Transmission Electron Microscopy (TEM) was used to investigate
the phase separation into HDs and SDs but for that purpose,
the samples had to be stained prior to the observation. The choice
of the staining agent used in a given polymer system is guided
largely by empirical success [10]. OsO4 and RuO4 are probably
among the best known staining agents and are routinely used to
stain polymers. OsO4 is believed to act as a covalent crosslinker
between unsaturated carbon double bonds [11]. RuO4 is mainly
used to differentially label aromatic moieties from aliphatic ones
[12,13]. However due to similar chemical structures between HSs
and SSs in the PUs studied here, a significant differential staining
could not be obtained with OsO4 or RuO4 alone. Indeed for each PU
type, a piece of filmwas stained in a 4% aqueous solution of OSO4 for
24 h. After rinsing with ethanol, the films of PU60 and PU75 turned
to colors which clearly indicates incomplete staining see Fig. 1.
RuO4 led to similar colors on the PUs studied here (not shown).
Thus, a protocol similar to that of Janina Foks [14] was used, since
they demonstrated that it was possible to localize the HDs of pure
PU by using a double staining technique based on OsO4 and
formaldehyde. By adding OsO4, Os stained double bonds on both
segments as a first step. As a second step by adding formaldehyde
and OsO4, formaldehyde opened the NeH bonds and made it
accessible for osmium (the NeH bond exists only in the HS).
For each PU type, a piece of film was stained in a 4% aqueous
solution of OsO4 for 24 h. After rinsing with ethanol, the films were
stained in a 50:50 aqueous solution of OSO4 and HCHO4 (initial
concentrations of 4% and 37% in water, respectively) for 24 h [14].
After a second rinsing with ethanol, the films appeared to be
completely black, which is an indication of a successful staining
step (see Fig. 1). Thus, the films were embedded in an Epofix® resin
and ultrathin sections were obtained by cryoultramicrotomy with a
Reichert Ultracut S. The sample temperature was set to 200 K and
the 35 diamond knife speed to 1 mm/s.
TEM observations were carried out at acceleration voltage of
200 KV with a JEOL 2010F microscope, operating at 200 kV. Bright
field images were recorded with a Gatan Orius 200 camera.
Transmission Electron Microscopy (TEM) was used to investigatethe phase separation into HDs and SDs but for that purpose,the samples had to be stained prior to the observation. The choiceof the staining agent used in a given polymer system is guidedlargely by empirical success [10]. OsO4 and RuO4 are probablyamong the best known staining agents and are routinely used tostain polymers. OsO4 is believed to act as a covalent crosslinkerbetween unsaturated carbon double bonds [11]. RuO4 is mainlyused to differentially label aromatic moieties from aliphatic ones[12,13]. However due to similar chemical structures between HSsand SSs in the PUs studied here, a significant differential stainingcould not be obtained with OsO4 or RuO4 alone. Indeed for each PUtype, a piece of filmwas stained in a 4% aqueous solution of OSO4 for24 h. After rinsing with ethanol, the films of PU60 and PU75 turnedto colors which clearly indicates incomplete staining see Fig. 1.RuO4 led to similar colors on the PUs studied here (not shown).Thus, a protocol similar to that of Janina Foks [14] was used, sincethey demonstrated that it was possible to localize the HDs of purePU by using a double staining technique based on OsO4 andformaldehyde. By adding OsO4, Os stained double bonds on bothsegments as a first step. As a second step by adding formaldehydeand OsO4, formaldehyde opened the NeH bonds and made itaccessible for osmium (the NeH bond exists only in the HS).For each PU type, a piece of film was stained in a 4% aqueoussolution of OsO4 for 24 h. After rinsing with ethanol, the films werestained in a 50:50 aqueous solution of OSO4 and HCHO4 (initialconcentrations of 4% and 37% in water, respectively) for 24 h [14].After a second rinsing with ethanol, the films appeared to becompletely black, which is an indication of a successful stainingstep (see Fig. 1). Thus, the films were embedded in an Epofix® resinand ultrathin sections were obtained by cryoultramicrotomy with aReichert Ultracut S. The sample temperature was set to 200 K andthe 35 diamond knife speed to 1 mm/s.TEM observations were carried out at acceleration voltage of200 KV with a JEOL 2010F microscope, operating at 200 kV. Brightfield images were recorded with a Gatan Orius 200 camera.
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