Microscopes are easy to use because any child can do it, and other myths will be exposed.
What is light?
Light is a form of electromagnetic radiation covering wavelengths between 400 and 700nm. (that is 4 x 10-7 and 7 x 10-7m)Very small numbers! The overall spectrum covers wavelengths between 10-12 and 103 metres. Other wavelengths cover Gamma Rays, X-Rays, Ultra Violet, Infra Red, and Radio waves.
What is a microscope?
It is an instrument that is used to magnify objects
• To allow examination of items that cannot be seen by the naked eye
• To look at small features on surfaces
It consists of:
• An objective lens that magnifies the object
• An eyepiece that magnifies the image produced by the objective
• Other parts to assist in illumination that will be coved later
The metallurgical or compound microscope:
• Metals are not transparent so we cannot shine light through them to examine the surfaces
• Uses a half silvered mirror to transfer light to the surface of the object and then transfer the light back to the eyepiece
• There are a vast number of types of metallurgical microscope but they all follow the same basic principle
Components of the microscope
• The light source illuminates the sample.
• There are a number of different types of illumination each producing its own colour temperature
• Field diaphragm that is placed in front of the condensing lens to minimise glare
• Condensing lens to image the light at a position in the optical system
• Aperture diaphragm alters the amount of light and the angle of the cone entering the objective lens
Half mirrored glass
• This reflects light from the condensing system onto the sample and allows reflected light from the sample to enter the eyepiece
• In both cases approximately 50% of the available light is utilised.
• This focuses the light onto the sample and then gathers the reflected light that in turn is transmitted to the eyepiece and is the most important part of the microscope.
• This lens magnifies the image produced by the objective lens.
Now lets look more at the light!
Illumination is by both transmitted and reflected light although reflected light is used most frequently in the stereomicroscope and macroscope as they are mainly used for examination of larger solid specimens.
• For reflected light almost any light source can be used depending upon the quality and magnification of the image.
• For higher quality images both the intensity and angle of the light source should be adjusted to the optimum levels.
• Flexible fibre optic sources are easily adjustable for angle, can be used with multiple sources and convey little heat to the specimen.
The light interacts with surfaces in a number of different ways that if not utilised correctly can reduce the quality of the image but if understood and used correctly can produce quality images.
Originally reflected ambient light was used for the light source which was super ceded by the use of standard lighting with reflectors. This type of lighting was not very efficient producing a lot of heat at higher lighting levels.
Both external and internal light sources can be used.
• External – Can use one or a number of diaphragms to control the illuminated field. Alternatively a focusable collector lens can be used
• Internal – The lamps can be pre-aligned which does not allow any movement, or in a pre centred holder which can allow movement which facilitates focussing of the image in the front focal plane of the condenser.
The light sources were generally tungsten filament lamps with a transformer controlling the light output that produced a light of 300 to 1500nm wavelength giving a light in the red region.
• These have been replaced by tungsten halogen that have a more constant light output throughout their lives and when operated at a colour temperature of 3200 – 3250 K emit a continuous colour spectrum.
• These lamps operate at a high temperature and need heat filters and a ventilated light box.
• Special discharge lamps can be used to produce spectra at specific wavelengths that can be used to produce fluorescence in specimens.
There are a number of different types of illumination that can be used each with its own advantages and disadvantages. These include:
Quartz – Iodine
Interactions between light and surfaces
• Light striking a perfectly smooth surface the angle of reflection is equal to the angle of incidence giving specular reflection.
• In practice few surfaces are perfectly smooth with any incident light being reflected in a number of directions.
• The severity of the dispersion of the light is dependant upon the roughness of the object.
• Additionally the absorption of some wavelengths can affect the colour of the reflected light giving a means of, for example, identifying phases in metals
• This is the process by which the amplitude of light is reduced as it passes through the object.
• This phenomenon is mainly used in biological microscopy where the absorption of light of different wavelengths is used in identification of objects.
• Most biological cells and tissues are transparent requiring staining before examination.
The metallurgical microscope does not utilise transmitted light
• Occurs when light passes from one medium into another of a different refractive index for example from air into glass.
• The refractive index is the ratio of the phase velocity of light in a vacuum to the phase velocity in the medium.
• The difference in the velocity of the light causes the light to change angle (essentially bend) at the interface between the two media – the effect is governed by Snell’s Law:-
I x n1 = sinR x n2
Where I and R are the angles of incidence and refraction and n1 and n2 the refractive indices of the two media.
• Refraction is essential for the operation of glass lenses and hence essential for microscopy.
• Light generally contains waves of many different wavelengths with various phase differences vibrating in all possible planes.
• If the vibration can be restricted to one particular plane the light is termed as plane polarised.
• The speed of light differs from light in one plane and that in another plane at 90 degrees, hence there are two refractive indices and such materials are termed as birefringent.
• The scattering of light at the edge of an object that is related to the wavelength of the light.
• This is larger for longer wavelengths.
• Diffraction causes a halo of rings around a single hole and hence it limits resolution for it is not possible to produce an absolutely sharp image of an object.
• This is the process by which light from shorter wavelengths is absorbed by an object and re emitted as light of longer wavelengths.
• The difference between two paths of light, one that has gone around the object and one that has travelled through it is called the phase change or optical path difference.
• This results in waves of light that can be in phase or out of phase by various degrees.
• If the waves are in phase the light will be at it’s strongest and if 90 degrees out of phase the waves cancel resulting in cancellation. The figures below show this effect.
Source Focussed and Köhler Illumination
Control of the illumination of the specimen is essential as overlighting of the specimen can produce glare that reduces image quality.
• In source focussed illumination an opal lamp, field and aperture irises and a condenser lens are used to focus the light onto the specimen surface.
• Tungsten halogen lamps have an exposed visible filament that does not allow source focussed illumination.
• In Köhler illumination the image of the filament is focussed onto the front focal plane of the condenser where the iris is located.
• This focus is repeated at other conjugate planes in the aperture series up to the eyepiece.
• This type of illumination is not used for solid metallurgical samples and is generally used for biological and histological specimens.
• At its simplest this type of illumination can simply use external oblique lighting although this is limited to low magnifications.
• When used in specially designed microscopes the light source is mounted at 90 degrees to the optical plane and is focussed onto the specimen through the objective lens using a partially silvered mirror mounted at 45 degrees.
Resolution and Magnification
Resolution and magnification are often confused:
• Resolution – This is the minimum resolved distance i.e. the least separation between two points which can be distinguished as separate.
• Magnification – The increase in the apparent size of the object of the object.
• Although it is possible to magnify images to high levels (10,000 times) this is of little use if the eye cannot resolve the image.
• The actual eye can only resolve items when the points on the image are separated by above 1 minute of arc.
• Although different wavelengths of light are used to enhance the image the practical resolution is limited to approximately 0.25µm.