Collaborative Research Center (SFB 1278) Investigation of cellular uptake mechanisms by correlative TEM and SIM Rainer Heintzmann, Fengjiao Ma, Institute of Physical Chemistry Stephanie Höppener, Martin Reifarth, Daniel Friedrich Jena Center for Soft Matter SFB 1278 PhD Seminar on January 19, 2018
Outline Motivation: What is C04 doing? Methods we are using First Practicing Summary and Outlook Acknowledgements 2
Outline POLYTARGET aims on tailored and guided drug delivery to cells by designing smart polymer nanoparticles. For efficient drug delivery uptake and release mechanisms are very important. These are: particle-membrane interactions, localization of drug delivery vehicles in the cell, release of cargos from cellular compartments into the cytosol (e.g., by the proton sponge effect) or transport of cargos to the nucleus (e.g. for gene transfection). M. Reifarth, S. Hoeppener, U.S. Schubert, Adv. Mater. 2018, DOI: 10.1002/adma.201703704. Inside cellular compartments 3
What is C04 doing? Correlative approach: Combination of the information obtained by two different microscopy methodes to investigate one and the same object (cell). Sample preparation: Finding the best working routine. Staining: Finding stains which are optimized for both methodes. Combination of both data sets: How to combine both data sets? Image editing. Finding suitable markers. For statistical analysis and model development: Image-based systems biology (Z01 AG Figge). 4
Methods CLEM: Bridging the gap in the resolution efficiency of light and electron microscopy. Obtaining complementary information that none of the techniques would provide on its own. 5
What to expect from SIM & TEM Transmission Electron Microscopy Mitochondria ER Nuclear membrane Ribosomes 6
Advantages of CLEM Example: Polymer nanoparticles in cells Polymer NPs in a cell + Fluorescent marked NPs are easily identified. + Judgment of their localization after uptake. + Suitable dyes can help to identify their target compartments (MitoTracker, LysoTracker, ) + Live-cell studies on uptake. - Aggregation stage and number of individual particles is difficult to determine. - Exact localization (e.g. membrane interaction) cannot be evaluated. + Number of particles can be determined. + Changes in the shape or size of the nanoparticles in the cell can be monitored. + Interactions with membranes (membrane rupture, ) can be seen. - Needs additional contrasting. - Thin film sectioning is required. - No live-cell imaging. 7
Practicing with Saccharomyces cerevisiae (yeast) Because yeast cells ar easily available, easy to cultivate and the inward cell structure is related to other eukaryotes. TEM sample preparation Cell culture Fixation and staining Dehydration Embedding in resin Ultramicrotome slicing Preparation of the pre-form Slicing (< 150 nm) TEM imaging Images of yeast cells 8
TEM sample preparation After the cell cultivation the sample will be prepared for TEM: Protein-fixation with glutaraldehyde. Membrane-fixation and staining with osmium tetroxide. Stepwise substitution of water by ethanol. Immersing the cell pellet in embedding medium; substitution of ethanol by resin/ethanol mixture Substitution of the blend by pure epoxy resin. Addition of cross linker. 9
Ultramicrotome Slicing Slicing with the Ultramicrotome: Preparation of pre-form with razor blade or glass knife. Slicing of thin slices (80 to 150 nm) with diamond knife. 10
TEM Imaging Thick cell membranes. Growth by budding. Nucleus and vacuoles are weakly visible. 11
TEM Imaging after additional staining by uranyl acetate and lead citrate will ad more contrast. Now Showing nucleus, Mitochrondria and vacuoles. 12
TEM Imaging after additional staining, done with Scanning Transmission Electron Microscopy (STEM). 13
Aspects of Particle Uptake Polystyrene fluoro beads (100 nm) 14
Summary and Outlook What has been done yet: Preparation of yeast cells for TEM imaging. TEM imaging of yeast cells. Polymer-metal complex What will be done: Metal-polymer hybrid Correlative imaging. Establishing routine for correlative imaging (preparation, dyes, imaging, finding marker, ) Fluorescent polymer Nanoparticle 15
Acknowledgements Dr. Stephanie Höppener, Prof. Dr. Rainer Heintzmann, Steffi Stumpf, Pelin Sungur, Martin Reifarth, Prof. Dr. Ulrich S. Schubert SFB 1268 16