Microscopy: Types of Microscopy, Facts, and Resources
Microscopy is the technical field of using microscopes to view samples or objects that cannot be seen by the naked eye. There are three well-known branches of microscopy which are optical, electron and scanning probe microscopy. Twenty different types of microscopes exist, with the most important and recognizable being the compound microscope, scanning probe microscope and the dissection microscope.
An optical microscope is commonly known as a “compound microscope” or as a “light microscope”. The compound microscopes are light-illuminated and are the most commonly used. They use visible light and a system of lenses to magnify images of small samples. Two-dimensional images are seen in this manner. This type of microscope uses two lenses; the first lens is an objective lens and the second is an ocular lens. These lens are positioned at opposing sides of a closed tube. The objective lens is made up of a variety of lens elements, which form an amplified real image of the object that is being analyzed. Optical microscopes are the oldest and simplest of the microscopes. A simple microscope is a microscope that uses only one lens for magnification and is the original light microscope, while a compound microscope uses multiple lenses to gather light, using a mechanism to change lens distances. A compound optical microscope can produce a magnified image of a specimen up to 1000 times. At high magnifications, these enlargements are used to study thin specimens as they have a very limited depth of field. Optical microscopy is used extensively in microelectronics, nanophysics, biotechnology, pharmaceutical research and microbiology.
The electron microscope produces an electronically magnified image of a specimen for detailed observation. It is very expensive, is often quite large and the machinery differs greatly from an optical microscope. The electron microscope uses a particle beam of electrons to illuminate the specimen and to create a magnified image of it. It uses electrostatic and electromagnet “lenses” to control the electron beam, which when focused forms an image. There are two types of electron microscopes; the first type is called the transmission electron microscope (TEM) and the second type is the scanning electron microscope (SEM).
TEMs work by passing electrons through a specimen that is no more than one micrometer thick. The electrons in the specimen are diffracted and a fluorescent screen then picks up the electrons. An image is formed from the interaction of the electrons transmitted through the specimen. The image is then magnified and focused into an imaging device. Such imaging devices include a fluorescent screen or a layer of photographic film. It could also be detected by a sensor such as a CCD camera. The SEM is a type of electron microscope that images the sample surface by scanning it with a high-energy beam of electrons in a raster scan pattern.
Three dimensional images are seen once electrons are illuminated through the scanning probe microscope. A probe is used to scan the surface of a sample and this then provides a three-dimensional picture of molecules or atoms on the top of the object. The probe is a very honed point and this point can be as slim as one atom at the tip. The advantage of the SEM is that it creates more three-dimensional images, but the resolution and magnification is less.
The dissection microscope is also referred to as a stereoscope and it too is light-illuminated. Images with this microscope also appear in three-dimensions and the scope is used for dissection to obtain a better look at a larger specimen. A dissecting microscope manages the enlargement and analysis of three dimensional objects and differs from a compound microscope, which provides a very high level of magnification for two-dimensional objects. This type of microscope has two lens arrays and these lenses are aligned to create a three-dimensional or stereoscopic image.
Questions and Fun Facts about Microscopy
What does the inscription 0.17 on the objective signify?
0.17 refers to the thickness, in millimeters, of the cover glass that was assumed by the lens designer in computing the corrections for the objective.
What is the significance of the N.A. number inscribed on the outer barrel of an objective?
This number is the numerical aperture of the objective, a measure of the light-gathering capacity of the objective. The higher the numerical aperture, other things being equal, the better the objective is able to separate the details of the specimen in forming an image.
What does the objective inscription 160 mean?
This number identifies a finite tube length objective. 160 millimeters is the distance from the opening of the nosepiece to the top of the observation tube.
What does the objective inscription signify?
This inscription identifies the objective as an infinity-corrected objective. Light rays emerging from such an objective are in parallel bundles projected toward infinity.
Some objectives have the inscription Plan. What does that mean?
Plan designates an objective that projects, at the eyepiece diaphragm plane, an image which is flat from edge to edge of the field of view.
If an objective carries the inscription Planapo, what does that mean?
The term Planapo signifies a planapochromat, an objective of the highest correction, corrected for four colors chromatically and spherically. Such an objective, for its magnification, will have a higher numerical aperture than objectives of lesser correction.
What does the inscription PlanFl mean?
PlanFl denotes an objective that is a planfluorite also called a plan-semi-apochromat.
Some objectives have no inscription about their corrections or only the inscription Plan. What kinds are these?
Such objectives are achromats or planachromats. They are now corrected for three wavelengths chromatically, and one or two wavelengths spherically. They give their best images in green light. However, in white light, the planachromats will yield satisfactory images for color photomicrography but not as good as objectives of better correction.
Objectives usually have a color ring inscribed. What do these colors signify?
The 4x or 5x has a red ring; the 10x yellow; the 20x green; the 40x or 50x or 60x blue; the 100x white. These rings make it easier to visually identify the magnification of the objective; the colors are standard for most manufacturers.
What is a No-cover glass or NCG or NC objective?
Such an objective has been designed to look at a specimen which is not covered by a cover glass; e.g. a smear. At numerical apertures above 0.45, such an objective will yield images free of spherical aberration when smears or other uncovered specimens are examined.
Why would one use an objective marked LWD or ULWD?
The letters identify a long working or ultra-long working distance objective. The vertical distance from the front lens of the objective to the focused specimen is much longer than that for a similar magnification standard objective.
Some objectives are marked NIC or DIC. Why?
This designates an objective that is preferred for use in Nomarski (NIC) or differential interference microscopy (DIC).
What does the inscription oil or oel or WI on the objective mean?
Such an objective is an immersion objective, requiring a drop of immersion oil between the front lens of the objective in contact with the cover glass or top of a smear. Unless there is oil contact with such an objective, the image will be very poor.
Why are some objectives marked UV?
Ordinary glass is relatively opaque to ultraviolet light wave lengths below 400 nanometers. UV objectives contain specially formulated glass elements and coatings to transmit a relatively high percentage of light of such wave lengths.
What do the letters PL or NH mean on a phase objective?
The letters PL stand for positive low, a phase contrast in which the specimen appears darker than the background of the field of view.
Why are some objectives marked P or POL or SF?
These letters designate the objective as being relatively strain-free. Such objectives are required for high quality polarized light qualitative and/or quantitative investigations.
Eyepieces, in addition to having the magnification inscribed, are marked 20, or 22, or 26.5. What do these numbers mean?
The designation is the field number of the eyepiece. The higher the field number of the eyepiece being used with a particular objective, the more specimen area will appear in the field of view. The diameter of the field of view, in millimeters, is calculated by dividing the field number of the eyepiece by the magnification of the objective. For many microscopists, e.g. hematologists, it saves time to be able to see more of the specimen at a given time. Eyepieces with a field number of 26.5 are called super-wide eyepieces.
What do the inscriptions C or K or WF or H on the eyepiece mean?
C or k identify a compensating eyepiece. Some microscope objectives do not include correction for lateral chromatic aberration.
Why do some observation tubes have notches cut into them.
The notch is meant for placement of the projecting “locator” pin on eyepieces which have a reticle installed.
What are the outer dimensions of the most common observation tubes?
These are usually 25 millimeters or 30 millimeters.
Resources about Microscopy
Microscopy.org: Microscopy Society of America. What is the Microscopy Society of America? The Microscopy Society of America is an organization dedicated to the promotion and advancement of the knowledge of the science and practice of all microscopical imaging, analysis and diffraction techniques useful for elucidating the ultrastructure and function of materials in diverse areas of biological, materials, medical and physical sciences.
Journal of Microscopy and Microanalysis. Editor(s): Robert L. Price, University of South Carolina, USA
FSU.edu questions and answers About Optical Microscopy Answered by Mortimer Abramowitz, Technical Consultant at Olympus America, Inc.
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