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.
In optics, the refractive index of an optical medium is a dimensionless number that gives the indication of the light bending ability of that medium.
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.
Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very-high-resolution type of scanning probe microscopy (SPM), with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.
Optical tweezers are scientific instruments that use a highly focused laser beam to hold and move microscopic and sub-microscopic objects like atoms, nanoparticles and droplets, in a manner similar to tweezers. If the object is held in air or vacuum without additional support, it can be called optical levitation.
A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.
Fluorescence correlation spectroscopy (FCS) is a statistical analysis, via time correlation, of stationary fluctuations of the fluorescence intensity. Its theoretical underpinning originated from L. Onsager's regression hypothesis. The analysis provides kinetic parameters of the physical processes underlying the fluctuations. One of the interesting applications of this is an analysis of the concentration fluctuations of fluorescent particles (molecules) in solution. In this application, the fluorescence emitted from a very tiny space in solution containing a small number of fluorescent particles (molecules) is observed. The fluorescence intensity is fluctuating due to Brownian motion of the particles. In other words, the number of the particles in the sub-space defined by the optical system is randomly changing around the average number. The analysis gives the average number of fluorescent particles and average diffusion time, when the particle is passing through the space. Eventually, both the concentration and size of the particle (molecule) are determined. Both parameters are important in biochemical research, biophysics, and chemistry.
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.
Dynamic light scattering (DLS) is a technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution. In the scope of DLS, temporal fluctuations are usually analyzed using the intensity or photon auto-correlation function. In the time domain analysis, the autocorrelation function (ACF) usually decays starting from zero delay time, and faster dynamics due to smaller particles lead to faster decorrelation of scattered intensity trace. It has been shown that the intensity ACF is the Fourier transform of the power spectrum, and therefore the DLS measurements can be equally well performed in the spectral domain. DLS can also be used to probe the behavior of complex fluids such as concentrated polymer solutions.
Photothermal spectroscopy is a group of high sensitivity spectroscopy techniques used to measure optical absorption and thermal characteristics of a sample. The basis of photothermal spectroscopy is the change in thermal state of the sample resulting from the absorption of radiation. Light absorbed and not lost by emission results in heating. The heat raises temperature thereby influencing the thermodynamic properties of the sample or of a suitable material adjacent to it. Measurement of the temperature, pressure, or density changes that occur due to optical absorption are ultimately the basis for the photothermal spectroscopic measurements.
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.
Differential dynamic microscopy (DDM) is an optical technique that allows performing light scattering experiments by means of a simple optical microscope. DDM is suitable for typical soft materials such as for instance liquids or gels made of colloids, polymers and liquid crystals but also for biological materials like bacteria and cells.
Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique with an intermediate-to-high optical resolution, but good optical sectioning capabilities and high speed. In contrast to epifluorescence microscopy only a thin slice of the sample is illuminated perpendicularly to the direction of observation. For illumination, a laser light-sheet is used, i.e. a laser beam which is focused only in one direction. A second method uses a circular beam scanned in one direction to create the lightsheet. As only the actually observed section is illuminated, this method reduces the photodamage and stress induced on a living sample. Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. Because light sheet fluorescence microscopy scans samples by using a plane of light instead of a point, it can acquire images at speeds 100 to 1,000 times faster than those offered by point-scanning methods.
Atmospheric lidar is a class of instruments that uses laser light to study atmospheric properties from the ground up to the top of the atmosphere. Such instruments have been used to study, among other, atmospheric gases, aerosols, clouds, and temperature.
Interferometric scattering microscopy (iSCAT) refers to a class of methods that detect and image a subwavelength object by interfering the light scattered by it with a reference light field. The underlying physics is shared by other conventional interferometric methods such as phase contrast or differential interference contrast, or reflection interference microscopy. The key feature of iSCAT is the detection of elastic scattering from subwavelength particles, also known as Rayleigh scattering, in addition to reflected or transmission signals from supra-wavelength objects. Typically, the challenge is the detection of tiny signals on top of large and complex, speckle-like backgrounds. iSCAT has been used to investigate nanoparticles such as viruses, proteins, lipid vesicles, DNA, exosomes, metal nanoparticles, semiconductor quantum dots, charge carriers and single organic molecules without the need for a fluorescent label.
The operation of a photon scanning tunneling microscope (PSTM) is analogous to the operation of an electron scanning tunneling microscope, with the primary distinction being that PSTM involves tunneling of photons instead of electrons from the sample surface to the probe tip. A beam of light is focused on a prism at an angle greater than the critical angle of the refractive medium in order to induce total internal reflection within the prism. Although the beam of light is not propagated through the surface of the refractive prism under total internal reflection, an evanescent field of light is still present at the surface.
Stimulated Raman spectroscopy, also referred to as stimulated Raman scattering (SRS) is a form of spectroscopy employed in physics, chemistry, biology, and other fields. The basic mechanism resembles that of spontaneous Raman spectroscopy: a pump photon, of the angular frequency , which is scattered by a molecule has some small probability of inducing some vibrational transition, as opposed to inducing a simple Rayleigh transition. This makes the molecule emit a photon at a shifted frequency. However, SRS, as opposed to spontaneous Raman spectroscopy, is a third-order non-linear phenomenon involving a second photon—the Stokes photon of angular frequency —which stimulates a specific transition. When the difference in frequency between both photons resembles that of a specific vibrational transition the occurrence of this transition is resonantly enhanced. In SRS, the signal is equivalent to changes in the intensity of the pump and Stokes beams. The signals are typically rather low, of the order of a part in 10^5, thus calling for modulation-transfer techniques: one beam is modulated in amplitude and the signal is detected on the other beam via a lock-in amplifier. Employing a pump laser beam of a constant frequency and a Stokes laser beam of a scanned frequency allows for the unraveling of the spectral fingerprint of the molecule. This spectral fingerprint differs from those obtained by other spectroscopy methods such as Rayleigh scattering as the Raman transitions confer to different exclusion rules than those that apply for Rayleigh transitions.
Photoacoustic microscopy is an imaging method based on the photoacoustic effect and is a subset of photoacoustic tomography. Photoacoustic microscopy takes advantage of the local temperature rise that occurs as a result of light absorption in tissue. Using a nanosecond pulsed laser beam, tissues undergo thermoelastic expansion, resulting in the release of a wide-band acoustic wave that can be detected using a high-frequency ultrasound transducer. Since ultrasonic scattering in tissue is weaker than optical scattering, photoacoustic microscopy is capable of achieving high-resolution images at greater depths than conventional microscopy methods. Furthermore, photoacoustic microscopy is especially useful in the field of biomedical imaging due to its scalability. By adjusting the optical and acoustic foci, lateral resolution may be optimized for the desired imaging depth.
The scanning helium microscope (SHeM) is a novel form of microscopy that uses low-energy (5–100 meV) neutral helium atoms to image the surface of a sample without any damage to the sample caused by the imaging process. Since helium is inert and neutral, it can be used to study delicate and insulating surfaces. Images are formed by rastering a sample underneath an atom beam and monitoring the flux of atoms that are scattered into a detector at each point.
