What is Microscopy?
Microscopy is the technical field of using microscopes to view samples & objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye).
Over the past decades, microscopy has become an invaluable tool in observing molecular changes at the sub-cellular level in order to make important discoveries across a range of life science disciplines.
There are three main branches of microscopy techniques that scientists use in their research: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.
The History of Microscopy
The origins of microscopy can be traced to around 1000 AD when a glass sphere was used to magnify text. In 1021 Iqbal al Haytham wrote a book on “Optics” which increased the understanding of how light behaved but it wasn’t until 1590 that Hans and Zacharia Janssen placed lenses in a tube to create the forerunner of modern microscopes. In 1609, Galileo famously developed the compound microscope which was not named until 1625 by Giovanni Faber.
In the late 17th Century, Anthony von Leeuwenhoek from Holland invented a single lens, the hand-held microscope that could achieve a magnification of 270x. Using this lens, he went on to develop the first microscope that could actually be made use of. Leeuwenhoek found he was able to see structures that no one had seen before such as blood cells and bacteria.
In the same century, Englishman Robert Hooke was acknowledged as having discovered the smallest most basic unit of an organism – the cell. He was also recognized as the first person to use a microscope with three lenses, the configuration used in today’s microscopes.
In 1874, Ernst Abbe developed a formula that allowed the maximum resolution of a microscope to be calculated. In 1931, Ruske and Knoll built the first Transmission Electron Microscope using an idea from Sziland. Throughout the 20th and early 21st Century, there have been continued innovations in all branches of microscopy.
The Nobel Prize has been awarded to microscopy work twice; In 1986 it was awarded jointly to Ruske for work on the electron microscope and Bing and Rohrer for work on scanning and tunneling microscopy. In 2014, the prize was awarded to Betzig, Hell, and Moernerfor the development of super fluorescent microscopy which allows for resolution down to two micrometers.
Principle of Microscopy
To use the microscope efficiently and with minimal frustration, you should understand the basic principles of microscopy: magnification, resolution, illumination, and focusing.
Magnification
Enlargement or magnification of a specimen is the function of a two-lens system; the ocular lens is found in the eyepiece, and the objective lens is situated in a revolving nose-piece.
These lenses are separated by the body tube. The objective lens is nearer the specimen and magnifies it, producing the real image that is projected up into the focal plane and then magnified by the ocular lens to produce the final image.
The most commonly used microscopes are equipped with a revolving nosepiece containing four objective lenses possessing different degrees of magnification. When these are combined with the magnification of the ocular lens, the total or overall linear magnification of the specimen is obtained.
Resolving Power or Resolution
In addition to magnification, a microscope must also have an adequate resolution or resolving power. Resolution defines the capacity of an optical system to distinguish two adjacent objects or points from one another.
For example, at a distance of 25 cm (10 in), the lens in the human eye can resolve two small objects as separate points just as long as the two objects are no closer than 0.2 mm apart. The eye examination given by optometrists is in fact a test of the resolving power of the human eye for various-size letters read at a distance of 20 feet.
Because microorganisms are extremely small and usually very close together, they will not be seen with clarity or any degree of detail unless the microscope’s lenses can resolve them.
A simple equation in the form of a fraction expresses the main mathematical factors that influence the expression of resolving power.
Resolving power = Wavelength of Light/ 2 x Numerical Aperture of an objective lens.
From this equation, it is evident that the resolving power is a function of the wavelength of light that forms the image, along with certain characteristics of the objective. The light source for optical microscopes consists of a band of colored wavelengths in the visible spectrum.
The shortest visible wavelengths are in the violet-blue portion of the spectrum (400 nm), and the longest is in the red portion (750 nm). Because the wavelength must pass between the objects that are being resolved, shorter wavelengths (in the 400–500 nm range) will provide better resolution
Illumination
Effective illumination is required for efficient magnification and resolving power. Since the intensity of daylight is an uncontrolled variable, artificial light from a tungsten lamp is the most commonly used light source in microscopy. The light is passed through the con-denser located beneath the stage.
The condenser contains two lenses that are necessary to produce a maximum numerical aperture. The height of the condenser can be adjusted with the con-denser knob. Always keep the condenser close to the stage, especially when using the oil-immersion objective.
Between the light source and the condenser is the iris diaphragm, which can be opened and closed by means of a lever; thereby regulating the amount of light entering the condenser. Excessive illumination may actually obscure the specimen because of lack of contrast.
The amount of light entering the microscope differs with each objective lens used. A rule of thumb is that as the magnification of the lens increases, the distance between the objective lens and slide, called working distance, decreases, whereas the numerical aperture of the objective lens increases.
Type Of Microscopy
- Optical or Light Microscopy
- Ultraviolet Microscopy
- Infrared Microscopy
- Electron Microscopy
- Scanning Probe Microscopy
- X-ray Microscopy
Optical microscopy
Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single lens or multiple lenses to allow a magnified view of the sample. The resulting image can be detected directly by the eye, imaged on a photographic plate, or captured digitally.
The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage, and support, makes up the basic light microscope.
The most recent development is the digital microscope, which uses a CCD camera to focus on the exhibit of interest. The image is shown on a computer screen, so eye-pieces are unnecessary.
Techniques
To improve specimen contrast or highlight structures in a sample, special techniques must be used. A huge selection of microscopy techniques is available to increase contrast or label a sample.
- Bright field microscopy
- Oblique illumination microscopy
- Dark field microscopy
- Dispersion staining microscopy
- Phase contrast microscopy
- Differential interference contrast microscopy
- Interference reflection microscopy
- Fluorescence microscopy
- Confocal microscopy
- Two-photon microscopy
- Single plane illumination microscopy and light sheet fluorescence microscopy
- Wide-field multiphoton microscopy
- Deconvolution microscopy
Ultraviolet microscopy
UV microscopy is a type of light microscopy that uses UV light to view samples at a greater resolution than is possible with visible light. The light source typically ranges from deep blue to UV light wavelengths (180-400 nm) to give a magnification approximately double that which can be achieved with white light.
Infrared microscopy
Infrared (IR) microscopy, also known as infrared microspectroscopy, is a type of light microscopy that uses a source that transmits infrared wavelengths of light to view an image of the sample.
Electron microscopy
Until the invention of sub-diffraction microscopy, the wavelength of the light-limited the resolution of traditional microscopy to around 0.2 micrometers. In order to gain higher resolution, the use of an electron beam with a far smaller wavelength is used in electron microscopes.
Transmission electron microscopy (TEM):
is quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. The resolution limit in 2005 was around 0.05[dubious – discuss] nanometer and has not increased appreciably since that time.
Scanning electron microscopy (SEM):
visualizes details on the surfaces of specimens and gives a very nice 3D view. It gives results much like those of the stereo light microscope. The best resolution for SEM in 2011 was 0.4 nanometer.
Scanning probe microscopy
This is a sub-diffraction technique. Examples of scanning probe microscopes are the atomic force microscope (AFM), the Scanning tunneling microscope, the photonic force microscope, and the recurrence tracking microscope. All such methods use the physical contact of a solid probe tip to scan the surface of an object, which is supposed to be almost flat.
Ultrasonic force microscopy (UFM)
Ultrasonic force microscopy (UFM) has been developed in order to improve the details and image contrast on “flat” areas of interest where AFM images are limited in contrast. The combination of AFM-UFM allows a near-field acoustic microscopic image to be generated.
The AFM tip is used to detect the ultrasonic waves and overcomes the limitation of wavelength that occurs in acoustic microscopy. By using the elastic changes under the AFM tip, an image of much greater detail than the AFM topography can be generated.
Ultrasonic force microscopy allows the local mapping of elasticity in atomic force microscopy by the application of ultrasonic vibration to the cantilever or sample. To analyze the results of ultrasonic force microscopy in a quantitative fashion, a force-distance curve measurement is done with ultrasonic vibration applied to the cantilever base, and the results are compared with a model of the cantilever dynamics and tip-sample interaction based on the finite-difference technique.
X-ray microscopy
X-ray microscopy is a non-destructive technique that generates an image of the internal features of the examined materials, with the image contrast being determined by the difference in absorption of X-rays by the different components in the materials.
