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PLNT3140 Introductory Cytogenetics
Lecture 1, part 2 of 2

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INTRODUCTION TO THE MICROSCOPE



Typical Compound Microscope

This is the most important tool of the cytologist. Its purpose is to allow observer to see greater detail in small objects which is achieved by means of resolution and magnification.

The principle of a microscope can be summarised as follows:

1. Light is focused to a specific focal plane in the sample, resulting in an image.
2. The image is magnified such that a focused image is projected to a focal plane at the observer's eye.

Magnification. A 10x lens will furnish an image ten times the diameter of the object as it naturally appears when held at a given distance from the eye.

The compound microscope is a combination of two simple microscopes, the objective (the lens closer to the object) and the eyepiece (the lens closer to the eye).

The microscope consists of the following parts (numbers from 1 to 13 refer to the light path) :



3. Fine focusing control and coarse focusing control.

2. Diaphragm insert with luminous field iris diaphragm. Lets you control the amount of light illuminating the sample.

1. Base with built-in 6V 15W low-voltage illumination.

The eyepiece has two functions: to magnify and correct. The eyepiece can be over- or under-corrected. If it is under corrected, a blue ray of light from the objective will be bent in more than a red ray. Looking into the microscope, the blue ray will appear outside the red ray and a blue fringe will be seen around the edge of the eyepiece diaphragm. Overcorrected eyepiece system show a reddish orange fringe around the periphery of the field of view as a red ray of light is bent more than a blue one.

An over-corrected eyepiece may be balanced by an under corrected objective or vice-versa. An eyepiece may show a slight colour fringe around the edge of the diaphragm.

Types of simple lenses

The objective lenses are the most important component of the microscope. Modern objective lenses are parfocal so the image remains visible when objective lenses are switched, and only the fine focus has to be used for optimum sharpness.

There are six simple lenses of two types:



The Six Simple Lenses.  The biconvex, plano-convex and convex (converging) meniscus are positive lenses. The biconcave, plano-concave and concave (diverging) meniscus are negative lenses.

positive which are thicker in the middle and therefore capable of converging light to a focus. These are termed biconvex, plano-convex, and convex (converging) meniscus.

negative which are thinner in the middle so that rays of light passing through them are made divergent termed biconcave, plano-concave, concave (diverging) meniscus

The refraction or bending of rays of light by a lens is the basis of image formation and magnification with a microscope. By changing the thickness, curvature, refractive index and dispersion of the glass of the above six lenses, two or more lenses can be combined to make corrected lens systems.

The condenser combines simple lenses to focus illumination precisely in the specimin

The substage condenser functions to bring concentrated light onto the specimen, bringing the light rays from the illumination source into focus in the plane of the specimen. Without a condenser, the microscope would be a magnifying glass with no resolving power of its own. The student microscopes are fitted with Abbe (chromatic) condensers. It is simply constructed and transmits a large amount of light.

A. Oil Abbe Chromatic, N.A.  1.20 or N.A. 1.40 (Two Lenses).  B. Oil Aplanatic, N.A.  1.40.  C. Dry Achromatic, N.A. 1.00.



As illustrated in the examples, the condenser is designed to converge light from the light source so that the entire field of the specimen is evenly illuminated. Note that the lowermost face of the lowest lens is convex, collecting light from a wide area. The the light path converges to a smaller area as you go up the light path. Concurrently, you should notice that the lenses become more narrow from bottom to top. The upper face of the top lens is always flat, causing the light collected by the lenses to exit the lens in a uniform fashion.

On the microscopes in our lab, the working distance from the top lens of the condenser to the specimen is 1.2-1.6 mm. The slides vary from 0.9-1.5 mm (1.1 mm nominal), so that the focal point of condenser may be within slide or on the specimen. The cover glass thickness is also critical and the microscope is adjusted for the standard 0.17mm. It is therefore important to adjust the focal point for illumination for each slide when you are doing critical work such as photography.

Numerical aperture of the objective

The numerical aperture (NA) is a measure of the resolving power of the objective. The resolving power is determined by the wavelength of the light passing through the microscope, the NA of the objective and the index of visibility or refractive index of the medium which fills the space between the cover glass of the slide on the stage and the front lens of the objective.

resolving power ::= minimum separation of two objects such that they appear distinct and separate when viewed through a microscope or telescope.

Resolving power can never be greater than the wavelength of light used.

The image we see through the eyepiece is the aerial image formed by the microscope objective in the tube. This image has a limit, where useful magnification ends and the empty magnification begins. There is a good parallel with the grain of a photographic film. As soon as the image details reach the same size as the image grain, the details cannot be recognised. In the same way, as you move closer and closer to the photographic image on a projector screen, you reach the point where you can no longer see the actual details on the photograph. The performance limit of the microscope is determined by the NA, so that the total magnification of the microscope (objective magnification multiplied by the eyepiece magnification) cannot exceed 1000 X NA.
 

NA is calculated using a mathematical formula devised by Ernst Abbe for the direct comparison of the resolving power of dry and all types of immersion objectives.

NA =  n sin( µ)

where

n ::= the refractive index of the medium between the front lens of the objective and the cover slip.
When a ray of light passes from a rare medium (air) to a denser medium (oil) it is bent and refracted. Air has a refractive index of 1; immersion oil has a refractive index of 1.5.
µ ::=  the aperture angle defined by the optical axis and the outermost rays still covered by the objective


Thus, the numerical aperture is the sine of half the angular aperture of an objective lens.

Comparison of dry and oil immersion objectives. The values for NA range from 0.1 to 0.95 for dry objectives and up to 1.5 for oil immersion lenses. Air has a refractive index of 1. So for air, the image scatters beyond the aperture angle.  Immersion oil fills the space between the cover glass and the front lens of the microscope has a refractive index of 1.5. Oil keeps the image within the aperture angle of the objective lens.

Angular Apertures of Objectives Compared. The 3x objective is at a longer focal length, taking in a larger area at a smaller angle. The 95x objective is at a shorter focal length, taking in a smaller area in a larger angle.

 

Take home message: By increasing the refractive index, the NA of the objective is also increased. The higher the refractive index, the greater the useful magnification which can be achieved with the microscope objective ie., the greater the resolving power.


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previous  page

PLNT3140 Introductory Cytogenetics
Lecture 1, part 2 of 2

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