Further applications and future developments in cryo-STET
For many of the samples studied in cryo-STET, they can be directly
deposited (or grown) and frozen on cryo-EM grids. This allows the cells
to be studied using light and fluorescence microscopy before
cryo-fixation. This workflow is referred to as the correlative light and
electron microscopy method (also known as CLEM). The correlative study
can be performed on the whole sample because the cells do not require
cutting before observation at the electron microscope as the thickness
requirement of cryo-STET allows it. The cryo-fixation method for such
samples is easily performed using plunge-freezing, where the grid is
rapidly plunged inside a cryogen (usually ethane) cooled-down at about
-175°C by liquid nitrogen. It must be noted that such samples are the
thickest ones that can be properly frozen using plunge-freezing.
Interestingly, the thickness limitation of cryo-STET is equivalent to
the thickness limitation of plunge-freezing. For thicker samples,
plunge-freezing can no longer be used without the formation of ice
crystals. These crystals would be deleterious for imaging as they would
diffract electrons which would no longer be detected on the BF detector.
This means that thicker samples must be frozen at high-pressure. This
cryo-fixation method, far more complex than plunge-freezing, involves an
equipment which is more expansive than a simple plunge-freezer and
generates a thicker layer of vitrified ice which is at least several
tens of µm thick. It is possible to image this slab of frozen sample
using a cryo-FIB SEM. But if cryo-STET is required (e.g. for resolution
purposes since cryo-STET offers higher resolutions compared to cryo-FIB
SEM), then a cryo-lamella could be prepared to thin-down the thick slab
of ice down to 1 µm using a cryo-FIB. This process adds even more
complexity to the realisation of the experiment.
The examples presented in this review all used BF STEM, yet this is not
the only modality available. As previously briefly presented, ADF and
HAADF imaging can inform about the presence of atoms heavier than the
most abundant organic atoms (H, C and O) such as K, Ca, Fe. Another
method, differential phase contrast (DPC) has recently been applied in
biology39–41. This method uses a modified ADF
detector which is composed of 4 quadrants, each representing a 90°
portion of the full detector. It is then possible to split the electrons
arriving on the detector in 4 different populations (top, bottom, left
and right) and their analysis allows to retrieve the sample phase
information, hence the name of the method. As the application of this
method to biological samples is extremely recent, its successful
application on thick samples still has to be demonstrated.
Current cryo-STET resolution and sample thickness limits are associated
to the electron dose used during the experiment. To achieve high
resolutions, the sampling (pixel size) has to be small, however a dense
sampling of the specimen mechanically increases the electron dose. To
achieve a two-fold resolution increase, the pixel size must be two times
smaller, leading in a four-fold increase of the electron dose. Since
images are already collected with the maximum electron dose the specimen
can sustain, experiments aiming at resolution increases are dose-limited
and must deal with the collection of low-dose, low signal-to-noise ratio
images. To image thick specimens in BF STEM, the electron dose must be
high as thick samples scatter electrons more than thin ones. Thus, there
is a limit in the maximum achievable sample thickness which is when the
electron dose becomes too high and starts generating visual damages to
the sample. Sparse imaging has the potential to tackle both limitations
at once. Sparse imaging consists in the collection of a limited number
of pixels (as opposed to collecting the whole image pixels), which
mechanically reduces the electron dose applied to the area of interest.
This method is possible in STEM as the point-to-point image generation
allows it. The uncollected missing pixels must be somehow reconstructed
to generate the image of the area of interest. This can be performed
using inpainting algorithms42. The sparse imaging +
inpainting reconstruction workflow can be applied to room temperature
and cryo-samples in 2D and 3D experiments43,44. By
reducing the electron dose, it opens the way to higher resolution or
thicker sample studies.