Critical Point Drying and Sputter Coating for SEM
Prior to conventional SEM imaging, the sample must be carefully dehydrated and coated with a layer of heavy metal, often gold, only a few atoms thick. The dehydration is best performed using a device called a critical point dryer, which replaces water with miscible alcohol and then liquid CO2. Finally, the CO2 is evaporated under pressure at its critical point. This process avoids surface tension forces that can otherwise destroy ultrastructural details. Next, the dry sample is placed in a high vacuum coater in order to coat it with a layer of (conductive) metal only a few atoms thick. The coating is achieved by resistively heating the metal in a moderate vacuum until it evaporates. Some of the atoms then condense onto the sample.
Sample Preparation for Focused Ion Beam-SEM
FIB-SEM is a block face SEM imaging method that uses an ion beam to iteratively mill the block surface, creating a 3D image stack. Sample prep for FIB-SEM is similar to the methods used for TEM since the goal is to visualize internal structures based on back-scattered electrons. Following standard protocol, sample is fixed in glutaraldehyde and stained with osmium and other heavy metals. It is then embedded in Durcupan resin, which well-infiltrates that sample and is most compatible with ion beam milling. Before final imaging, which is practically limited to a very small region (~4 x 4 x 2 um), the resin block must be manually trimmed to find the desired region, which can be a very painstaking process.
Ultramicrotomy and Array Tomography
Conventional TEM is based on imaging electrons that transmit through the sample. Thus, the sample must be very thin, in the range of 50-100 nm. Larger samples must first be fixed, stained with osmium, and then embedded in an epoxy resin. The resin block is then manually trimmed to find the region of interest and finally the desired region is sliced into ultra-thin sections for imaging.
In a technique called array tomography, a large sequence of hundreds of serial sections are cut from a resin block and placed in order (‘arrayed’) onto a support. The sections are then imaged in an SEM and the many 2D images from each section are digitally reassembled into single 3D image. This method is also useful for super resolution light microscopy.
Processing for Conventional TEM
Biological samples must be strongly fixed, embedded in a resin for cutting, and stained with heavy metals prior to imaging with electrons in a high vacuum. The core can work with a range of resins and stains, depending on the sample. Macro-molecular samples can also be cast into a heavy metal salt and their imprint imaged, a process called negative staining. Our standard methods and protocols can be found here:
Protocol for TEM of suspensions
High Pressure Freezing – Freeze Substitution
For very high resolution TEM imaging where ultrastructural details are critical or to capture transient feature that are perturbed by chemical fixation, an alternative is to freeze the sample very rapidly (<1 msec), in vitreous ice. This rapid freezing is accomplished in a high-pressure freezer. After freezing, the sample is then chemically fixed and embedded while still frozen using a processes called freeze substitution. Finally, the sample is brought back to room temperature for imaging. Alternatively, high pressure freezing is used to prepare tissue or larger cells for cryo-electron microscopy. In this case, the sample is kept frozen in ice through out the imaging process.
Tokuyasu method for immunogold labeling
Immunogold labeling is an antibody labeling method for electron microscopy, where small (5 nm) gold particles are used for contrast. Due to ease of use, we recommend to first attempt immunogold labeling using post-embed staining of acrylic resin sections (LR White). Though less technically challenging to perform, post-resin embed staining often does not work very well because the antigen may be damaged during the fixation/embedding process or antibody may not be able to access the antigen. An alternative approach is to infiltrate the sample with a saturated sucrose solution and then freeze it in liquid nitrogen. While still frozen, ultrathin sections are cut on a cryo-ultramicrotome. The sections are then immunostained prior to embedding. Cutting frozen sections is extremely challenging, but when successful the immunostaining steps are much more likely to work well.