied by dynamically varying the amount of ECM proteins deposited per spatial location and the number of spatial locations on the substrate. The method is applicable to a broad range of biological and biomedical studies including cell-cell communications, cellular microenvironment, migration, and stimulus response.</p></div

Characteristics of cell populations obtained under the four different deposition patterns shown in Fig 4.

<p>A) Average number of cells per population; B) Average surface area of populations; C) Average population perimeter. All three characteristics were significantly different (p = 0.05, two-way ANOVA, Tukey test)) among the two experimental parameters–number of drops and locations–indicating a robust performance and high level of control of the population size. Error bars represent standard deviation (SD).</p

Patterning human esophageal epithelial cell types on substrates deposited with the ECM mixture.

<p>A) The four different cell types (CP-A, CP-B, CP-C, and CP-D) representing the metaplastic and dysplastic stages of premalignant BE progression showed similar population sizes and densities when seeded on custom ECM protein mixture. The micrographs were taken at 15 minutes after seeding. Scale bar 100 μm. B) Small populations of dysplastic (CP-D cell line) BE epithelial cells created using the four different patterning configurations detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176079#pone.0176079.g004" target="_blank">Fig 4</a>. Simple manipulations of the ECM mixture volume and the spatial arrangement of the deposition locations results in significantly different population sizes and numbers of cells per population. A circle has been drawn on the image to show that there is a difference in number between the cell populations. Custom deposition patterns with varying population sizes can be readily created by varying the distance between the spots and/or volume of the ECM mixture and its local deposition pattern. Scale bar 200 μm.</p

Cell seeding on patterned substrates workflow.

<p>Procedural steps for cell seeding and removal to produce spatially confined small populations (10–50 cells/population) of cells are shown.</p

Conceptual approach of the cell micropatterning method used in this study.

<p>Subnanoliter volumes of an ECM proteins mixture are deposited on a flat substrate with pre-defined custom spatial patterns. The amount of the deposited mixture as well as its spatial location can be controlled precisely via customizable dispensing parameters for flexibility in controlling the population size and proximity to other adjacent populations.</p

Spatial patterns of ECM mixture deposition used in this study to produce differently sized small populations of cells.

<p>The panels in the first row depict a pattern where one drop (~40 pL volume) of the ECM mixture was deposited on one (upper left) or two adjacent (upper right) locations on the substrate. The bottom two panels represent the same spatial patterns as above, but with two droplets of the mixture being deposited per location.</p