Trouble Shooting on Microscopes

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Trouble Shooting on Microscopes

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:
• 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

Light source:
• The light source illuminates the sample
• There are a number of different types of illumination each producing its own colour temperature

Condensing system:
• 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.

Light source:
• The light source illuminates the sample
• There are a number of different types of illumination each producing its own colour temperature

Objective lens:
• 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

Eyepiece:
• This lens magnifies the image produced by the objective lens

Now lets look more at the light

Illumination

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.

Light Sources

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:

• Tungsten Halogen
• Xenon Arc
• Quartz – Iodine
• Carbon Arc

Interactions between light and surfaces

Reflection

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.

Absorption

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.

Refraction

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.

Polarisation

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.

Diffraction

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.

Fluorescence

This is the process by which light from shorter wavelengths is absorbed by an object and re emitted as light of longer wavelengths.

Phase Change

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.

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