Microscopes

Microscopes are among the most expensive items in the laboratory. They are used for magnifying. There are many types of microscopes each suited for a specific purpose. They include,

Light or optical microscope
Electron microscope
Phase contrast microscope
Fluorescent microscope
UV microscope
Dark ground illumination microscope
- THE LIGHT MICROSCOPE

Light microscopes were invented in the 17th century. They operate by allowing light rays from a light source beneath the stage to be transmitted through the eye piece lens and objective lens. Depending on their strength, these two lenses can routinely provide a magnification of over X400.

The light microscope has had profound influence in microbiology and cell biology, however, they have a limit in the amount of details they can show. This limit is set by its resolving power. The resolving power is the minimum distance by which two points must be separate for them to be perceived as two separate points rather than a single fused point .For light microscope these image should be 2 μm apart .The limited resolution of light microscope is imposed by the wavelength of the visible light .

These put a limit in the amount of structural details that can be detected within the cell. Higher magnification can be achieved by using a sppecial objective lens with a fluid i.e. oil emersion, placed between the lenses. But even then it is not possible to achieve magnification of 2000

Types of light microscopes

Monocular microscopes
Binocular microscopes

Monocular microscopes are those microscopes having one eye piece lens fixed to the microscope. They include:

The standard microscope

This is the ordinary common light microscope. Its stage is not permanently fixed and it can be inclined or and it’s movable.

Fixed Inclined Limb Microscope

The stage is maintained in a horizontal plane and the body tube is inclined towards the user, in some models, the rotation of the body tube in a horizontal plane is possible. These may be useful in teaching as the teacher can share the instrument with students without moving or exchanging places with students.

STRUCTURAL COMPONENTS OF MICROSCOPES

The three basic, structural components of a compound microscope are the head, base and arm.

Head/Bodyhouses the optical parts in the upper part of the microscope
Base of the microscope supports the microscope and houses the illuminator
Armconnects to the base and supports the microscope head. It is also used to carry the microscope.

When carrying a compound microscope always take care to lift it by both the arm and base, simultaneously.

OPTICAL COMPONENTS OF MICROSCOPES

There are two optical systems in a compound microscope: Eyepiece Lenses and Objective Lenses:

Eyepiece or Ocular is what you look through at the top of the microscope. Typically, standard eyepieces have a magnifying power of 10 xs. Optional eyepieces of varying powers are available, typically from 5x-30x.

Eyepiece Tube holds the eyepieces in place above the objective lens. Binocular microscope heads typically incorporate a diopter adjustment ring that allows for the possible inconsistencies of our eyesight in one or both eyes. The monocular (single eye usage) microscope does not need a diopter. Binocular microscopes also swivel (Interpupillary Adjustment) to allow for different distances between the eyes of different individuals.

Objective Lenses are the primary optical lenses on a microscope. They range from 4x-100x and typically, include, three, four or five on lens on most microscopes. Objectives can be forward or rear-facing.

Nosepiece houses the objectives. The objectives are exposed and are mounted on a rotating turret so that different objectives can be conveniently selected. Standard objectives include 4x, 10x, 40x and 100x although different power objectives are available.

Coarse and Fine Focus knobs are used to focus the microscope. Increasingly, they are coaxial knobs – that is to say they are built on the same axis with the fine focus knob on the outside. Coaxial focus knobs are more convenient, the viewer does not have to grope for a different knob.

Stage is where the specimen to be viewed is placed. A mechanical stage is used when working at higher magnifications where delicate movements of the specimen slide are required.

Stage Clips are used when there is no mechanical stage. The viewer is required to move the slide manually to view different sections of the specimen.

Aperture is the hole in the stage through which the base (transmitted) light reaches the stage.

Illuminator is the light source for a microscope, typically located in the base of the microscope. Most light microscopes use low voltage, halogen bulbs with continuous variable lighting control located within the base.

Condenser is used to collect and focus the light from the illuminator on to the specimen. It is located under the stage often in conjunction with an iris diaphragm.

Iris Diaphragm controls the amount of light reaching the specimen. It is located above the condenser and below the stage. Most high quality microscopes include an Abbe condenser with an iris diaphragm. Combined, they control both the focus and quantity of light applied to the specimen.

Condenser Focus Knob moves the condenser up or down to control the lighting focus on the specimen.

Magnification and resolving power of a light microscopes

Magnification is simply the ability to make tiny things appear big. Increase in magnification power can be achieved by using a wide range of objective lens. Magnifying power is always linked to the resolving power. The higher the magnification , the higher the resolving power and hence the more the details of the specimen that can be seen .The resolving power of an objective lens depends on the numerical aperture (NA) of that objective lens .

The following are the usual NA values of the commonly used objective lens

X10 objective lens = NA 0. 25

X20 objective lens = NA 0.45

X40 objective lens = NA 0.65

X100 objective lens = NA 1.25

Immersion oil

In a light microscope, beam of light passes from the specimen on the stage and moves through air towards the objective lens and from the objective lens, this light will also have to travel through an air space between the objective lens and the eye piece lens before reaching the eye

Movement of light through different medium as describe above will cause the light to be refracted or to bend. This bending of light have little effect on low power objective lens i.e. X10,X20 or X40 objectives, but it have significant limitation to the amount of light which enter through the high power objective lens i.e. X100 objective lens and consequently its resolving power.

Such bedding can be avoided by replacing the air between the specimen and the lens with an oil Immersion which have the same optical property as that of glass. These makes light to pass in a straight line as though it was passing through the same media i.e. glass all the way. These therefore will enhance a better resolving power

Chromatic and spherical aberration

Chromatic aberration occurs when a biconcave lens splits white light into its component colors and in the process blue light is magnified more than red light so that blue comes into focus near to the lens. Spherical aberration occurs due to the edges of the lens giving slightly higher magnification than its center

Illumination system of the light microscopes

Daylight illumination should be discouraged because it is not reliable, difficult to use and rarely adequate for oil emulsion work. Good microscopy requires an adequate well controllable illumination system. These can be achieved by using a microscope with an inbuilt illumination system provided by a special bulb installed below the stage instead of relying on the reflective mirror.

Glare

Glare in microscopes is simply that inconvenience caused as a result of light reaching the eye which does not go into making up the perfect image , instead, it only interferes with the image and the ability of the objective to distinguish details in a specimen . To test for glare, remove the eyepiece and check to see if the inside of the tube is illuminated. These indicate presence of glare.

glare can be reduced in the following ways:

Positioning a microscope with an inbuilt illumination in a subdued light i.e. not in front of a window.
Avoid using a large source of illumination than its necessary.
Reducing the condenser glare by reducing the condenser aperture i.e. by adjusting the iris diaphragm when using low power objective.

Microscope filters

Filters are special devises in microscopes are used to:

Reduce the intensity of light when it is required.
To increase contrast and resolution.
To transmit light of selected wavelength.
To protect the eyes from injuries caused by UV light.

Procedure for using light microscope

Place the microscope on a firm bench.
Turn on, adjust or switch on the in-built bulb of the microscope to direct light into the microscope.
Switch or rock the lowest objective (x10) into the optical train.
Place the slide containing the specimen on the stage.
Rock down the objective using coarse adjustment until it comes to stop. Do so while your eyes are looking at the objective, not into the microscope.
Now look into the microscope and using the coarse adjustment knob, rock up the objective until you see the image then rock the fine adjustment knob to focus properly.
If you need higher magnification, switch the x40 objective into train and then use the adjustment to focus the image and if you need still higher magnification, apply oil immersion on the specimen placed on the stage and switch the x100 objective into train.
Rock down the objective to touch the slide while the eyes are out of the microscope.
Rock up the condenser to the maximum.
Focus clearly using fine adjustment.

Care and maintenance of light microscopes

Carry microscopes using both hands by the limb and the base and should be in upright position so as to avoid dropping loose parts e.g. mirror and eye- pieces.
Do not touch the microscopes lens using fingers
Clean microscope lens using lens cleansing tissues
Microscopes should be kept clean and covered to avoid dust
Keep microscopes away from direct sunlight and dust i.e. always cover microscopes and keep then in locked cupboards
The optical parts should be cleaned using a little xylene on a lens cleaning tissue or soft cloth. Do not use sleeves or lab coats
Never place microscopes at the edge of benches
Avoid using a high power objective when a low power objective is satisfactory
It is dangerous to rock down the objective while you are looking into the microscope. You risk breaking the slide, the objective lens or the condenser
Repair should be left only to a trained expert
Do not place wet preparation on the stage without wiping the undersurface of the slide.

b) THE ELECTRON MICROSCOPE

The development of electron microscope has revolutionized microscopic studies since 1950s. These microscope uses electron beams instead of light and electromagnet instead of glass lens. The electrons are recorded in efflorescent screen which then forms a viewable image on the screen (photomicrograph)

Electrons travel in cathode ray tube which is a vacuum chamber. The specimens are mounted in total vacuum in order to enable the electrons to travel with high velocity without colliding with air or any atoms in space within. These electrons pass through an electromagnet which acts as lenses where they produce electric fields

Also the specimens are prepared by sectioning using an extremely delicate microtome which produces extremely thin slices which are fixed using osmic acid or glutaraldehyde for cytoplasm components

Electron microscope have very high resolving power that is 1000 times more than an optical microscope , thus specimens can be magnified much more without loss of clarity . With this microscope, materials which were initially described as structureless have been shown to have elaborate internal organization and the so called homogeneous fluids have now shown to contain a variety of complex structures. These microscopes have had a greater impact in biology and have helped in opening up a new world whose existence was barely realized in the 1950s

The electron microscope however has some disadvantages which include:

The materials for examination have to be mounted in a vacuum and therefore only dead specimens can be viewed.
The materials have to be fixed and stained, these preliminary treatments may distort the delicate structures inside the cells and create images that are not real (artifacts). These problem can be overcome by using electron microscopes together with other types of microscopes.

Types of electron microscopes

Transmission electron microscope –images are derived from electrons which have passed through the specimens
Scanning electron microscope –solid specimens are bombarded with a beam of electrons which causes secondary electrons to be emitted from the surface of specimen. These electrons are recorded on a photoelectric plate as in transmission electron microscope. The scanning electron microscope enables some details on the surface to be seen more clearly. Images seen on these microscopes are three dimensional.

PHASE CONTRAST MICROSCOPE

This microscope was developed in 1940s. It enables transparent objects to be seen and it is ideal for studying unstained living cells. In appreciation of living cells, there may be sudden changes of refractive index between the inside and the outside of the cell or between the nucleus and the cytoplasm. An annulus is used to give a halo cone of rays since full cone illumination will cause direct or diffracted rays to be superimposed

The region on the center has a low refractive index and the outside have a high refractive index. The transmitted rays are diffracted and will not enter the annulus. The region will appear dark. The diffracted and the undiffracted rays strike different parts of the phase plate where the phase contrast as well as the amplitude can be altered. The diffraction patterns are produced by interfering and reinforcement of light waves. Variation in brightness occur at boundaries of different diffractive indexes in the specimen which gives the characteristic of a halo appearance

THE UV MICROSCOPE

The visible region of the electromagnetic spectrum extends from 650 Ao (deep red) –450Ao . Wavelengths shorter than 400Ao are called UV. In this region, some cellular compounds e.g. nucleic acid and proteins absorb a particular wavelength. If the UV microscope is used in conjunction with visible light microscopy, much useful information may be obtained. UV microscopes use quartz lenses to transmit the UV light. These microscopes however have limited use in living cells because the UV rays rapidly kill the living cell. They also require the use of filters to protect the eyes from the harmful effect of the UV radiations.

FLUORESCENT MICROSCOPE

When certain chemical substances are irradiated by UV light, they absorb the radiation and emit visible light. Objects which emit such chemicals within living cells can therefore be absorbed as fluorescent areas when illuminated with UV light. The chemicals absorbed are known as fluorochromes. The method is extremely sensitive and can detect minute quantities of materials. It is particularly useful for studying how proteins and other molecules enter or are adsorbed into cells. The proteins are labeled by coupling with the molecules of fluorescent dyes. The fluorescent substances may be naturally present within the cell or e.g. norepinephrine within certain neurons or it may be artificially introduced into the cell as a marker.e.g. administration of antibodies tagged with fluorescent substances. The fluorescent substances will be visible wherever there are antigens

e) DARK FIELD (ILLUMINATION) MICROSCOPE

This type of microscope is appropriate for observing microorganisms suspended in fluid .These method enables clear viewing of microorganisms structures and motility, it makes some living organisms visible which cannot be seen by ordinary transmitted light

In this type of microscope, light enter special condensers, which have a central ‘blacken-out’ area so that light entering cannot pass out directly through it to enter the objective. Instead, the light is reflected to pass through the outer edge of the condenser edge at wide angle so that the only light entering the eyes come from the specimen themselves with no light entering directly from the light source. In this way, specimens are seen brightly illuminated against a black background like stars in a night sky or dust in a shaft of sunlight across a darkened room

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