Its invention dates back to the 14th century to the Italian art of spectacle lenses.
This technology was taken up by Dutch lens makers Hans and Zacharias Janssen in 1590 to make the first microscope by placing two lenses in a tube.
In 1675, Anton van Leeuwenhoek used a simple microscope with a single lens to examine blood in detail and became the first person to describe cells, the red blood cells that carry oxygen throughout the body.
Throughout the centuries, microscopes have been essential to the development of immunological research, but perhaps the most formative moment came in 1878 when Paul Ehrlich, a young scientist from Strzelin in Poland, described in his doctoral thesis the discovery of a new constituent of blood that he called mast cells.
He discovered that the granulated protoplasm of what were thought to be simply plasma cells could be made visible under a microscope by adding an alkaline dye.
He thought these granulated cells were a sign of good nutrition, therefore he named them after the German word for an animal fattening food called Mast.
In fact, with the help of his microscope, Ehrlich discovered a key cell type that belongs to the human immune system, the first of many.
Mast cells are now known to release histamine and other substances during inflammatory and allergic reactions.
Ehrlich’s interest in microscopic dyes that could differentially stain tissues also led him to be the first person to distinguish between lymphocytes and leukocytes, the two main groups of white blood cells, vital elements in the immune system.
His work led to a 1908 Nobel Prize in recognition of his work in immunology and in his acceptance lecture he recognized the importance of the microscope in understanding life.
Subsequently, other advances in labeling, such as immunofluorescence, and in microscopic technology, such as the electron microscope and the scanning microscope, shed even more light on the complex workings of the immune system.
The microscope is an instrument that produces magnified images of small objects, allowing the observer a very close view of minute structures at a convenient scale for examination and analysis.
The microscope can provide a dynamic image (as with conventional optical instruments) or one that is static (as with scanning electron microscopes).
The magnifying power of a microscope is an expression of the number of times the object under examination appears enlarged and is a dimensionless relationship.
It is generally expressed in the form 10 × (for an image magnified 10 times).
The resolution of a microscope is a measure of the smallest detail of the object that can be observed.
Resolution is expressed in linear units, generally micrometer (μm).
Types of microscopes
The most common type of microscope is the light or light microscope, in which glass lenses are used to form the image.
Optical microscopes can be simple, consisting of a single lens, or compound, consisting of several optical components in line.
The handheld magnifier can magnify from 3 to 20 ×. Simple single-lens microscopes can magnify up to 300 × and are capable of displaying bacteria.
While compound microscopes can magnify up to 2,000 ×.
A simple microscope can have a resolution down to 1 micrometer (μm, one millionth of a meter), a compound microscope can have a resolution down to about 0.2 μm.
The compound microscope is also called a light microscope.
There are other alternative microscopes such as the dark field microscope. With this type of microscope you can see a light object on a dark background.
It is used for the observation of live spirochetes.
The phase contrast microscope can clearly show living and unstained organisms and the internal cellular structures such as mitochondria, lysosomes and Golgi body can be observed.
In the fluorescent microscope, one uses ultraviolet light as the light source.
Images of interest can be captured by photography through a microscope, a technique known as photomicrography.
Since the 19th century, this has been done with film, but now digital imaging is widely used.
Some digital microscopes have dispensed with an eyepiece and provide images directly on the computer screen.
Other types of microscopes use the wave nature of various physical processes.
The transmission electron microscope has magnification powers of more than 1,000,000 × where the energy source used is a beam of electrons.
Transmission electron microscopes form images of thin specimens.
A scanning electron microscope, which creates a raised reflected image on a contoured specimen, generally has a lower resolution than a transmission electron microscope.
But it can show solid surfaces in a way that the conventional electron microscope cannot.
There are also microscopes that use lasers, sound, or X-rays.
The tunneling microscope, which can create images of atoms, and the environmental scanning electron microscope, which generates images using electrons from specimens in a gaseous environment, use other physical effects that further expand the types of objects that can be examined.
Several types of microscopes are available for use in the microbiology laboratory.
Microscopes have various applications and modifications that contribute to their usefulness.
A fixed platform with an opening in the center allows light to pass from a downward illuminated source to the lens system on stage.
This platform provides a surface for the placement of a slide with its specimen over the central opening.
In addition to the fixed stage, most microscopes have a mechanical stage that can be moved vertically or horizontally using adjustment controls.
Less sophisticated microscopes have clips on the fixed stage, and the slider must be manually positioned over the center aperture.
Some microscopes are equipped with a built-in light source to provide direct illumination.
Others are provided with a mirror; one side flat and the other concave.
An external light source, such as a lamp, is placed in front of the mirror to direct the light up into the lens system.
The flat side of the mirror is used for artificial light and the concave side for sunlight.
The condenser is located under the stage and has lens sets that concentrate the light that passes from the light source to the lens system.
The condenser is equipped with an iris diaphragm, a lever controlled shutter used to regulate the amount of light entering the lens system.
The tube is located on the stage and is attached to the microscope arm.
In this structure is the lens system that magnifies the preparation or sample.
At the top end of the tube is the ocular lens. The lower part consists of a movable revolver that contains the objective lenses.
And it allows the rotation of the objectives of the position of the piece located on the opening of the stage.
The body tube can be raised or lowered with the help of the coarse and fine adjustment knobs located above or below the stage, depending on the type and make of the instrument.
Theoretical principles of microscopy
To use the microscope efficiently and with minimal frustration, one must understand the basic principles of microscopy: magnification, resolution, numerical aperture, illumination, and focus.
Magnifying or magnifying a sample is the function of a two-lens system; the ocular lens is located in the eyepiece and the objective lens is located in a rotating nose piece.
These lenses are separated by the tube from the body.
The objective lens is closer to the sample, producing the actual image that is projected upward in the focal plane and then magnified by the ocular lens to produce the final image.
The most commonly used microscopes are equipped with an objective nosepiece that contains four lenses that have different degrees of magnification.
When these are combined with the magnification of the ocular lens, the total linear magnification of the sample is obtained.
Although scaling is important, it should be noted that unlimited scaling is not possible.
Simply increasing the magnifying power of the lenses or using additional lenses, as the lenses are limited by a property called resolving power.
By definition, resolving power is the ability of a lens to display two adjacent objects as discrete entities.
When a lens cannot discriminate, that is, when the two objects appear as one, it loses resolution.
The resolution of the lens will depend on the wavelength of the light that is used and the numerical aperture.
Numerical aperture is defined as a function of the diameter of the objective lens in relation to its focal length.
It doubles with the use of the substation capacitor; that illuminates the object with rays of light that pass through the specimen obliquely as well as directly.
The shorter the wavelength, the higher the resolving power of the lens.
Therefore, the short wavelengths of the electromagnetic spectrum are more suitable than the longer wavelengths in terms of numerical aperture.
However, as with magnification, the resolving power also has limits.
The relationship between wavelength and numerical aperture is valid only for higher resolving power when the light rays are parallel.
Therefore, the resolving power depends on another factor, the refractive index.
This is the bending power of the light that passes through the air from the glass slide to the objective lens.
The index of refraction of air is lower than that of glass, and as light rays pass through the glass and slide into the air, they are bent or refracted so that they do not pass the objective lens.
This would cause a loss of light, which would reduce the numerical aperture and decrease the resolving ability of the objective lens.
The loss of refracted light can be compensated for by interposing mineral oil, which has the same refractive index as glass, between the slide and the objective lens.
In this way, there is a decrease in the refraction of light and more rays of light enter directly into the objective lens, producing a vivid image with high resolution.
Effective lighting 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 passes through the condenser located under 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 condenser knob.
Always keep the condenser close to the stage, especially when using the oil immersion lens.
Between the light source and the condenser is the iris diaphragm, which can be opened and closed by means of a lever; therefore, it regulates the amount of light that enters the condenser.
Excessive lighting may darken the sample due to 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 the slide, called the working distance, decreases, while the numerical aperture of the objective lens increases.
Use and care of the microscope
The correct way to move a microscope is to firmly grasp the microscope arm with your right hand and the base with your left hand, and gently place it on the work table.
This will prevent collision with furniture and protect the instrument from damage.
Once the microscope is to be used the following rules must be observed:
- Clean all lens systems – The slightest bit of dust, oil, lint, or eyelash will decrease the effectiveness of the microscope.
- The Eyepiece: High-power, energy-saving, scanning lenses can be cleaned by wiping them several times with a lens cloth.
- Do not use paper or cloth to clean the lens surface: If the oil immersion lens is sticky, use a lens cloth moistened with methanol to clean it.
- If the lens is very dirty, it can be cleaned with xylene, however, the xylene cleaning procedure should only be performed if necessary. Consistent use of xylene can loosen the lens.
The following routine procedures should be followed to ensure correct and efficient use of the microscope while focusing.
- Place the microscope slide with the sample into the stage clips on the fixed stage. The slide is moved to center the specimen over the opening in the stage directly over the light source.
- Rotate the scan lens or low power lens into position. While looking from the side to ensure that the lens does not touch the specimen, the coarse focus knob is turned to move the stage as close to the objective as possible without touching the lens.
- While looking through the eyepiece lens, carefully turn the coarse focus knob, and slowly move the stage away from the lens until the specimen is out of focus. The fine focus knob is then used to focus the specimen well.
- The light source should be adjusted regularly by setting the light source transformer, and the iris diaphragm, for optimal illumination for each new slide and for each change in magnification.
- Once the specimen has been well focused with a low-power lens, you can prepare to view the specimen under oil immersion. A drop of oil is placed on the slide directly over the area to be viewed. Rotate the revolver until the oil immersion target is locked in position.
- During the microscopic examination of microbial organisms, it is always necessary to observe various areas of the preparation. This is accomplished by scanning the slide without the application of additional immersion oil.