that are tens of microns are at 5 nm isotropic voxel size [10]. FIB-SEM has been
applied to numerous samples and its use has extended into many different fields [7,
64, 66–73]. The diversity of applications is evidence of a successful system which
has significantly contributed to answering fundamental cell biology questions [69].
It also permits direct analysis of cell-substrate interfaces which is not possible with
traditional ultramicrotomy [74] as the ion beam can cut both hard and soft materials.
5.4.1 Sample Processing
Processing of biological material for FIB-SEM has been successful with many
different protocols (Fig. 5.3d–f). One such example is a dissected piece of mouse
brain that was processed according to Knott et al. [7]. Here the primary fixation was
by perfusion (2.5% glutaraldehyde, 2% paraformaldehyde in phosphate buffer),
followed by 1.5% potassium ferrocyanide with 1% osmium tetroxide, 1% osmium
tetroxide and then 1% uranyl acetate in water [7]. This is in stark contrast to the
extensive heavy metals necessary for SBEM. The following Sects. (5.4.1.1, 5.4.1.2,
5.4.1.3) outline processing protocols for cell monolayers by conventional bench,
microwave and high-pressure freezing/freeze substitution.
5.4.1.1 Conventional Bench Processing
Tissue culture cells are grown on a MatTek dish (MatTek Corp., USA,#
P35G-2-14-CGRD or P35G-2/1.5-14-CGRD) with coordinate pattern to facilitate
finding back cells at the FIB-SEM and processed with the following protocol.
1. Primary fixation with 2x concentration of fixative in equal proportions to the
cell media—final concentration 2.5% glutaraldehyde 0.05% malachite green
oxalate in 0.1 M PHEM buffer left 1 min
2. Immediately followed by same 1x fixative 20 min on ice.
3. Buffer rinses, 5 Â 3 min
4. 1% osmium tetroxide and 0.8% potassium ferricyanide (in water on ice for 1 h)
5. Water rinses, 5 Â 3 min
6. Aqueous 1% tannic acid 20 min on ice
7. Water rinses, 5 Â 3 min
8. 0.5% uranyl acetate in water 1 h room temperature
9. Dehydration in a series of ethanol solutions on ice
10. Resin infiltration was done by adding 100% Durcupan resin to the dish and
leaving the dishes overnight in the fumehood
11. The following day, the resin was reduced and a coverslip was placed over the
top of the well
12. Dish was polymerized at 60 °C for 3 days
5 Volume Scanning Electron Microscopy: Serial Block-Face …
135
applied to numerous samples and its use has extended into many different fields [7,
64, 66–73]. The diversity of applications is evidence of a successful system which
has significantly contributed to answering fundamental cell biology questions [69].
It also permits direct analysis of cell-substrate interfaces which is not possible with
traditional ultramicrotomy [74] as the ion beam can cut both hard and soft materials.
5.4.1 Sample Processing
Processing of biological material for FIB-SEM has been successful with many
different protocols (Fig. 5.3d–f). One such example is a dissected piece of mouse
brain that was processed according to Knott et al. [7]. Here the primary fixation was
by perfusion (2.5% glutaraldehyde, 2% paraformaldehyde in phosphate buffer),
followed by 1.5% potassium ferrocyanide with 1% osmium tetroxide, 1% osmium
tetroxide and then 1% uranyl acetate in water [7]. This is in stark contrast to the
extensive heavy metals necessary for SBEM. The following Sects. (5.4.1.1, 5.4.1.2,
5.4.1.3) outline processing protocols for cell monolayers by conventional bench,
microwave and high-pressure freezing/freeze substitution.
5.4.1.1 Conventional Bench Processing
Tissue culture cells are grown on a MatTek dish (MatTek Corp., USA,#
P35G-2-14-CGRD or P35G-2/1.5-14-CGRD) with coordinate pattern to facilitate
finding back cells at the FIB-SEM and processed with the following protocol.
1. Primary fixation with 2x concentration of fixative in equal proportions to the
cell media—final concentration 2.5% glutaraldehyde 0.05% malachite green
oxalate in 0.1 M PHEM buffer left 1 min
2. Immediately followed by same 1x fixative 20 min on ice.
3. Buffer rinses, 5 Â 3 min
4. 1% osmium tetroxide and 0.8% potassium ferricyanide (in water on ice for 1 h)
5. Water rinses, 5 Â 3 min
6. Aqueous 1% tannic acid 20 min on ice
7. Water rinses, 5 Â 3 min
8. 0.5% uranyl acetate in water 1 h room temperature
9. Dehydration in a series of ethanol solutions on ice
10. Resin infiltration was done by adding 100% Durcupan resin to the dish and
leaving the dishes overnight in the fumehood
11. The following day, the resin was reduced and a coverslip was placed over the
top of the well
12. Dish was polymerized at 60 °C for 3 days
5 Volume Scanning Electron Microscopy: Serial Block-Face …
135