Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.
Cathodoluminescence is an optical and electromagnetic phenomenon in which electrons impacting on a luminescent material such as a phosphor, cause the emission of photons which may have wavelengths in the visible spectrum. A familiar example is the generation of light by an electron beam scanning the phosphor-coated inner surface of the screen of a television that uses a cathode-ray tube. Cathodoluminescence is the inverse of the photoelectric effect, in which electron emission is induced by irradiation with photons.
Raman spectroscopy is a spectroscopic technique typically used to determine vibrational modes of molecules, although rotational and other low-frequency modes of systems may also be observed. Raman spectroscopy is commonly used in chemistry to provide a structural fingerprint by which molecules can be identified.
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast.
Scanning probe microscopy (SPM) is a branch of microscopy that forms images of surfaces using a physical probe that scans the specimen. SPM was founded in 1981, with the invention of the scanning tunneling microscope, an instrument for imaging surfaces at the atomic level. The first successful scanning tunneling microscope experiment was done by Gerd Binnig and Heinrich Rohrer. The key to their success was using a feedback loop to regulate gap distance between the sample and the probe.
Near-field scanning optical microscopy (NSOM) or scanning near-field optical microscopy (SNOM) is a microscopy technique for nanostructure investigation that breaks the far field resolution limit by exploiting the properties of evanescent waves. In SNOM, the excitation laser light is focused through an aperture with a diameter smaller than the excitation wavelength, resulting in an evanescent field on the far side of the aperture. When the sample is scanned at a small distance below the aperture, the optical resolution of transmitted or reflected light is limited only by the diameter of the aperture. In particular, lateral resolution of 6 nm and vertical resolution of 2–5 nm have been demonstrated.
Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin had patented the STED idea. This patent was unknown to Hell and Wichmann in 1994.
A scanning helium ion microscope is an imaging technology based on a scanning helium ion beam. Similar to other focused ion beam techniques, it allows to combine milling and cutting of samples with their observation at sub-nanometer resolution.
Fluorescence interference contrast (FLIC) microscopy is a microscopic technique developed to achieve z-resolution on the nanometer scale.
Phase-contrast microscopy (PCM) is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations.
Johan Sebastiaan Ploem is a Dutch microscopist and digital artist. He made significant contribution to the field of fluorescence microscopy, and invented reflection interference contrast microscopy.
Lipid bilayer characterization is the use of various optical, chemical and physical probing methods to study the properties of lipid bilayers. Many of these techniques are elaborate and require expensive equipment because the fundamental nature of the lipid bilayer makes it a very difficult structure to study. An individual bilayer, since it is only a few nanometers thick, is invisible in traditional light microscopy. The bilayer is also a relatively fragile structure since it is held together entirely by non-covalent bonds and is irreversibly destroyed if removed from water. In spite of these limitations dozens of techniques have been developed over the last seventy years to allow investigations of the structure and function of bilayers. The first general approach was to utilize non-destructive in situ measurements such as x-ray diffraction and electrical resistance which measured bilayer properties but did not actually image the bilayer. Later, protocols were developed to modify the bilayer and allow its direct visualization at first in the electron microscope and, more recently, with fluorescence microscopy. Over the past two decades, a new generation of characterization tools including AFM has allowed the direct probing and imaging of membranes in situ with little to no chemical or physical modification. More recently, dual polarisation interferometry has been used to measure the optical birefringence of lipid bilayers to characterise order and disruption associated with interactions or environmental effects.
Interference reflection microscopy (IRM), also called Reflection Interference Contrast Microscopy (RICM) or Reflection Contrast Microscopy (RCM) depending on the specific optical elements used, is an optical microscopy technique that leverages thin-film interference effects to form an image of an object on a glass surface. The intensity of the signal is a measure of proximity of the object to the glass surface. This technique can be used to study events at the cell membrane without the use of a (fluorescent) label as is the case for TIRF microscopy.
Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.
Photoconductive atomic force microscopy (PC-AFM) is a variant of atomic force microscopy that measures photoconductivity in addition to surface forces.
The technique of vibrational analysis with scanning probe microscopy allows probing vibrational properties of materials at the submicrometer scale, and even of individual molecules. This is accomplished by integrating scanning probe microscopy (SPM) and vibrational spectroscopy. This combination allows for much higher spatial resolution than can be achieved with conventional Raman/FTIR instrumentation. The technique is also nondestructive, requires non-extensive sample preparation, and provides more contrast such as intensity contrast, polarization contrast and wavelength contrast, as well as providing specific chemical information and topography images simultaneously.
Quantum microscopy allows microscopic properties of matter and quantum particles to be measured and imaged. Various types of microscopy use quantum principles. The first microscope to do so was the scanning tunneling microscope, which paved the way for development of the photoionization microscope and the quantum entanglement microscope.
Tip-enhanced Raman spectroscopy (TERS) is a variant of surface-enhanced Raman spectroscopy (SERS) that combines scanning probe microscopy with Raman spectroscopy. High spatial resolution chemical imaging is possible via TERS, with routine demonstrations of nanometer spatial resolution under ambient laboratory conditions, or better at ultralow temperatures and high pressure.
Super-resolution dipole orientation mapping (SDOM) is a form of fluorescence polarization microscopy (FPM) that achieved super resolution through polarization demodulation. It was first described by Karl Zhanghao and others in 2016. Fluorescence polarization (FP) is related to the dipole orientation of chromophores, making fluorescence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. In addition to fluorescence intensity, wavelength, and lifetime, the fourth dimension of fluorescence—polarization—can also provide intensity modulation without the restriction to specific fluorophores; its investigation in super-resolution microscopy is still in its infancy.
Libai Huang is a Chinese-American chemist who is a professor at Purdue University. She is interested in unravelling the structure-property relationships of next-generation solar materials.
