How cryo will solve your problems

Transcription

1 How cryo will solve your problems Nadejda B. Matsko FELMI, TU Graz, and ZFE Graz

2 What for do we need cryo microscopy? Cryo fixation Conventional fixation (rapid rapid dehydration)

3 High resolution microscopy techniques which are compatible with cryo preparation/analysis Transmission electron microscopy (TEM) Atomic force microscopy (AFM) Scanning electron microscopy (SEM)

4 TEM SEM AFM 1000 nm

5 Cryo (a) versus conventional (b) microscopy. Hydrated objects Plunge freezing High pressure freezing Nanostructured Lipid Carriers (NLC), loaded with Itraconazole Nematode C. Eleganse

6 a Cryo (a) versus conventional (b) microscopy. Soft polymers. b Polyamide 6/acrylonitrile-butadiene-styrene copolymer

7 Cryo TEM of liquids

8 Comprehensive information about size and droplet morphology of the sample 200 nm Nanoemulsion Monolinolein (MLO)/water nanostructured dispersions

9 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

10 Agregation tendensy Liposome /protein formulation

11 Unilamellar or multilamellar liposomes

12 Ageing of the sample 2 days after synthesis 6 month after synthesis 200 nm 200 nm Monolinolein (MLO)/water nanostructured dispersions. Hexagonal phase

13 Intercalation of proteins into the liposome membrane 20 nm Liposome /protein formulation

14 Self-assembled structures: liquid-crystalline properties 20 nm 20 nm Monolinolein (MLO)/water nanostructured dispersions. Hexagonal phase

15 Analytical TEM of liquids

16 Ice or drug crystal? Nanostructured lipid carriers loaded with the Itraconazole after autoclaving Placebo nanostructured lipid carriers after autoclaving

17 EDXS spectra of Itraconazole crystals STEM dark field image

18 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

19 TEM of the frozen tissues and cells

20 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

21 Analytical TEM Si (red) _ Ca (blue) _C (green) EDXS

22 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.

23 Atomic force microscopy (AFM)

24 AFM applications: polymers and polymer nanocomposites Elastomers (rubbers) Thermoplastic/elast omers composites Soft polymers Nanocomposite Copolymer blocks Pharmaceutical substances

25 Mechanical property of soft materials PE _ graphene _ compatibilizer 1 PE _ silica _ compatibilizer 2

26 omplementary sets of AFM and TEM (inc. ATEM) images 250 nm 2000 nm 100 nm C 100 nm 200 nm 200 nm

27 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

28 Freeze fracture technique, offered by cryo AFM Polyamide 6/styrene- acrylonitrile copolymer (30/70w/w) a, sample was cryo-fractured and immediately scanned at 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.

29 Cryo scanning electron microscopy (SEM)

30 Cryo SEM of PNIPAAM grafted latex particles. Sample was plunge frozen, partially freeze dried, platinum coated and investigated at -80 C.

31 Cryo SEM of a lotus leaf 1000 nm 50 nm Sample was plunge frozen, partially freeze dried, platinum coated and investigated at -80 C.

32 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