How cryo will solve your problems

(1)

Nadejda B. Matsko

FELMI, TU Graz, and ZFE Graz

How cryo will solve your problems

(2)

Cryo fixation

_{Conventional fixation}

(

rapid dehydration

)

What for do we need cryo microscopy?

(3)

High resolution microscopy techniques

which are compatible with cryo

preparation/analysis

¾

Transmission electron microscopy (TEM)

¾

Atomic force microscopy (AFM)

¾

(4)

TEM

SEM

AFM

(5)

Cryo (a) versus conventional (b) microscopy.

Hydrated objects

Plunge freezing

(6)

Cryo (a) versus conventional (b) microscopy.

Soft polymers.

Polyamide 6/acrylonitrile-butadiene-styrene copolymer

(7)

Cryo TEM of liquids

(8)

Comprehensive information about size and

droplet morphology of the sample

Monolinolein (MLO)/water

nanostructured dispersions

Nanoemulsion

(9)

Structural transition due to

formulation/temperature/pressure change

Cryo-TEM images of Itraconazole-loaded Nanostructured Lipid Carriers

50 nm

50 nm 50 nm

(10)

Agregation tendensy

(11)

Unilamellar or multilamellar liposomes

(12)

Ageing of the sample

Monolinolein (MLO)/water nanostructured dispersions. Hexagonal phase

2 days after synthesis

6 month after synthesis

(13)

Intercalation of proteins into the

liposome membrane

20 nm

(14)

Self

-

assembled structures: liquid

-

crystalline

properties

Monolinolein (MLO)/water nanostructured dispersions. Hexagonal phase

(15)

Analytical TEM of liquids

(16)

Ice or drug crystal?

Placebo nanostructured lipid

carriers after autoclaving

Nanostructured lipid carriers loaded

with the Itraconazole

after autoclaving

(17)

EDXS spectra of Itraconazole crystals

(18)

100 nm

Autoclaved

Itraconazole-loaded NLC.

EELS spectra providing

evidence for the

enclosure of Itraconazole

within the maltilamellar

layers.

Cryo TEM

Conventional TEM

(19)

TEM of the frozen tissues and cells

(20)

High pressure freezing

–

freeze substitution

-

resin

embedding. Sectioning at room temperature

(21)

Analytical TEM

EDXS

(22)

Immunocytochemical analysis of the high

Immunocytochemical analysis of the high-

-

pressure

frozen and freeze

frozen and freeze-

-substituted tissues

substituted tissues

TEM micrographs of antennal sensillas

Placodea

of the parasitic wasp

Cotesia glomerata

, which were

high-pressure 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.

200 nm

(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)

PE _ silica _ compatibilizer 2

PE _ graphene _ compatibilizer 1

Mechanical property of soft materials

(26)

Complementary sets of AFM and TEM (inc. ATEM) images

200 nm 2000 nm

C

250 nm 200 nm

C

100 nm

(27)

Morphological changes of a NBR rubber, which occurred

during warm up from

-

120

120 °

°

C till RT

a

,

Topographical AFM image of

an epoxy embedded

nitrile

butadiene

butadiene-

-rubber

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

Cryo AFM

(28)

Freeze fracture technique, offered by cryo AFM

Polyamide 6/styrene

-

-acrylonitrile copolymer

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

(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

Sample was plunge frozen, partially freeze dried, platinum coated and investigated at