|Year : 2021 | Volume
| Issue : 2 | Page : 59-64
Microscope in periodontics
Suman Mukherjee1, Sharmistha Dasgupta2
1 Periodontist and Oral Implantology, Arogya Bardini Dental Clinic, Puruliya, West Bengal, India
2 Periodontist and Oral Implantology, Spartan Dental Clinic, Nirman Vihar, Delhi, India
|Date of Submission||18-Jan-2021|
|Date of Decision||12-May-2021|
|Date of Acceptance||02-Jun-2021|
|Date of Web Publication||26-Dec-2021|
2nd Floor, Shakuntala Garden, Ranchi Road, Puruliya - 723 101, West Bengal.
Source of Support: None, Conflict of Interest: None
The microscope has been one of the oldest yet most exquisite inventions in human history. The lenses changed the future of medical science and its abstraction forever. Previously, humans never know much about the source of disease, but today, we know that the universe of microbes is vaster and more limitless than it ever was. However, the microscope is not just limited to laboratory in vitro research and study, it has remodeled dentistry more today than ever. This article describes the various types of microscope used in periodontics, endodontics, and oral pathology in dentistry.
Keywords: Bacteria, dental, electron, microbes, microscope
|How to cite this article:|
Mukherjee S, Dasgupta S. Microscope in periodontics. D Y Patil J Health Sci 2021;9:59-64
| Introduction|| |
The microscope has been one of the oldest yet most exquisite inventions in human history. The lenses changed the future of medical science and its abstraction forever. Previously, humans never know much about the source of disease, but today, we know that the universe of microbes is vaster and more limitless than it ever was. However, the microscope is not just limited to laboratory in vitro research and study; it has remodeled dentistry more today than ever. In periodontics, surgical microscopes and loupes have been playing a key role in microsurgery. Since, its advent in root coverage procedures and flap procedures, surgical loupes are gaining attention and interest. As such, we need to know the past of this novel discovery which changed the beliefs of medicine in general and dental science in particular forever.
Timeline of the microscope
1st century AD (year 100) – Romans invented Glass
1300 AD – Concave and Convex lenses first came into general use for spectacles
Robert Bacon (1212–1294) – believed to be the first person to combine to lenses
14th AD spectacles first made in Italy
1590 – Two Dutch spectacle-makers and father-and-son team, Hans and Zacharias Janssen, create the first microscope
1666 – Italian scientist Marcello Malpighi, called the father of histology by some historians of biology, began his analysis of biological structures with the lungs
1667: Robert Hooke’s famous “Micrographia” is published, which outlines Hooke’s various studies using the microscope
1675: Anton van Leeuwenhoek, who used a microscope with one lens to observe insects and other specimens. Leeuwenhoek was the first to observe bacteria
18th century: As technology improved, microscopy became more popular among scientists. Part of this was due to the discovery that combining two types of glass reduced the chromatic effect
1830: Joseph Jackson Lister discovers that using weak lenses together at various distances provided clear magnification
1873: Ernst Leitz microscope was introduced with a revolving mount (turret) for 5 objectives
1878: A mathematical theory linking resolution to light wavelength is invented by Ernst Abbe
1878: Oil immersion lens (cedar oil) was introduced that resulted in a homogeneous optical path
1903– Richard Zsigmondy won Nobel prize for his phase contrast microscope
1904: The first commercial ultraviolet microscope by Zeiss
1930: Fritz Zernike discovered that he could view unstained cells using the phase angle of rays. It took until 1941 to bring a commercial microscope to market
1931: Ernst Ruska co-invented an electron microscope, for which he won the Nobel Prize in Physics in 1986
1937: First scanning electron microscope was built
1939-Siemens supplied the first commercially available electron microscope
1953: Zernike was awarded the Nobel Prize for his phase-contrast work
1981: Gerd Binnig and Heinrich Rohrer invented a scanning tunneling microscope that gives three-dimensional images of objects down to the atomic level. Binnig and Rohrer won the Nobel Prize in Physics in 1986
1982 – A scanning probe microscope was invented that works by measuring current
1983-Scanning laser confocal microscope was commercially available
1986-The atomic force microscope was invented that measures force instead of current
1986-Gerard Bining Heinrich Rohrer Discovered atomic microscope in 1981 and won Nobel prize for it in 1986
Cryo-electron microscopy innovators win 2017 Nobel Prize in Chemistry.
The word Microscope is derived from the Ancient Greek word:
μικρός, mikrós, meaning “small” and σκοπεῖν, skopeîn, meaning “to look” or “see.”
It was Giovanni Faber who coined the term “microscope” for the compound microscope Galileo submitted to the Accademia dei Lincei in 1625 (Galileo had called it the “occhiolino” or “little eye”).
A microscope can be defined as an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called Microscopy. Microscopic means invisible to the eye unless aided by a microscope.
The ability to distinguish detail is called resolution or resolving power and depends on the wavelength of light used and on a value, called the numerical aperture (NA) a characteristic of microscopes that determines how much light enters the lens.
The limit of resolution of an objective (d) is the distance between any two closest points on the microscopic object, which can be resolved into two separate and distinct points on the enlarged image.
Limit of resolution = d = λ/2 NA.
The magnification power measures how much larger an object appears after magnification. It is calculated by dividing the focal length of the scanning object (lens) by the focal length of the eyepiece.
A 1x magnification power is a 100% increase in the magnified object’s size, for example, a 1-inch object at ×1 would appear to be 2 inches. At ×2 power, the same object would appear to be 3 inches. Magnification power is reported on scientific reports as a means of standardization.
There are three well-known branches of microscopy [Table 1] and [Table 2]:
|Table 1: In addition to the above categories, optical microscopes can be classified as follows|
Click here to view
Scanning Probe Microscopy.
Optical microscopy is a technique employed to closely view a sample through the magnification of a lens with visible light. This is the traditional form of microscopy, which was first invented before the 18th century and is still in use today.
Binocular stereoscopic microscope
It uses a magnification in range of ×10–×100. It allows easy observation of 3D objects at low magnification.
Polarizing microscope/petrographic microscope
It has a magnification range of ×4–×100. It uses different light transmission characteristics of materials, such as crystalline structures, to produce an image. Materials that can be examined under a polarized microscope include minerals, ceramics, polymers, urea, and funguses. It is also used to study the property of collagen and amyloid.
Differential interference contrast microscope
It uses a magnification in range of × 400–×1500. This microscope, similar to the phase contrast, is used to observe minute surface irregularities but at a higher resolution. However, the use of polarized light limits the variety of observable specimen containers.,
Total internal reflection fluorescence microscope
It uses an evanescent wave to only illuminate near the surface of a specimen. The region that is viewed is generally very thin compared to conventional microscopes. Observation is possible in molecular units due to reduced background light.
Multiphoton excitation microscope
It uses multiple excitation lasers that reduce damage to cells and allow high-resolution observation of deep areas. It is used to observe nerve cells and blood flow in the brain.,
Structured illumination microscope
It is a high-resolution microscope with advanced technology to overcome limited resolution found in optical microscopes that is caused by the diffraction of light.
Scanning probe microscope/atomic force microscope
It has a magnification of ×1,000,000. In 1986, Binnig and Quate demonstrated for the first time the ideas of Atomic force microscopy (AFM), which used an ultra-small probe tip at the end of a cantilever. This microscope scans the surface of samples with a probe, and this interaction is used to measure fine surface shapes or properties. The optical and electron microscopes can easily generate two dimensional images of a sample surface; However, these microscopes cannot measure the vertical dimension (z-direction) of the sample, the height (e.g., particles) or depth (e.g., holes, pits) of the surface features. AFM, which uses a sharp tip to probe the surface features by raster scanning, can image the surface topography with extremely high magnifications, up to ×1,000,000, comparable or even better than electronic microscopes. The measurement of an AFM is made in three dimensions, the horizontal X-Y plane and the vertical Z dimension. Resolution (magnification) at Z-direction is normally higher than X-Y.,
Scanning near-field optical microscope
NSOM/SNOM is a microscopic technique used for nanostructure investigation that breaks the far field resolution limit by exploiting the properties of evanescent waves.
It is ideally suited to quickly and effortlessly image the optical properties of a sample with resolution below the diffraction limit. SNOM is significant in nanotechnology research, nano-photonics and nano-optics. In Life Science and materials research, it is used for the optical detection of the most miniscule surface. With SNOM, single molecule detection is easily achievable. Dynamic properties can also be studied at a subwavelength scale. It provides 70 times better resolution than atomic force microscope.
The compound microscope
The word compound means multiple, mix, or a combination of both. A compound light microscope is a microscope with more than one lens and its own light source. Because it contains its own light source in its base, a compound light microscope is also considered a bright field microscope.
Parts of a compound microscope
Mechanical parts – support and adjustment
Magnifying parts – for enlargement of the specimen
Illuminating parts – to provide light.
What can be viewed
Using Stained Prepared slides, you should see bacteria, chromosomes, organelles, protist or metazoans, smears, blood, negative-stained bacteria, and thick tissue sections.
Utilizing unstained wet mounts for living preparations should enable you to see pond water, living protists or metazoans, and plant cells such as algae.
It can be used for blood analysis which is of great use in pathology labs so as to identify diseases. In forensic laboratories, it can be used to detect the presence or absence of minerals or metals at a crime scene, thereby aid in criminal investigation.
The phase contrast microscope
The phase contrast microscope is able to show components in a cell or bacteria, which would be very difficult to see in an ordinary light microscope. Frits Zernike (1888–1966) received a Nobel prize in 1953 for his discovery of phase contrast.
Altering the light waves
The phase contrast microscope uses the fact that the light passing through a transparent part of the specimen travels slower and due to this is shifted compared to the uninfluenced light. This difference in phase is not visible to the human eye. However, the change in phase can be increased to half a wavelength by a transparent phase-plate in the microscope and thereby causing a difference in brightness. This makes the transparent object shine out in contrast to its surroundings.
| “Invisible Can Be Seen”|| |
Transparent cells can be observed without staining them because the phase contrast can be converted into brightness differences because it is not necessary to stain cells, cell division, and other processes can be observed in a living state.
The sharp contrast in certain cases can only be seen through a phase-contrast microscope
The high-contrast images of transparent specimens such as microorganisms, thin tissue slices, living cells in culture, latex dispersions, lithographic patterns, glass fragments, and subcellular particles, such as nuclei and organelles, can be viewed in detail.
Dark field microscope
This technique is used to observe unstained samples causing them to appear brightly lit against a dark, almost purely black, background. When light hits an object, rays are scattered in all azimuths or directions. The design of the dark field microscope is such that it removes the dispersed light, or zeroth order, so that only the scattered beams hit the sample.
The introduction of a condenser and/or stop below the stage ensures that these light rays will hit the specimen at different angles, rather than as a direct light source above/below the object. The result is a “cone of light” where rays are diffracted, reflected, and/or refracted off the object, ultimately, allowing you to view a specimen in dark field.,
Advantages of dark-field microscopy
It is uses unstained slides, is transparent, and absorbs little or no light
The specimens often have similar refractive indices as their surroundings, making them hard to distinguish with other illumination techniques
It is used to study marine organisms such as algae and plankton, diatoms, insects, fibers, hairs, yeast, and protozoa as well as some minerals and crystals, thin polymers, and some ceramics
It is used in the research of live bacterium, as well as mounted cells and tissues
It is useful in examining external details, such as outlines, edges, grain boundaries, and surface defects than internal structure
Dark field microscopy is often dismissed for more modern observation techniques such as phase contrast and DIC, which provide more accurate, higher contrasted images and can be used to observe a greater number of specimens. However, recently, dark field has regained some of its popularity when combined with other illumination techniques, such as fluorescence, which widens its possible use in certain fields.
The dark field microscopy images are prone to degradation, distortion, and inaccuracies. A specimen that is not thin enough or its density differs across the slide may appear to have artifacts throughout the image. The preparation and quality of the slides can grossly affect the contrast and accuracy of a dark field image. One need to take special care that the slide, stage, nose, and light source are free from small particles such as dust, as these will appear as a part of the image. We have to use oil or water on the condenser and/or slide, it is almost impossible to avoid all air bubbles. These liquid bubbles will cause images degradation, flare, and distortion and even decrease the contrast and details of the specimen.
It uses a magnification of range ×1500. On October 8, 2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, William Moerner, and Stefan Hell for “the development of super-resolved fluorescence microscopy,” which brings “optical microscopy into the nanodimension. It was British scientist Sir George G. Stokes first described fluorescence in 1852.
In fluorescence microscopy, the sample you want to study is itself the light source. The technique is used to study specimens, which can be made to fluoresce.
The fluorescence microscope is based on the phenomenon that certain material emits energy detectable as visible light when irradiated with the light of a specific wavelength. A fluorescence microscope uses a much higher intensity light source which excites a fluorescent species in a sample of interest. This fluorescent species in turn emits a lower energy light of a longer wavelength that produces the magnified image instead of the original light source.,,
Applications: These microscopes are often used for:
Imaging structural components of small specimens such as cells
Conducting viability studies on cell populations (are they alive or dead?)
Imaging the genetic material within a cell (DNA and RNA)
Viewing specific cells within a larger population with techniques such as FISH.
Confocal microscopy is a specialized form of standard fluorescence microscopy (also called widefield fluorescence microscopy) that uses particular optical components to generate high-resolution images of material stained with fluorescent probes. It is rapidly gaining acceptance as an important technology because of its capability to produce images free of out-of-focus information. It provides a significant improvement in lateral resolution and the capacity for direct, noninvasive serial optical sectioning of intact, and thick living specimens.
Confocal microscopy offers several advantages over conventional optical microscopy, including shallow depth of field, elimination of out-of-focus glare, and the ability to collect serial optical sections from thick specimens.
In the biomedical sciences, a major application of confocal microscopy involves imaging either fixed or living cells and tissues that have usually been labeled with one or more fluorescent probes.,,,
A wide variety of studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues. Other applications include resonance energy transfer, stem cell research, photobleaching studies, lifetime imaging, multiphoton microscopy, total internal reflection, DNA hybridization, membrane and ion probes, bioluminescent proteins, and epitope tagging.
Many of these powerful techniques are described in these reviews. CLSM is widely used in numerous biological sciences disciplines, from cell biology and genetics to microbiology and developmental biology.
An electron microscope is a type of microscope that uses electrons to illuminate a specimen and create an enlarged image. It can magnify specimens up to 2 million times, while the best light microscopes are limited to magnifications of 2000 times. The greater resolution and magnification of the electron microscope is due to the wavelength of an electron, its de Broglie wavelength, being much smaller than that of a light photon, electromagnetic radiation.
Transmission electron microscope
The way the image is created is similar to how a shadow is created with visible light. When the electron beam is transmitted through the sample, not all the electrons make it out. Some electrons are absorbed or deflected as they try to pass through the sample. The areas where more electrons made it through create bright spots on the screen below, and the areas where fewer electrons came through create darker spots. This in turn creates a magnified, shadow-like, black and white image of the sample.
SEM images are created by electrons that bounce off or are ejected from the sample. Because of this, the SEM gets surface images of the sample, whereas the TEM gets images of the internal composition of the sample. The downside of this in a TEM is that the sample must be cut very thin for the electrons to pass through, making sample preparation much harder than that of a sample used in an SEM.,
The main application of a transmission electron microscope is to provide high magnification images of the internal structure of a sample. Being able to obtain an internal image of a sample opens new possibilities for what sort of information can be gathered from it.
A TEM operator can investigate the crystalline structure of an object, see the stress or internal fractures of a sample, or even view contamination within a sample through the use of diffraction patterns, to name just a few kinds of studies.
Scanning electron microscope
When a SEM fires electron at the sample you want to magnify several different signals can be given off as the electrons strike the sample. Among the various signals given off, three of the most important are backscattered electrons, secondary electrons, and X-rays. The backscattered electrons occur when the collision is elastic. The backscattered electrons are actually the electrons that were originally shot at the sample bouncing back off of it. Conversely, secondary electrons occur when the collision is inelastic. Unlike backscattered electrons, secondary electrons originate from the sample itself. They are electrons that have been jarred loose from inside the sample.
We use these two types of electrons to make an image of the sample by scanning a beam of the fired electrons across the whole sample, hence the “scanning” in scanning electron microscope. As the electron beam is scanned across the sample, detectors inside the microscope pick up the signals given off by this interaction. The detectors then use these signals to create the magnified image of the sample. The secondary electrons produce the highest quality images with the greatest possible magnification in the SEM The backscattered electrons not only produce a worse quality image but also give information of the sample’s composition.,
The STEM provides structural and chemical information of a specimen at atomic-scale resolution and complements conventional transmission electron microscopy techniques. Mass measurements can now be performed routinely on a wide range of molecular and supramolecular structures using elastically scattered electrons. The recent progress in the acquisition and analysis of electron energy-loss spectroscopy data indicates that the scanning transmission electron microscope is an efficient tool for mapping the chemical composition of biological samples.
Reflection electron microscope
In REM, the reflected beam of elastically scattered electrons is detected. It is used for looking at the microstructure of magnetic domains.
Scanning transmission electron microscope
It provides structural and chemical information of a specimen at atomic-scale resolution and complements conventional transmission electron microscopy techniques. Mass measurements can now be performed routinely on a wide range of molecular and supramolecular structures using elastically scattered electrons. The recent progress in the acquisition and analysis of electron energy-loss spectroscopy data indicates that the scanning transmission electron microscope is an efficient tool for mapping the chemical composition of biological samples.
Disadvantages of electron microscopy
It is Expensive to buy and maintain
Dynamic rather than static
The specimen is specially prepared by sometimes lengthy and difficult techniques to withstand the environment inside an electron microscope.
Modern day microscopes
In the present day, the modest utility of the microscope as a tool in dental treatment has played a colossal role in building its usefulness.
Apotheker and Jako first introduced a commercial operating microscope to dentistry in 1981. (1) Shanelec and Tibbetts took a step forward to introduce it in periodontics. (2) Microscope in periodontics includes most commonly loupes, digital, and surgical microscopes. Dinolite is one of the original innovations of the 21st century, with a handy size of a fat pen that offers low power zoom capabilities with magnification up to ×500.
With the digital microscope, a live image transmission to a Tv or computer can be done. Plain assimilation of a microscope and digital camera helped in advancement and revolutionizing microphotography.
The dental loupes are simple combinations of two or more lenses. It is available as simple, compound, and prism loupes. It helps to alleviate the eye strain by magnifying the image when you are working on tiny objects and you need precision in surgery. The approach and concepts of “Minimally Invasive Surgery” and “Microsurgery” are based on the utility of the microscope in surgery.
Dental microscopes, as a highly sophisticated structure of lenses, give magnification between ×4 and ×24.
The magnification recommended for periodontal surgery is between ×10 and ×20. The dental microscope provides an ergonomic working posture, optimal, coaxial lighting of the operation region, and quite freely selectable magnification levels.
During surgical intervention, the surgeon uses both hands to perform the treatment procedure. For this reason, a motor-driven magnification changer, operated by a foot pedal, seems to be more ergonomic.
Conversely, if the magnification needs frequent change, it can be accomplished faster with the manual changer. To visualize lingual or palatal sites that are difficult to access, the microscope must have sufficient maneuverability. Recent technical advancement has further enabled direct viewing of oral operation aspects.
| Conclusion|| |
It is safe to say that microscopes have played a central part in life sciences.
This has positively contributed to the enhancement of quality of life since a lot of discoveries directly contributed to the development of drugs and cures used in the treatment of diseases and conditions that were previously misunderstood or not well understood.
A cell is the single unit of life, and to understand and study it, the microscope is necessary. The discovery of cells and genes were major milestones in the medical sciences and were a great influence to the development of new effective cures and a reduction of mortality cases among populations.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
McNamara G, Difilippantonio MJ, Ried T. Microscopy and image analysis. Curr Protocols Human Genet 2005;46:4.4.1-4.4.34.
Agocs E, Attota RK. Enhancing optical microscopy illumination to enable quantitative imaging. Sci Rep 2018;8:4782.
Kwon K-C, Lim Y-T, Kim N, Yoo K-H, Hong J-M, Park G-C. High-definition 3D stereoscopic microscope display system for biomedical applications. EURASIP J Image Video Proc 2010;2010:1-8.
Shribak M. Polychromatic polarization microscope: Bringing colors to a colorless world. Sci Rep 2015;5:17340.
Trịnh H-X, Lin S-T, Chen L-C, Yeh S-L, Chen C-S. Differential interference contrast microscopy using Savart plates. J Opt 2017;19:045601.
Lee S, Kim Y, Kang SH. Differential interference contrast microscopy for real-time dynamics and manipulation of single cells in microchannels. Microchem J 2005;80:107-12.
Fish KN. Total internal reflection fluorescence (TIRF) microscopy. Curr Protoc Cytom 2009;Chapter 12:Unit12.18.
Larson, A. Multiphoton microscopy. Nat Photon 2011;5:1.
Diaspro A, Bianchini P, Vicidomini G, Faretta M, Ramoino P, Usai C. Multi-photon excitation microscopy. Biomed Eng Online 2006;5:36.
Krull A, Hirsch P, Rother C, et al
. Artificial-intelligence-driven scanning probe microscopy. Commun Phys 2020;3:54.
Vahabi S, Nazemi Salman B, Javanmard A. Atomic force microscopy application in biological research: A review study. Iran J Med Sci 2013;38:76-83.
Vobornik D, Vobornik S. Scanning near-field optical microscopy. Bosn J Basic Med Sci 2008;8:63-71.
Encyclopaedia Britannica. Available from: https://www.britannica.com/technology/microscope. [Last accessed on 2021, Jan 18].
Ueno H, Nishikawa S, Iino R, Tabata KV, Sakakihara S, Yanagida T, et al
. Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution. Biophys J 2010;98:2014-23.
Heon Kim J, Su Park J. Partial dark-field microscopy for investigating domain structures of double-layer microsphere film. Sci Rep 2015;5:10157.
Sanderson MJ, Smith I, Parker I, Bootman MD. Fluorescence microscopy. Cold Spring Harb Protoc 2014;2014:pdb.top071795.
Young MR. Principles and technique of fluorescence microscopy. J Cell Sci 1961;s3-102:419-49.
Dai B, Jiao Z, Zheng L, Bachman H, Fu Y, Wan X, et al
. Colour compound lenses for a portable fluorescence microscope. Light Sci Appl 2019;8:75.
St. Croix CM, Shand SH, Watkins SC. Confocal microscopy: Comparisons, applications, and problems. BioTechniques 2005;39:S2-5.
Elliott AD. Confocal microscopy: Principles and modern practices. Curr Protoc Cytom 2020;92:e68.
Jonkman J, Brown CM, Wright GD, Anderson KI, North AJ. Tutorial: Guidance for quantitative confocal microscopy. Nat Protoc 2020;15:1585-611.
Nwaneshiudu A, Kuschal C, Sakamoto FH, Rox Anderson R, Schwarzenberger K, Young RC. Introduction to confocal microscopy. J Investig Dermatol 2012;132:1-5.
Thiberge S, Nechushtan A, Sprinzak D, Gileadi O, Behar V, Zik O, et al
. Scanning electron microscopy of cells and tissues under fully hydrated conditions. Proc Natl Acad Sci U S A 2004;101:3346-51.
Goldstein JI, Newbury DE, Echlin P, Joy DC, Romig AD, Lyman CE, et al
. Scanning Electron Microscopy and X-Ray Microanalysis. 2nd ed. New York: Plenum; 1992.
Tang CY, Yang Z. Transmission electron microscopy (TEM). In: Hilal N, Ismail AF, Matsuura T, Oatley-Radcliffe D, editors. Membrane Characterization. Amsterdam: Elsevier; 2017. p. 145-59.
[Table 1], [Table 2]