and EM in general; Typically require less sample than in CryoEM,
enabling the finding and visualization of rare/sparse particles; Does
not have some of the drawbacks of CryoEM, for example, air-water
interface and electron dose destruction of the particles [13]; Can be
used more efficiently and can provide insight at any point of a
biochemical experiment—including stages prior to purification;
Can ascertain if all components of a macromolecule are present and
at what orientations and stoichiometries and if any flexibility is
present [14]; Can screen for binding of antibodies and other binders
or co-complexes [11]; etc.
In CryoEM, the sample appears darker than the surrounding
ice while stained samples appear white giving rise to the term
“negative-stain” [6]. The stain surrounds the sample, covering it,
causing drying, and therefore likely flattening it (Fig. 1e). If the
stain is too thick, it may also break the sample, so damage and
particle breakage can be common. The most widely used stains are
the heavy metal stains Uranyl Acetate (UA) and Uranyl Formate
(UF), each having their own benefits and drawbacks [6]. Both
stains are electron-dense and provide great contrast, are radioactive,
toxic, light-sensitive, and low in pH. Solutions of UA can be stored
for long periods of time ready to be used while UF needs to be
freshly prepared every few days. UF, however, has a smaller grain
size, which can help with the visualization of finer details—especially advantageous in the case of smaller proteins and thin crystals.
In the following sections, I give a detailed list of the materials
and equipment required and a primer for a robust Uranyl-based
negative-stain workflow, much of which is shared among other
methods of staining. I also show examples of different macromolecules in stain (Figs. 1, 2, and 3), comparisons with CryoEM (Figs. 1
and 3) and provide a notes section which includes discussing
changes users can make to optimize the protocol. One of the
most powerful aspects of negative-stain is its versatility. The ideal
result is to get thinly stained samples on a carbon support. As such,
many microscopists have made tweaks and found their own methods of staining and some can vary wildly from the method I outline
below. Once a user gets familiar with the basics of staining and
relating that to results from the microscope, they can easily tailor
their use of negative-stain to their own samples.
2 Materials
2.1 Grid Handling
and Preparation
1. Carbon-coated grids (200–400 copper mesh) (see Notes
2 and 3).
2. Tweezers (e.g., Dumont high-precision) for general grid
handling.
3. Anti-capillary tweezers (e.g., Dumont #5) for staining.
118
Shane Gonen
enabling the finding and visualization of rare/sparse particles; Does
not have some of the drawbacks of CryoEM, for example, air-water
interface and electron dose destruction of the particles [13]; Can be
used more efficiently and can provide insight at any point of a
biochemical experiment—including stages prior to purification;
Can ascertain if all components of a macromolecule are present and
at what orientations and stoichiometries and if any flexibility is
present [14]; Can screen for binding of antibodies and other binders
or co-complexes [11]; etc.
In CryoEM, the sample appears darker than the surrounding
ice while stained samples appear white giving rise to the term
“negative-stain” [6]. The stain surrounds the sample, covering it,
causing drying, and therefore likely flattening it (Fig. 1e). If the
stain is too thick, it may also break the sample, so damage and
particle breakage can be common. The most widely used stains are
the heavy metal stains Uranyl Acetate (UA) and Uranyl Formate
(UF), each having their own benefits and drawbacks [6]. Both
stains are electron-dense and provide great contrast, are radioactive,
toxic, light-sensitive, and low in pH. Solutions of UA can be stored
for long periods of time ready to be used while UF needs to be
freshly prepared every few days. UF, however, has a smaller grain
size, which can help with the visualization of finer details—especially advantageous in the case of smaller proteins and thin crystals.
In the following sections, I give a detailed list of the materials
and equipment required and a primer for a robust Uranyl-based
negative-stain workflow, much of which is shared among other
methods of staining. I also show examples of different macromolecules in stain (Figs. 1, 2, and 3), comparisons with CryoEM (Figs. 1
and 3) and provide a notes section which includes discussing
changes users can make to optimize the protocol. One of the
most powerful aspects of negative-stain is its versatility. The ideal
result is to get thinly stained samples on a carbon support. As such,
many microscopists have made tweaks and found their own methods of staining and some can vary wildly from the method I outline
below. Once a user gets familiar with the basics of staining and
relating that to results from the microscope, they can easily tailor
their use of negative-stain to their own samples.
2 Materials
2.1 Grid Handling
and Preparation
1. Carbon-coated grids (200–400 copper mesh) (see Notes
2 and 3).
2. Tweezers (e.g., Dumont high-precision) for general grid
handling.
3. Anti-capillary tweezers (e.g., Dumont #5) for staining.
118
Shane Gonen