PLNT3140 Introductory
Cytogenetics |

**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 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) :

13. Eyepiece which is slipped into the upper end of the tube. The engraved magnification indicates the magnification of the eyepiece alone.

12. The tube which has a standard length of 160 mm.

11. The revolving nosepiece for a quick exchange of objective lenses.

10. The objective lenses, each of which have a magnification engraved on the side. The initial magnification of the objective multiplied by the magnification of the eyepiece gives the total magnification in diameters, providing the draw-tube of the microscope is set at the standard mechanical tube length of 160mm (Leiz 170 mm). Example: Plan 40/0.65 160/0.17 - describes the 4x by 10x Planachromat lenses, and the numerical aperture (NA) 0.65, which will be explained later, computed for a tube length of 160 mm and 0.17 mm cover-glass thickness.

9. Specimen stage which holds the slide

8. Controls for
moving stage on X and Y axes.

7. Iris lever
controlling the condenser or aperture diaphragm.

6. Condenser - focuses light to control the area of the specimen that is illuminated.

5. Knobs which control the condenser centering.

4. Swing-out holders
for filters and auxiliary lens

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.

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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PLNT3140 Introductory
Cytogenetics |