Explore the available techniques within the ALL-MICRO network

SEM is a technique used to obtain details regarding the surface morphology of samples of any nature up to a nanometric resolution. In a scanning electron microscope, a nanometer-sized beam of electrons is focused on successive points, scanning point-bypoint the sample to be analyzed. For each position, different detectors placed inside the microscope allow information to be collected from various phenomena caused by the impact of electrons on the sample. Among them, secondary electrons expelled from the surface layers of the sample show its morphology, down to details smaller than 1 nm (10-9m, or a thousandth of a μm). Backscattered electrons are higher energy electrons that come from more internal layers and allow for the chemically different areas of the sample to be distinguished.

Due to the impact of electrons, X-rays are also ejected from the sample revealing the chemical composition present at each point of the sample. Energy Dispersive X-ray Spectroscopy (EDS) analysis performed with an SEM instrument can reveal the elemental composition using the X-rays emitted by the sample upon electron impact. The technique allows to obtain quantitative or semiquantitative determination of the chemical composition in precise locations of the area analyzed.In general, specimens used in SEM microscopy must be able to conduct electrons. For this purpose, the samples are often covered with a metal layer. In addition, samples must be dry, and fresh biological samples must be fixed and dehydrated. To obtain the highest resolution and magnification the sample must be flat or very thin, e.g. a monolayer of nanomaterial or a polished section.

SEM works with a wide range of samples, including (but not limited to) composites, rocks, minerals, and biological samples. Analyzing the backscattered electrons it is possible to easily detect differences between elements with different atomic weights, e.g. organic contaminations on metal materials, or vice versa metal nanoparticles on organic materials.

The Low Vacuum and Environmental SEM techniques allow the imaging of nonconductive samples in low vacuum conditions, without the need for metal coating. This modality can be used to analyze samples that are wet or dry and not conductive and for which fixing and dehydration would cause structural damage, or for which the metal coating cannot be applied. For example, cell-seeded materials, samples derived from plants, and unicellular organisms can be analyzed with ESEM, albeit with a lower resolution than for the high vacuum conditions.

FIB-SEM instruments couple the electron beam scanning of the sample of the SEM instrument with an additive ion beam, usually using gallium ions. Unlike electrons, the ion beam is destructive for the sample, allowing milling and cutting to expose the crosssection of the material or to prepare small sections of the material. The FIB column, coupled with the SEM column, can be used to analyze the microscopic structure of the bulk of the sample, by carving out different sections.

The Scanning Probe Microscopy (SPM) is a family of imaging techniques that allow for high-resolution visualization of material surfaces at the atomic level. Unlike optical microscopy, which uses light to illuminate samples, or electron microscopy, which uses an electron beam, SPM techniques rely on the interactions between a probe that scans the surface and the sample itself. These interactions can involve forces or quantum phenomena, enabling detailed imaging and characterization of materials.

Atomic Force Microscopy (AFM) is one of the most widely used SPM techniques. In AFM, an extremely fine probe is mounted on a flexible cantilever, positioned just a few nanometers above the sample surface. As the probe scans the surface, it measures the forces of interaction—such as van der Waals forces, electrostatic forces, and contact forces—which cause deflection of the cantilever. This deflection is recorded to create a three-dimensional image of the surface.

AFM can operate in various environments: air, solution, or vacuum, making it versatile for different types of materials, from conductors to biological substances. Its applications are extensive, including the characterization of nanotubes, measuring the mechanical properties of materials, and studying biomolecules and cell membranes.

Scanning Tunneling Microscopy (STM) is another key SPM technique that utilizes the principle of quantum tunneling. In STM, a conductive probe is positioned very close to the surface of a conductive or semiconductive material. When the probe approaches within a few angstroms of the surface, a tunneling current is generated between the probe and the sample, proportional to the distance between them. By measuring this current while the probe scans the surface, high-resolution images of the sample's topography can be obtained.

STM is particularly valuable for studying conductive surfaces and gaining detailed insights into the electronic structure of materials at the atomic level. Applications of STM include research in nanotechnology, analysis of semiconductor materials, and study of molecular interactions.

Scanning Probe Microscopy techniques are applied across a wide range of fields. In scientific research, they are used for characterizing new materials, nanofabrication, and analyzing complex biological systems. In industrial settings, SPM can be employed for quality control of surfaces and material research, with applications extending from microelectronics to nanotechnology.

Optical microscopy utilizes visible light to magnify and visualize samples, providing essential insights in fields like biology, materials science, and medicine. Key optical microscopy techniques include linear (e.g., confocal microscopy) and non-linear methods (e.g., multiphoton microscopy).

Confocal microscopy improves resolution and contrast by using a spatial pinhole to exclude out-of-focus light. This technique allows for the collection of high-resolution, three-dimensional images from thick specimens by scanning the sample point by point.

Confocal microscopy has applications in cell biology, as the visualization of cellular structures and dynamics, in the detailed imaging of complex tissues and organs. Fluorescence Imaging allows the analysis of interactions between proteins and other biomolecules.

Multiphoton microscopy is a non-linear technique that uses two or more low-energy photons to excite fluorescent molecules, enabling deeper tissue penetration and minimizing phototoxicity. This method provides high-resolution three-dimensional images of live samples and has applications in neuroscience (e.g., imaging neuronal structures and synaptic activities in live brains), developmental biology (e.g., studying embryonic development and cellular interactions in real-time), and cancer research (e.g., monitoring of tumor growth and response to therapies).

In addition to these more common techniques, the ALL-MICRO network comprises a Digital Holographic Microscope. This instrument captures the interference pattern of light reflected from a sample, allowing for quantitative phase imaging. This technique enables a detailed assessment of morphology and refractive index without staining or labeling.

Micro-Raman spectroscopy combines optical microscopy with Raman spectroscopy to provide detailed information about the molecular composition and structure of materials at a microscopic scale. In this technique, a laser is focused on a small area of the sample, and the scattered light is analyzed to identify the vibrational modes of the molecules. This provides chemical information without the need for extensive sample preparation or labeling.