Comprehensive Notes on Scanning Electron Microscopy (SEM)

Scanning Electron Microscope (SEM)

Overview

The scanning electron microscope (SEM), also known as SCM, is a versatile instrument used in material and biological sciences.
It provides detailed topographic images of a sample surface and chemical contrast based on the atomic number of elements present.
The Hitachi SU N00 SEM features a secondary electron detector, backscattered electron detector, and a detector for low vacuum observation of charging samples.
It also has a STEM detector which functions similarly to a TEM but running at much lower accelerating voltage.
Resolution ranges from 500 nanometers to smaller than 1 nanometer, depending on the configuration.
It informs about the chemical composition of samples using backscatter electrons and electron dispersive spectroscopy (EDS).
Typically operates between 1 and 30 kilowatts.
The electron beam is focused into a small spot and scanned across the sample.
The image is built up pixel by pixel, line by line, using a raster scan.
Small angles of convergence (a few milliradians, approximately 0.05 degrees) give a large depth of field.
Data generated during the scan can be analyzed, allowing for spatially resolved chemical analysis.
The distance between the bottom of the objective lens and the sample is typically 5 to 10 millimeters.

Imaging Modes

Two main imaging modes: secondary electron imaging and backscattered electron imaging.
Secondary electrons produce topographic images that are visually appealing and easy to interpret.
Backscatter electrons are sensitive to the chemical composition of the sample.
The likelihood of a backscatter event is proportional to the atomic number of the sample.
A brighter signal indicates a higher atomic number.
For example, a darker area may be an aluminum alloy, while a lighter region is a copper alloy.
If the sample isn't smooth, the backscatter image will have shadows that highlight topography.
Large differences in composition can create compositional contrast in secondary electron images.

Key Definitions

Working Distance: The distance from the sample to the objective lens, usually measured in millimeters.
Objective Aperture: A strip of platinum with holes of different sizes (typically 10 to 100s of micrometers in diameter).
Probe Size: The diameter of the beam at the sample surface.
Interaction Volume: The volume of the sample that the beam interacts with.
Microscope resolution is defined by the volume of the sample that the signal is generated from

User-Controlled Parameters

Accelerating Voltage: Ranges from 30 kV down to 1 kV, with some instruments reaching 0.5 kV or lower.
Excitation of the Condenser System: Controls the intensity of the electron beam.
Objective Aperture: Used to define the convergence angle.
Working Distance: The distance between the objective lens and the sample.
Detectors: Choice depends on whether topography, chemistry, or both are of interest.

Accelerating Voltage Considerations

Higher voltage gives a shorter wavelength, which can lead to higher resolution.
Higher voltages can achieve smaller probe sizes.
At higher voltages, increased scattering over a larger volume of the sample can reduce resolution.
Voltage selection depends on sensitivity, elemental analysis needs, and excitation voltage of the sample.
Reducing voltage can minimize beam-induced damage on delicate samples.
For biology samples, 2 to 5 kV is common, while hard materials may require more than 15 kV.
For EDS, the voltage should be at least twice the excitation voltage of the element being analyzed.

Condenser System Excitation

Controls the electron beam as it's focused onto the sample using a series of lenses.
Controls the current density of the electron beam, also known as spot size or beam current.
Larger spot sizes equate to more electrons and a stronger signal but reduced resolution.
Smaller spot sizes provide high resolution but a weaker signal.

Objective Aperture

Changing the aperture size alters the angle of convergence.
Smaller apertures reduce the angle of convergence, improving the depth of field.
With a small enough angle, a significant range of the sample can appear in focus simultaneously.
Using smaller apertures can cause diffraction of the electron beam, spreading out the probe and reducing resolution.

Working Distance

Provides control over convergence angle and depth of focus.
Shorter working distances typically improve the signal for backscatter detectors.
Too short a working distance can reduce the amount of signal for secondary electronic detectors.
EDS detectors have a very specific working distance requirement.
Longer walking distances tend to reduce the resolution of the microscope.

Detectors

Fundamental detectors: secondary electron and backscattering electron detectors.
Secondary electrons are generated by interactions between the incident beam and weakly bound conduction band electrons.
They are emitted in random directions and have low energy (2 to 5 EV).
A positive voltage attracts the secondary electrons.
Collection depends on the topography of the sample; sharp edges facing the detector yield a brighter signal.
Backscattered electrons are primary beam electrons that have scattered enough to leave the sample again.
The probability of this happening is higher with higher atomic numbers.
For example, an aluminum-rich phase appears darker, while a nickel-rich phase is brighter.

Interaction Volume

Secondary electrons have low energy, so they can't travel far through the sample before being reabsorbed.
The resolution of a secondary electron image is related to the probe size of the beam.
Backscattered electrons have as much energy as the incident electrons, so they can escape from deeper within the sample.
The interaction volume is a more realistic indicator of resolution when using backscattered electrons.
Using a low voltage will reduce the interaction volume but increase the probe size and signal strength.

Energy Dispersive Spectroscopy (EDS)

A qualitative and quantitative X-ray microanalytical technique that provides information about the chemical composition of a sample.
Generally works for elements above atomic number 3, with specialized detectors that can detect lithium too.
Incident X-rays cause an electron from the sample to be ejected. Subsequently is replaced by an electron from the higher energy and causes the emission of a photon from the sample.
The spectra is generated with energy on the X-axis and frequency on the Y-axis.
Characteristic X-rays appear as strong, well-defined peaks.

Impact of Accelerating Voltage on EDS Spectrum

The incident electron needs to have enough energy to eject the core shell electron.
The incident beam electrons must have at least as much energy as the X-ray being observed.
Typically, double the energy of the peak being observed is used as a threshold.
If the beam was 10 KV, the frequency of peaks would start to decrease, and at 5 KV, certain peaks might disappear entirely.
The possible energy transitions should be considered and balance the voltage required to see the X-rays.

EDS Mapping

The SCM generates a megapixel by pixel image, allowing it to do this with all signals.
Every signal of the image has a spectra associated with it.
We can create an image of elemental distribution in the sample.

Sample Preparation

Samples are mounted on a sample stub (aluminum sample holder).
Samples need to be dry due to the high vacuum in the SEM.
They also need to be conductive to allow electrons to escape and prevent imaging problems.
A flow chart summarizes the sample preparation process, ending in a dry and conductive sample.
Fresh living tissue requires substantial processing, while a lump of metal may not need any prep.
Tissue samples are typically fixed chemically, then dried using critical point drying with liquid carbon dioxide or chemical drying with HMDS.
The sample is then coated with a thin layer of conducting material like carbon, gold, or platinum.

Specialized SCM Techniques

Cryo-SEM: Snap freezing a sample and imaging it under cryogenic temperatures allows imaging without fixation and dehydration.
This introduces its own complications, such as damage from ice crystals.
Transfer chambers with spot coaters and articulated arms are used to fracture and coat the surface.
Ultramicrotome in SEM: A miniaturized version of a device used in TEM is placed inside the SCM to cut slices less than 100 nanometers thick.
Repeated cutting and imaging allows for building a 3D volume.
Backscatter detectors and heavy metal staining are used to see the details.
Focused Ion Beam (FIB): Uses a beam of ions to cut layers of the sample.

Vacuum Modes

High Vacuum Mode: The normal mode of operation minimizes scattering of the electron beam.
Low Vacuum Mode: Reduces the vacuum in the chamber, allowing electrons to dissipate.
Since backscattering electrons and characteristic X-rays are generally higher energy than secondary electrons, their detection is not critically dependent on a high vacuum being maintained in the specimen chamber.
Secondary Electron Detection in Low Vacuum: Secondary electrons travel through the cloud of low-pressure air, leading to photon emission.
SCMs with VP mode are equipped with a detector to measure the intensity of this glow to create a topographic image.
Environmental SCM: Pressure is high enough to sustain liquid water in the microscope, enabling imaging of fresh wet biological samples without special preparation.

Limitations of SEM

Imaging wet samples requires special setups like low vacuum, environmental SCM, special detectors, or cryogenic conditions.
SEM require samples to be dry
Non-conductive samples require coating and preparation.
SEM only produces grayscale images.
SEM cannot accurately measure height like AFM does.
Sub-surface imaging is limited due to the interaction volume.
Imaging through fluid is limited due to interaction volume.
Atomic imaging is not possible due to resolution and probe size limitations.
Elemental analysis below the micrometer scale is difficult because the sharp probe not possible to make.
Charged molecules can interfere with electron detection.