The research in our group is focused on the use of advanced Bioimaging techniques to answer fundamental questions in biological systems. Fluorescence based imaging techniques have been a fundamental tool in histology, cell biology, and molecular biology, for the many years. Although the ability to specifically label and image different molecules, membranes or organelles in cells and tissue has provided invaluable information, we can with the techniques we have available in our group extract more information than was previously available from simple images. For example we can measure the diffusion and transport of single molecules in cells and tissue, as well as pH and other ion concentrations and lipid membrane packing. (see figure 1)
Figure 1: Laurdan GP can be used to indirectly monitor the packing in lipid membranes. A higher GP value suggest a more packed membrane. In situ study of lysosome phase behavior. LAURDAN GP of lysosome membranes was measured in live HEK293 cells using multiphoton excitation microscopy (a-c) (a) Lysosomes labeled with quinacrine in HEK293cells. Image size 78 x 78 _m2. (b) Fluorescence intensity image (c) LAURDAN GP image of HEK293 cells labeled with LAURDAN for 3h. For both b and c, image size is 52 x 52 _m2. The average LAURDAN GP of the lysosomes was 0:07±0:07, whereas the average LAURDAN GP for the plasma membrane was 0:3±0:04
Among others techniques, we have used multiphoton excitation microscopy related techniques as a tool for skin imaging studies (See figure 2). Combinations of multiphoton excitation with techniques, such as fluorescence lifetime imaging (FLIM), fluorescence correlation spectroscopy (FCS), Raster image correlation spectroscopy (RICS) and second harmonic generation microscopy (SHG), offer very powerful experimental tools to simultaneously explore structural and dynamical aspects of biological specimens. We have via collaborations with world leading research groups brought new technology and expertise to Denmark such as digital frequency domain FLIM and RICS. Recenly thanks to grants given to DaMBIC and to international collaborations we have and built Denmark’s first STED microscopy. The STED microscope is state of the art; the microscope can via point spread function engineering circumvent the diffraction limit and can deliver images with a resolution 5-10 times better than traditional microscopes. The other equipment has been developed through grants and collaboration with MEMPHYS and the Membrane Biophysics and Biophotonics group to.
Figure 2: Images comparing healthy skin with diseased skin from cholesteatoma. images of Nile red labeled normal skin and cholesteatoma at different depths measured relative to the specimen surface. Column a) Human retro-auricular skin. The image size is 82µm x 82µm. Column b) Cholesteatoma imaged from the stratum basale/outer side of the cholesteatoma sac. The image size is 81µm x 81µm. The structures seen in the bottom image of column b) are due to the orientation of the corneocytes relative to the plane of the image. Column c) Cholesteatoma imaged from the SC of the cholesteatoma sac. The image size is 128µm x 128µm. The images are representative of at least 3 individual samples.
Head of research: Associate professor Jonathan R. Brewer
Researchers and research group:
PhD fellow Stéphanie Yuki Kolbeck Hotta
One of our ongoing projects is to study diffusion of different types of particles in human skin. We have as the first group utilized Raster Image Correlation Spectroscopy to study spatially resolved molecular diffusion in skin.We have previously shown that liposomes used for transdermal drug delivery burst on contact with the skin and do not as function as vehicle to transport drugs through the skins barrier. Results from these experiments have shown that although both hydrophilic and hydrophobic molecules seem to accumulate in the same intercellular spaces in the stratum corneum, they have different local diffusion properties. See figure 1. An one going project is to use the enhanced resolution of the STED microscope to resolve the different penetration pathways of hydrophilic and hydrophobic molecules and directly measure the diffusion in the intact tissue using. The goal of the project is to increase our understanding of the skin barrier and for use in developing successful methods for transdermal drug delivery.
Figure 3: Diffusion maps of human skin labeled with RhB-DDDP (hydrophobic) ATTO-647N-Streptavadin (hydrophilic) obtained 4µm under the skin SC surface. The diffusion maps are positioned on top of the corresponding fluorescence intensity images. From this data it is clear that i) the diffusion is not homogeneous across the image and ii) it is clear that spatially resolving the two different dyes is not possible with the available techniques.
• Brewer, Jonathan, Maria Bloksgaard, Jakub Kubiak, Jens Ahm Sørensen, and Luis A. Bagatolli. “Spatially Resolved Two-Color Diffusion Measurements in Human Skin Applied to Transdermal Liposome Penetration.” Journal of Investigative Dermatology (00/01/0000). http://www.nature.com/jid/journal/vaop/ncurrent/full/jid2012461a.html.
• Bloksgaard, M., Svane-Knudsen, V., Sorensen, J. A., Bagatolli, L., and Brewer, J., Structural Characterization and Lipid Composition of Acquired Cholesteatoma: A Comparative Study with Normal Skin, Otology & Neurotology, 33, 177 (2012).
• Brewer, J., de la Serna, J. B., Wagner, K., and Bagatolli, L. A., Multiphoton excitation fluorescence microscopy in planar membrane systems, Biochimica Et Biophysica Acta-Biomembranes, 1798, 1301 (2010).
• Iwai, I., Han, H., Hollander, L. D., Svensson, S., Ofverstedt, L. G., Anwar, J., Brewer, J., Bloksgaard, M., Laloeuf, A., Nosek, D., Masich, S., Bagatolli, L. A., Skoglund, U., and Norlen, L.,“The Human Skin Barrier Is Organized as Stacked Bilayers of Fully Extended Ceramides with Cholesterol Molecules Associated with the Ceramide Sphingoid Moiety.” Journal of Investigative Dermatology 132, no. 9 (April 26, 2012): 2215–2225.
• Bloksgaard, M., Bek, S., Neess, D., Brewer, J., Haninibal-Bach, H. K., Helledie, T., Ejsing, C. S., Fenger, C., Due, M., Chemnitz, J., Finsen, B., Clemmensen, A., Wilbertz, J., Saxtorph, H., Knudsen, J., Bagatolli, L. A., and S., M., “The acyl-CoA Binding Protein Is Required for Normal Epidermal Barrier Function in Mice.” Journal of Lipid Research 53, no. 10 (October 1, 2012): 2162–2174.
• Kubiak, J., Brewer, J., Hansen, S., and Bagatolli, L. A., Lipid Lateral Organization on Giant Unilamellar Vesicles Containing Lipopolysaccharides, Biophysical Journal, 100, 978 (2011).
• Stock, R. P., Brewer, J., Wagner, K., Ramos-Cerrillo, B., Duelund, L., Jernshoj, K. D., Olsen, L. F., and Bagatolli, L. A., Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate, PLoS One, 7, e36003 (2012).
• Arnspang, Eva C., Jonathan R. Brewer, and B. Christoffer Lagerholm. “Multi-Color Single Particle Tracking with Quantum Dots.” PLoS ONE 7, no. 11 (November 14, 2012): e48521.
• Bernchou, U., Brewer, J., Midtiby, H. S., Ipsen, J. H., Bagatolli, L. A., and Simonsen, A. C., Texture of Lipid Bilayer Domains, Journal of the American Chemical Society, 131, 14130 (2009).