How cryo will solve your problems Nadejda B. Matsko FELMI, TU Graz, and ZFE Graz
What for do we need cryo microscopy? Cryo fixation Conventional fixation (rapid rapid dehydration)
High resolution microscopy techniques which are compatible with cryo preparation/analysis Transmission electron microscopy (TEM) Atomic force microscopy (AFM) Scanning electron microscopy (SEM)
TEM SEM AFM 1000 nm
Cryo (a) versus conventional (b) microscopy. Hydrated objects Plunge freezing High pressure freezing Nanostructured Lipid Carriers (NLC), loaded with Itraconazole Nematode C. Eleganse
a Cryo (a) versus conventional (b) microscopy. Soft polymers. b Polyamide 6/acrylonitrile-butadiene-styrene copolymer
Cryo TEM of liquids
Comprehensive information about size and droplet morphology of the sample 200 nm Nanoemulsion Monolinolein (MLO)/water nanostructured dispersions
Structural transition due to formulation/temperature/pressure change 50 nm 50 nm 50 nm 200 nm 200 nm Cryo-TEM images of Itraconazole-loaded Nanostructured Lipid Carriers
Agregation tendensy Liposome /protein formulation
Unilamellar or multilamellar liposomes
Ageing of the sample 2 days after synthesis 6 month after synthesis 200 nm 200 nm Monolinolein (MLO)/water nanostructured dispersions. Hexagonal phase
Intercalation of proteins into the liposome membrane 20 nm Liposome /protein formulation
Self-assembled structures: liquid-crystalline properties 20 nm 20 nm Monolinolein (MLO)/water nanostructured dispersions. Hexagonal phase
Analytical TEM of liquids
Ice or drug crystal? Nanostructured lipid carriers loaded with the Itraconazole after autoclaving Placebo nanostructured lipid carriers after autoclaving
EDXS spectra of Itraconazole crystals STEM dark field image
Cryo TEM Autoclaved Itraconazoleloaded NLC. EELS spectra providing evidence for the enclosure of Itraconazole within the maltilamellar layers. Conventional TEM Experimental support of Ilse etofsky-papst is highly acknowledged 100 nm
TEM of the frozen tissues and cells
High pressure freezing freeze substitution-resin embedding. Sectioning at room temperature 2 µm 100 nm Cross section of the nerve tissue, stained with UrAc/LC
Analytical TEM Si (red) _ Ca (blue) _C (green) EDXS
Immunocytochemical analysis of the high- pressure frozen and freeze-substituted tissues 20 nm 200 nm 200 nm TEM micrographs of antennal sensillas Placodea of the parasitic wasp Cotesia glomerata, which were highpressure frozen and freeze-substituted. (a,b), immunocytochemical analysis of the location of tubulin (the major building block of microtubules). Ch chitin, id inner dendrites, od outer dendrites bear microtubules (mt), reaching cuticle pores.
Atomic force microscopy (AFM)
AFM applications: polymers and polymer nanocomposites Elastomers (rubbers) Thermoplastic/elast omers composites Soft polymers Nanocomposite Copolymer blocks Pharmaceutical substances
Mechanical property of soft materials PE _ graphene _ compatibilizer 1 PE _ silica _ compatibilizer 2
omplementary sets of AFM and TEM (inc. ATEM) images 250 nm 2000 nm 100 nm C 100 nm 200 nm 200 nm
Cryo AFM Morphological changes of a NBR rubber, which occurred during warm up from -120 C C till RT 200 nm 200 nm a, Topographical AFM image of an epoxy embedded nitrile butadiene-rubber latex stripe, cryo-sectioned and immediately scanned at -120 C. b, the same sample was warmed up to room temperature and examined using the same AFM adjustment parameters c, TEM image of the last thin section of the NBR latex sample. 200 nm
Freeze fracture technique, offered by cryo AFM Polyamide 6/styrene- acrylonitrile copolymer (30/70w/w) a, sample was cryo-fractured and immediately scanned at - 120 C. b, sample was sectioned and examined at room temperature. c, TEM image of the thin section of the same sample, which was sectioned at -80 C and observed at room temperature.
Cryo scanning electron microscopy (SEM)
Cryo SEM of PNIPAAM grafted latex particles. Sample was plunge frozen, partially freeze dried, platinum coated and investigated at -80 C.
Cryo SEM of a lotus leaf 1000 nm 50 nm Sample was plunge frozen, partially freeze dried, platinum coated and investigated at -80 C.
THANKS!! Prof. Vikas Mittal Chemical Engineering Department, The Petroleum Institute, Abu Dhabi, UAE Prof. Claudia Valenta Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna Dr. Raimund Schaller Semperit, Wimpassing, Austria Dr. Jana Pardeike Institute of Pharmaceutical Sciences, KFU, Graz Prof. Andreas Zimmer Institute of Pharmaceutical Sciences, KFU, Graz Prof. Otto Glatter Department of Chemistry, University of Graz Martin Müller Ferdinand Hofer ETH Zürich FELMI-ZfE, Graz