Scanning Transmission Electron Microscopy (STEM) is an advanced imaging technique that utilizes a finely focused electron beam scanned across a thin specimen to provide high-resolution images and detailed insights into material structures at the nano to sub-atomic scale. It involves several components including an electron source, electromagnetic lenses, scanners, and various detectors that capture and analyze electron interactions, enabling detailed elemental composition analysis via techniques such as X-ray microanalysis and electron energy loss spectroscopy. Applications of STEM span various fields including biological sciences, semiconductor development, and materials science, allowing for precise imaging and characterization of diverse specimen types.

1. SCANNING TRANSMISSION ELECTRON
MICROSCOPY
Dr Pruthvi Raj D
MVM 23051
First Yr MVSc

2. TEM - images are formed by electrons passing through
a sufficiently thin specimen.
STEM - the electron beam is focused to a fine spot (with
the typical spot size 0.05 – 0.2 nm) which is then
scanned over the sample in a raster illumination system
constructed so that the sample is illuminated at each
point with the beam parallel to the optical axis.

4. RASTER PATTERN

5. Scanning transmission electron microscopy (STEM)
 Advanced imaging technique that is used to visualize
the structure and composition of materials at
nanometer to sub-atomic length scales.
 It provides high-resolution images

6. The Core Principle of STEM
 It scans a very finely focused beam of electrons across
the sample in a raster pattern.
 Interactions between the beam electrons and sample
atoms generate a serial signal stream.
 signal level at any location in the sample is represented
by the gray level at the corresponding location in the
image.
 Its primary advantage over conventional SEM imaging
is the improvement in spatial resolution.

7. Visualization journey
 Electron source - tungsten filament within Field
Emission Gun (FEG).
 Electron optical coloumn -The heart of the STEM
where electromagnetic lenses, such as condenser,
objective, and projector lenses, converge and steer
the electron beam with remarkable precision.

9.  Vacuum chamber -sample holder is the stage for
the specimen, allowing intricate manipulation across
multiple axes.
 The deflection coils, or scanners, are the essence of
the STEM's scanning capability. They meticulously
sweep the electron beam in a raster pattern over the
specimen, ensuring every segment is thoroughly
scanned.
 Detectors are the sentinels that capture the signals
from the electron beam after passing through
sample

10.  The Bright Field (BF) detector captures electrons that
have traversed the specimen with minimal deviation.
 Dark Field (DF) detectors, including the specialized
High-Angle Annular Dark Field (HAADF) variety, gather
electrons scattered at steep angles
 For surface detail, Secondary Electron (SE) detectors
offer topographical insights.

13. X ray microanalysis
 Electrons bombarding the specimen cause it to emit X-
rays whose energy is characteristic of the elemental
composition of the sample. It uses an energy dispersive
dispersive X-ray (EDX) spectrometer to count and sort
characteristic X-rays according to their energy.
 Wavelength dispersive X-ray (WDX) spectrometry
measures and counts X-rays by their wavelength (a
correlate of energy).
 Electron energy loss spectrometry (EELS) analyzes
transmitted electrons to determine the amount of
energy they have lost in interactions with the sample.

14.  It provides information about the interacting atoms,
including elemental identity, chemical bonding,
valence, conduction properties and surface
properties.
 Able to identify the element or specimen

15. SAMPLE PREPARATION FOR STEM
Fixation - fixed with chemical products (e.g.
glutaraldehyde)
Rinsing and 'staining" - treated with heavy metal
compounds.
Dehydration-washing with increasing ethanol
concentration, followed by final wash
Embedding in resin - material is gradually infiltrated with
the still unpolymerized resin.
Trimming of resin block and ultrathin sectioning -
sections with a thickness of about 70 nm are cut with
special knifes of cleaved glass. The cutting is done with a
ultra-microtome.

16. Specimen Thinning
Preparation done in two steps
1)Pre-Thinning:
Reducing the thickness to about 0.1mm
2)Final Thinning:
Reducing the thickness to about 100nm involve
 Ion Milling
 Electrolytic Thinning
 Ultramicrotomy

17. Ion Milling
Uses a beam of energetic ions to bombard specimen
surfaces to reduce the thickness by knocking atoms out of a
specimen
ion beam of 1-10 keV bombarded
specimen is placed in the center at certain angle
Electrolytic Thinning
Reducing specimen thickness to 100nm
A specimen placed in an electrochemical cell as anode
Common technique is jet polishinging
Electrolytic thinning completed in 3-15 minutes

19. Cryo-ultramicrotomy
 slicing the specimen at subzero temperatures to
preserve its native state and avert thermal degradation.
 solution for temperature-sensitive samples
 staining steps into the spotlight to enhance image
contrast.
 Biological specimens, inherently low in electron density,
density, benefit from the application of heavy metal
stains such as osmium, lead, uranium, or gold.
 These agents bind selectively to specific structures
within the sample, thereby increasing electron density
and enhancing contrast in the STEM imagery.
 The choice of stain hinges on the structures or
molecules of interest and the contrast required.

20. Typical STEM samples
tissue sections, lamella, and nanoparticles on the
order of 100 nm thickness or less, meet the criteria
for electron transparency.

21. Advantages
 Enabling the use of secondary electrons, scattered beam
electrons, characteristic X-rays, and electron energy loss
 High spatial resolution
 Non-destructive technique
 ability to image at much lower accelerating voltages to
prevent sample degradation.
Disadvantage
 STEM must be conducted in a vacuum so not suitable for
certain materials, particularly some biological samples,
which are not vacuum-compatible.
 The images produced are inherently monochrome and
thus tend to require a substantial level of interpretation.

22. Applications of STEM
It can provide sample’s physical structure, electronic
structure, and composition.
Biological sciences:
 Mass determination of macromolecules can be
precisely measured with HAADF imaging since the
HAADF signal is directly proportional to mass-thickness
 It has been most widely used for filamentous structures
like DNA.
 Both the ADF and HAADF signals can also be used to
visualize specific proteins that are labeled with heavy
atoms.

23. Electron tomography acquired in the STEM can yield 3D
reconstruction of cells and cell sections up to one
micrometer in thickness.
Ceramic grain boundaries: In the study of grain
boundaries in ceramics, the grain boundary character
significantly influences bulk materials properties such as
ionic conductivity.
Semiconductor interfaces: STEM is a major analytical
technique used in the semiconductor development

24. TEM STEM
Convergent Beam Parallel At certain angle
Magnification Post specimen intermediate lenses Detectors and digital imagfe software

25. References:
 https://lifesciences.danaher.com/us/en/products/microscope
s/topics/scanning-transmission-electron-microscopy.html
 https://search.app.goo.gl/P3Necxf
 https://measurlabs.com/methods/scanning-transmission-
electron-microscopy/

26. THANK YOU