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.
A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope.
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.
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.
Two-photon excitation microscopy is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image. Due to the non-linearity of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.
A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution. With it the typical range of the axial resolution of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy.
ICFO – The Institute of Photonic Sciences is a research center devoted to the science and technology of light. Located in Castelldefels, ICFO was created in 2002 by the Government of Catalonia and the Technical University of Catalonia.
Caesar Aya-ay Saloma is a professor of the National Institute of Physics (NIP) at the University of the Philippines College of Science and a member of the National Academy of Science and Technology.
Andor Technology Ltd is a global developer and manufacturer of high-performance scientific cameras, microscopy systems and spectrographs for academic, government, and industrial applications. Founded in 1989, the company's products play a central role in the advancement of research in the fields of life sciences, physical sciences, and industrial applications. Andor was purchased for £176 million in December 2013 by Oxford Instruments. The company is based in Belfast, Northern Ireland and now employs over 400 staff across the group at its offices in Belfast, Japan, China, Switzerland and the US.
Bruce J. Tromberg is an American photochemist and a leading researcher in the field of biophotonics. He is the director of the National Institute of Biomedical Imaging and Bioengineering (NIBIB) within the National Institutes of Health (NIH). Before joining NIH, he was Professor of Biomedical Engineering at The Henry Samueli School of Engineering and of Surgery at the School of Medicine, University of California, Irvine. He was the principal investigator of the Laser Microbeam and Medical Program (LAMMP), and the Director of the Beckman Laser Institute and Medical Clinic at Irvine. He was a co-leader of the Onco-imaging and Biotechnology Program of the NCI Chao Family Comprehensive Cancer Center at Irvine.
Second-harmonic imaging microscopy (SHIM) is based on a nonlinear optical effect known as second-harmonic generation (SHG). SHIM has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. A second-harmonic microscope obtains contrasts from variations in a specimen's ability to generate second-harmonic light from the incident light while a conventional optical microscope obtains its contrast by detecting variations in optical density, path length, or refractive index of the specimen. SHG requires intense laser light passing through a material with a noncentrosymmetric molecular structure, either inherent or induced externally, for example by an electric field.
Winfried Denk is a German physicist. He built the first two-photon microscope while he was a graduate student in Watt W. Webb's lab at Cornell University, in 1989.
Christoph Cremer is a German physicist and emeritus at the Ruprecht-Karls-University Heidelberg, former honorary professor at the University of Mainz and was a former group leader at Institute of Molecular Biology (IMB) at the Johannes Gutenberg University of Mainz, Germany, who has successfully overcome the conventional limit of resolution that applies to light based investigations by a range of different methods. In the meantime, according to his own statement, Christoph Cremer is a member of the Max Planck Institute for Chemistry and the Max Planck Institute for Polymer Research.
The Raman microscope is a laser-based microscopic device used to perform Raman spectroscopy. The term MOLE is used to refer to the Raman-based microprobe. The technique used is named after C. V. Raman, who discovered the scattering properties in liquids.
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.
Endomicroscopy is a technique for obtaining histology-like images from inside the human body in real-time, a process known as ‘optical biopsy’. It generally refers to fluorescence confocal microscopy, although multi-photon microscopy and optical coherence tomography have also been adapted for endoscopic use. Commercially available clinical and pre-clinical endomicroscopes can achieve a resolution on the order of a micrometre, have a field-of-view of several hundred µm, and are compatible with fluorophores which are excitable using 488 nm laser light. The main clinical applications are currently in imaging of the tumour margins of the brain and gastro-intestinal tract, particularly for the diagnosis and characterisation of Barrett’s Esophagus, pancreatic cysts and colorectal lesions. A number of pre-clinical and transnational applications have been developed for endomicroscopy as it enables researchers to perform live animal imaging. Major pre-clinical applications are in gastro-intestinal tract, toumour margin detection, uterine complications, ischaemia, live imaging of cartilage and tendon and organoid imaging.
Wide-field multiphoton microscopy refers to an optical non-linear imaging technique tailored for ultrafast imaging in which a large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as two-photon fluorescence or second harmonic generation. In scanning multiphoton microscopes the high intensities are achieved by tightly focusing the light, and the image is obtained by beam scanning. In wide-field multiphoton microscopy the high intensities are best achieved using an optically amplified pulsed laser source to attain a large field of view (~100 µm). The image in this case is obtained as a single frame with a CCD without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest. With wide-field multiphoton microscopy the frame rate can be increased up to a 1000-fold compared to multiphoton scanning microscopy. Wide-field multiphoton microscopes are not yet commercially available, but working prototypes exist in several optics laboratories.
Elizabeth M. C. Hillman is a British-born academic who is Professor of Biomedical Engineering and Radiology at Columbia University. She was awarded the 2011 Adolph Lomb Medal from The Optical Society and the 2018 SPIE Biophotonics Technology Innovator Award.
Kristen Carlson Maitland is an associate professor at Texas A&M University. She develops optical instrumentation for the detection and diagnosis of diseases, including infection and cancer. She has served on the Board of Directors of SPIE.
