Chapter 4: Optimization of a protocol for high-resolution imaging
4.2 Methodology and optimization of SBF imaging for the human limbus
4.2.3 Sample loading, serial block-face imaging and data analysis
Once mounted on cryopins, the surface of resin blocks was sputter coated with a thin layer of gold palladium in order to generate a conductive surface and limit the charging effect. Samples were then carefully inserted on the ultramicrotome of the 3view system (Figure 4.4A) and loaded inside the chamber of a Zeiss Zigma scanning electron microscope. Approach of the diamond knife to the sample was initially made manually using a binocular and the light reflection at the surface of the resin block and then automatically by making a 100nm step- by-step approach.
Numerous settings can be adjusted in order to obtain the best imaging quality. Typically, acceleration voltages (AVs) ranging between 2kV and 20kV are used for SEM imaging. For biological samples, more details are visible when using a high AV as more BSEs are generated from the sample. However, a high AV involves an increased interaction of the electron beam with the specimen and can be at the origin of melting of the surface of the resin block. Magnification is set by the size of the raster of the electron beam on the sample surface and is typically ranging between 30X and 30,000X. High magnification gives better details of what is seen but reduces the field of view and might generate resin softening. The pressure inside the chamber of the SEM is maintained by nitrogen and is also adjustable. A better signal to noise is generally obtained with a higher vacuum. However, a higher vacuum generates more charging and thus affects the quality of the micrograph. The dwell time corresponds to the length of time the electron beam dwells on one pixel of the sample. A long dwell time increases the
amount of BSEs that can be collected and thus increases quality of the image. However, a long dwell time involves a longer ‘scanning’ time that is directly associated with charging and melting of sample. Dwell time is a setting to consider when larger pieces of tissue are analyzed as it could drastically increase duration of the imaging run. Diameter of the aperture controls the amount of electrons hitting the surface of the sample. A high aperture is proportional to the amount of BSEs emitted and thus to the quality of the image generated. A high aperture however also increases the risk of charging and resin softening. Resolution of the image generated can also be adjusted and reach up to 4K x 4K. However, the amounts of details observed on the final image mostly depend on the quality of the sample (preservation, embedding, staining…). Using the highest resolution generates fundamental problem in the storage and subsequent analysis of large amounts of data that can routinely reach hundreds of gigabytes in one single overnight run. For this reason, setting a reasonable resolution for the amount of details required is essential when considering the storage of the vast amounts of data that volume EM involves. Advantages and disadvantages of changing settings of the 3View imaging are summarized in table 4.1.
Description Increasing Decreasing
Advantage Disadvantage Advantage Disadvantage
Acceleration
voltage The voltage at which
electrons are pulled from the anode
More backscattered electrons (BSEs) therefore better signal to noise ratio
Increased interaction volume can mean more melting of sample, but also possible over sampling of image
Smaller interaction volume –
can cut thinner sections Fewer BSEs so signal to noise can be poor
Magnification
Set by the size of the raster area of the electron beam on the sample surface
Increases the detail of what is seen
Decreases the field of view. Because of nature of SEM the electron beam is now scanning over a smaller area and melting can occur.
Increases field of view Decreases the detail/ resolution
Variable Pressure
Use of a gas (in our VP SEM this is nitrogen) within the chamber of the SEM
Decreasing the vacuum,
decreases the charging Decreases signal to noise ratio, thus interference
and noisy image. Increases charging Better signal to noise
Dwell time Length of time the
electron beam dwells on one pixel worth of sample.
Increases the number of BSEs that can be collected = better image
Increases the chance of charging and melting of sample.
Longer acquisition time.
Shorter acquisition time =more sections cut in same
number of hours. Fewer BSE collected = image could be noisy
Aperture
The final aperture of the SEM
Increasing the diameter increases the width of the electron beam and thus the number of electrons hitting the sample. = more BSEs
More electrons = more charging and heating of sample = chance of melting
Smaller beam diameter =
better resolution Fewer BSEs, lower signal to noise ratio
Resolution By this we mean pixel
resolution of the image, not actual resolution of the sample
Depending on sample may get more details
within the sample Larger file sizes
Less interaction of electron beam with sample = less charging/heating/melting
Fewer details within sample
e.g. the same sort of data could conceivably be obtained from these 2 scenarios (with the other parameters staying the same): (1) High accelerating voltage Low vacuum (more gas) Short dwell time;
(2) Low voltage High vacuum (less gas) Long dwell time;
Table 4.1 Advantages/disadvantages of increasing or decreasing
settings in the 3View.
The table illustrates what would happen when one parameter is
changed and the others kept the same. Thus there is a fine balance
for the setting of all the parameters to retrieve the information
wanted from of a sample. Note that to make the point with each of
these, the worst-case outcome was put in and the increase/decrease
may have to be considerable (depending on the sample) to visualise
the change.
For imaging of the limbal basal epithelial layer shown in chapter 5, the following settings were used:
o Magnification: x6.000. o Accelerating voltage: 4 kV. o Dwell time: 2 s. o Pressure: 20 Pa. o Aperture: 60 mm. o Resolution: 4k x 4k. o Slice thickness 100 nm.
Because the sectioning process of SBF imaging takes approximately 30sec; with a dwell time set to s at a resolution of 4k x4k, the total duration of an imaging-sectioning cycle is about 1min. The total duration time of SBFSEM imaging would be thus about 16-17 hours to cover 100m of the sample in Z direction with an ultrathin sectioning thickness set to 100nm.
The automated process of sectioning-imaging was repeated for up to 999 cycles generating a large data stack of 999 serial images (figure 4.4B). Serial images were collected as .Dm3 file format and converted into .tiff files using Digital Micrograph™ (Gatan, UK). The complete data stack was then transferred into a Wacom Cintiq workstation and loaded into AMIRA 3D Software for Life Sciences for conversion into voxels (volumetric picture elements). Noise reduction median filter was applied to the entire data stack, and area of interest manually segmented on every single slice using the interactive pen (figure 4.4C). Finally, once the area of interest was entirely manually segmented, 3D volumes were generated (figure 4.4D).
Figure 4.4 Serial block face imaging, manual segmentation and 3D
reconstruction.
A. Gatan 3view serial block face imaging system within the specimen
chamber of a Zeiss Sigma FESEM. Inbox shows the ultramicrotome,
the diamond knife and the specimen loaded inside the chamber of
the microscope.
B. Serial imaging and sectioning generate a large data stack of the
area of interest. Here, the interface is between the limbal basal
epithelial layer and the limbal stroma.
C. Converted files were transferred into a workstation and converted
into voxels using AMIRA imaging software. Area of interest was
manually segmented (purple and pink areas).
D. Manual segmentation of the area of interest on the entire data
stack generated 3D volumes in x, y and z directions.
Serial block face imaging theoretically allows 3D reconstruction of a specimen in great detail, including subcellular structures as small as collagen fibrils. In practice, the resolution of images collected was affected by the quality of limbal biopsies prior to fixation. As discussed previously, rims stored in Optisol were generally not suitable for EM imaging, as these tissues were usually only available between 5 and 10 days post mortem. Fresh tissues unsuitable for corneal transplantation and usually available within 48 hours post mortem had a greater preservation as seen on semi-thin and hematoxylin-eosin sections. However, at very high-magnification, these tissues could also show some artifacts that limited imaging of small organelles and subcellular structures. Even if considered as relatively fresh, these tissues were not immediately fixed post enucleation, as it is the case for animal tissues, cells in culture or other model organisms. For this reason, the amount of details observed was limited when
imaging at a magnification higher than x6.000. Figure 4.5 compares the interface between the limbal basal epithelial layer and the limbal stroma imaged by both TEM and SBFSEM. Details of the limbal epithelium, limbal stromal cells and the basement membrane are clearly revealed by both imaging techniques. However, resolution of SBF imaging is marked by the absence of details of the collagen network within the limbal stroma. The resolution of SBF imaging however remains sufficient to image the basement membrane at the interface between the basal epithelial layer and the limbal stroma and also cell-to-cell interactions that might occur in this specific area.